NZ755780B2

NZ755780B2 - Rna cancer vaccines

Info

Publication number: NZ755780B2
Application number: NZ755780A
Authority: NZ
Inventors: Ted Ashburn; Kristen Hopson; Nicholas Valiante
Original assignee: Modernatx Inc
Filing date: 2017-10-26
Publication date: 2024-01-30

Abstract

The disclosure relates to cancer ribonucleic acid (RNA) vaccines, as well as methods of using the vaccines and compositions comprising the vaccines. In particular, the disclosure relates to concatemeric mRNA cancer vaccines encoding several cancer epitopes on a single mRNA construct, i.e. poly-epitope mRNA constructs or poly-neo-epitope constructs. The disclosure further relates to p53 and KRAS mutations, as well as incorporation of immune enhancers such as STING, e.g. mRNA constructs further encoding an immune stimulator or adjuvant. The disclosure further relates to inclusion of universal T cell epitopes, such as tetanus or diphtheria toxins to elicit an enhanced immune response.

Description

RNA CANCER VACCINES RELATED APPLICATIONS This application claims the bene?t under 35 U.S.C. 119(e) of the ?ling date of US. ional Application Serial Number 62/453,444, ?led February 1, 2017, entitled "RNA CANCER VACCINES", ofUS. Provisional Application Serial Number 62/453,465, ?led February 1, 2017, entitled NOMODULATORY THERAPEUTIC MRNA COMPOSITIONS ENCODING ACTIVATING ONCOGENE ON PEPTIDES", and of US. Provisional Application Serial Number 62/558,23 8, ?led September 13, 2017, entitled "CONCATAMERIC RNA CANCER VACCINES", the entire contents of each of which are incorporated herein by nce.

BACKGROUND OF INVENTION Recent theories in cancer evolution have focused on three steps including stress- induced genome instability, tion diversity or heterogeneity, and genome-mediated macroevolution. The theory explains why most of the known molecular mechanisms can bute to cancer yet there is no single dominant ism for the majority of al cases. However, the common mechanisms suggest that cancer vaccines may provide a universal solution in the treatment of cancer. 2O Cancer vaccines include tive or prophylactic vaccines, which are intended to prevent cancer from developing in healthy people, and therapeutic vaccines, which are ed to treat an eXisting cancer by strengthening the body’s natural defenses against the cancer. Cancer preventive es may, for instance, target infectious agents that cause or contribute to the development of cancer in order to prevent infectious diseases from causing cancer. Gardasil® and CervariX®, are two examples of commercially available prophylactic vaccines. Each vaccine protects against HPV infection. Other preventive cancer vaccines may target host ns or fragments that are predicted to increase the likelihood of an individual developing cancer in the future.

Most commercial or developing es (e.g., cancer vaccines) are based on whole microorganisms, protein antigens, peptides, polysaccharides or deoxyribonucleic acid (DNA) vaccines and their combinations. DNA vaccination is one technique used to stimulate humoral and cellular immune ses to antigens. The direct injection of genetically engineered DNA (e.g., naked plasmid DNA) into a living host results in a small number of its cells directly producing an antigen, resulting in a protective immunological response.

With this technique, however, comes potential problems ofDNA integration into the vaccine’s genome, including the possibility of insertional mutagenesis, which could lead to the activation of oncogenes or the inhibition of tumor suppressor genes.

SUMMARY OF INVENTION Provided herein is a cleic acid (RNA) cancer vaccine of an RNA (e.g., messenger RNA (mRNA)) that can safely direct the body’s cellular machinery to produce nearly any cancer protein or nt thereof of interest. In some embodiments, the RNA is a modi?ed RNA. The RNA vaccines of the present disclosure may be used to induce a balanced immune response against cancers, comprising both cellular and humoral immunity, without risking the possibility of insertional mutagenesis, for example.

The RNA vaccines may be utilized in various settings ing on the prevalence of the cancer or the degree or level of unmet medical need. The RNA es may be utilized to treat and/or prevent a cancer of various stages or degrees of metastasis. The RNA vaccines have superior properties in that they produce much larger antibody titers and produce responses earlier than ative anti-cancer therapies including cancer vaccines. While not wishing to be bound by theory, it is believed that the RNA vaccines, as mRNA polynucleotides, are better designed to produce the appropriate protein conformation upon ation as the RNA vaccines co-opt natural cellular machinery. Unlike traditional therapies and vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the RNA vaccines are presented to the ar system in a more native fashion.

The RNA vaccines may include a ribonucleic acid (RNA) polynucleotide having an open g frame encoding at least one cancer antigenic polypeptide or an genic fragment thereof (e.g., an immunogenic fragment e of inducing an immune response to cancer). Other embodiments include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame ng two or more antigens or epitopes capable of inducing an immune response to .

The invention in some aspects is an mRNA cancer vaccine of one or more mRNA each having an open reading frame encoding a cancer antigen peptide epitope formulated in a lipid nanoparticle, n the mRNA vaccine encodes 5-100 peptide epitopes and at least two of the peptide epitopes are personalized cancer ns, and a pharmaceutically acceptable carrier or excipient.

The disclosure, in some aspects, provides an mRNA cancer vaccine comprising a lipid nanoparticle sing one or more mRNA each having one or more open reading frames encoding 1-500 peptide epitopes which are personalized cancer antigens and a universal type II T-cell epitope.

The sure, in some aspects, provides an mRNA cancer vaccine comprising a lipid nanoparticle comprising one or more of the following: (a) one or more mRNA each having one or more open reading frames encoding 1-500 peptide epitopes which are personalized cancer antigens and a universal type II T-cell e, (b) one or more mRNA each having an open reading frame encoding an activating oncogene mutation peptide, optionally wherein the mRNA further comprises a universal type II T-cell epitope, (c) one or more mRNA each having an open reading frame encoding a cancer antigen peptide epitope, wherein the mRNA vaccine encodes 5-100 peptide es and at least two of the peptide epitopes are personalized cancer antigens, optionally wherein the mRNA further ses a universal type II T-cell epitope, and/or (d) one or more mRNA each having an open g frame encoding a cancer antigen e epitope, wherein the mRNA vaccine encodes 5-100 e es and at least three of the peptide epitopes are compleX variants and at least two of the peptide epitopes are point ons, optionally wherein the mRNA further ses a universal type II T-cell epitope.In some embodiments, the mRNA cancer vaccine encodes 1-20 universal type II T-cell epitopes. In other embodiments, the universal type II T- cell e is selected from the group consisting of: ILMQYIKANSKFIGI (Tetanus toxin, SEQ ID NO: 226), FNNFTVSFWLRVPKVSASHLE, (Tetanus toxin, SEQ ID NO: 227), 2O QYIKANSKFIGITE (Tetanus toxin, SEQ ID NO: 228) QSIALSSLMVAQAIP (Diptheria toxin, SEQ ID NO: 229), and AKFVAAWTLKAAA (pan-DR epitope, SEQ ID NO: 230).

In some embodiments, the universal type II T-cell epitope is the same universal type II T-cell epitope throughout the mRNA. In other emebodiments, the universal type II T-cell epitope is repeated 1-20 times in the mRNA. In one embodiment, the universal type II T-cell epitopes are different from one another throughout the mRNA. In some embodiments, the universal type II T-cell epitope is located n every cancer antigen peptide epitope. In another embodiment, the universal type II T-cell epitope is located between every other cancer antigen peptide epitope. In one embodiment, the universal type II T-cell epitope is located between every third cancer antigen peptide epitope.

In some embodiments, one or more of the following conditions are met: (i) the activating ne mutation is a KRAS mutation, (ii) the KRAS mutation is a G12 mutation, optionally n the G12 KRAS mutation is selected from a Gl2D, Gl2V, G12S, G12C, G12A, and a G12R KRAS mutation, (iii) the KRAS mutation is a G13 mutation, optionally n the G13 KRAS mutation is a G13D KRAS on; and/or (iv) the activating oncogene mutation is a H-RAS or N—RAS mutation.

In some ments, one or more of the following conditions are met: (A) the mRNA has an open reading frame encoding a concatemer of two or more activating oncogene mutation peptides; (B) at least two of the peptide epitopes are separated from one another by a single Glycine, optionally wherein all of the peptide epitopes are separated from one another by a single Glycine, (C) the concatemer comprises 3-10 activating oncogene mutation peptides, and/or (D) at least two of the peptide epitopes are linked directly to one another t a linker.

In certain embodiments, one or more of the following conditions are met: (i) at least one of the peptide epitopes is a ional cancer n, (ii) at least one of the e epitopes is a recurrent polymorphism, (iii) the recurrent polymorphism comprises a recurrent somatic cancer mutation in p53, (iv) the recurrent somatic cancer mutation in p53 is selected from the group consisting of: (A) mutations at the canonical 5’ splice site neighboring codon p.T125, inducing a retained intron having peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) that contains epitopes AVSPCISFVW (SEQ ID NO: 233) (HLA-B*57:Ol, HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) (HLA-A*02:01, 02:O6, HLA-B*35:01), (B) 2O ons at the canonical 5’ splice site neighboring codon p.33 1, inducing a retained intron having peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) that ns epitopes LQVLSLGTSY (SEQ ID NO: 237) (HLA-B*15:Ol), FQSNTQNAVF (SEQ ID NO: 23 8) (HLA-B*15:Ol), (C) mutations at the canonical 3’ splice site neighboring codon p. 126, inducing a c alternative exonic 3’ splice site producing the novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 239) that contains epitopes CTMFCQLAK (SEQ ID NO: 240) (HLA-A*1 1 :01), KSVTCTMF (SEQ ID NO: 241) (HLA- B*58:Ol), and/or (D) mutations at the canonical 5’ splice site neighboring codon p.224, inducing a cryptic alternative intronic 5’ splice site producing the novel spanning peptide ce VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) that contains epitopes VPYEPPEVW (SEQ ID NO: 243) (HLA-B*53:Ol, HLA-B*5l:Ol), LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:Ol, HLA-B*57 :01), wherein the transcript codon positions refer to the canonical full-length p53 transcript ENSTOOOOO269305 (SEQ ID NO: 245) from the l V83 human genome annotation; and/or (V) the mRNA cancer vaccine does not comprise a izing agent.

In some embodiments, the one or more mRNA further comprise an open reading frame encoding an immune potentiator. In other embodiments, the immune potentiator is formulated in the lipid nanoparticle. In one embodiment, the immune potentiator is formulated in a separate lipid rticle. In some ments, the immune potentiator is a constitutively active human STING polypeptide. In one embodiment, the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1. In another embodiment, the mRNA encoding the constitutively active human STING polypeptide comprises the tide sequence shown in SEQ ID NO: 170. In some embodiments, the mRNA encoding the tutively active human STING polypeptide comprises a 3’ UTR having a miR-122 microRNA binding site. In one ment, the miR-122 microRNA binding site comprises the nucleotide sequence shown in SEQ ID NO: In some embodiments, the one or more mRNA each comprise a 5’ UTR comprising the nucleotide ce set forth in SEQ ID NO: 176. In one embodiment, the one or more mRNA each comprise a poly A tail. In one embodiment, the poly A tail comprises about 100 nucleotides. In some embodiments, the one or more mRNA each comprise a 5’ Cap 1 structure.

In some embodiments, the one or more mRNA comprise at least one chemical modification. In one embodiment, the chemical modi?cation is Nl-methylpseudouridine. In another embodiment, the one or more mRNA is fully modi?ed with N1- methylpseudouridine.

In some embodiments, the one or more mRNA encode 45-55 alized cancer antigens. In one embodiment, the one or more mRNA encode 52 personalized cancer ns. In some embodiments, each of the personalized cancer antigens is encoded by a separate open reading frame. In another embodiment, the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 2-100 peptide epitopes, optionally wherein the concatemeric cancer antigen is comprised of 5-100 peptide epitopes.

In some embodiments, the concatemeric cancer antigen comprises one or more of: a) the 2-100 peptide es, or the 5-100 peptide epitopes, are interspersed by cleavage sensitive sites, b) the mRNA encoding each peptide epitope is linked directly to one another without a linker, c) the mRNA encoding each e epitope is linked to one or another with a single tide linker, d) each peptide epitope comprises 25-35 amino acids and includes a centrally located SNP mutation; e) at least 30% of the peptide epitopes have a t af?nity for class IMHC molecules from a t; f) at least 30% of the peptide epitopes have a highest af?nity for class II MHC molecules from a subject; g) at least 50% of the peptide epitopes have a predicated binding af?nity of IC >500nM for HLA-A; HLA-B and/or DRBl; h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) 50% of the peptide epitopes have a binding af?nity for class I MHC and 50% of the peptide epitopes have a binding af?nity for class II MHC; k) the mRNA encoding the peptide epitopes is arranged such that the peptide epitopes are ordered to minimize pseudoepitopes ; l) at least 30% of the e es are class I MHC binding peptides of 15 amino acids in length; and/or m) at least 30% of the peptide epitopes are class II MHC binding peptides of 21 amino acids in .

In some aspects; the disclosure es an mRNA cancer vaccine comprising one or more mRNA each having one or more open reading frames encoding 45-55 peptide epitopes which are personalized cancer antigens ated in a lipid nanoparticle.

In some aspects; the disclosure provides an mRNA cancer vaccine; comprising one or more mRNA each having one or more open reading frames encoding 45-55 peptide epitopes which are personalized cancer antigens formulated in a lipid nanoparticle; optionally wherein at least one of the peptide es is an activating oncogene mutation peptide or a traditional cancer antigen; and optionally n at least three of the peptide epitopes are compleX 2O variants and at least two of the e epitopes are point mutations.

In some embodiments; the one or more mRNA encode 48-54 personalized cancer antigens. In one embodiment; the one or more mRNA encode 52 personalized cancer antigens. In some embodiments; each of the personalized cancer antigens is d by a separate open reading frame.

In another embodiment; the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 2-100 peptide es; ally wherein the concatemeric cancer antigen is comprised of 5-100 peptide epitopes. In some embodiments; the concatemeric cancer antigen ses one or more of: a) the 2-100 peptide epitopes; or the 5-100 peptide epitopes; are interspersed by cleavage sensitive sites; b) the mRNA encoding each peptide epitope is linked directly to one another without a linker; c) the mRNA encoding each peptide epitope is linked to one or another with a single nucleotide linker; d) each peptide epitope comprises 25-35 amino acids and es a centrally located SNP mutation; e) at least 30% of the peptide epitopes have a highest af?nity for class I MHC molecules from a t; f) at least 30% of the peptide epitopes have a highest af?nity for class II MHC molecules from a subject; g) at least 50% of the peptide epitopes have a predicated binding af?nity of IC >500nM for HLA-A, HLA-B and/or DRBl, h) the mRNA encodes 45-55 peptide epitopes, i) the mRNA s 52 peptide epitopes, j) 50% of the peptide epitopes have a binding af?nity for class IMHC and 50% of the e epitopes have a binding af?nity for class II MHC, k) the mRNA encoding the peptide epitopes is arranged such that the peptide epitopes are ordered to minimize pseudo-epitopes, l) at least 30% of the peptide epitopes are class I MHC binding es of 15 amino acids in length; and/or m) at least 30% of the peptide epitopes are class II MHC binding peptides of 21 amino acids in length.

In some embodiments, at least two of the peptide epitopes are separated from one another by a universal type II T-cell epitope. In one embodiment, all of the peptide epitopes are ted from one another by a universal type II T-cell epitope. In another embodiment, the mRNA cancer vaccine s 1-20 universal type II T-cell epitopes.

In some embodiments, the universal type II T- cell epitope is selected from the group consisting of: ILMQYIKANSKFIGI (Tetanus toxin, SEQ ID NO: 226), FNNFTVSFWLRVPKVSASHLE, (Tetanus toxin, SEQ ID NO: 227), QYIKANSKFIGITE (Tetanus toxin, SEQ ID NO: 228) QSIALSSLMVAQAIP (Diptheria toxin, SEQ ID NO: 229), and AKFVAAWTLKAAA (pan-DR epitope, SEQ ID NO: 23 O).

In one ment, the universal type II T-cell e is the same universal type II T-cell epitope throughout the mRNA. In some embodiments, the universal type II T-cell 2O e is repeated 1-20 times in the mRNA. In another embodiment, the universal type II T- cell epitopes are different from one another throughout the mRNA. In one embodiment, the universal type II T-cell epitope is located between every peptide epitope. In some embodiments, the universal type II T-cell epitope is located between every other peptide epitope. In one embodiment, the universal type II T-cell epitope is located between every third e epitope.

In some embodiments, the one or more mRNA r comprise an open reading frame encoding an immune potentiator. In one embodiment, the immune iator is formulated in the lipid rticle. In another embodiment, the immune iator is formulated in a separate lipid nanoparticle. In some embodiments, the immune potentiator is a constitutively active human STING polypeptide. In one embodiment, the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1. In r embodiment, the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 170.

In some embodiments, one or more of the following conditions are met: (i) the activating ne on is a KRAS mutation; (ii) the KRAS mutation is a G12 mutation, optionally wherein the G12 KRAS mutation is ed from a G12D, G12V, G12S, G12C, G12A, and a G12R KRAS mutation, (iii) the KRAS mutation is a G13 mutation, optionally wherein the G13 KRAS mutation is a G13D KRAS mutation, and/or (iv) the activating oncogene mutation is a H-RAS or N—RAS mutation.

In certain embodiments, one or more of the following conditions are met: (A) the mRNA has an open g frame encoding a concatemer of two or more activating oncogene mutation peptides, (B) at least two of the peptide es are separated from one r by a single e, optionally wherein all of the peptide epitopes are separated from one another by a single Glycine, (C) the concatemer comprises 3-10 activating ne mutation peptides, and/or (D) at least two of the e epitopes are linked directly to one another without a linker.

In speci?c ments, one or more of the following conditions are met: (i) at least one of the peptide es is a traditional cancer antigen, (ii) at least one of the peptide epitopes is a recurrent polymorphism, (iii) the recurrent polymorphism comprises a recurrent somatic cancer mutation in p53, (iv) the recurrent somatic cancer mutation in p53 is selected from the group consisting of: (A) mutations at the canonical 5’ splice site neighboring codon p.T125, inducing a retained intron having peptide sequence 2O TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) that contains epitopes AVSPCISFVW (SEQ ID NO: 233) (HLA-B*57:01, HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) (HLA-A*02:01, HLA-A*02:O6, HLA-B*35:01), (B) mutations at the canonical 5’ splice site neighboring codon p.331, inducing a retained intron having peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) that contains epitopes LQVLSLGTSY (SEQ ID NO: 237) (HLA-B*15:01), FQSNTQNAVF (SEQ ID NO: 23 8) (HLA-B*15:01), (C) mutations at the canonical 3’ splice site neighboring codon p. 126, ng a cryptic alternative exonic 3’ splice site producing the novel spanning peptide ce AKSVTCTMFCQLAK (SEQ ID NO: 239) that contains epitopes LAK (SEQ ID NO: 240) (HLA-A*1 1 :01), KSVTCTMF (SEQ ID NO: 241) (HLA- B*58:01), and/or (D) mutations at the canonical 5’ splice site neighboring codon p.224, inducing a cryptic alternative intronic 5’ splice site producing the novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) that contains epitopes EVW (SEQ ID NO: 243) (HLA-B*53:Ol, HLA-B*5l:Ol), LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:Ol, HLA-B*57 :01), wherein the transcript codon positions refer to the canonical full-length p53 transcript ENSTOOOOO269305 (SEQ ID NO: 245) from the Ensembl V83 human genome annotation; and/or (V) the mRNA cancer vaccine does not comprise a stabilizing agent.

Another aspect of the present disclosure is an mRNA cancer vaccine, comprising a lipid nanoparticle comprising (i) one or more mRNA each having one or more open reading frames encoding 1-500 peptide epitopes which are personalized cancer antigens, and (ii) an mRNA having an open reading frame encoding a polypeptide that enhances an immune response to the personalized cancer antigens, optionally wherein (i) and (ii) are present at mass ratio of approximately 5: 1.

Another aspect of the present disclosure is an mRNA cancer vaccine, comprising: a lipid nanoparticle comprising: (i) one or more mRNA each having one or more open reading frames encoding 1-500 e epitopes which are personalized cancer antigens, and (ii) an mRNA having an open g frame encoding a ptide that enhances an immune response to the alized cancer antigens, optionally wherein (i) and (ii) are present at mass ratio of approximately 5: l, optionally wherein at least one of the peptide es is an activating ne mutation peptide or a traditional cancer antigen, and optionally wherein at least three of the peptide epitopes are compleX variants and at least two of the peptide 2O epitopes are point mutations.

In some embodiments, the immune response comprises a cellular or humoral immune se characterized by: (i) stimulating Type I interferon pathway signaling, (ii) stimulating NFkB pathway signaling, (iii) stimulating an in?ammatory response, (iv) stimulating cytokine production, or (v) ating tic cell development, activity or mobilization, and (vi) a combination of any of (i)-(vi).

In one embodiment, the mRNA cancer e comprises a single mRNA construct encoding both the peptide epitopes and the ptide that enhances an immune response to the personalized cancer antigens. In another ment the peptide epitopes are in the form of a concatemeric cancer antigen comprised of 2-100 peptide es, optionally wherein the concatemeric cancer antigen is comprised of 5-100 peptide epitopes.

In some ments, the concatemeric cancer antigen comprises one or more of: a) the 2-100 peptide epitopes, or the 5-100 peptide epitopes, are interspersed by cleavage sensitive sites, b) the mRNA encoding each peptide epitope is linked directly to one another without a linker, c) the mRNA encoding each peptide epitope is linked to one or another with a single nucleotide linker; d) each peptide epitope ses 25-35 amino acids and includes a centrally located SNP mutation; e) at least 30% of the e es have a highest af?nity for class IMHC molecules from a subject; f) at least 30% of the peptide epitopes have a highest af?nity for class II MHC molecules from a t; g) at least 50% of the peptide es have a predicated binding af?nity of IC >500nM for HLA-A; HLA-B and/or DRBl; h) the mRNA encodes 45-55 peptide epitopes; i) the mRNA encodes 52 peptide epitopes; j) 50% of the peptide epitopes have a binding af?nity for class I MHC and 50% of the peptide epitopes have a binding af?nity for class II MHC; k) the mRNA encoding the peptide epitopes is arranged such that the peptide epitopes are ordered to minimize pseudo- es; l) at least 30% of the peptide epitopes are class I MHC binding peptides of 15 amino acids in length; and/or m) at least 30% of the peptide epitopes are class II MHC g peptides of 21 amino acids in length.

In some embodiments; each peptide epitope comprises a centrally located SNP mutation with 15 ?anking amino acids on each side of the SNP mutation.

In one embodiment; the polypeptide that enhances an immune response to at least one personalized cancer antigens in a subject is a constitutively active human STING polypeptide. In one embodiment; the constitutively active human STING polypeptide comprises one or more mutations selected from the group consisting of V147L; N154S; V155M; R284M; R284K; R284T; E315Q; R375A; and combinations thereof. In another 2O embodiment; the constitutively active human STING polypeptide comprises a V155M mutation. In another embodiment; the tutively active human STING ptide comprises mutations R284M/V147L/N154S/V155M.

In some embodiments; each mRNA is formulated in the same or different lipid rticle. In another embodiment; each mRNA encoding a cancer alized cancer antigens is formulated in the same or different lipid nanoparticle. In some embodiments; each mRNA encoding a polypeptide that enhances an immune response to the personalized cancer antigens is formulated in the same or ent lipid nanoparticle.

In some embodiments; each mRNA encoding a personalized cancer antigen is formulated in the same lipid nanoparticle; and each mRNA encoding a polypeptide that enhances an immune response to the personalized cancer antigen is formulated in a different lipid nanoparticle. In another embodiment; each mRNA encoding a personalized cancer n is formulated in the same lipid nanoparticle; and each mRNA encoding a polypeptide that enhances an immune response to the personalized cancer antigen is formulated in the same lipid nanoparticle as each mRNA encoding a personalized cancer antigen. In some embodiments, each mRNA ng a personalized cancer antigen is formulated in a different lipid nanoparticle, and each mRNA encoding a ptide that enhances an immune response to the personalized cancer antigen is formulated in the same lipid rticle as each mRNA encoding each personalized cancer antigen.

In some embodiments, the peptide epitopes are T cell epitopes and/or B cell epitopes.

In other embodiments, the peptide epitopes comprise a combination of T cell epitopes and B cell epitopes. In one embodiment, at least 1 of the peptide epitopes is a T cell epitope. In another embodiment, at least 1 of the e epitopes is a B cell epitope.

In some embodiments, the peptide epitopes have been optimized for binding strength to a MHC of the subject. In other embodiments, a TCR face for each epitope has a low similarity to endogenous proteins.

In another embodiment, the mRNA cancer vaccine further comprises a recall antigen.

In some embodiments, the recall antigen is an infectious disease antigen.

In one embodiment, the mRNA cancer vaccine further comprises an mRNA having an open reading frame encoding one or more traditional cancer ns.

In one embodiment, one or more of the following conditions are met: (i) the activating oncogene mutation is a KRAS mutation, (ii) the KRAS mutation is a G12 mutation, optionally wherein the G12 KRAS mutation is selected from a G12D, G12V, G12S, Gl2C, G12A, and a G12R KRAS mutation, (iii) the KRAS mutation is a G13 mutation, optionally 2O wherein the G13 KRAS mutation is a G13D KRAS mutation, and/or (iv) the ting oncogene mutation is a H-RAS or N—RAS on.

In one embodiment, one or more of the following conditions are met: (A) the mRNA has an open reading frame ng a concatemer of two or more activating oncogene on peptides, (B) at least two of the peptide epitopes are separated from one another by a single Glycine, optionally wherein all of the e es are separated from one another by a single Glycine, (C) the emer comprises 3-10 activating oncogene mutation peptides, and/or (D) at least two of the peptide epitopes are linked directly to one another without a linker.

In one embodiment, one or more of the following conditions are met: (i) at least one of the peptide es is a traditional cancer antigen, (ii) at least one of the e epitopes is a recurrent polymorphism, (iii) the recurrent polymorphism comprises a recurrent somatic cancer mutation in p53, (iv) the recurrent c cancer mutation in p53 is selected from the group consisting of: (A) mutations at the canonical 5’ splice site neighboring codon p.T125, inducing a retained intron having peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) that ns epitopes AVSPCISFVW (SEQ ID NO: 233) (HLA-B*57 :01 7 HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) (HLA-A*02:01, HLA-A*02:06, HLA-B*35:01), (B) mutations at the canonical 5’ splice site neighboring codon p.331, inducing a retained intron having peptide sequence YFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) that contains epitopes LQVLSLGTSY (SEQ ID NO: 237) (HLAB *15:01), FQSNTQNAVF (SEQ ID NO: 238) (HLA-B*15:01), (C) mutations at the canonical 3’ splice site neighboring codon p. 126, ng a cryptic alternative exonic 3’ splice site producing the novel ng peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 239) that contains epitopes CTMFCQLAK (SEQ ID NO: 240) (HLA-A*11:01), KSVTCTMF (SEQ ID NO: 241) (HLA-B*58:01), and/or (D) mutations at the canonical 5’ splice site neighboring codon p.224, inducing a cryptic ative intronic 5’ splice site producing the novel spanning peptide ce VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) that contains epitopes EVW (SEQ ID NO: 243) (HLA-B*53 :01, HLA- B*51:01), LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:01, HLA-B*57:01), wherein the transcript codon positions refer to the canonical full-length p53 transcript ENST000002693 05 (SEQ ID NO: 245) from the Ensembl V83 human genome annotation, and/or (V) the mRNA cancer vaccine does not comprise a stabilizing agent.

In some embodiments, the lipid nanoparticle ses a molar ratio of about 20-60% ionizable amino lipid: 5-25% neutral lipid: 25-55% sterol, and 05-15% PEG-modi?ed lipid, optionally wherein the ionizable amino lipid is a cationic lipid. In one embodiment, the lipid rticle comprises a molar ratio of about 50% compound 25: about 10% DSPC: about 38.5% cholesterol, and about 1.5% PEG-DMG. In another embodiment, the ble amino lipid is selected from the group consisting of for example, 2,2-dilinoleyl dimethylaminoethyl-[1,3]—dioxolane (DLin-KC2-DMA), dilinoleyl-methyl dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-nonenyl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the lipid rticle comprises a compound of Formula (I). In one embodiment, the compound of Formula (I) is Compound 25. In another embodiment, the lipid nanoparticle has a polydispersity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH value.

In one embodiment, a TCR face for each epitope has a low rity to endogenous proteins.

In another embodiment, the mRNA further comprises an open g frame encoding an immune checkpoint modulator. In one embodiment, the mRNA cancer vaccine further comprises an additional cancer therapeutic agent; optionally wherein the additional cancer therapeutic agent is an immune checkpoint modulator. In another embodiment, the immune checkpoint modulator is an tory checkpoint ptide. In some embodiments, the inhibitory oint polypeptide inhibits PDl, PD-Ll, CTLA4, THVI—3, VISTA, AZAR, B7- H3, B7-H4, BTLA, IDO, KIR, LAG3, or a ation thereof.

In some embodiments, the checkpoint inhibitor polypeptide is an antibody. In one ment, the inhibitory checkpoint ptide is an antibody selected from an anti- CTLA4 antibody or antigen-binding fragment thereof that speci?cally binds CTLA4, an anti- PDl antibody or antigen-binding fragment thereof that speci?cally binds PDl, an anti-PD-Ll antibody or antigen-binding fragment thereof that speci?cally binds PD-Ll, and a combination thereof. In one embodiment, the checkpoint inhibitor polypeptide is an anti-PD- Ll antibody selected from izumab, avelumab, or durvalumab. In another embodiment, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody ed from tremelimumab or umab. In some embodiments, the checkpoint inhibitor polypeptide is an anti-PDl antibody selected from nivolumab or pembrolizumab.

In some embodiments, the chemical modi?cation is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- methylcytosine, -l -methyldeaza-pseudouridine, 2-thio- l -methyl-p seudouridine, 2- thioaza-uridine, -dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxythio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, yluridine, 5- methyluridine, 5-methoxyuridine, and 2’-O-methyl uridine.

The present disclosure, in another aspect, provides a method for vaccinating a subject, comprising administering to a subject having cancer the mRNA cancer vaccine described above.

In some embodiments, the mRNA vaccine is administered at a dosage level suf?cient to deliver between 10 ug and 400 ug of the mRNA vaccine to the subject. In one embodiment, the mRNA vaccine is administered at a dosage level ent to deliver 0.033mg, O. lmg, 0.2 mg, or 0.4 mg to the subject. In another embodiment, the mRNA vaccine is administered to the subject twice, three times, four times or more. In some embodiments, the mRNA vaccine is administered once a day every three weeks. In one embodiment, the mRNA vaccine is administered by intradermal, intramuscular, and/or subcutaneous administration. In another embodiment, the mRNA vaccine is administered by intramuscular administration.

In some embodiments, the method further comprises administering an additional cancer therapeutic agent; ally wherein the additional cancer eutic agent is an immune checkpoint modulator to the subject. In one embodiment, the immune checkpoint modulator is an inhibitory oint polypeptide. In another embodiment, the inhibitory checkpoint polypeptide inhibits PD1,PD-L1, CTLA4, TlM-3, VISTA, AZAR, B7-H3, B7- H4, BTLA, IDO, KIR, LAG3, or a combination thereof. In some embodiments, the checkpoint inhibitor polypeptide is an dy. In other ments, the inhibitory checkpoint polypeptide is an antibody selected from an anti-CTLA4 antibody or antigenbinding nt thereof that speci?cally binds CTLA4, an Dl antibody or antigen- binding nt thereof that speci?cally binds PDl, an anti-PD-Ll antibody or antigen- binding fragment thereof that speci?cally binds PD-Ll, and a combination thereof. In some embodiments, the checkpoint inhibitor polypeptide is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab. In another embodiment, the checkpoint inhibitor ptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In other embodiments, the checkpoint inhibitor polypeptide is an anti-PDl antibody selected from nivolumab or pembrolizumab.

In one embodiment, the immune checkpoint modulator is administered at a dosage level suf?cient to deliver 100-300 mg to the subject. In some embodiments, the immune checkpoint modulator is stered at a dosage level suf?cient to deliver 200 mg to the subject. In some embodiments, the immune checkpoint modulator is administered by enous infusion. In one embodiment, the immune checkpoint modulator is administered to the subject twice, three times, four times or more. In some embodiments, the immune checkpoint modulator is administered to the subject on the same day as the mRNA e administration.

In some embodiments, the cancer is selected from the group ting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell oma (HNSCC), and a solid malignancy that is microsatellite high (MSI H) / mismatch repair (MMR) de?cient. In one embodiment, the NSCLC lacks an EGFR izing mutation and/or an ALK ocation. In another embodiment, the solid malignancy that is microsatellite high (MSI H) / ch repair (MMR) de?cient is selected from the group consisting of colorectal cancer, stomach adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from cancer of the pancreas, peritoneum, large intestine, small intestine, y tract, lung, endometrium, ovary, genital tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid s.

The invention in some aspects is an mRNA cancer vaccine of one or more mRNA each having an open reading frame encoding a cancer antigen e epitope formulated in a lipid nanoparticle, wherein the mRNA vaccine encodes 5-100 peptide epitopes and at least two of the peptide epitopes are alized cancer antigens, and a pharmaceutically acceptable carrier or excipient.

In other s the invention is an mRNA cancer vaccine, having one or more mRNA each having an open reading frame encoding a cancer antigen peptide epitope, wherein the mRNA vaccine encodes 5-100 peptide epitopes and at least three of the peptide epitopes is a compleX variant and at least two of the peptide epitopes are point ons, and a pharmaceutically acceptable carrier or excipient.

In some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol, and 05-15% dified lipid. In some embodiments, the cationic lipid is selected from the group ting of for example, 2,2-dilinoleyldimethylaminoethyl-[ l ,3]—dioxolane (DLin-KCZ-DMA), dilinoleyl-methyl- 4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-nonen-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In other embodiments, the lipid nanoparticle comprises a nd of a (I). In some embodiments, the compound of Formula (I) is Compound 25.

In some embodiments, the lipid nanoparticle has a polydispersity value of less than 0.4. In some embodiments, the lipid rticle has a net l charge at a neutral pH value.

The vaccine in some embodiments is an mRNA having an open reading frame encoding a concatemeric cancer antigen comprised of the 5-100 peptide es. In other embodiments at least two of the peptide epitopes are separated from one another by a single Glycine. In other embodiments the concatemeric cancer antigen comprises 20-40 peptide epitopes. In some embodiments all of the peptide epitopes are separated from one another by a single Glycine. In some embodiments at least two of the peptide epitopes are linked directly to one another without a linker.

Each peptide epitope in embodiments comprises a 25-35 amino acids and es a centrally located SNP mutation.

In some embodiments at least 30% of the peptide epitopes have a highest af?nity for class IMHC molecules from the subject. In other embodiments at least 30% of the peptide epitopes have a highest af?nity for class II MHC molecules from the t. In yet other ments at least 50% of the peptide epitopes have a predicted binding af?nity of IC >500nM for HLA-A, HLA-B and/or DRB 1.

In some ments, one or more mRNAs of the invention encode up to 20 peptide epitopes. In some ments, one or more mRNAs of the invention encode up to 50 epitopes. In some embodiments, one or more mRNAs of the invention encode up to 100 epitopes.

According to other embodiments the mRNA ng the peptide epitopes is arranged such that the peptide epitopes are ordered to minimize pseudo-epitopes.

Each peptide epitope may comprise 31 amino acids and includes a centrally located SNP mutation with 15 ?anking amino acids on each side of the SNP mutation.

In some ments a TCR face for each epitope has a low similarity to endogenous proteins.

In yet other embodiments the mRNA further comprises a recall antigen. The recall antigen may be an infectious disease antigen.

In other embodiments, at least one of the peptide es is a traditional cancer n. The vaccine in some embodiments includes an mRNA having an open g 2O frame encoding one or more ent polymorphisms. The one or more recurrent polymorphisms may comprise a recurrent somatic cancer mutation in p53. The one or more recurrent somatic cancer mutation in p53 in some ments are selected from the group consisting of: (A) mutations at the canonical 5’ splice site neighboring codon p.T125, inducing a retained intron having peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) that contains epitopes AVSPCISFVW (SEQ ID NO: 233) (HLA-B*57 :01, HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) *02:01, HLA-A*02:O6, HLA-B*35:01), (B) mutations at the canonical 5’ splice site neighboring codon p.331, inducing a retained intron having peptide sequence VLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) that contains epitopes LQVLSLGTSY (SEQ ID NO: 237) (HLA-B*15:01), FQSNTQNAVF (SEQ ID NO: 23 8) (HLA-B*15:01), (C) mutations at the canonical 3’ splice site neighboring codon p. 126, inducing a cryptic alternative exonic 3’ splice site producing the novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 239) that contains epitopes CTMFCQLAK (SEQ ID NO: 240) (HLA-A*11:01), KSVTCTMF (SEQ ID NO: 241) (HLA- B*58:Ol), and/or (D) ons at the canonical 5’ splice site neighboring codon p.224, inducing a cryptic alternative intronic 5’ splice site producing the novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) that contains epitopes EVW (SEQ ID NO: 243) (HLA-B*53:Ol, HLA-B*5l:Ol), LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:Ol, HLA-B*57 :01), wherein the transcript codon positions refer to the canonical full-length p53 transcript ENSTOOOOO269305 (SEQ ID NO: 245) from the Ensembl V83 human genome annotation.

In some embodiments, the mRNA further comprises an open reading frame encoding an immune checkpoint modulator. In some ments, the mRNA cancer vaccine comprises an immune checkpoint tor. In some embodiments, the immune checkpoint modulator is an inhibitory checkpoint ptide. In some embodiments, the inhibitory checkpoint polypeptide is an dy or fragment thereof that specifically binds to a molecule selected from the group ting of PD-l, THVI-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. In some embodiments, the inhibitory checkpoint polypeptide is an anti-CTLA4 or anti-PDl antibody. In some embodiments, the D-l antibody is pembrolizumab.

In some embodiments, the mRNA cancer vaccine does not comprise a stabilization 2O agent.

In some ments the mRNA includes at least one chemical modification. The chemical modi?cation may be selected from the group ting of pseudouridine, Nl- methylpseudouridine, 2-thiouridine, 4’ -thiouridine, 5-methylcytosine, 2-thiomethyl deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thioaza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio—pseudouridine, 4-methoxythiopseudouridine , 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thiopseudouridine , 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5- methoxyuridine, and 2’-O-methyl uridine.

In other aspects a method for vaccinating a t is provided. The method involves stering to a subject having cancer an mRNA vaccine sed herein.

In some embodiments, the mRNA vaccine is administered at a dosage level sufficient to deliver between 10 ug and 400 ug of the mRNA vaccine to the subject. In some embodiments, the mRNA vaccine is administered at a dosage level suf?cient to deliver 0.033mg, O. lmg, 0.2 mg, or 0.4 mg to the subject. In some ments, the mRNA vaccine is administered to the subject twice, three times, four times or more. In some ments, the mRNA vaccine is administered once a day every three weeks.

In some embodiments, the mRNA vaccine is administered by intradermal, intramuscular, and/or subcutaneous administration. In some embodiments, the mRNA vaccine is administered by intramuscular administration.

In some embodiments, the method further includes administering an additional cancer therapeutic agent, optionally wherein the additional cancer therapeutic agent is an immune checkpoint modulator to the subject. In some embodiments, the immune checkpoint modulator is an inhibitory checkpoint polypeptide. In some embodiments, the inhibitory checkpoint polypeptide is an antibody or fragment thereof that speci?cally binds to a molecule ed from the group consisting of PD-l, THVI—3, VISTA, AZAR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. In some embodiments, the inhibitory checkpoint polypeptide is an anti-PDl antibody. In some embodiments, the anti-PD-l antibody is pembrolizumab.

In some embodiments, the immune checkpoint modulator is administered at a dosage level suf?cient to deliver 100-300 mg to the t. In some embodiments, the immune checkpoint modulator is administered at a dosage level suf?cient to deliver 200 mg to the subject.

In some embodiments, the immune oint modulator is administered by intravenous infusion.

In some embodiments, the immune checkpoint modulator is administered to the subject twice, three times, four times or more. In some embodiments, the immune checkpoint modulator is stered to the subject on the same day as the mRNA vaccine administration.

In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, HPV-negative head and neck us cell carcinoma (HNSCC), and a solid ancy that is microsatellite high (MSI H) / mismatch repair (MMR) de?cient. In some embodiments, the NSCLC lacks an EGFR sensitizing mutation and/or an ALK ocation.

In some ments, the solid malignancy that is microsatellite high (MSI H) / mismatch repair (MMR) de?cient is ed from the group consisting of colorectal cancer, stomach adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from cancer of the as, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, genital tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues.

A method for preparing an mRNA cancer vaccine is provided in other aspects. The method involves isolating a sample from a subject, fying a plurality of cancer ns in the sample, determining immunogenic epitopes from the plurality of cancer antigens, preparing an mRNA cancer vaccine having an open reading frame encoding the cancer antigens. A method of producing an mRNA encoding a concatemeric cancer antigen comprising between 1000 and 3000 nucleotides, is provided in other aspects of the invention.

The method involves (a) binding a ?rst polynucleotide comprising an open reading frame encoding the cancer antigen of any one of the preceding claims and a second polynucleotide sing a '-UTR to a polynucleotide conjugated to a solid t, (b) ligating the 3 '-terminus of the second polynucleotide to the 5'-terminus of the first polynucleotide under suitable conditions, wherein the suitable ions comprise a DNA Ligase, thereby producing a ?rst ligation product, (c) ligating the 5’ terminus of a third polynucleotide comprising a 3'-UTR to the 3’- terminus of the first ligation product under suitable conditions, wherein the suitable conditions se an RNA Ligase, thereby producing a second ligation product, and (d) releasing the second on product from the solid support, thereby producing an mRNA encoding the concatemeric cancer antigen comprising between 1000 and 3000 tides.

In other aspects the ion is an mRNA cancer vaccine comprising a concatemeric cancer n able according to the methods described herein.

A method for treating a subject with a personalized mRNA cancer vaccine is provided according to other s of the invention. The method involves fying a set of neoepitopes by analyzing a patient transcriptome and/or a patient exome from the sample to produce a patient specif1c mutanome, selecting a set of neoepitopes for the vaccine from the mutanome based on MHC binding strength, MHC g diversity, ted degree of immunogenicity, low self reactivity, presence of activating oncogene mutations, and/or T cell vity, preparing the mRNA vaccine to encode the set of neoepitopes, and administering the mRNA vaccine to the subject within two months of isolating the sample from the t.

In some embodiments, the identifying comprises analyzing a patient transcriptome and/or a patient exome from a sample from the subject. In some embodiments, the sample from the subject is a biological sample, e.g., a biopsy. In some embodiments, the method further comprises isolating the sample from the subject. In some embodiments, the identifying comprises analyzing tissue-specif1c expression in available databases.

A method of identifying a set of neoepitopes for use in a personalized mRNA cancer vaccine having one or more polynucleotides that encode the set of topes is provided in other aspects of the invention. The method involves: a. identifying a patient specific mutanome by analyzing a patient transcriptome and a t exome; b. selecting a subset of 15-500 neoepitopes from the mutanome using a weighted value for the neoepitopes based on at least three of: an assessment of gene or transcript-level expression in patient RNA-seq; variant call con?dence score; q -specif1c expression; conservative vs. non-conservative amino acid substitution; position of point mutation (Centering Score for increased TCR engagement); on of point mutation (Anchoring Score for differential HLA binding); Selfness: with patient WES data; HLA-A and —B IC50 for 8mers-l lmers; HLA-DRBl IC50 for 15mers-20mers; promiscuity Score (i.e. number of patient HLAs predicted to bind); HLA-C IC50 for 8mers-l lmers;HLA-DRB3-5 IC50 for lSmers-20mers; HLA-DQBl/Al IC50 for -20mers; HLA-DPBl/Al IC50 for 15mers-20mers; Class Ivs Class II proportion; Diversity of patient HLA-A; -B and DRBl allotypes covered; proportion of point mutation vs complex epitopes (e.g. frameshifts); pseudo-epitope HLA binding ; presence and/or abundance of RNAseq reads; and c. selecting the set of neoepitopes for use in a personalized mRNA cancer vaccine from the subset based on the t weighted value; wherein the set of topes comprise 15-40 neoepitopes.

The invention in some aspects is an mRNA cancer vaccine of one or more mRNA each having an open reading frame encoding a cancer antigen peptide epitope; wherein the mRNA the further comprises a miRNA binding site. In some embodiment the vaccine s 5-100 peptide epitopes.

In some embodiments the nucleic acid es described herein are chemically modified. In other embodiments the nucleic acid vaccines are unmodi?ed.

Yet other aspects provide compositions for and methods of vaccinating a subject sing administering to the subject a nucleic acid vaccine comprising one or more RNA cleotides having an open reading frame encoding a cancer antigen epitope; wherein the RNA polynucleotide does not include a stabilization element; and wherein an adjuvant is not coformulated or co-administered with the vaccine.

In other aspects the invention is a composition for or method of vaccinating a t comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open g frame encoding a ?rst cancer antigen epitope wherein a dosage of between 10 pg/kg and 400 pg/kg of the nucleic acid vaccine is administered to the subject. In some embodiments the dosage of the RNA polynucleotide is 1—5 pg, 5—10 pg, 10—15 pg, 15—20 pg, 10—25 pg, 2025 pg, 2060 pg, 30—50 pg, 40—50 pg, 40— 60 pg, 60-80 pg, 60-100 pg, 50—100 pg, 80-120 pg, 40—120 pg, 40—150 pg, 50—150 pg, 50— 200 pg, 80-200 pg, 100—200 pg, 120—250 pg, 150—250 pg, 180-280 pg, 200—300 pg, 50—300 pg, 80-300 pg, 100—300 pg, 40—300 pg, 50—350 pg, 0 pg, 200—350 pg, 300—350 pg, 320-400 pg, 40-380 pg, 40-100 pg, 0 pg, 200-400 pg, or 300-400 pg per dose. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.

In some ments, a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is ed in the nucleic acid vaccine administered to the subject. In some 2O ments, a dosage of 75 micrograms of the RNA polynucleotide is included in the nucleic acid e administered to the subject. In some embodiments, a dosage of 150 micrograms of the RNA polynucleotide is included in the c acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine stered to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the nucleic acid e administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the nucleic acid e is chemically modi?ed and in other embodiments the nucleic acid vaccine is not chemically modified.

In some embodiments, the effective amount is a total dose of 1-100 pg. In some ments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a dose of 25 pg administered to the subject a total of one or two times. In some embodiments, the effective amount is a dose of 100 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 1 pg -10 pg, 1 pg -20 Mg, 1 Mg -30 Mg, 5 Mg -10 Mg, 5 Mg -20 Mg, 5 Mg -30 Mg, 5 Mg -40 Mg, 5 Mg -50 Mg, 10 Mg - Mg, 10 Mg -20 Mg, 10 Mg -25 Mg, 10 Mg -30 Mg, 10 Mg -40 Mg, 10 Mg -50 Mg, 10 Mg -60 Mg, Mg -20 Mg, 15 Mg -25 Mg, 15 Mg -30 Mg, 15 Mg -40 Mg, 15 Mg -50 Mg, 20 Mg -25 Mg, 20 Mg - Mg, 20 Mg -40 Mg 20 Mg -50 Mg, 20 Mg -60 Mg, 20 Mg -70 Mg, 20 Mg -75ug, 30 Mg -35 Mg, pg -40 pg, 30 pg -45 pg 30 pg -50 pg, 30 pg -60 pg, 30 pg -70 pg, 30 pg -75pg which may be administered to the subject a total of one or two times or more.

Aspects of the ion provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open g frame encoding a ?rst antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and a pharmaceutically able carrier or excipient, wherein an adjuvant is not included in the vaccine. In some embodiments, the stabilization element is a histone stem-loop. In some embodiments, the ization element is a nucleic acid sequence having increased GC t relative to wild type sequence.

Aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modi?cation or optionally no chemical modi?cation, the open reading frame encoding a ?rst antigenic ptide, wherein the RNA polynucleotide is present in the ation for in vivo administration to a subject such that the level of antigen sion in the subject signi?cantly exceeds a level of antigen expression ed by an mRNA vaccine having a stabilizing element or formulated 2O with an adjuvant and encoding the ?rst antigenic polypeptide.

Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one al modi?cation or optionally no chemical modi?cation, the open reading frame encoding a ?rst antigenic polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodi?ed mRNA vaccine to produce an equivalent antibody titer.

Aspects of the invention also provide a unit of use vaccine, comprising between lOug and 400 ug of one or more RNA polynucleotides having an open reading frame comprising at least one chemical ation or optionally no chemical modi?cation, the open g frame encoding a ?rst antigenic polypeptide, and a pharmaceutically acceptable r or excipient, ated for delivery to a human subject. In some embodiments, the vaccine further comprises a cationic lipid nanoparticle.

Aspects of the invention provide kits including a vial comprising the mRNA cancer vaccine disclosed herein. In some embodiments, the vial contains 0.1 mg to 1 mg of mRNA.

In some embodiments, the vial contains 0.35 mg of mRNA. In some embodiments, the concentration of the mRNA is 1 mg/mL.

In some embodiments, the vial contains 5-15 mg of total lipid. In some embodiments, the vial contains 7 mg of total lipid. In some embodiments, the concentration of total lipid is mg/mL.

In some embodiments, the mRNA cancer vaccine is a liquid.

In some embodiments, the kit further includes a syringe. In some embodiments, the syringe is suitable for intramuscular stration.

Aspects of the invention provide methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 ug/kg and 400 ug/kg of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame ng a first antigenic polypeptide in an effective amount to vaccinate the subject.

The invention in some aspects is an mRNA cancer vaccine which may include an activating oncogene mutation as an n. In some embodiments, the activating oncogene mutation is a KRAS mutation. In some embodiments, the KRAS mutation is a G12 mutation.

In some embodiments, the G12 KRAS mutation is selected from a G12D, G12V, G12S, G12C, G12A, and a G12R KRAS on, e.g., the G12 KRAS mutation is selected from a G12D, G12V, and a G12S KRAS mutation. In other embodiments, the KRAS mutation is a G13 mutation, e.g., the G13 KRAS on is a G13D KRAS on. In some 2O embodiments, the activating oncogene mutation is a H—RAS or N—RAS mutation.

In some embodiments the skilled artisan will select a KRAS mutation, a HLA subtype and a tumor type based on the guidance provided herein and prepare a KRAS vaccine for therapy. In some embodiments the KRAS mutations is selected from: G12C, G12V, G12D, G13D. In some ments the HLA e is selected from: A*02:01, C*O7:01, 1, C*O7:02. In some embodiments the tumor type is selected from colorectal, pancreatic, lung, and endometrioid.

In some embodiments, the HRAS mutation is a mutation at codon 12, codon 13, or codon 61. In some embodiments, the HRAS mutation is a 12V, 61L, or 61R mutation.

In some embodiments, the NRAS mutation is a mutation at codon 12, codon 13, or codon 61. In some embodiments, the NRAS mutation is a 12D, 13D, 61K, or 61R mutation.

Some embodiments of the t disclosure provide an mRNA cancer vaccine that include an mRNA having an open reading frame encoding a concatemer of two or more activating oncogene mutation peptides. In some embodiments, at least two of the peptide epitopes are separated from one another by a single e. In some embodiments, the concatemer comprises 3-10 activating oncogene mutation peptides. In some such embodiments, all of the peptide es are separated from one another by a single Glycine.

In other embodiments, at least two of the peptide epitopes are linked directly to one another without a linker.

In some embodiments, the mRNA cancer vaccine further comprises a cancer therapeutic agent. In some embodiments, the mRNA cancer e further comprises an inhibitory checkpoint polypeptide. For e, in some embodiments, the inhibitory checkpoint polypeptide is an antibody or fragment thereof that speci?cally binds to a molecule selected from the group consisting of PD-l, TlM-3, VISTA, AZAR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. In other embodiments, the mRNA cancer vaccine further comprises a recall n. For example, in some embodiments, the recall antigen is an infectious disease n.

In some embodiments, the mRNA cancer vaccine does not comprise a stabilization agent.

In some ments the mRNA is formulated in a lipid nanoparticle carrier such as a lipid nanoparticle carrier comprising a molar ratio of about 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol, and 05-15% di?ed lipid. The cationic lipid may be selected from the group consisting of for example, 2,2-dilinoleyldimethylaminoethyl- [l,3]—dioxolane (DLin-KCZ-DMA), dilinoleyl-methyldimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-nonen-l-yl) (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

In some ments the mRNA includes at least one al modification. The chemical modi?cation may be selected from the group consisting of pseudouridine, Nl- methylpseudouridine, 2-thiouridine, 4’ -thiouridine, 5-methylcytosine, 2-thiomethyl deaza-pseudouridine, -l-methyl-pseudouridine, 2-thioaza-uridine, 2-thiodihydropseudouridine , 2-thio-dihydrouridine, 2-thio—pseudouridine, 4-methoxythiopseudouridine , 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, yluridine, 5- methoxyuridine, and 2’-O-methyl uridine.

In other aspects, a method for ng a subject is provided. The method involves administering to a subject having cancer an mRNA cancer vaccine of any one of the foregoing embodiments. In some embodiments, the mRNA cancer vaccine is administered in combination with a cancer therapeutic agent. In some embodiments, the mRNA cancer vaccine is administered in combination with an inhibitory checkpoint polypeptide. For example, in some embodiments, the mRNA cancer vaccine is an antibody or nt thereof that speci?cally binds to a molecule selected from the group consisting of PD-l, THVI-3, VISTA, AZAR, B7-H3, B7-H4, BTLA, , IDO, KIR and LAG3.

Methods provided herein may be used for treating a subject having cancer. In some embodiments, the cancer is selected from cancer of the as, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, genital tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, , and hematopoietic and lymphoid tissues. In some embodiments, the cancer is colorectal cancer.

In some embodiments the dosage of the mRNA cancer e administered to a IO subject is 1—5 pg, 5—10 pg, 10—15 pg, 15—20 pg, 10—25 pg, 20—25 pg, 20—50 pg, 30—50 pg, 40— 50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50—100 pg, 80-120 pg, 40—120 pg, 40—150 pg, 50—150 pg, 50—200 pg, 80-200 pg, 100—200 pg, 120—250 pg, 150—250 pg, 180-280 pg, 200—300 pg, 50—300 pg, 80-300 pg, 100—300 pg, 40—300 pg, 50—350 pg, 100—350 pg, 200—350 pg, 300—350 pg, 0 pg, 40-380 pg, 40-100 pg, 100-400 pg, 200-400 pg, or 300-400 pg per dose. In some embodiments, the mRNA cancer vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the mRNA cancer vaccine is administered to the subject on day zero. In some embodiments, a second dose of the mRNA cancer vaccine is administered to the subject on day twenty one.

In some embodiments, a dosage of 25 micrograms of the mRNA cancer vaccine is 2O administered to the subject. In some embodiments, a dosage of 100 micrograms of the mRNA cancer vaccine is administered to the subject. In some embodiments, a dosage of 50 micrograms of the mRNA cancer vaccine is administered to the subject. In some embodiments, a dosage of 75 micrograms of the mRNA cancer e is administered to the subject. In some embodiments, a dosage of 150 micrograms of the mRNA cancer vaccine is administered to the subject. In some embodiments, a dosage of 400 micrograms of the mRNA cancer vaccine is administered to the subject. In some embodiments, a dosage of 200 micrograms of the mRNA cancer e is stered to the subject. In some embodiments, the mRNA cancer vaccine lates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the mRNA cancer vaccine is chemically modi?ed and in other embodiments the mRNA cancer vaccine is not chemically modi?ed.

In some embodiments, the effective amount is a total dose of 1-100 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a dose of 25 pg administered to the subject a total of one or two times. In some embodiments, the effective amount is a dose of 100 ug stered to the subject a total of two times. In some embodiments, the effective amount is a dose of 1 pg -10 pg, 1 ug -20 Mg, 1 Mg -30 Mg, 5 Mg -10 Mg, 5 Mg -20 Mg, 5 Mg -30 Mg, 5 Mg -40 Mg, 5 Mg -50 Mg, 10 Mg - Mg, 10 Mg -20 Mg, 10 Mg -25 Mg, 10 Mg -30 Mg, 10 Mg -40 Mg, 10 Mg -50 Mg, 10 Mg -60 Mg, Mg -20 Mg, 15 Mg -25 Mg, 15 Mg -30 Mg, 15 Mg -40 Mg, 15 Mg -50 Mg, 20 Mg -25 Mg, 20 Mg - Mg, 20 Mg -40 Mg 20 Mg -50 Mg, 20 Mg -60 Mg, 20 Mg -70 Mg, 20 Mg -75ug, 30 Mg -35 Mg, pg -40 pg, 30 pg -45 pg 30 pg -50 pg, 30 pg -60 pg, 30 pg -70 pg, 30 ug -75ug which may be administered to the subject a total of one or two times or more.

Aspects of the ion provide methods of producing an mRNA encoding a concatemeric cancer antigen comprising n 1000 and 3000 nucleotides, the method comprising: (a) binding a ?rst polynucleotide comprising an open reading frame encoding the cancer antigen of any one of claim l-103 and a second polynucleotide comprising a 5'-UTR to a polynucleotide conjugated to a solid support, (b) ligating the 3 '-terminus of the second cleotide to the 5 '-terminus of the ?rst polynucleotide under suitable conditions, wherein the suitable conditions se a DNA Ligase, thereby producing a ?rst ligation product, (c) ligating the 5’ terminus of a third polynucleotide comprising a 3'-UTR to the 3’- terminus of the ?rst ligation product under suitable conditions, wherein the suitable conditions comprise an RNA Ligase, thereby producing a second ligation product, and (d) releasing the second ligation product from the solid support, thereby producing an mRNA encoding the concatemeric cancer antigen comprising between 1000 and 3000 nucleotides. s of the invention provide s for ng a subject with a personalized mRNA cancer vaccine, comprising identifying a set of topes to produce a patient speci?c mutanome, selecting a set of neoepitopes for the e from the mutanome based on MHC binding th, MHC binding diversity, predicted degree of immunogenicity, low self vity, and/or T cell reactivity, preparing the mRNA vaccine to encode the set of neoepitopes, and administering the mRNA vaccine to the subject within two months of isolating the sample from the t.

Aspects of the invention provide methods of identifying a set of neoepitopes for use in a alized mRNA cancer vaccine having one or more polynucleotides that encode the set of neoepitopes comprising: (a) identifying a patient speci?c mutanome by analyzing a patient transcriptome and a patient exome, (b) selecting a subset of 15-500 neoepitopes from the mutanome using a weighted value for the neoepitopes based on at least three of: an assessment of gene or transcript-level expression in patient RNA-seq, variant call con?dence score, RNA-seq allele-speci?c expression, conservative vs. non-conservative amino acid substitution; position of point mutation (Centering Score for increased TCR engagement); on of point mutation (Anchoring Score for ential HLA binding); Selfness: <100% core epitope homology with patient WES data; HLA-A and —B IC50 for 8mers-11mers; HLA-DRBl IC50 for -20mers; promiscuity Score; HLA-C IC50 for 8mers- llmers;HLA-DRB3-5 IC50 for 15mers-20mers; HLA-DQBl/Al IC50 for 15mers-20mers; HLA-DPBl/Al IC50 for 15mers-20mers; Class Ivs Class II proportion; Diversity of patient HLA-A; -B and DRBl allotypes covered; proportion of point mutation vs complex epitopes; pseudo-epitope HLA binding scores; ce and/or abundance of RNAseq reads; and (c) selecting the set of neoepitopes for use in a alized mRNA cancer vaccine from the subset based on the highest weighted value; wherein the set of neoepitopes comprise 15-40 neoepitopes.

Aspects of the invention provide methods of identifying a set of neoepitopes for use in a personalized mRNA cancer vaccine having one or more polynucleotides that encode the set of neoepitopes comprising: (a) generating a RNA-seq sample from a t tumor to e a set of RNA-seq reads; (b) compiling overall counts of tide sequences from all RNA- seq reads; (c) comparing sequence information n the tumor sample and a corresponding database of normal tissues of the same tissue type; and(d) selecting a set of neoepitopes for use in a personalized mRNA cancer vaccine from the subset based on the highest ed value; wherein the set of neoepitopes comprise 15-40 neoepitopes. 2O The details of various embodiments of the invention are set forth in the description below. Other features; obj ects; and advantages of the invention will be apparent from the description and the drawings; and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS The ing and other obj ects; features and advantages will be apparent from the following description of particular embodiments of the invention; as illustrated in the anying gs in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis instead being placed upon illustrating the principles of various embodiments of the invention. shows con?rmation of full read through of the concatamer EKL is SEQ ID NO: 231). shows antigen-speci?c responses to Class I epitopes found in both constructs. shows antigen-speci?c responses to Class I epitopes found exclusively in 52mer constructs. shows antigen-speci?c responses to Class II epitopes found in both constructs (left) and found exclusively in the 52mer constructs (right). is a block diagram of an exemplary computer system on which some embodiments may be ented. shows n-speci?c responses from mice immunized with mRNA encoding a concatemer of 52 murine epitopes (adding epitopes_4a_DX_RX_perm) in combination with a STING immunopotentiator mRNA at varying antigen and STING dosages and antigen:STING ratios. Data shown is for in vitro restimulation with the peptide sequence corresponding to the Class II epitope RNA 2, encoded within the concatemer. shows antigen-speci?c responses from mice zed with mRNA encoding a concatemer of 52 murine epitopes (adding epitopes_4a_DX_RX_perm) in combination with a STING immunopotentiator mRNA at g n and STING dosages and antigen:STING ratios. Data shown is for in vitro restimulation with the peptide sequence corresponding to the Class II epitope RNA 3, encoded within the concatemer. shows antigen-speci?c responses from mice immunized with mRNA encoding a concatemer of 52 murine epitopes (adding epitopes_4a_DX_RX_perm) in combination with a STING immunopotentiator mRNA at varying antigen and STING dosages and antigen:STING . Data shown is for in vitro restimulation with the peptide sequence corresponding to Class I epitope RNA 7, d within the concatemer. 2O shows antigen-speci?c responses from mice immunized with mRNA encoding a concatemer of 52 murine epitopes (adding epitopes_4a_DX_RX_perm) in combination with a STING immunopotentiator mRNA at varying antigen and STING dosages and antigen:STING ratios. Data shown is for in vitro ulation with the peptide ce corresponding to Class I epitope RNA 13, encoded within the concatemer. shows antigen-speci?c responses from mice immunized with mRNA encoding a concatemer of 52 murine epitopes (adding epitopes_4a_DX_RX_perm) in combination with a STING immunopotentiator mRNA at g antigen and STING dosages and antigen:STING ratios. Data shown is for in vitro restimulation with the peptide sequence corresponding to Class I epitope RNA 22, encoded within the concatemer. shows antigen-speci?c responses from mice zed with mRNA ng a concatemer of 52 murine epitopes (adding epitopes_4a_DX_RX_perm) in combination with a STING immunopotentiator mRNA at varying antigen and STING s and antigen:STING ratios. Data shown is for in vitro restimulation with the peptide sequence corresponding to Class II epitope RNA 10, d within the concatemer. is a bar graph showing antigen-speci?c IFN-y T responses from mice immunized with mRNA encoding a concatemer of 20 murine epitopes (RNA 31) in combination with a STING immunopotentiator mRNA, as compared to standard adj uvants, or unformulated (not encapsulated in LNP). Data shown is for in vitro peptide restimulation with Class II epitopes (RNA 2 and RNA 3) encoded within the concatemer.. is a bar graph showing antigen-speci?c IFN-y T responses from mice immunized with mRNA encoding a emer of 20 murine epitopes (RNA 31) in combination with a STING immunopotentiator mRNA, as compared to standard adj uvants, or unformulated (not encapsulated in LNP). Data shown is for in vitro peptide restimulation with Class I epitopes (RNA 7, RNA 10, and RNA 13) encoded within the concatemer.. is a bar graph showing antigen-speci?c IFN-y T responses from mice zed with mRNA encoding a concatemer of 20 murine epitopes (RNA 31) in combination with a STING immunopotentiator mRNA, wherein the STING construct was administered either simultaneously with the vaccine, 24 hours later or 48 hours later. Data shown is for in vitro peptide restimulation with either Class II epitopes (RNA 2 and RNA 3) or Class I epitopes (RNA 7, RNA 10, RNA 13) encoded within the concatemer. depicts KRAS mutations in colorectal cancer as identi?ed in COSMIC, 2012 data set. s m-speci?c point mutation speci?city for HRAS. Data 2O representing total number of tumors with each point mutation were collated from COSMIC V52 release. Single base mutations generating each amino acid substitution are indicated.

The most frequent ons for each m for each cancer type are ghted with grey shading. H/L: hematopoietic/lymphoid tissues. (Prior et al. Cancer Res. 2012 May 15, 72(10): 2457—2467). depicts isoform-speci?c point mutation speci?city for KRAS. Data representing total number of tumors with each point mutation were ed from COSMIC V52 release. Single base mutations generating each amino acid substitution are indicated.

The most frequent mutations for each isoform for each cancer type are highlighted with grey shading. H/L: hematopoietic/lymphoid tissues. (Prior et al. Cancer Res. 2012 May 15, : 2457—2467). s m-speci?c point mutation speci?city for NRAS. Data representing total number of tumors with each point mutation were collated from COSMIC V52 release. Single base mutations generating each amino acid substitution are indicated.

The most frequent mutations for each isoform for each cancer type are highlighted with grey shading. H/L: hematopoietic/lymphoid tissues. (Prior et al. Cancer Res. 2012 May 15, 72(10): 2457—2467). depicts secondary KRAS mutations after acquisition of EGFR blockade resistance. (Diaz et al The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers, Nature 486:537 (2012)). depicts secondary KRAS mutations after EGFR blockade. (Misale et al.

Emergence ofKRAS muations and ed resistance to anti-EGFR y in colorectal cancer, Nature 486:532 (2012)). depicts NRAS and KRAS on frequency in colorectal cancer as identi?ed using cBioPortal.

DETAILED DESCRIPTION ments of the present disclosure provide RNA (e.g., mRNA) vaccines that e a polynucleotide encoding a cancer antigen. Cancer RNA vaccines, as provided herein may be used to induce a balanced immune response, comprising cellular and/or humoral immunity, without many of the risks ated with DNA vaccination. In some embodiments, a e ses at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a cancer antigen. In some embodiments, a e comprises at least one RNA (e.g., mRNA) polynucleotide having at least one open reading frame encoding 2O a cancer antigen and at least one open reading frame encoding a universal type II T-cell epitope. In another embodiment, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having at least one open reading frame ng a cancer antigen and at least one open reading frame encoding an immune iator (e.g., an adj uvant). In some embodiments, a vaccine comprises at least one RNA (e.g., an mRNA) polynucleotide having an open reading frame encoding a cancer antigen (e.g., an activating oncogene mutation peptide).

Although attempts have been made to produce functional RNA vaccines, including mRNA cancer vaccines, the therapeutic efficacy of these RNA vaccines have not yet been fully established. Quite surprisingly, the inventors have ered a class of formulations for delivering mRNA vaccines that results in signi?cantly ed, and in many respects synergistic, immune responses ing enhanced T cell responses. The vaccines of the invention include traditional cancer vaccines as well as personalized cancer vaccines. The invention involves, in some aspects, the surprising finding that lipid nanoparticle formulations signi?cantly enhance the effectiveness of mRNA vaccines, including chemically modi?ed and ?ed mRNA vaccines.

The lipid nanoparticle used in the studies described herein has been used previously to deliver siRNA various in animal models as well as in humans. In view of the observations made in association with the siRNA delivery of lipid nanoparticle formulations, the fact that the lipid nanoparticle, in contrast to liposomes, is useful in cancer vaccines is quite surprising. It has been ed that eutic delivery of siRNA formulated in lipid nanoparticle causes an undesirable in?ammatory response associated with a transient IgM response, typically leading to a reduction in antigen production and a compromised immune response. In st to the ?ndings observed with siRNA, the lipid nanoparticle-mRNA cancer vaccine formulations described herein are demonstrated to generate enhanced IgG levels, ent for prophylactic and therapeutic methods rather than transient IgM responses. The lipid nanoparticles of the invention are not liposomes. A liposome as used herein is a lipid based structure having a lipid bilayer or monolayer shell with a nucleic acid payload in the core.

The generation of cancer antigens that elicit a desired immune response (e.g. T-cell responses) against ed polypeptide sequences in vaccine development remains a challenging task. The invention involves logy that overcome s associated with such development. Through the use of the technology of the invention, it is possible to tailor the desired immune response by selecting appropriate T or B cell cancer epitopes and formulating the es or antigens for ive delivery in viva. onally or alternatively, the immune response may be further augmented by selecting one or more universal type II T-cells eptiopes to be delivered in addition to appropriate T and/or B cell cancer epitopes or antigens.

Additionally or atively, the mRNA vaccines may include an activating ne mutation peptide (e.g., a KRAS mutation peptide). Prior research has shown limited ability to raise T cells speci?c to the oncogenic mutation. Much of this research was done in the context of the most common HLA allele (A2, which occurs in ~50% of Caucasians). More recent work has explored the generation of speci?c T cells against point mutations in the context of less common HLA alleles (Al 1, C8). These ?ndings have signi?cant implications for the treatment of cancer. Oncogenic mutations are common in many cancers. The ability to target these mutations and generate T cells that are suf?cient to kill tumors has broad applicability to cancer therapy. It is quite sing that delivery of antigens using mRNA would have such a signi?cant advantage over the delivery of peptide vaccines. Thus the invention involves, in some aspects, the surprising ?nding that activating oncogenic mutation antigens delivered in vivo in the form of an mRNA signi?cantly enhances the effectiveness of cancer therapy.

HLA class I molecules are highly polymorphic trans-membrane glycoproteins composed of two polypeptide chains (heavy chain and light chain). Human leucocyte antigen, the major histocompatibility compleX in humans, is speci?c to each individual and has hereditary features. The class I heavy chains are encoded by three genes: HLA-A, HLA-B and HLA-C. HLA class I les are important for establishing an immune response by presenting nous ns to T lymphocytes, which initiates a chain of immune reactions that lead to tumor cell elimination by cytotoxic T cells. Altered levels of tion ofHLA class I antigens is a widespread phenomenon in malignancies and is accompanied by signi?cant inhibition of anti-tumor T cell function. It represents one of the main isms used by cancer cells to evade immuno-surveillance. Down regulated levels ofHLA class I antigens were detected in 90% ofNSCLC tumors (n=65). A reduction or loss ofHLA was detected in 76% of pancreatic tumor samples (n=l9). The expression ofHLA class I antigens in colon cancer was dramatically reduced or undetectable in 96% of tumor s (n=25).

Mounting evidence suggests that two general strategies are utilized by tumor cells to escape immune llance: immunoselection (poorly immunogenic tumor cell variants) and immunosubversion (subversion of the immune system). A correlation between changes in 2O HLA class I antigens and the presence of KRAS codon 12 mutations was demonstrated, which suggests a possible inductive effect of KRAS codon 12 mutations on HLA class I antigen regulation in cancer progression. Many frequent cancer mutations are predicted to bind HLA Class I alleles with high-af?nity (ICSO <= 50 nM)7 and may be le for prophylactic cancer ation.

The therapeutic mRNA can be delivered alone or in combination with other cancer therapeutics such as checkpoint tors to provide a signi?cantly enhanced immune response against . The oint inhibitors can enhance the effects of the mRNA encoding activing oncogenic peptides by ating some of the obstacles to promoting an immune response, thus allowing the activated T cells to ef?ciently promote an immune se against the tumor.

It has been discovered that the mRNA vaccines described herein are superior to current vaccines in several ways. First, the lipid nanoparticle (LNP) delivery is superior to other formulations including liposome or protamine based approaches described in the literature. The use of LNPs enables the effective delivery of ally modi?ed or unmodi?ed mRNA vaccines. Both modi?ed and unmodi?ed LNP formulated mRNA vaccines are superior to conventional vaccines by a signi?cant degree. In some ments the mRNA vaccines of the invention are superior to conventional vaccines by a factor of at least 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000 fold.

Although attempts have been made to produce onal RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic ef?cacy of these RNA es have not yet been fully established. Quite surprisingly, the inventors have discovered, according to aspects of the invention a class of formulations for delivering mRNA vaccines in vivo that results in signi?cantly enhanced, and in many ts synergistic, immune responses including enhanced antigen generation and functional dy production with neutralization capability. These results can be achieved even when signi?cantly lower doses of the mRNA are administered in comparison with mRNA doses used in other classes of lipid based formulations. The formulations of the ion have demonstrated signi?cant cted in vivo immune ses suf?cient to establish the ef?cacy of onal mRNA vaccines as prophylactic and therapeutic agents. Additionally, self-replicating RNA vaccines rely on viral replication pathways to deliver enough RNA to a cell to produce an immunogenic response. The formulations of the invention do not require viral replication to produce enough protein to result in a strong immune response. Thus, the mRNA of the invention are not self-replicating RNA and do not include components ary for viral replication.

The invention involves, in some aspects, the surprising ?nding that lipid nanoparticle (LNP) formulations cantly enhance the effectiveness of mRNA vaccines, including chemically modi?ed and unmodi?ed mRNA vaccines. Furthermore, it was found that immunogenicity to epitopes is r, independent of the total number of epitopes contained within the uct. Epitopes contained in a 52mer constructs have similar immunogenicity compared to 20mer ucts as measured by epitope-speci?c IFNy responses. It was quite unexpected that the increased mRNA length was demonstrated to have no deleterious effect on immunogenicity of epitopes.The last epitope encoded in the 20mer and 52mer (SIINFEKL, SEQ ID NO: 231) was comparable, this also indicates a full read through of the concatamers. Also surprisingly, it was found that antigen-speci?c responses to Class I epitopes increased when the vaccines were formulated with a constitutively active immune potentiator.

The LNP used in the studies described herein has been used previously to deliver siRNA in various animal models as well as in humans. In view of the observations made in association with the siRNA delivery of LNP formulations, the fact that LNP is useful in vaccines is quite surprising. It has been observed that therapeutic delivery of siRNA formulated in LNP causes an undesirable in?ammatory response associated with a transient IgM response, lly leading to a reduction in antigen production and a compromised immune response. In st to the gs observed with siRNA, the LNP-mRNA formulations of the invention are demonstrated herein to generate enhanced IgG levels, suf?cient for prophylactic and therapeutic s rather than transient IgM responses.

The mRNA cancer vaccines provide unique eutic alternatives to peptide based or DNA es. When the mRNA cancer vaccine is delivered to a cell, the mRNA will be processed into a polypeptide by the intracellular machinery which can then process the polypeptide into immunosensitive fragments capable of stimulating an immune response against the tumor.

In some embodiments, the mRNA cancer vaccine may be administered with an anti- cancer eutic agent, ing but not limited to, a traditional cancer vaccine. The mRNA cancer vaccine and anti-cancer therapeutic can be combined to enhance immune therapeutic responses even further. The mRNA cancer vaccine and other therapeutic agent may be administered aneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are stered at the same time. The other eutic agents are administered sequentially with one another and with the mRNA cancer vaccine, when the administration of the other therapeutic agents and the mRNA cancer vaccine is temporally separated. The separation in time n the administration of these compounds may be a matter of minutes or it may be longer, e.g. hours, days, weeks, months. Other therapeutic agents e but are not limited to anti-cancer therapeutic, adjuvants, cytokines, antibodies, antigens, etc.

The cancer vaccines described herein include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to cancer). The antigenic peptide may be a alized cancer antigen e, and/or a recurrent antigen. In some preferred embodiments the vaccine is multiple epitopes of a mixture of each of the above. Thus the cancer vaccines may be traditional or personalized cancer vaccines or mixtures thereof. A traditional cancer e is a vaccine including a cancer antigen that is known to be found in cancers or tumors generally or in a specific type of cancer or tumor. Antigens that are expressed in or by tumor cells are referred to as "tumor associated antigens". A particular tumor associated antigen may or may not also be expressed in non-cancerous cells. Many tumor mutations are known in the art.

It has been discovered surprisingly that RNA based multiepitopic cancer vaccines, whether formulated as individual epitopes or as a concatemer, can produce optimal immune stimulation through a careful balance ofMHC class I epitopes and MHC class II es.

RNA vaccines which encode both components have enhanced immunogenicity.

Personalized vaccines, for instance, may e RNA encoding for one or more known cancer antigens speci?c for the tumor or cancer antigens speci?c for each subject, referred to as neoepitopes or subject speci?c epitopes or antigens (referred to as personalized antigens). A "subject speci?c cancer antigen" is an antigen that has been identi?ed as being sed in a tumor of a particular patient. The subject speci?c cancer antigen may or may not be typically present in tumor samples generally. Tumor associated antigens that are not expressed or rarely expressed in non-cancerous cells, or whose sion in non-cancerous cells is suf?ciently reduced in comparison to that in cancerous cells and that induce an immune response induced upon vaccination, are referred to as neoepitopes. topes, like tumor associated antigens, are tely foreign to the body and thus would not produce an immune response against healthy tissue or be masked by the protective components of the immune system. In some embodiments personalized es based on neoepitopes are 2O desirable because such vaccine formulations will maximize speci?city against a t’s speci?c tumor. Mutation-derived neoepitopes can arise from point mutations, non- synonymous mutations leading to different amino acids in the protein, read-through mutations in which a stop codon is modi?ed or deleted, leading to translation of a longer protein with a novel tumor-speci?c sequence at the C-terminus, splice site mutations that lead to the inclusion of an intron in the mature mRNA and thus a unique tumor-speci?c protein sequence, chromosomal rearrangements that give rise to a chimeric protein with tumor- speci?c sequences at the junction of 2 proteins (1'.e., gene fusion), frameshift mutations or deletions that lead to a new open g frame with a novel tumor-speci?c protein sequence, and ocations. Thus, in some embodiments the mRNA cancer vaccines include at least 2 cancer antigens ing mutations ed from the group consisting of frame-shift mutations and recombinations or any of the other ons described herein. s for ting personalized cancer vaccines generally involve identi?cation of mutations, e.g., using deep nucleic acid or protein sequencing techniques, identi?cation of topes, e.g., using application of validated e-MHC binding prediction algorithms or other analytical techniques to generate a set of candidate T cell epitopes that may bind to t HLA s and are based on mutations present in tumors, optional demonstration of antigen-speci?c T cells against ed neoepitopes or demonstration that a candidate tope is bound to HLA proteins on the tumor surface and development of the vaccine.

The mRNA cancer vaccines of the invention may include multiple copies of a single neoepitope, le different neoepitopes based on a single type of mutation, 1'. e. point mutation, multiple different neoepitopes based on a variety of mutation types, neoepitopes and other antigens, such as tumor associated antigens or recall antigens.

Examples of techniques for identifying mutations include but are not limited to dynamic allele-speci?c hybridization (DASH), microplate array diagonal gel ophoresis (MADGE), pyrosequencing, oligonucleotide-speci?c ligation, the TaqMan system as well as various DNA "chip" technologies i.e. AffymetriX SNP chips, and methods based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling-circle ampli?cation.

The deep nucleic acid or n sequencing techniques are known in the art. Any type of sequence analysis method can be used. Nucleic acid sequencing may be performed on whole tumor genomes, tumor exomes (protein-encoding DNA), tumor transcriptomes, or exosomes. Real-time single molecule sequencing-by-synthesis technologies rely on the ion of ?uorescent nucleotides as they are incorporated into a nascent strand ofDNA that is complementary to the template being sequenced. Other rapid high throughput sequencing methods also eXist. Protein sequencing may be performed on tumor proteomes.

Additionally, n mass spectrometry may be used to identify or validate the presence of mutated peptides bound to MHC proteins on tumor cells. Peptides can be acid-eluted from tumor cells or from HLA les that are immunoprecipitated from tumor, and then identi?ed using mass spectrometry. The results of the sequencing may be compared with known control sets or with sequencing is med on normal tissue of the patient.

Accordingly, the present invention relates to methods for identifying and/or detecting neoepitopes of an n. Speci?cally, the invention provides methods of identifying and/or detecting tumor speci?c neoepitopes that are useful in inducing a tumor c immune response in a subject. Optionally, some of these neoepitopes bind to class I HLA proteins with a greater af?nity than the ype peptide and/or are capable of activating anti-tumor CD8 T-cells. Others bind to class II and activate CD4+ T helper cells. While the important role that class I antigens play in a vaccine have been recognized it has been discovered herein that vaccines composed of a balance of class I and class II antigens actually produce a more robust immune response than a vaccine based on class I or class II alone.

Proteins ofMHC class I are t on the surface of almost all cells of the body, including most tumor cells. The proteins ofMHC class I are loaded with antigens that usually originate from nous proteins or from pathogens present inside cells, and are then presented to xic T-lymphocytes (CTLs). T-Cell receptors are capable of recognizing and g peptides complexed with the molecules ofMHC class I. Each cytotoxic T- lymphocyte expresses a unique T-cell receptor which is capable of binding speci?c MHC/peptide complexes.

Using computer algorithms, it is possible to predict potential neoepitopes, 1'. e. peptide sequences, which are bound by the MHC molecules of class I or class II in the form of a peptide-presenting complex and then, in this form, recognized by the T-cell receptors of T- lymphocytes. Examples of programs useful for identifying peptides which will bind to MHC include for instance: Lonza Epibase, THI (Rammensee et al, Immunogenetics, 50 (1999),213-219)andIILfrgBIhH)(ParkeretaL,J.Inununol,152(1994),163-175) Once putative neoepitopes are selected, they can be further tested using in vitro and/or in vivo assays. Conventional in vitro lab assays, such as Elispot assays may be used with an isolate from each patient, to refine the list of neoepitopes selected based on the algorithm's predictions.

The mRNA cancer vaccines of the invention are compositions, including pharmaceutical compositions. The invention also encompasses methods for the selection, design, ation, manufacture, formulation, and/or use of mRNA cancer vaccines. Also provided are systems, processes, devices and kits for the ion, design and/or utilization of the mRNA cancer es described herein.

The mRNA vaccines of the ion may include one or more cancer antigens. In some embodiments the mRNA vaccine is composed of 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, or 55 or more antigens. In other embodiments, the mRNA vaccine is composed of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or 9 or more antigens. In other embodiments the mRNA vaccine is ed of 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less cancer antigens. In yet other embodiments the mRNA vaccine has 3-100, 5-100, 10-100, 15-100, , 25- 100,30-100,35-100,40-100,45-100,50-100,55-100,60-100,65-100,70-100,75-100,80- 100, 90—100, 560, 10—50, 15—50, 20—50, 25—50, 30—50, 35—50, 40—50, 45—50, 100—150, 100— 200, 100—300, 100—400, 0, 50—500, 50-800, 501,000, or 100—1,000 cancer antigens.

In some ments the mRNA cancer vaccines and vaccination methods include epitopes or antigens based on speci?c mutations (neoepitopes) and those expressed by cancer-germline genes (antigens common to tumors found in multiple patients).

An epitope, also known as an antigenic determinant, as used herein is a portion of an antigen that is recognized by the immune system in the appropriate context, speci?cally by antibodies, B cells, or T cells. Epitopes include B cell epitopes and T cell epitopes. B-cell epitopes are e sequences which are required for recognition by speci?c dy ing B-cells. B cell epitopes refer to a speci?c region of the antigen that is recognized by an antibody. The portion of an antibody that binds to the epitope is called a paratope. An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope. A linear, or continuous, epitope is de?ned by the y amino acid sequence of a particular region of a protein. The sequences that ct with the antibody are ed next to each other sequentially on the protein, and the epitope can usually be mimicked by a single e. Conformational epitopes are epitopes that are de?ned by the conformational structure of the native protein. These epitopes may be continuous or discontinuous, 1'. e. components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure.

T-cell epitopes are peptide sequences which, in association with proteins on APC, are required for recognition by c T-cells. T cell epitopes are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including MHC class II and MHC class I. The peptide epitope may be any length that is reasonable for an epitope. In some embodiments the peptide epitope is 9-30 amino acids. In other ments the length is 9—22, 9—29, 9-28, 9—27, 9-26, 9—25, 9—24, 9—23, 9—21, 9—20, 9—19, 9— 18, 10—22, 10—21, 10—20, 11—22, 22—21, 11—20, 12—22, 12—21, 12—20,13—22, 13—21, 13—20, 14— 19, 15-18, or 16-17 amino acids.

In some ments, the peptide epitopes comprise at least one MHC class I epitope and at least one MHC class II epitope. In some embodiments, at least 10% of the epitopes are MHC class I epitopes. In some embodiments, at least 20% of the epitopes are MHC class I es. In some embodiments, at least 30% of the epitopes are MHC class I epitopes. In some embodiments, at least 40% of the epitopes are MHC class I epitopes. In some ments, at least 50%, 60%, 70%, 80%, 90% or 100% of the epitopes are MHC class I epitopes. In some embodiments, at least 10% of the epitopes are MHC class II epitopes. In some embodiments, at least 20% of the epitopes are MHC class II epitopes. In some embodiments, at least 30% of the epitopes are MHC class II epitopes. In some ments, at least 40% of the epitopes are MHC class II epitopes. In some embodiments, at least 50%, 60%, 70%, 80%, 90% or 100% of the epitopes are MHC class II epitopes. In some embodiments, the ratio ofMHC class I epitopes to MHC class II epitopes is a ratio selected from about 10%:about 90%, about 20%:about 80%, about 30%:about 70%, about out 60%, about 50%:about 50%, about 60%:about 40%, about 70%:about 30%, about 80%: about 20%, about 90%: about 10% MHC class 1: MHC class II epitopes. In one embodiment, the ratio ofMHC class I : MHC class II epitopes is 3:1. In some embodiments, the ratio of MHC class II epitopes to MHC class I epitopes is a ratio selected from about 10%:about 90%, about 20%:about 80%, about 30%:about 70%, about 40%:about 60%, about 50%:about 50%, about 60%:about 40%, about 70%:about 30%, about 80%: about 20%, about 90%: about 10% MHC class II: MHC class I epitopes. In one embodiment, the ratio ofMHC class II : MHC class I es is 1:3. In some embodiments, at least one of the peptide es of the cancer vaccine is a B cell epitope. In some embodiments, the T cell epitope of the cancer vaccine comprises n 8-11 amino acids. In some embodiments, the B cell epitope of the cancer vaccine comprises between 13-17 amino acids.

In other aspects, the cancer vaccine of the invention comprises an mRNA vaccine ng multiple peptide epitope antigens, arranged with one or more interspersed universal type II T-cell epitopes. The universal type II T-cell epitopes, include, but are not limited to ILMQYIKANSKFIGI (Tetanus toxin, SEQ ID NO: 226), FNNFTVSFWLRVPKVSASHLE, (Tetanus toxin, SEQ ID NO: 227), QYIKANSKFIGITE (Tetanus toxin, SEQ ID NO: 228) QSIALSSLMVAQAIP (Diptheria toxin, SEQ ID NO: 229), and AKFVAAWTLKAAA (pan-DR epitope ), SEQ ID NO: 230). In some embodiments, the mRNA vaccine comprises the same universal type II T-cell e. In other embodiments, the mRNA vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 different universal type II T-cell epitopes. In some embodiments, the one or more universal type II T-cell e(s) are interspersed between every cancer n. In other embodiments, the one or more universal type II T-cell epitope(s) are interspersed between every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 cancer antigens.

The cancer vaccine of the invention, in some aspects comprises an mRNA vaccine encoding multiple peptide e antigens arranged with a single nucleotide spacer between the epitopes or directly to one another without a spacer n the epitopes. The multiple e antigens es a mixture ofMHC class I epitopes and MHC class II epitopes. For instance, the multiple peptide epitope antigens may be a polypeptide having the structure: (X-G—X)1.10 (G—Y-G—Y)1.10(G—X-G—X)0.10(G—Y-G—Y)0.10, (X-G)1.10 (G—Y)1.10(G-X)0.

(G—Y)0.10, (X-G—X-G-X)1.10(G—Y-G—Y)1.10(X-G—X)0.10(G—Y-G—Y)0.10, (X-G-X)1.10(G-Y-GY-G —Y)1_10(X-G—X)0_10(G—Y-G—Y)0_10, (X-G—X-G-X-G—X)1_10(G—Y-G—Y)1.10(X-G-X)0_10(G-Y- G—Y)0_10, (X-G-X)1_10 (G—Y-G—Y-G—Y-G—Y)1.10(X-G—X)0_10(G-Y-G-Y)0_10, (X)1_10 (Y)1_10 (X)0_ (Y)0-10, 001-10 001-10 (Y)0-10(X)0-10, 10 (Y)1-10(X)0-10(Y)0-10, (YY)1-10(XX)1-10 (Y)0-10 000-10, 001-10 (YY)1-10(X)0-10(Y)0-10, (XXX)1-10 (YYY)1-10(XX)0-10(YY)0-10, (YYY)1- (XXX)1-1o (YY)o-10(XX)o-1o, (XY)1-10 (Y)1-10(X)1-10(Y)1-10, (YX)1-1o 0(X)1-10(Y)1-10, (YX)1_10 (X)1_10(Y)1_10(Y)1_10, (Y-G—Y)1_10(G—X-G—X)1.10(G—Y-G—Y)0_10(G—X-G—X)0.10, (YG )1.10 (G—X)1.10(G-Y)0.10(G-X)0.10, (Y-G—Y-G—Y)1.10 (G—X-G—X)1.10(Y-G-Y)0.10(G—X-G—X)0.

, (Y-G—Y)1.10(G—X-G—X-G—X)1.10(Y-G—Y)0.10(G—X-G—X)0.10, (Y-G—Y-G—Y-G—Y)1.1o(G—X-G— X)1.10(Y-G—Y)0_10(G—X-G—X)0_10, (Y-G—Y)1_10(G—X-G—X-G—X-G—X)1.10(Y-G-Y)0.10(G-X-GX )o-1o, (XY)1-10 (YX)1-10 (XY)o-10(YX)o-lo, (YX)1-1o (XY)1-1o (Y)o-10(X)o-1o, (YY)1-10 (X)1- 10(Y)0-10(X)0-10, (XY)1-10(XY)1-10 000-10 000-10, 001-10 10(X)0-10(Y)0-10, -10 -10(YX)0-10(YY)0-10, Of (YYX)1-10(XXY)1-10 (YX)0-10(XY)0-10, X is an MHC class I epitope of 10-40 amino acids in length, Y is an MHC class II epitope of 10-40 amino acids in length, and G is glycine.

The cancer vaccine of the invention, in some aspects, comprises an mRNA vaccine 2O encoding le peptide epitope antigens arranged with a centrally located single nucleotide rphism (SNP) mutation with g amino acids on each side of the SNP mutation. In some embodiments, the number of ?anking amino acids on each side of the centrally located SNP mutation is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30. In one embodiment, an epitope of the cancer vaccine comprises an SNP ?anked by two Class I sequences, each sequence comprising seven amino acids. In another embodiment, an epitope of the cancer vaccine comprises a SNP ?anked by two Class II sequences, each sequence sing 10 amino acids. In some embodiments, an epitope may comprise a centrally located SNP and ?anks which are both Class I sequences, both Class II sequences, or one Class I and one Class II sequence.

Immune iator mRNAs One aspect of the disclosure pertains to mRNAs that encode a polypeptide that stimulates or enhances an immune response against one or more of the cancer antigens of interest. Such mRNAs that enhance immune responses to the cancer antigen(s) of interest are referred to herein as immune potentiator mRNA constructs or immune potentiator mRNAs, including chemically modi?ed mRNAs (mmRNAs). An immune potentiator of the sure enhances an immune response to an antigen of interest in a subject. The enhanced immune response can be a cellular response, a humoral response or both. As used herein, a "cellular" immune response is intended to encompass immune responses that involve or are mediated by T cells, whereas a "humoral" immune response is intended to encompass immune responses that involve or are mediated by B cells. An immune potentiator may enhance an immune response by, for example, (i) stimulating Type I interferon pathway signaling, (ii) stimulating NFkB pathway signaling, (iii) stimulating an in?ammatory response, (iv) stimulating ne tion, or (v) stimulating dendritic cell development, activity or mobilization, and (vi) a combination of any of i).

As used herein, lating Type I eron pathway signaling" is intended to encompass activating one or more components of the Type I interferon signaling pathway (e.g., modifying phosphorylation, dimerization or the like of such components to thereby te the pathway), stimulating transcription from an eron-sensitive response element (ISRE) and/or stimulating production or secretion of Type I interferon (e.g., lFN—oc, lFN—B, lFN—s, IFN-K and/or lFN—oa). As used herein, "stimulating NFkB pathway signaling" is intended to ass activating one or more components of the NFkB signaling pathway (e.g., modifying phosphorylation, dimerization or the like of such ents to thereby activate the y), stimulating transcription from an NFkB site and/or stimulating production of a gene product whose expression is regulated by NFkB. As used herein, "stimulating an in?ammatory response" is intended to encompass stimulating the production of in?ammatory cytokines (including but not limited to Type I interferons, 1L-6 and/or TNFOL). As used herein, "stimulating dendritic cell development, activity or zation" is intended to ass directly or indirectly stimulating dendritic cell tion, proliferation and/or functional activity.

In some aspects, the disclosure provides an mRNA encoding a ptide that stimulates or enhances an immune response in a subject in need thereof (e.g., potentiates an immune response in the t) by, for e, inducing adaptive immunity (e.g., by stimulating Type I eron tion), stimulating an in?ammatory response, ating NFkB signaling and/or stimulating dendritic cell (DC) pment, actiVity or mobilization in the subject. In some s, administration of an immune potentiator mRNA to a subject in need thereof enhances cellular immunity (e.g., T cell-mediated immunity), humoral immunity (e.g., B cell-mediated immunity) or both cellular and humoral immunity in the subject. In some aspects, administration of an immune potentiator mRNA stimulates cytokine production (e.g., in?ammatory cytokine production), stimulates cancer antigen - speci?c CD8+ or cell responses, ates antigen-speci?c CD4+ helper cell responses, increases the or memory CD62L10 T cell population, stimulates B cell actiVity or stimulates antigen-speci?c antibody production, including ations of the foregoing responses. In some aspects, administration of an immune potentiator mRNA stimulates cytokine production (e.g., in?ammatory cytokine production) and ates antigen-speci?c CD8+ effector cell responses. In some aspects, administration of an immune potentiator mRNA stimulates cytokine production (e.g., in?ammatory cytokine production), and stimulates antigen-speci?c CD4+ helper cell responses. In some aspects, administration of an immune potentiator mRNA stimulates cytokine production (e.g., in?ammatory cytokine production), and increases the effector memory CD62L10 T cell population. In some aspects, administration of an immune potentiator mRNA stimulates cytokine production (e.g., in?ammatory cytokine production), and stimulates B cell actiVity or stimulates antigen- speci?c antibody production.

In one embodiment, an immune potentiator increases cancer antigen-speci?c CD8+ effector cell responses (cellular immunity). For example, an immune potentiator can se one or more indicators of antigen-speci?c CD8+ effector cell actiVity, ing but not limited to CD8+ T cell proliferation and CD8+ T cell ne production. For e, in one embodiment, an immune potentiator increases production of IFN—y, TNFoc and/or IL-2 by antigen-speci?c CD8+ T cells. In various embodiments, an immune potentiator can increase CD8+ T cell cytokine production (e.g., IFN—y, TNFoc and/or IL-2 production) in response to an antigen (as compared to CD8+ T cell cytokine production in the absence of the immune potentiator) by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%. For example, T cells obtained from a treated subject can be ated in vitro with the cancer antigens and CD8+ T cell cytokine production can be assessed in vitro. CD8+ T cell cytokine production can be determined by standard methods known in the art, including but not limited to measurement of secreted levels of cytokine production (e.g., by ELISA or other suitable method known in the art for determining the amount of a cytokine in supernatant) and/or determination of the percentage of CD8+ T cells that are positive for ellular staining (IC S) for the cytokine. For example, intracellular staining (ICS) of CD8+ T cells for expression of IFN—y, TNFoc and/or IL-2 can be carried out by methods known in the art (see e.g., the Examples). In one embodiment, an immune potentiator increases the percentage of CD8+ T cells that are positive by ICS for one or more cytokines (e.g., IFN—y, TNFoc and/or IL-2) in response to an antigen (as compared to the percentage of CD8+ T cells that are positive by ICS for the cytokine(s) in the absence of the immune potentiator) by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%.

In yet another embodiment, an immune iator increases the percentage of CD8+ T cells among the total T cell population (e.g., splenic T cells and/or PBMCs), as compared to the percentage of CD8+ T cells in the e of the immune potentiator. For example, an immune potentiator can increase the percentage of CD8+ T cells among the total T cell tion by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%, as compared to the percentage of CD8+ T cells in the e of the immune potentiator. The total percentage of CD8+ T cells among the total T cell population can be determined by standard methods known in the art, ing but not limited to ?uorescent activated cell sorting (FACS) or ic activated cell g (MACS).

In r embodiment, an immune potentiator ses a tumor-speci?c immune cell response, as determined by a decrease in tumor volume in vivo in the presence of the immune potentiator as compared to tumor volume in the absence of the immune potentiator.

For example, an immune potentiator can decrease tumor volume by at least 5% or at least % or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%, as compared to tumor volume in the absence of the immune potentiator. Measurement of tumor volume can be determined by methods well established in the art.

In another embodiment, an immune iator increases B cell activity (humoral immune se), for example by sing the amount of antigen-speci?c antibody production, as compared to antigen-specif1c aantibody production in the absence of the immune iator. For example, an immune potentiator can increase antigen-speci?c antibody production by at least 5% or at least 10% or at least 15% or at least 20% or at least % or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%, as compared to antigen-speci?c antibody tion in the absence of the immune iator.

In one embodiment, antigen-specif1c IgG production is evaluated. Antigen-speci?c antibody production can be evaluated by methods well established in the art, including but not limited to ELISA, RIA and the like that measure the level of antigen-speci?c antibody (e.g., IgG) in a sample (e.g., a serum sample).

In another embodiment, an immune potentiator increases the effector memory CD62L10 T cell population. For example, an immune potentiator can increase the total % of CD62L10 T cells among CD8+ T cells. Among other functions, the effector memory CD62L10 T cell population has been shown to have an important function in lymphocyte traff1cking (see e.g., Schenkel, J.M. and Masopust, D. (2014) Immunity 41 :886-897). In various embodiments, an immune potentiator can se the total percentage of effector memory CD62L10 T cells among the CD8+ T cells in response to an n (as compared to the total percentage of CD62L10 T cells among the CD8+ T cells population in the absence of the immune potentiator) by at least 5% or at least 10% or at least 15% or at least 20% or at least % or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%. The total percentage of effector memory CD62L10 T cells among the CD8+ T cells can be determined by standard methods known in the art, including but not limited to cent activated cell sorting (FAC S) or magnetic activated cell sorting (MACS).

The ability of an immune potentiator mRNA construct to enhance an immune response to a cancer antigen can be evaluated in mouse model systems known in the art. In one embodiment, an immune competent mouse model system is used. In one ment, the mouse model system comprises C57/Bl6 mice (e.g., to evaluate antigen-specif1c CD8+ T cell responses to a cancer antigen, such as described in the Examples). In another embodiment, the mouse model system comprises Bale mice or CD1 mice (e.g., to evaluate B cell ses, such an antigen-specif1c antibody responses).

In one embodiment, an immune potentiator polypeptide of the disclosure functions downstream of at least one Toll-like receptor (TLR) to thereby enhance an immune response.

Accordingly, in one embodiment, the immune potentiator is not a TLR but is a molecule within a TLR ing pathway downstream from the receptor itself.

In one embodiment, an mRNA of the disclosure encoding an immune potentiator can comprises one or more modi?ed bases. Suitable ations are discussed further below.

In one embodiment, an mRNA of the disclosure encoding an immune potentiator is formulated into a lipid rticle. In one embodiment, the lipid nanoparticle r comprises an mRNA encoding a cancer antigen. In one embodiment, the lipid nanoparticle is administered to a subject to enhance an immune response against the cancer antigen in the subject. Suitable nanoparticles and methods of use are discussed further below.

Immune Potentiator mRNAs that Stimulate Type [Interferon In some aspects, the disclosure provides an immune potentiator mRNA encoding a polypeptide that stimulates or enhances an immune response against an antigen of st by simulating or enhancing Type I interferon pathway signaling, thereby stimulating or enhancing Type I interferon (IFN) production. It has been established that sful ion of umor or anti-microbial adaptive immunity requires Type I IFN signaling (see e.g., Fuertes, MB. et al (2013) Trends Immunol. 34:67-73). The production of Type I IFNs (including IFN—oc, IFN—B, IFN—s, lFN-K and IFN—oa) plays a role in clearance of microbial infections, such as viral infections. It has also been appreciated that host cell DNA (for example d from damaged or dying cells) is e of inducing Type I interferon tion and that the Type I IFN signaling pathway plays a role in the development of adaptive anti-tumor immunity. However, many pathogens and cancer cells have d mechanisms to reduce or inhibit Type I interferon responses. Thus, activation (including stimulation and/or enhancement) of the Type I IFN signaling pathway in a subject in need thereof, by ing an immune potentiator mRNA of the disclosure to the subject, stimulates or enhances an immune se in the subject in a wide variety of clinical situations, including treatment of cancer and pathogenic infections, as well as in potentiating vaccine responses to provide protective immunity.

Type I interferons (IFNs) are pro-in?ammatory cytokines that are rapidly produced in multiple different cell types, typically upon viral infection, and are known to have a wide variety of effects. The canonical consequences of type I IFN production in vivo is the activation of antimicrobial cellular programs and the development of innate and adaptive immune responses. Type I IFN induces a cell-intrinsic antimicrobial state in infected and neighboring cells that limits the spread of infectious agents, particularly viral pathogens.

Type I IFN also modulates innate immune cell activation (e.g., maturation of dendritic cells) to promote antigen presentation and nature killer cell functions. Type I IFN also es the development of high-aff1nity antigen-speci?c T and B cell responses and immunological memory (Ivashkiv and Donlin (2014) Nat Rev Immunol l4(l):36-49).

Type I IFN activates dendritic cells (DCs) and promotes their T cell atory capacity through autocrine ing (Montoya et al., (2002) Blood 99:3263-3271). Type I IFN exposure facilitates maturation of DCs via increasing the expression of chemokine receptors and adhesion molecules (e.g., to promote DC ion into draining lymph nodes), co-stimulatory molecules, and MHC class I and class II antigen presentation. DCs that mature following type I IFN exposure can effectively prime protective T cell responses (Wijesundara et al., (2014) Front Immunol ) and references therein).

Type I IFN can either promote or inhibit T cell activation, proliferation, differentiation and survival depending largely on the timing of type I IFN signaling relative to T cell receptor signaling e et al., (2015) Nat Rev Immunol 15:231-242). Early studies revealed that MHC-I expression is upregulated in response to type I IFN in multiple cell types (Lindahl et al., (1976), JInfect Dis 133(Suppl):A66-A68, Lindahl et al., (1976) Proc Natl/lead Sci USA 17: 1284-1287) which is a requirement for optimal T cell stimulation, differentiation, expansion and cytolytic activity. Type I IFN can exert potent co-stimulatory effects on CD8 T cells, enhancing CD8 T cell eration and differentiation (Curtsinger et al. JImmunol 174:4465-4469, Kolumam et al. Med 7-650). , (2005) , (2005) JExp Similar to effects on T cells, type I IFN signaling has both positive and negative effects on B cell responses ing on the timing and t of re (Braun et al, (2002)]nl1mmunol 14(4):411-419, Lin et al, (1998) 187(1):79-87). The survival and maturation of immature B cells can be inhibited by type I IFN signaling. In contrast to re B cells, type I IFN exposure has been shown to promote B cell activation, dy production and e switch following viral infection or following experimental immunization (Le Bon et al, (2006) JImmunol 176:4:2074-2078, Swanson et al., (2010) J EXp Med 207: 1485-1500).

A number of components involved in Type I IFN pathway signaling have been established, including STING, Interferon tory Eactors, such as IRFl, IRF3, IRFS, IRF7, IRF8, and IRF9, TBKl, IKKi, MyD88 and TRAM. Additional components involved in Type I IFN pathway signaling include TRAF3, TRAF6, IRAK-l, , TRIF, IPS-l, TLR-3, TLR-4, TLR-7, TLR-8, TLR-9, RIG-1, DAI, and 1FIl6.

Accordingly, in one embodiment, an immune potentiator mRNA encodes any of the foregoing components involved in Type I IFN pathway signaling.

Immune Potentialor mRNA Encoding STING The t disclosure encompasses mRNA (including mmRNA) encoding STING, including constitutively active forms of STING, as immune potentiators. STING (S_Timulator of Eterferon Genes, also known as transmembrane n 173 73), mediator of IRF3 tion (MITA), methionine-proline-tyrosine-serine (MPYS), and ER IFN stimulator (ERIS)) is a 379 amino acid, endoplasmic reticulum (ER) resident transmembrane protein that functions as a signaling molecule controlling the transcription of immune se genes, including type I IFNs and pro-in?ammatory cytokines (Ishikawa & Barber, (2008) Nature 455:647-678, Ishikawa et al., (2009) Nature 461 :788-792, Barber (2010) Nat Rev Immunol 15(12):760-770).

STING functions as a signaling adaptor linking the cytosolic detection ofDNA to the TBKl/IRF3/Type I IFN signaling axis. The signaling adaptor functions of STING are ted through the direct sensing of cyclic dinucleotides . Examples of CDNs include cyclic di-GMP (guanosine 5'—monophosphate), cyclic di-AMP (adenosine 5'- monophosphate) and cyclic GMP-AMP (cGAMP). Initially characterized as ubiquitous bacterial secondary messengers, CDNs are now known to tute a class of pathogen- associated molecular pattern molecules (PAMPs) that activate the TBKl/IRF3/type I IFN signaling aXis via direct interaction with STING. STING is capable of sensing aberrant DNA species and/or CDNs in the cytosol of the cell, including CDNs derived from bacteria, and/or from the host protein cyclic GMP-AMP synthase (cGAS). The cGAS protein is a DNA sensor that produces cGAMP in response to detection ofDNA in the cytosol (Burdette et al, (201 1) Nature 478:515-518; Sun et al, (2013) Science 339:786-791; Diner et al, (2013) Cell Rep -1361; Ablasser et al., (2013) Nature 498:380-384).

Upon binding to a CDN, STING dimerizes and oes a conformational change that promotes formation of a complex with TANK-binding kinase 1 (TBK1) (Ouyang et al., (2012) Immunity 36(6): 1073-1086). This complex translocates to the perinuclear Golgi, resulting in ry of TBKl to endolysosomal compartments where it phosphorylates IRF3 and NF-KB transcription factors (Zhong et al., (2008) Immunity 29:53 . A recent study has shown that STING functions as a scaffold by binding to both TBKl and IRF3 to speci?cally promote the phosphorylation of IRF3 by TBKl (Tanaka & Chen, (2012) Sci Signal 5(214):ra20). Activation of the IRF3-, IRF7- and NF-KB-dependent signaling pathways induces the production of cytokines and other immune se-related proteins, such as type I lFNs, which e anti-pathogen and/or anti-tumor activity.

A number of studies have investigated the use of CDN agonists of STING as potential vaccine adjuvants or immunomodulatory agents to elicit humoral and cellular immune responses (Dubensky et al , (2013) Ther Adv Vaccines 1(4): 13 1-143 and references therein).

Initial studies demonstrated that administration of the CDN c-di-GMP attenuated Staphylococcus aureus infection in vivo, reducing the number of recovered bacterial cells in a mouse infection model yet c-di-GMP had no observable tory or bactericidal effect on bacterial cells in vitro suggesting the reduction in bacterial cells was due to an effect on the host immune system (Karaolis et al crob Agents Chemother 49: 1029-1038; , (2005) Karaolis et al., (2007) Infect Immun 75:4942-4950). Recent studies have shown that synthetic CDN derivative molecules formulated with granulocyte-macrophage colony-stimulating factor (GM-C SF)—producing cancer vaccines (termed STINGVAX) elicit enhanced in vivo antitumor effects in therapeutic animal models of cancer as compared to immunization with GM-CSF e alone (Fu et al., (2015) Sci TranslMed 7(283):283ra52), suggesting that CDN are potent vaccine adjuvants.

Mutant STING proteins ing from polymorphisms mapped to the human IMEMI 73 gene have been bed exhibiting a gain-of function or constitutively active phenotype. When expressed in vitro, mutant STING alleles were shown to ly stimulate induction of type I IFN (Liu et al., (2014) NEngl JMea’ 371 :507-518; Jeremiah et al., (2014) J Clin Invest 16-5520; Dobbs et al., (2015) Cell HostMicrobe 18(2): 157-168; Tang & Wang, (2015) PLOS ONE 10(3):e0120090; Melki et al. , (2017) JAllergy Clin Immunol In Press, Konig et al, (2017) Ann Rheum Dis 76(2):468-472, Burdette et al (201 1) Nature 478:515-518). ed herein are modi?ed mRNAs (mmRNAs) encoding constitutively active forms of STING, including mutant human STING isoforms for use as immune potentiators as described herein. mmRNAs encoding constitutively active forms of STING, including mutant human STING isoforms are set forth in the Sequence Listing herein. The amino acid residue numbering for mutant human STING polypeptides used herein corresponds to that used for the 379 amino acid residue wild type human STING (isoform 1) available in the art as Genbank Accession Number NP_93 8023.

Accordingly, in one aspect, the disclosure provides a mmRNA encoding a mutant human STING protein having a mutation at amino acid residue 155, in particular an amino acid substitution, such as a V155M mutation. In one embodiment, the mmRNA encodes an amino acid sequence as set forth in SEQ ID NO:1. In one embodiment, the STING V155M mutant is encoded by a nucleotide sequence shown in SEQ ID NO: 199. In one embodiment, the mmRNA comprises a 3’ UTR sequence as shown in SEQ ID NO: 209, which es an miR122 binding site.

In other aspects, the disclosure es a mmRNA encoding a mutant human STING protein having a mutation at amino acid residue 284, such as an amino acid substitution.

Non-limiting examples of residue 284 substitutions include R284T, R284M and R284K. In n embodiments, the mutant human STING protein has as a R284T mutation, for example has the amino acid sequence set forth in SEQ ID NO: 2 or is encoded by an the nucleotide sequence shown in SEQ ID NO 200. In n embodiments, the mutant human STING protein has a R284M mutation, for example has the amino acid sequence as set forth in SEQ ID NO: 3 or is encoded by the nucleotide sequence shown in SEQ ID NO: 201. In certain embodiments, the mutant human STING protein has a R284K mutation, for example has the amino acid ce as set forth in SEQ ID NO: 4 or 224, or is d by the nucleotide sequence shown in SEQ ID NO: 202 or 225.

In other s, the disclosure provides a mmRNA encoding a mutant human STING protein having a mutation at amino acid e 154, such as an amino acid substitution, such as a N154S mutation. In certain embodiments, the mutant human STING protein has a N154S mutation, for example has the amino acid sequence as set forth in SEQ ID NO: 5 or is encoded by the nucleotide sequence shown in SEQ ID NO: 203.

In yet other aspects, the disclosure provides a mmRNA encoding a mutant human STING protein having a mutation at amino acid residue 147, such as an amino acid substitution, such as a V147L mutation. In certain embodiments, the mutant human STING protein having a V147L mutation has the amino acid sequence as set forth in SEQ ID NO: 6 or is encoded by the nucleotide sequence shown in SEQ ID NO: 204.

In other aspects, the sure provides a mmRNA encoding a mutant human STING protein having a mutation at amino acid residue 315, such as an amino acid substitution, such as a E315Q mutation. In certain ments, the mutant human STING protein having a E315Q mutation has the amino acid sequence as set forth in SEQ ID NO: 7 or is encoded by the tide sequence shown in SEQ ID NO: 205.

In other aspects, the disclosure es a mmRNA encoding a mutant human STING protein having a mutation at amino acid residue 375, such as an amino acid substitution, such as a R375A mutation. In n embodiments, the mutant human STING protein having a R375A mutation has the amino acid sequence as set forth in SEQ ID NO: 8 or is encoded by the nucleotide sequence shown in SEQ ID NO: 206.

In other aspects, the disclosure provides a mmRNA encoding a mutant human STING n having a one or more or a combination of two, three, four or more of the foregoing mutations. ingly, in one aspect the disclosure provides a mmRNA encoding a mutant human STING protein having one or more mutations selected from the group consisting of: V147L, N154S, V155M, R284T, R284M, R284K, E315Q and R375A, and combinations thereof. In other aspects, the disclosure provides a mmRNA encoding a mutant human STING protein having a combination of mutations selected from the group ting of: V155M and R284T, V155M and R284M, V155M and R284K, V155M and V147L, V155M and N154S, V155M and E3 15Q, and V155M and R375A.

In other aspects, the disclosure provides a mmRNA encoding a mutant human STING protein having a V155M and one, two, three or more of the following mutations: R284T, R284M, R284K, V147L, N154S, E315Q, and R375A. In other aspects, the disclosure provides a mmRNA encoding a mutant human STING protein having V155M, V147L and N154S mutations. In other aspects, the sure provides a mmRNA encoding a mutant human STING protein having V155M, V147L, N154S mutations, and, optionally, a mutation at amino acid 284. In yet other aspects, the disclosure provides a mmRNA encoding a mutant human STING protein having V155M, V147L, N154S mutations, and a mutation at amino acid 284 selected from R284T, R284M and R284K. In other aspects, the disclosure provides a mmRNA encoding a mutant human STING n having V155M, V147L, N154S, and R284T mutations. In other aspects, the disclosure provides a mmRNA encoding a mutant human STING protein having V155M, V147L, N154S, and R284M mutations. In other aspects, the disclosure provides a mmRNA encoding a mutant human STING protein having V155M, Vl47L, N154S, and R284K mutations.

In other embodiments, the sure provides a mmRNA encoding a mutant human STING protein having a combination of mutations at amino acid residue 147, 154, 155 and, optionally, 284, in particular amino acid substitutions, such as a V147L, N154S, V155M and, optionally, R284M. In certain ments, the mutant human STING protein has V147N, N154S and V155M ons, such as the amino acid sequence as set forth in SEQ ID NO: 9 or encoded by the nucleotide sequence shown in SEQ ID NO: 207. In certain embodiments, the mutant human STING protein has R284M, V147N, N154S and V155M mutations, such as the amino acid sequence as set forth in SEQ ID NO: 10 or encoded by the nucleotide sequence shown in SEQ ID NO: 208.

In another embodiment, the sure provides a mmRNA encoding a mutant human STING protein that is a constitutively active truncated form of the full-length 379 amino acid wild type n, such as a constitutively active human STING polypeptide consisting of amino acids 137-379.

Agentsfor Promotion ofAntigen Presenting Cells In some embodiments the RNA vaccines can be combined with agents for ing the production of antigen presenting cells (APCs), for instance, by converting non-APCs into -APCs. Antigen presentation is a key step in the initiation, ampli?cation and duration of an immune response. In this process fragments of antigens are presented through the Major Histocompatibility x (MHC) or Human yte Antigens (HLA) to T cells driving an antigen-specif1c immune response. For immune prophylaxis and therapy, enhancing this response is important for improved efficacy. The RNA vaccines of the invention may be designed or ed to drive ef?cient antigen presentation. One method for enhancing APC sing and presentation, is to provide better targeting of the RNA vaccines to antigen presenting cells (APC). Another approach involves activating the APC cells with immune-stimulatory formulations and/or components.

Alternatively, methods for reprograming non-APC into becoming APC may be used with the RNA vaccines of the invention. Importantly, most cells that take up mRNA formulations and are targets of their therapeutic actions are not APC. Therefore, designing a way to convert these cells into APC would be ial for efficacy. Methods and approaches for delivering RNA vaccines, e.g., mRNA vaccines to cells while also promoting the shift of a non-APC to an APC are provided herein. In some embodiments a mRNA ng an APC reprograming molecule is included in the RNA vaccine or coadministered with the RNA vaccine.

An APC reprograming molecule, as used herein, is a molecule that promotes a transition in a non APC cell to an APC-like phenotype. An APC-like phenotype is property that enables MHC class II processing. Thus, an APC cell having an APC-like phenotype is a cell having one or more exogenous molecules (APC raming molecule) which has enhanced MHC class II processing capabilities in comparison to the same cell not having the one or more exogenous molecules. In some embodiments an APC reprograming molecule is a CIITA (a central regulator ofMHC Class II expression), a chaperone protein such as CLIP, HLA-DO, HLA-DM etc. (enhancers of loading of antigen fragments into MHC Class 11) and/or a costimulatory molecule like CD40, CD80, CD86 etc. (enhancers of T cell antigen recognition and T cell activation).

A CIITA protein is a ctivator that enhances activation of transcription ofMHC Class 11 genes le et al, 1993, Cell 75: 135-146) by interacting with a conserved set of DNA binding proteins that associate with the class 11 promoter region. The riptional activation function of CIITA has been mapped to an amino terminal acidic domain (amino acids 26-137). A c acid molecule encoding a protein that interacts with CIITA, termed CIITA-interacting protein 104 (also ed to herein as CIP104). Both CITTA and CIP104 have been shown to enhance transcription from MHC class II promoters and thus are useful as APC reprograming molecule of the invention. In some embodiments the APC reprograming molecule are full length CIITA, C1P104 or other related molecules or active fragments thereof, such as amino acids 26-137 of CIITA, or amino acids having at least 80% sequence identity o and maintaining the ability to enhance activation of transcription of MHC Class 11 genes.

In preferred ments the APC raming le is red to a subject in the form of an mRNA encoding the APC reprograming molecule. As such the RNA es of the invention may include an mRNA encoding an APC reprograming molecule. In some embodiments the mRNA in monocistronic. In other embodiments it is polycistronic. In some embodiments the mRNA encoding the one or more ns is in a separate formulation from the mRNA encoding the APC reprograming molecule. In other embodiments the mRNA encoding the one or more antigens is in the same formulation as the mRNA encoding the APC reprograming molecule. In some embodiments the mRNA ng the one or more antigens is administered to a subject at the same time as the mRNA encoding the APC raming molecule. In other embodiments the mRNA encoding the one or more antigens is administered to a subject at a ent time than the mRNA encoding the APC reprograming molecule. For instance, the mRNA encoding the APC reprograming molecule may be administered prior to the mRNA encoding the one or more antigens. The mRNA ng the APC reprograming molecule may be administered immediately prior to, at least 1 hour prior to, at least 1 day prior to, at least one week prior to, or at least one month prior to the mRNA encoding the antigens.

Alternatively, the mRNA ng the APC reprograming molecule may be administered after the mRNA encoding the one or more antigens. The mRNA encoding the APC reprograming molecule may be administered immediately after, at least 1 hour after, at least 1 day after, at least one week after, or at least one month after the mRNA ng the antigens. In some ments the antigen is a cancer antigen, such as a patient speci?c antigen. In other embodiments the antigen is an infectious disease antigen.

In some embodiments the mRNA vaccine may include a recall antigen, also sometimes ed to as a memory antigen. A recall antigen is an antigen that has usly been encountered by an individual and for which there are pre-eXistent memory lymphocytes.

In some embodiments the recall n may be an infectious disease antigen that the individual has likely encountered such as an in?uenza antigen. The recall antigen helps promote a more robust immune response.

The antigens or neoepitopes ed for inclusion in the mRNA vaccine typically will be high af?nity binding peptides. In some aspects the antigens or neoepitopes binds an HLA protein with r af?nity than a wild-type peptide. The antigen or tope has an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less in some embodiments. Typically, peptides with predicted IC50<50 nM, are generally considered medium to high af?nity binding peptides and will be selected for testing their af?nity empirically using biochemical assays of HLA-binding. The cancer antigens can be personalized cancer antigens. Personalized RNA cancer vaccine, for instance, may include RNA encoding for one or more known cancer antigens speci?c for the tumor or cancer antigens speci?c for each subject, referred to as neoepitopes or subject speci?c epitopes or antigens. A "subject speci?c cancer n" is an antigen that has been identi?ed as being expressed in a tumor of a particular patient. The subject speci?c cancer antigen may or may not be lly present in tumor samples generally. Tumor associated antigens that are not expressed or rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is suf?ciently reduced in comparison to that in ous cells and that induce an immune response induced upon vaccination, are referred to as neoepitopes. Neoepitopes, like tumor associated antigens, are completely foreign to the body and thus would not produce an immune response against healthy tissue or be masked by the tive components of the immune system. In some embodiments personalized RNA cancer vaccines based on neoepitopes are desirable because such vaccine formulations will maximize speci?city against a patient’s speci?c tumor. Mutation-derived neoepitopes can arise from point mutations, non-synonymous ons leading to different amino acids in the protein, read- through mutations in which a stop codon is modi?ed or deleted, g to translation of a longer protein with a novel tumor-speci?c sequence at the C-terminus, splice site mutations that lead to the inclusion of an intron in the mature mRNA and thus a unique tumor-speci?c protein sequence, chromosomal rearrangements that give rise to a chimeric protein with tumor-speci?c sequences at the junction of 2 proteins (1'.e., gene fusion), frameshift ons or deletions that lead to a new open reading frame with a novel tumor-speci?c protein sequence, and translocations. Thus, in some embodiments the RNA cancer vaccines include at least 1 cancer antigens ing mutations selected from the group consisting of frame- shift mutations and recombinations or any of the other mutations bed herein.

Methods for generating personalized RNA cancer vaccines generally involve identi?cation of mutations, e.g., using deep nucleic acid or protein sequencing techniques, identi?cation of neoepitopes, e.g., using application of validated e-MHC binding prediction algorithms or other ical techniques to generate a set of ate T cell epitopes that may bind to patient HLA s and are based on ons present in tumors, optional demonstration of antigen-speci?c T cells against selected neoepitopes or tration that a candidate neoepitope is bound to HLA proteins on the tumor surface and development of the vaccine. The RNA cancer vaccines of the ion may include multiple copies of a single neoepitope, multiple different topes based on a single type of mutation, 1'. e. point mutation, multiple different neoepitopes based on a variety of mutation types, neoepitopes and other antigens, such as tumor associated antigens or recall antigens.

Examples of ques for identifying mutations e but are not limited to c allele-speci?c hybridization (DASH), microplate array diagonal gel electrophoresis ), pyrosequencing, oligonucleotide-speci?c ligation, the TaqMan system as well as various DNA "chip" technologies i.e. Affymetrix SNP chips, and methods based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling-circle ampli?cation.

The deep nucleic acid or protein sequencing techniques are known in the art. Any type of sequence analysis method can be used. Nucleic acid sequencing may be performed on whole tumor genomes, tumor exomes (protein-encoding DNA), tumor transcriptomes, or exosomes. Real-time single molecule sequencing-by-synthesis technologies rely on the detection of ?uorescent nucleotides as they are incorporated into a t strand ofDNA that is complementary to the template being sequenced. Other rapid high throughput sequencing s also exist. Protein sequencing may be performed on tumor proteomes.

Additionally, protein mass spectrometry may be used to identify or validate the presence of mutated peptides bound to MHC proteins on tumor cells. Peptides can be acid-eluted from tumor cells or from HLA molecules that are immunoprecipitated from tumor, and then identi?ed using mass spectrometry. The results of the cing may be compared with known control sets or with sequencing analysis performed on normal tissue of the patient.

Accordingly, the present invention relates to methods for identifying and/or detecting neoepitopes of an antigen, such as T-cell epitopes. cally, the invention provides methods of identifying and/or ing tumor speci?c neoepitopes that are useful in inducing a tumor c immune response in a subject. Optionally, these neoepitopes bind to class I HLA proteins with a greater af?nity than the wild-type peptide and/or are capable of activating anti-tumor CD8 T-cells. Identical mutations in any particular gene are rarely found across tumors.

Proteins ofMHC class I are present on the surface of almost all cells of the body, including most tumor cells. The proteins ofMHC class I are loaded with antigens that y originate from endogenous proteins or from pathogens t inside cells, and are then presented to cytotoxic T-lymphocytes (CTLs). T-Cell receptors are capable of recognizing and binding peptides complexed with the molecules ofMHC class I. Each cytotoxic T- cyte expresses a unique T-cell receptor which is capable of binding speci?c MHC/peptide complexes.

Using er algorithms, it is possible to predict potential neoepitopes such as T- cell es, 1'.e. peptide sequences, which are bound by the MHC molecules of class I or class II in the form of a peptide-presenting complex and then, in this form, recognized by the T-cell receptors of T-lymphocytes. Examples of programs useful for identifying peptides which will bind to MHC include for instance: Lonza Epibase, SYFPEITHI (Rammensee et al., Irnmunogenetics, 50 (1999), 9) and HLA_BIND (Parker et al., J. Immunol., 152 , 163-175).

Once ve neoepitopes are ed, they can be further tested using in vitro and/or in vivo assays. Conventional in vitro lab , such as Elispot assays may be used with an isolate from each patient, to re?ne the list of topes selected based on the algorithm's predictions. Neoepitope vaccines, methods of use thereof and methods of preparing are all described in PCT/USZOl6/044918 which is hereby incorporated by reference in its entirety.

The activating oncogene on peptides selected for inclusion in the RNA cancer vaccines typically will be high af?nity binding peptides. In some aspect the activating oncogene mutation peptide binds an HLA protein with greater af?nity than a wild-type peptide. The activating oncogene mutation peptides have an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less in some embodiments. Typically, peptides with predicted IC50<50 nM, are generally considered medium to high af?nity binding peptides and will be selected for testing their af?nity empirically using biochemical assays of HLA-binding.

In a personalized cancer vaccine, the subject speci?c cancer antigens may be identi?ed in a sample of a patient. For instance, the sample may be a tissue sample or a tumor sample. For instance, a sample of one or more tumor cells may be examined for the presence of subject speci?c cancer ns. The tumor sample may be examined using whole genome, exome or transcriptome analysis in order to identify the t speci?c cancer antigens.

Alternatively the subject speci?c cancer antigens may be identi?ed in an e of the subject. When the antigens for a vaccine are ?ed in an exosome of the t, such antigens are said to be representative of exosome antigens of the subject.

Exosomes are small microvesicles shed by cells, typically having a diameter of approximately 30-100 nm. Exosomes are classically formed from the inward invagination and pinching off of the late endosomal membrane, resulting in the ion of a multivesicular body (MVB) laden with small lipid bilayer vesicles, each of which contains a sample of the parent cell's cytoplasm. Fusion of the MVB with the cell membrane results in the release of these exosomes from the cell, and their delivery into the blood, urine, cerebrospinal ?uid, or other bodily ?uids. es can be recovered from any of these biological ?uids for further analysis.

Nucleic acids within exosomes have a role as biomarkers for tumor ns. An advantage of analyzing exosomes in order to identify subject speci?c cancer antigens, is that the method circumvents the need for biopsies. This can be particularly advantageous when the patient needs to have several rounds of y including identi?cation of cancer ns, and vaccination.

A number of methods of isolating exosomes from a biological sample have been bed in the art. For example, the following methods can be used: differential centrifugation, low speed centrifugation, anion exchange and/or gel permeation chromatography, sucrose density gradients or organelle ophoresis, magnetic activated cell sorting (MACS), nanomembrane ultra?ltration concentration, Percoll nt isolation and using uidic devices. Exemplary methods are described in US Patent Publication No. 20l4/O2l287l for instance.

The term "biological sample" refers to a sample that contains biological materials such as a DNA, a RNA and a protein. In some embodiments, the biological sample may suitably comprise a bodily ?uid from a subject. The bodily ?uids can be ?uids isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, , spinal ?uid, cerebrospinal ?uid, 2O l ?uid, nipple aspirates, lymph ?uid, ?uid of the respiratory, intestinal, and genitourinary tracts, tear ?uid, saliva, breast milk, ?uid from the lymphatic system, semen, cerebrospinal ?uid, intra-organ system ?uid, ascitic ?uid, tumor cyst ?uid, amniotic ?uid and combinations thereof.

In some ments, the progression of the cancer can be monitored to identify changes in the sed antigens. Thus, in some embodiments the method also involves at least one month after the administration of a cancer mRNA e, identifying at least 2 cancer antigens from a sample of the subject to produce a second set of cancer ns, and administering to the subject a mRNA vaccine having an open reading frame encoding the second set of cancer antigens to the subject. The mRNA vaccine having an open reading frame encoding second set of antigens, in some embodiments, is administered to the subject 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 1 year after the mRNA vaccine having an open reading frame ng the ?rst set of cancer antigens. In other embodiments the mRNA vaccine having an open reading frame encoding second set of antigens is administered to the subject 1 1/2, 2, 2 1/2 3 , 3, 1/2, 4, 4 1/2, or 5 years after the mRNA vaccine having an open reading frame encoding the ?rst set of cancer antigens.

Holspol mutations as neoantigens In population analyses of , certain mutations occur in a higher tage of patients than would be expected by . These "recurrent" or "hotspot" mutations have often been shown to have a "driver" role in the tumor, producing some change in the cancer cell function that is important to tumor initiation, maintenance, or metastasis, and is therefore selected for in the evolution of the tumor. In addition to their importance in tumor biology and therapy, recurrent mutations provide the unity for ion medicine, in which the patient population is stratif1ed into groups more likely to respond to a particular therapy, including but not d to ing the d protein itself.

Much effort and research on recurrent mutations has focused on non-synonymous (or "missense") single nucleotide variants (SNVs), but population analyses have ed that a variety of more complex NV) variant classi?cations, such as synonymous (or "silent"), splice site, multi-nucleotide variants, insertions, and deletions, can also occur at high frequencies.

The p53 gene (off1cial symbol TP53) is d more frequently than any other gene in human cancers. Large cohort studies have shown that, for most p53 mutations, the 2O genomic position is unique to one or only a few patients and the mutation cannot be used as recurrent neoantigens for therapeutic vaccines designed for a specific population of patients.

Surprisingly, a small subset of p53 loci do, r, exhibit a "hotspot" pattern, in which several positions in the gene are mutated with relatively high frequency. Strikingly, a large portion of these recurrently mutated regions occur near exon-intron boundaries, disrupting the canonical nucleotide sequence motifs recognized by the mRNA splicing ery. Mutation of a splicing motif can alter the final mRNA sequence even if no change to the local amino acid sequence is predicted (1'.e., for mous or intronic mutations). Therefore, these mutations are often annotated as "noncoding" by common annotation tools and neglected for further analysis, even though they may alter mRNA ng in unpredictable ways and exert severe functional impact on the translated protein. If an alternatively spliced isoform produces an in-frame sequence change (1'.e., no PTC is produced), it can escape depletion by NMD and be readily expressed, sed, and presented on the cell surface by the HLA system. Further, mutation-derived alternative splicing is usually "cryptic", 1'. e., not expressed in normal tissues, and therefore may be recognized by T-cells as non-self neoantigens.

In some aspects, the present ion es neoantigen peptide sequences resulting from certain recurrent somatic cancer mutations in p53, not limited to missense SNVs and often resulting in alternative splicing, for use as targets for therapeutic vaccination.

In some embodiments, the mutation, mRNA splicing events, resulting neoantigen peptides, and/or HLA-restricted epitopes include mutations at the canonical 5’ splice site oring codon p.T125, inducing a retained intron having e sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) that contains epitopes AVSPCISFVW (SEQ ID NO: 233) (HLA-B*57:Ol 7 HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) *O2:Ol, HLA-A*O2:O6, HLA-B*35:Ol).

In some embodiments, the mutation, mRNA splicing events, resulting neoantigen peptides, and/or HLA-restricted epitopes include ons at the canonical 5’ splice site neighboring codon p.331, inducing a retained intron having peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) that contains epitopes LQVLSLGTSY (SEQ ID NO: 237) (HLA-B*15:Ol), NAVF (SEQ ID NO: 238) (HLA-B*15:01).

In some embodiments, the mutation, mRNA splicing events, ing neoantigen peptides, and/or HLA-restricted epitopes include mutations at the canonical 3’ splice site neighboring codon p. 126, ng a cryptic alternative exonic 3’ splice site producing the 2O novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 239) that contains epitopes CTMFCQLAK (SEQ ID NO: 240) (HLA-A*1 1 :01), KSVTCTMF (SEQ ID NO: 241) (HLA-B*58:01).

In some embodiments, the mutation, mRNA splicing events, resulting neoantigen peptides, and/or stricted epitopes include mutations at the canonical 5’ splice site neighboring codon p.224, inducing a cryptic alternative intronic 5’ splice site producing the novel ng peptide ce VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) that contains epitopes VPYEPPEVW (SEQ ID NO: 243) (HLA-B*53 :Ol, HLA-B*51:Ol), LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:01, HLA-B*57:01) In the foregoing sequences, the transcript codon positions refer to the canonical full- length p53 transcript ENSTOOOOO269305 (SEQ ID NO: 245) from the l v83 human genome tion.

Mutations are typically obtained from a patient’s DNA cing data to derive neo- epitopes for prior art peptide vaccines. mRNA expression, however, is a more direct measurement of the global space of possible neo-epitopes. For example, some tumor-speci?c neo-epitopes may arise from splicing changes, insertions/deletions (InDels) resulting in hifts, alternative promoters, or epigenetic modi?cations that are not easily ?ed using only the exome sequencing data. There is untapped value in identifying these types of compleX mutations for neoantigen vaccines because they will increase the number of epitopes capable of binding a patient’s unique HLA allotypes. Moreover, the compleX ts will be more immunogenic and likely lead to more effective immune responses against tumors due to their difference from self proteins compared to variants resulting from a single amino acid change.

In some aspects, the ion involves a method for fying patient speci?c compleX mutations and formulating these mutations into ive personalized mRNA es. The s involve the use of short read RNA-Seq. A major challenge inherent to using short reads for RNA-seq is the fact that multiple mRNA transcript isoforms can be ed from the same genomic locus, due to alternative splicing and other mechanisms.

Due to the sequencing reads being much shorter than the ength mRNA transcript, it becomes dif?cult to map a set of reads back to the correct corresponding isoform within a known gene annotation model. As a result, compleX variants that diverge from the known gene annotations (as are common in cancer) can be dif?cult to discover by standard approaches. The invention, however, involves the identi?cation of short peptides rather than the exact exon composition of the full-length transcript. The methods for identifying short 2O peptides that will be representative of these compleX mutations involves a short k-mer counting ch to neo-epitope prediction of X variants.

A typical next generation sequencing read is 150 base-pairs, which, if capturing a coding region, can resolve 50 codons, or 41 distinct peptide epitopes of length 9 (27 nucleotides). Therefore, using a simple, computationally scalable operation to count all 27- mers from an RNA-seq sample, the results can be compared versus normal tissue from the same sample, or to a puted database of 27-mers from RNA-seq of normal tissues (e.g., GTEX).

An mRNA vaccine containing neo-epitopes predicted from RNA-seq data can be created, whereby 1) all possible 27-mers are counted from all RNA-seq reads from a tumor , 2) the open reading frame for each read is predicted by aligning any part of the entire read to the riptome, and 3) 27-mer counts are compared to the corresponding 27-mer counts of the matched normal sample and/or a database of normal tissues from the same tissue type, and 4) DNA-seq data from the same tumor is used to add con?dence to the neo- epitope predictions, if there is a somatic mutation found in the same gene. Regarding point (4), often a mutation can cause transcriptional or splicing changes that result in a change of the mRNA sequence that is not directly predictable from the mutation itself. For e, a splice site mutation may be ted to cause exon skipping, but it is not possible to know with nty which ream exon will be chosen by the splicing machinery in its place.

In one ment, the invention provides an mRNA vaccine comprising a concatemeric polyepitope construct or set of individual epitope ucts containing open g frame (ORF) coding for igen peptides 1 through 4.

In one embodiment, the ion provides the selective administration of a vaccine containing or coding for peptides l-4, based on the patient’s tumor containing any of the above mutations.

In one embodiment, the invention provides the selective stration of the e based on the dual criteria of the l) patient’s tumor containing any of the above mutations and 2) the patient’s normal HLA type containing the corresponding HLA allele predicted to bind to the resulting neoantigen.

It has been discovered that the mRNA vaccines described herein are superior to current vaccines in several ways. First, the lipid nanoparticle (LNP) delivery is superior to other formulations including liposome or protamine based approaches described in the literature and no additional adjuvants are to be necessary. The use of LNPs enables the effective delivery of chemically modi?ed or unmodi?ed mRNA vaccines. Both modi?ed and unmodi?ed LNP ated mRNA vaccines are superior to conventional vaccines by a signi?cant degree. In some embodiments the mRNA vaccines of the invention are superior to conventional vaccines by a factor of at least 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000 fold.

Although attempts have been made to e functional RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic ef?cacy of these RNA vaccines have not yet been fully established. Quite surprisingly, the inventors have discovered, according to aspects of the invention a class of formulations for delivering mRNA vaccines in vivo that results in signi?cantly enhanced, and in many respects synergistic, immune responses including enhanced antigen generation and onal antibody production with neutralization capability. These results can be achieved even when signi?cantly lower doses of the mRNA are administered in comparison with mRNA doses used in other classes of lipid based formulations. The ations of the invention have demonstrated signi?cant unexpected in vivo immune responses suf?cient to establish the ef?cacy of functional mRNA vaccines as prophylactic and therapeutic agents. Additionally, self-replicating RNA vaccines rely on viral replication pathways to deliver enough RNA to a cell to produce an immunogenic response. The formulations of the invention do not require viral replication to produce enough protein to result in a strong immune response. Thus, the mRNA of the invention are not self-replicating RNA and do not include ents necessary for viral replication.

The invention involves, in some aspects, the surprising ?nding that lipid nanoparticle (LNP) formulations signi?cantly enhance the effectiveness of mRNA vaccines, including chemically modi?ed and unmodi?ed mRNA vaccines. The ef?cacy of mRNA vaccines formulated in LNP was examined in vivo using several distinct tumor antigens. In addition to providing an enhanced immune response, the formulations of the invention generate a more rapid immune response with fewer doses of antigen than other es . The mRNA- LNP formulations of the ion also e quantitatively and atively better immune responses than vaccines formulated in a different carriers. Additionally, the mRNA- LNP formulations of the ion are or to other vaccines even when the dose of mRNA is lower than other vaccines.

The LNP used in the s described herein has been used previously to deliver siRNA in various animal models as well as in humans. In view of the observations made in association with the siRNA delivery of LNP formulations, the fact that LNP is useful in es is quite surprising. It has been observed that therapeutic delivery of siRNA formulated in LNP causes an undesirable in?ammatory response associated with a transient IgM response, typically leading to a reduction in antigen production and a mised immune response. In contrast to the ?ndings observed with siRNA, the NA formulations of the invention are demonstrated herein to generate enhanced IgG levels, suf?cient for prophylactic and therapeutic methods rather than transient IgM responses.

Nucleic Acids/Polynucleolides Cancer vaccines, as provided herein, comprise at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigenic polypeptide. The term "nucleic acid," in its broadest sense, includes any compound and/or nce that comprises a polymer of tides. These polymers are referred to as polynucleotides.

Nucleic acids (also referred to as polynucleotides) may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a B- D-ribo con?guration, 0t-LNA having an 0t-L-ribo con?guration (a diastereomer of LNA), 2'-amino-LNA having a no functionalization, and 2'-amino- 0t-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure function as messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or ed polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.

The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their onal and/or structural design features which serve to overcome existing problems of effective ptide expression using nucleic-acid based therapeutics.

In some embodiments, a RNA polynucleotide of a cancer vaccine encodes 2-10, 2-9, 2-8, 2—7, 2-6, 2—5, 2—4, 2—3, 3—10, 3—9, 3-8, 3—7, 3-6, 3—5, 3—4, 4—10, 4—9, 4-8, 4—7, 4-6, 4—5, 5— , 5—9, 5-8, 5—7, 5-6, 6-10, 6-9, 6-8, 6-7, 7—10, 7—9, 7-8, 8-10, 8-9 or 9—10 antigenic polypeptides. In some embodiments, a RNA polynucleotide of a cancer e encodes at least 10, 20, 30, 40, 50 90 or 100 antigenic polypeptides. In some embodiments, , 60, 70, 80, a RNA polynucleotide of a cancer vaccine encodes at least 100 or at least 200 antigenic polypeptides. In some embodiments, a RNA polynucleotide of a cancer vaccine encodes l- , 5—15, 10—20, 15—25, 20—30, 25—35, 30—40, 35—45, 40—50, 55-65, 60-70, 65-75, 70-80, 75-85 7 80-90, 85-95, 90—100, 1—50, 1—100, 2—50 or 2—100 antigenic polypeptides.

In some embodiments, a RNA polynucleotide of a cancer vaccine s 2-10, 2-9, 2-8, 2—7, 2-6, 2—5, 2—4, 2—3, 3—10, 3—9, 3-8, 3—7, 3-6, 3—5, 3—4, 4—10, 4—9, 4-8, 4—7, 4-6, 4—5, 5— , 5—9, 5-8, 5—7, 5-6, 6-10, 6-9, 6-8, 6-7, 7—10, 7—9, 7-8, 8-10, 8-9 or 9—10 activating oncogene mutation peptides. In some ments, a RNA polynucleotide of a cancer vaccine encodes at least 10, 20, 30, 40, 50 90 or 100 activating ne , 60, 70, 80, mutation peptides. In some embodiments, a RNA polynucleotide of a cancer vaccine encodes at least 100 or at least 200 activating oncogene mutation peptides. In some ments, a RNA polynucleotide of a cancer e encodes 1-10, 5-15, 10-20, 15-25, —30, 25—35, 30—40, 35—45, 40—50, 55-65, 60-70, 65-75, 70-80, 75-85, 80-90, 85-95, , 1-50, 1-100, 2-50 or 2-100 activating oncogene mutation peptides.

Polynucleotides of the present disclosure, in some embodiments, are codon optimized.

Codon optimization methods are known in the art and may be used as ed herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA ity or reduce ary structures, minimize tandem repeat codons or base runs that may impair gene uction or expression, customize transcriptional and translational control regions, insert or remove protein traff1cking sequences, remove/add post translation modi?cation sites in encoded protein (e.g. glycosylation sites), add, remove or shuf?e protein domains, insert or delete restriction sites, modify ribosome g sites and mRNA degradation sites, adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the cleotide. Codon optimization tools, algorithms and services are known in the art — non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95% ce ty to a naturally-occurring or wild-type ce (e.g., a naturally-occurring or wild- type mRNA sequence ng a polypeptide or protein of interest (e.g., an antigenic protein or ptide)). In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or ype sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a ptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, a codon optimized sequence shares less than 85% ce identity to a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic n or polypeptide)). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, a codon zed sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of st (e.g., an antigenic protein or polypeptide)).

In some embodiments, a codon optimized sequence shares between 65% and 85% (e. g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild- type mRNA sequence encoding a polypeptide or n of st (e.g., an antigenic protein or polypeptide)). In some embodiments, a codon optimized sequence shares n 65% and 75% or about 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA ce encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

In some embodiments a codon optimized RNA may, for instance, be one in which the levels of G/C are enhanced. The G/C-content of nucleic acid molecules may in?uence the stability of the RNA. RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than nucleic acids ning a large amount of e (A) and thymine (T) or uracil (U) nucleotides. W002/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modi?cations in the translated region. Due to the degeneracy of the genetic code, the ations work by substituting eXisting codons for those that promote r RNA stability without changing the resulting amino acid. The ch is limited to coding regions of the RNA.

Antigens/Antigenic Polypeptides In some embodiments, a cancer polypeptide (e.g., an activating oncogene mutation peptide) is longer than 5 amino acids and shorter than 50 amino acids. In some embodiments, a cancer polypeptide is longer than 25 amino acids and shorter than 50 amino acids. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi- lar compleX such as a dimer, trimer or tetramer. Polypeptides may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most ly, disulf1de linkages are found in multichain ptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical ue of a corresponding naturally-occurring amino acid.

The term "polypeptide variant" refers to les which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess tutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a native or reference sequence.

In some embodiments "variant mimics" are provided. As used herein, the term "variant mimic" is one which contains at least one amino acid that would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or oro-serine. Alternatively, t mimics may result in vation or in an inactivated product containing the mimic, for example, phenylalanine may act as an inactivating substitution for tyrosine, or alanine may act as an inactivating substitution for serine.

"Orthologs" refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. ?cation of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.

"Analogs" is meant to include polypeptide variants which differ by one or more amino acid tions, for example, substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.

The t disclosure provides l types of compositions that are polynucleotide or polypeptide based, including ts and derivatives. These include, for example, substitutional, insertional, deletion and nt variants and derivatives. The term "derivative" is used mously with the term "variant" but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.

As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modif1cations with respect to reference sequences, in particular the polypeptide sequences disclosed , are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N—terrninal or C-terminal ends).

Sequence tags can be used for peptide detection, puri?cation or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N—terminal es) may alternatively be d depending on the use of the sequence, as for example, sion of the sequence as part of a larger sequence which is soluble, or linked to a solid support.

"Substitutional variants" when referring to polypeptides are those that have at least one amino acid e in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be , where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

As used herein the term rvative amino acid substitution" refers to the substitution of an amino acid that is normally present in the ce with a different amino acid of similar size, , or polarity. Examples of conservative substitutions include the substitution of a lar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, es of conservative substitutions include the tution of one polar (hydrophilic) residue for another such as between arginine and lysine, between ine and asparagine, and between glycine and serine. Additionally, the tution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic e are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar e for a non-polar residue.

"Features" when referring to polypeptide or polynucleotide are de?ned as ct amino acid sequence-based or nucleotide-based components of a molecule respectively.

Features of the ptides encoded by the polynucleotides include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.

As used herein when referring to polypeptides the term "domain" refers to a motif of a polypeptide having one or more identi?able structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the terms "site" as it pertains to amino acid based embodiments is used synonymously with "amino acid e" and "amino acid side chain." As used herein when referring to cleotides the terms "site" as it pertains to nucleotide based embodiments is used synonymously with "nucleotide." A site represents a position within a peptide or polypeptide or polynucleotide that may be modi?ed, lated, altered, derivatized or varied within the polypeptide or polynucleotide based molecules.

As used herein the terms "termini" or "terminus" when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide respectively. Such extremity is not limited only to the ?rst or ?nal site of the polypeptide or polynucleotide but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be terized as having both an N—terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group ). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces mers, oligomers). These proteins have multiple N— and C-termini. Alternatively, the termini of the polypeptides may be modi?ed such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic ate.

As recognized by those skilled in the art, n fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, ed herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue r than a reference polypeptide sequence but otherwise identical) of a reference protein 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In another e, any protein that includes a stretch of 10, 20, 30, 40, 50, or 100 amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than 80%, 75%, 70%, 65% or 60% identical to any of the sequences described herein can be utilized in accordance with the disclosure.

Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference les (e.g., reference ptides or reference polynucleotides), for example, with scribed les (e.g., ered or designed molecules or wild-type molecules). The term "identity" as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, ty also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid es or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., "algorithms"). ty of related peptides can be readily calculated by known methods. "% identity" as it applies to polypeptide or polynucleotide sequences is de?ned as the percentage of residues (amino acid residues or c acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by ce alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of n database search programs", Nucleic Acids Res. 25:3389-3402). r popular local ent technique is based on the Smith- Waterman algorithm , T.F. & Waterman, MS. (1981) "Identi?cation of common molecular subsequences." J. Mol. Biol. 147:195-197). A general global alignment technique based on dynamic programming is the Needleman—Wunsch algorithm (Needleman, S.B. & Wunsch, CD. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453.). More recently a Fast Optimal Global ce Alignment Algorithm (FOGSAA) has been developed that purportedly produces global ent of nucleotide and protein sequences faster than other l global alignment methods, ing the man—Wunsch algorithm. Other tools are bed , speci?cally in the de?nition of "identity" below.

As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA les) and/or between polypeptide molecules. ric molecules (e.g. nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or polypeptide les) that share a old level of similarity or identity determined by alignment of matching residues are termed homologous. Homology is a qualitative term that describes a relationship between molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that de?nes the degree of sequence match between two compared sequences. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% cal or similar. The term ogous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4—5 uniquely ed amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4— uniquely speci?ed amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least amino acids.

Homology implies that the compared sequences diverged in evolution from a common origin. The term "homolog" refers to a ?rst amino acid ce or nucleic acid sequence (e.g., gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ral sequence. The term "homolog" may apply to the relationship between genes and/or proteins separated by the event of speciation or to the relationship between genes and/or proteins separated by the event of genetic duplication. logs" are genes (or ns) in different species that evolved from a common ancestral gene (or n) by speciation. Typically, ogs retain the same function in the course of evolution. ogs" are genes (or ns) related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.

The term "identity" refers to the overall relatedness between ric molecules, for example, between cleotide les (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a ?rst and a second nucleic acid sequences for l alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the ?rst sequence is occupied by the same nucleotide as the ponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the t identity between two nucleic acid sequences can be determined using methods such as those described in ational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., ic Press, New York, 1993, Sequence Analysis in Molecular Biology, von Heinj e, G., Academic Press, 1987, Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994, and Sequence Analysis Primer, Gribskov, M. and Devereux, J ., eds., M Stockton Press, New York, 1991, each of which is incorporated herein by reference.

For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 1 1-17), which has been incorporated into the ALIGN m (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap y of 4. The percent identity n two c acid sequences can, alternatively, be determined using the GAP program in the GCG re package using an dna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988), incorporated herein by reference. ques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al, Nucleic Acids Research, 12(1), 387 (1984)), , BLASTN, and FASTA Altschul, S. F. et al, J.

Molec. Biol, 215, 403 (1990)).

ChemicalModifications Modified Nucleotide ces Encoding Epitope Antigen Polypeptides RNA (e.g., mRNA) vaccines of the t disclosure comprise, in some embodiments, at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one respiratory syncytial virus (RSV) nic polypeptide, wherein said RNA comprises at least one chemical modi?cation.

The terms cal modification" and "chemically modified" refer to modi?cation with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the cleotide modi?cations in naturally occurring 5'-terminal mRNA cap moieties.

Modi?cations of polynucleotides include, without limitation, those described , and include, but are expressly not limited to, those modi?cations that comprise chemical modi?cations. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may se modi?cations that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a ation of naturally-occurring and non-naturally-occurring modi?cations. Polynucleotides may include any useful modi?cation, for example, of a sugar, a nucleobase, or an intemucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).

With respect to a ptide, the term "modi?cation" refers to a modi?cation relative to the canonical set 20 amino acids. Polypeptides, as provided herein, are also considered "modi?ed" of they n amino acid substitutions, insertions or a combination of substitutions and insertions.

Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise various (more than one) different modi?cations. In some embodiments, a particular region of a polynucleotide contains one, two or more (optionally different) side or tide modi?cations. In some embodiments, a d RNA polynucleotide (e.g., a modi?ed mRNA polynucleotide), introduced to a cell or organism, eXhibits reduced degradation in the cell or organism, respectively, relative to an unmodi?ed polynucleotide. In some embodiments, a modi?ed RNA polynucleotide (e.g., a modi?ed mRNA polynucleotide), introduced into a cell or organism, may t reduced immunogenicity in the cell or organism, respectively (e.g., a d innate response).

Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modi?ed nucleotides that are uced during synthesis or ynthesis of the polynucleotides to achieve desired functions or properties. The modi?cations may be present on an intemucleotide linkages, purine or pyrimidine bases, or . The ation may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modi?ed.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a chemically modi?ed nucleobase. The invention includes modi?ed polynucleotides comprising a polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding one or more cancer epitope polypeptides). The modi?ed polynucleotides can be chemically d and/or structurally modi?ed. When the cleotides of the present invention are chemically and/or urally modi?ed the polynucleotides can be referred to as ed polynucleotides." The present disclosure provides for d nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides) encoding one or more cancer epitope polypeptides. A "nucleoside" refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). A "nucleotide" refers to a nucleoside including a phosphate group. Modi?ed nucleotides can by synthesized by any useful method, such as, for e, chemically, enzymatically, or inantly, to include one or more modi?ed or tural nucleosides. Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester es, in which case the polynucleotides would comprise regions of nucleotides.

The modi?ed polynucleotides disclosed herein can comprise various distinct modi?cations. In some embodiments, the d polynucleotides contain one, two, or more (optionally ent) nucleoside or nucleotide modi?cations. In some embodiments, a modi?ed polynucleotide, introduced to a cell can exhibit one or more desirable properties, e.g., improved protein expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodi?ed polynucleotide.

In some embodiments, a polynucleotide of the t ion (e.g., a polynucleotide comprising a nucleotide sequence encoding one or more cancer epitope polypeptides) is structurally modi?ed. As used herein, a "structura " modi?cation is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without cant chemical modi?cation to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modi?cation, structural modi?cations are of a chemical nature and hence are chemical modi?cations. However, structural modi?cations will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" can be chemically modi?ed to "AT-5meC-G". The same polynucleotide can be structurally modi?ed from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modi?cation to the polynucleotide.

In some ments, the polynucleotides of the present ion are chemically modi?ed. As used herein in nce to a polynucleotide, the terms "chemical modi?cation" or, as appropriate, "chemically modi?ed" refer to modi?cation with respect to adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribo- or ibonucleosides in one or more of their on, pattern, percent or population. Generally, herein, these terms are not ed to refer to the ribonucleotide modi?cations in naturally occurring 5'-terminal mRNA cap moieties.

In some embodiments, the polynucleotides of the present invention can have a m chemical modi?cation of all or any of the same nucleoside type or a population of modi?cations produced by mere downward titration of the same starting modi?cation in all or any of the same nucleoside type, or a measured percent of a chemical modi?cation of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment, the polynucleotides can have a uniform chemical modi?cation of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modi?ed in the same way).

Modi?ed nucleotide base g encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modi?ed nucleotides comprising non-standard or modi?ed bases, wherein the arrangement of hydrogen bond donors and en bond acceptors permits hydrogen g between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those polynucleotides haVing at least one chemical modi?cation. One example of such non-standard base pairing is the base pairing between the modi?ed nucleotide e and e, cytosine or uracil. Any combination of base/sugar or linker can be incorporated into polynucleotides of the present disclosure.

The d n will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite "T"s in a representative DNA sequence but where the sequence represents RNA, the "T"s would be substituted for "U"s.

Modi?cations of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), including but not limited to chemical modi?cation, that are useful in the compositions, methods and synthetic processes of the present disclosure include, but are not limited to the following:uniformly tides, nucleosides, and nucleobases: 2-methylthio-N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6- threonyl carbamoyladenosine, N6-glycinylcarbamoyladenosine, pentenyladenosine, N6- methyladenosine, N6-threonylcarbamoyladenosine, l,2'-O-dimethyladenosine, lmethyladenosine , 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), 2-methyladenosine, 2-methylthio-N6 isopentenyladenosine, ylthio-N6-hydroxynorvalyl carbamoyladenosine, 2'-O-methy1adenosine; 2'—O-ribosy1adenosine (phosphate); Isopentenyladenosine; N6-(cis— hydroxyisopenteny1)adenosine; N6;2'-O-dimethy1adenosine; N6;2'-O-dimethy1adenosine; N6;N6;2'-O-trimethy1adenosine; N6;N6-dimethy1adenosine; N6-acety1adenosine; N6- hydroxynorvalylcarbamoyladenosine; hy1-N6-threony1carbamoyladenosine; 2- methyladenosine; 2-methy1thio-N6-isopentenyladenosine; 7-deaza-adenosine; Nl-methyl- adenosine; N6; N6 (dimethy1)adenine; -hydroxy-isopentenyl-adenosine; u-thio-adenosine; 2 (amino)adenine; 2 (aminopropy1)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2- )adenine; 2-(aminoalky1)adenine; 2-(aminopropy1)adenine; 2-(halo)adenine; 2- (halo)adenine; 2-(propy1)adenine; 2'-Amino-2'—deoxy-ATP; 2'-Azido—2'—deoxy-ATP; 2'-Deoxy- 2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (a1ky1)adenine; 6 (methy1)adenine; 6-(a1ky1)adenine; 6-(methy1)adenine; 7 )adenine; 8 (alkeny1)adenine; 8 y1)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(a1keny1)adenine; 8-(a1ky1)adenine; 8-(a1kyny1)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxy1)adenine; 8-(thioa1ky1)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methy1)adenine; N6-(isopenty1)adenine; 7- deazaaza-adenosine; y1adenine; aadenosine TP; 2'F1uoro—N6-Bz- deoxyadenosine TP; 2'-OMeAmino—ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a- Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP; 2-Amino—ATP; 2'-a- Tri?uoromethyladenosine TP; 2-Azidoadenosine TP; 2'-b-Ethyny1adenosine TP; 2- denosine TP; 2'-b-Tri?uoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2';2'- 2O di?uoroadenosine TP; 2'-Deoxy-2'-a-mercaptoadenosine TP; 2'—Deoxy-2'—a- thiomethoxyadenosine TP; 2'-Deoxy-2'—b-aminoadenosine TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine TP; 2'—Deoxy-2'—b-chloroadenosine TP; 2'-Deoxy-2'—b- ?uoroadenosine TP; 2'—Deoxy-2'—b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine TP; 2'- Deoxy-2'—b-thiomethoxyadenosine TP; 2-F1uoroadenosine TP; adenosine TP; 2- Mercaptoadenosine TP; oxy-adenine; 2-methy1thio—adenine; 2-Tri?uoromethyladenosine TP; 3-Deazabromoadenosine TP; 3-Deazachloroadenosine TP; 3-Deaza?uoroadenosine TP; 3-Deazaiodoadenosine TP; 3-Deazaadenosine TP; 4'—Azidoadenosine TP; 4'—Carbocyclic adenosine TP; 4'—Ethyny1adenosine TP; 5'—Homo—adenosine TP; ATP; 8-bromo-adenosine TP; 8-Tri?uoromethy1adenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2;6- diaminopurine; 7-deazaaza-2;6-diaminopurine; 7-deazaazaaminopurine; 2,6- diaminopurine; 7-deazaaza-adenine; aaminopurine; 2-thiocytidine; 3-methy1cytidine; -for1ny1cytidine; 5-hydroxymethylcytidine; S-methylcytidine; N4-acetylcytidine; 2'-O- methylcytidine; 2'-O-methy1cytidine; 5,2'-O-dimethy1cytidine; 5-for1ny1-2'-O-methy1cytidine; Lysidine; N4;2'-O-dimethy1cytidine; N4-acety1-2'-O-methy1cytidine; N4-methylcytidine; N4;N4- Dimethy1-2'-OMe-Cytidine TP; 4-methy1cytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo- cytidine; 0t-thio-cytidine; 2-(thio)cytosine; 2'—Amino-2'—deoxy-CTP; 2'—Azido-2'—deoxy-CTP; 2'- Deoxy-2'-a-aminocytidine TP; 2'-Deoxy-2'—a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methy1)cytosine; 3-(a1ky1)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methy1)cytidine; 4,2'—O- dimethylcytidine; 5 (halo)cytosine; 5 (methy1)cytosine; 5 (propyny1)cytosine; 5 (tri?uoromethy1)cytosine; 5-(a1ky1)cytosine; 5-(a1kyny1)cytosine; 5-(halo)cytosine; 5- ny1)cytosine; 5-(tri?uoromethy1)cytosine; 5-bromo—cytidine; 5-iodo-cytidine; 5-propyny1 cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acety1)cytosine; 1- methyl-l-deaza-pseudoisocytidine; 1-methy1-pseudoisocytidine; 2-methoxymethy1-cytidine; 2-methoxy-cytidine; 2-thiomethy1-cytidine; 4-methoxymethy1-pseudoisocytidine; 4- methoxy-pseudoisocytidine; 4-thiomethy1deaza-pseudoisocytidine; 4-thiomethy1- pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; S-methyl-zebularine; pyrrolo- pseudoisocytidine; Zebularine; (E)(2-Bromo-Viny1)cytidine TP; 2,2'—anhydro-cytidine TP hydrochloride; 2'F1uor-N4-Bz-cytidine TP; 2'F1uoro-N4-Acety1-cytidine TP; ethy1-N4- Acetyl-cytidine TP; 2'O-methyl-N4-Bz-cytidine TP; thyny1cytidine TP; 2'—a- Tri?uoromethylcytidine TP; 2'—b-Ethyny1cytidine TP; 2'-b-Tri?uoromethy1cytidine TP; 2'- Deoxy-2';2'-di?uorocytidine TP; 2'-Deoxy-2'-a-mercaptocytidine TP; 2'—Deoxy-2'—a- thiomethoxycytidine TP; 2'—Deoxy-2'-b-aminocytidine TP; 2'—Deoxy-2'—b-azidocytidine TP; 2'- Deoxy-2'—b-bromocytidine TP; 2'—Deoxy-2'—b-chlorocytidine TP; 2'—Deoxy-2'-b-?uorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; xy-2'-b-mercaptocytidine TP; xy-2'—b- thiomethoxycytidine TP; ethy1(1-propyny1)cytidine TP; 3'-Ethyny1cytidine TP; 4'- Azidocytidine TP; 4'—Carbocyc1ic ne TP; 4'—Ethyny1cytidine TP; 5-(1-Propyny1)ara-cytidine TP; 5-(2-Ch1oro-pheny1)thiocytidine TP; 5-(4-Amino-pheny1)thiocytidine TP; 5- Aminoallyl-CTP; ocytidine TP; 5-Ethyny1ara-cytidine TP; 5-Ethyny1cytidine TP; 5'- Homo-cytidine TP; 5-Methoxycytidine TP; 5-Tri?uoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoy1-cytidine TP; isocytidine; 7-methy1guanosine; N2;2'-O- dimethylguanosine; NZ-methylguanosine; e; 1,2'-O-dimethylguanosine; 1- methylguanosine; 2'-O-methy1guanosine; 2'-O-ribosy1guanosine (phosphate); 2'—O- methylguanosine; 2'—O-ribosy1guanosine (phosphate); 7-aminomethy1deazaguanosine; 7- cyanodeazaguanosine; Archaeosine; Methylwyosine; N2;7-dimethy1guanosine; N2;N2;2'-O- trimethylguanosine; 7-trimethy1guanosine; N2;N2-dimethylguanosine; N2;7;2'-O- trimethylguanosine; -guanosine; 7-deaza-guanosine; 8-oxo-guanosine; Nl-methylguanosine ; a-thio-guanosine; 2 (propy1)guanine; 2-(a1ky1)guanine; 2'—Amino-2'—deoxy-GTP; 2'- Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'—Deoxy-2'—a-azidoguanosine TP; 6 (methy1)guanine; 6-(a1ky1)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alky1)guanine; 7 (deaza)guanine; 7 (methy1)guanine; 7-(a1ky1)guanine; 7-(deaza)guanine; 7-(methy1)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(a1keny1)guanine; 8- (alkyl)guanine; 8-(a1kynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxy1)guanine; 8- lky1)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; y1)guanine; N- (methy1)guanine; y1thio-guanosine; 6-methoxy-guanosine; —7-deazaazaguanosine ; 6-thiodeaza-guanosine; 6-thiomethy1-guanosine; 7-deazaaza-guanosine; 7- methyloxo-guanosine; N2;N2-dimethy1thio-guanosine; N2-methylthio-guanosine; l-Me- GTP; 2'F1uoro—N2-isobutyl-guanosine TP; 2'O-methy1-N2-isobutyl-guanosine TP; 2'—a- Ethynylguanosine TP; 2'-a-Tri?uoromethylguanosine TP; 2'-b-Ethyny1guanosine TP; 2'—b- Tri?uoromethylguanosine TP; 2'-Deoxy-2';2'-di?uoroguanosine TP; 2'—Deoxy-2'—a- mercaptoguanosine TP; xy-2'—a-thiomethoxyguanosine TP; 2'—Deoxy-2'-b-aminoguanosine TP; 2'—Deoxy-2'-b-azidoguanosine TP; 2'—Deoxy-2'—b-bromoguanosine TP; 2'—Deoxy-2'—b- chloroguanosine TP; xy-2'—b-?uoroguanosine TP; 2'-Deoxy-2'—b-iodoguanosine TP; 2'- Deoxy-Z'-b-mercaptoguanosine TP; 2'—Deoxy-2'—b-thiomethoxyguanosine TP; 4'—Azidoguanosine TP; 4'—Carbocyclic guanosine TP; 4'—Ethyny1guanosine TP; 5'—Homo—guanosine TP; 8-bromo— guanosine TP; 9-Deazaguanosine TP; N2-isobuty1-guanosine TP; 1-methy1inosine; Inosine; 1,2'- O-dimethylinosine; 2'-O-methylinosine; 7-methy1inosine; 2'—O-methy1inosine; Epoxyqueuosine; galactosyl-queuosine; quueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2'—O-methy1uridine; 2-thiouridine; 3-methyluridine; 5- carboxymethyluridine; 5-hydroxyuridine; S-methyluridine; 5-taurinomethy1thiouridine; 5- taurinomethyluridine; Dihydrouridine; Pseudouridine; (3-(3-aminocarboxypropy1)uridine; 1- methy1(3 -amino—5-carboxypropyl)pseudouridine; 1-methy1pseduouridine; 1-ethy1- pseudouridine; 2'-O-methy1uridine; 2'—O-methy1pseudouridine; 2'—O-methy1uridine; 2-thio-2'-O- methyluridine; 3-(3-aminocarboxypropy1)uridine; 3,2'-O-dimethyluridine; 3-Methy1-pseudo- Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethy1)uridine methyl ester; 5,2'-O-dimethy1uridine; hydro-uridine; 5-aminomethy1thiouridine; 5- carbamoylmethy1-2'-O-methy1uridine; 5-carbamoylmethyluridine; 5- carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5- carboxymethylaminomethyl-Z'-O-methy1uridine; 5-carboxymethylaminomethylthiouridine; 5- ymethylaminomethylthiouridine; 5-carboxymethylaminomethyluridine; 5- carboxymethylaminomethyluridine; 5-Carbamoy1methyluridine TP; S-methoxycarbonylmethyl- 2'-O-methyluridine; 5-methoxycarbonylmethylthiouridine; 5-methoxycarbonylmethyluridine; -methy1uridine,); S-methoxyuridine; 5-methy1thiouridine; S-methylaminomethyl-Z- selenouridine; 5-methylaminomethylthiouridine; 5-methylaminomethyluridine; 5- Methyldihydrouridine; 5-Oxyacetic acid- Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; Nl-methyl-pseudo-uracil; Nl-ethyl-pseudo-uracil; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3 -Amino—3-carboxypropyl)—Uridine TP; 5-(iso—Pentenylaminomethyl)- 2- thiouridine TP; 5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso- Pentenylaminomethyl)uridine TP; 5-propynyl uracil; d-thio-uridine; l (aminoalkylaminocarbonylethylenyl )-2(thio)-pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-2;4- (dithio)pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-pseudouracil; l (aminocarbonylethylenyl)-2(thio)— pseudouracil; 1 (aminocarbonylethylenyl)-2;4-(dithio)pseudouracil; 1 (aminocarbonylethylenyl)— 4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted )- pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 tuted 4 (thio)pseudouracil; 1 substituted pseudouracil; 1-(aminoalkylamino-carbonylethylenyl)(thio)-pseudouracil; 1- (3 -amino—3 -carboxypropyl) uridine TP; l-Methyl-3 -(3 -amino carboxypropyl)pseudo-UTP; yl-pseudo-UTP; l-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 2' ?uorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl; 2'amino; 2'azido; 2'?uro-guanosine; 2'-Amino-2'—deoxy-UTP; 2'-Azido—2'—deoxy-UTP; 2'-Azido— deoxyuridine TP; 2'-O-methylpseudouridine; 2' deoxy uridine; 2' ridine; 2'-Deoxy-2'—a- aminouridine TP; 2'-Deoxy-2'—a-azidouridine TP; ylpseudouridine; 3 (3 3 2O carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio )pseudouracil; 4-(thio)uracil; 4-thiouracil; 5 (l,3-diazolealkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 diniumalkyl)uracil; 5 (methoxycarbonylmethyl) (thio)uracil; 5 xycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 l) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2;4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (tri?uoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl) (thio)pseudouracil; 5-(alkyl)—2;4 (dithio)pseudouracil; 5-(alkyl)—4 (thio)pseudouracil; 5- (alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil‘u -(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5- (halo)uracil; -diazole-l-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl) (thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio )uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)(thio)pseudouracil; 5-(methyl)-2;4 (dithio)pseudouracil; 5-(methyl)—4 (thio)pseudouracil; 5-(methyl)pseudouracil; 5- (methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2;4(dithio )uracil; 5- (Inethylaminomethy1)(thio)uraci1; 5-(propynyl)uraci1; ?uoromethy1)uraci1; 5-aminoallyl- uridine; 5-bromo-uridine; -uridine; 5-uraci1; 6 (azo)uraci1; 6-(azo)uraci1; 6-aza-uridine; ino-uracil; aza uracil; deaza uracil; N3 (methy1)uraci1; P seudo-UTP-l-Z-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1-carboxymethy1-pseudouridine; l-methyl-l-deaza- pseudouridine; l-propynyl-uridine; inomethy1methy1-uridine; 1-taurinomethy1thio- uridine; 1-taurinomethy1-pseudouridine; 2-methoxythio—pseudouridine; 2-thiomethy1 deaza-pseudouridine; 2-thio—1-methy1-pseudouridine; 2-thioaza-uridine; 2-thio— opseudouridine; 2-thio-dihydrouridine; 2-thio—pseudouridine; 4-methoxythio— pseudouridine; 4-methoxy-pseudouridine; 4-thio-l-methyl-pseudouridine; —pseudouridine; 5-aza-uridine; Dihydropseudouridine; (::)1-(2-Hydroxypropy1)pseudouridine TP; (2R)(2- ypropyl)pseudouridine TP; (ZS)(2-Hydroxypropyl)pseudouridine TP; (E)(2-Bromo- Viny1)ara-uridine TP; (E)(2-Bromo-Viny1)uridine TP; (Z)(2-Bromo-Vinyl)ara-uridine TP; (Z)(2-Bromo-Vinyl)uridine TP; 1-(2;2;2-Tri?uoroethyl)-pseudo—UTP; 1-(2;2;3;3;3- Penta?uoropropyl)pseudouridine TP; 1-(2;2-Diethoxyethyl)pseudouridine TP; 1-(2;4;6- Trimethylbenzy1)pseudouridine TP; 1-(2;4;6-Trimethyl-benzy1)pseudo-UTP; 1-(2;4;6-Trimethy1- pheny1)pseudo-UTP; 1-(2-Amino—2-carboxyethyl)pseudo—UTP; 1-(2-Amino-ethy1)pseudo—UTP; 1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethy1)pseudouridine TP; 1-(3;4-Bis- tri?uoromethoxybenzyl)pseudouridine TP; 1-(3,4-Dimethoxybenzyl)pseudouridine TP; 1-(3- Aminocarboxypropyl)pseudo—UTP; 1-(3-Amino—propy1)pseudo—UTP; 1-(3-Cyclopropyl-prop- 2-yny1)pseudouridine TP; 1-(4-Amino—4-carboxybutyl)pseudo—UTP; 1-(4-Amino-benzy1)pseudo- UTP; 1-(4-Amino—buty1)pseudo-UTP; mino—phenyl)pseudo-UTP; 1-(4- Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzy1)pseudouridine TP; 1-(4- Chlorobenzy1)pseudouridine TP; 1-(4-F1uorobenzy1)pseudouridine TP; 1-(4- nzy1)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4- Methoxybenzy1)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxyphenyl )pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methy1-benzy1)pseudo-UTP; 1- (4-Nitrobenzy1)pseudouridine TP; 1-(4-Nitro-benzy1)pseudo-UTP; 1(4-Nitro-pheny1)pseudo- UTP; 1-(4-Thiomethoxybenzyl)pseudouridine TP; 1-(4-Tri?uoromethoxybenzyl)pseudouridine TP; ri?uoromethylbenzyl)pseudouridine TP; 1-(5-Amino—pentyl)pseudo-UTP; 1-(6- Amino-hexyl)pseudo-UTP; 1,6-Dimethy1-pseudo—UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]- ethoxy}-ethoxy)—propionyl]pseudouridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propiony1 } pseudouridine TP; ylpseudouridine TP; 1-A1ky1(1-propyny1)-pseudo—UTP; 1-A1ky1 (2-propynyl)-pseudo—UTP; 1-A1ky1a11y1-pseudo—UTP; 1-A1ky1ethyny1-pseudo-UTP; 1- Alkylhomoallyl-pseudo—UTP; 1-Alky1Viny1-pseudo-UTP; l-Allylpseudouridine TP; 1- Aminomethyl-pseudo—UTP; l-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; l-Benzyl-pseudo-UTP; 1-Biotinyl-PEGZ-pseudouridine TP; l-Biotinylpseudouridine TP; 1- Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP; obuty1methy1-pseudo—UTP; 1- Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP; 1-Cyclohepty1-pseudo-UTP; 1- Cyclohexylmethyl-pseudo—UTP; ohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1-Cycloocty1-pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopenty1-pseudo-UTP; 1- ropylmethyl-pseudo-UTP; 1-Cyclopropy1-pseudo-UTP; l-Ethyl-pseudo-UTP; 1-Hexy1- pseudo-UTP; 1-Homoa11y1pseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propy1- pseudo-UTP; l-Me-Z-thio-pseudo-UTP; 1-Methio—pseudo-UTP; l-Me-alpha-thio-pseudo- UTP; 1-Methanesulfonylmethylpseudouridine TP; 1-Methoxymethy1pseudouridine TP; 1- Methyl(2,2,2-Tri?uoroethy1)pseudo—UTP; 1-Methy1(4-morpholino)—pseudo-UTP; 1- Methyl(4-thiomorpholino)—pseudo—UTP; 1-Methy1(substituted pheny1)pseudo-UTP; 1- Methylamino—pseudo—UTP; y1azido—pseudo-UTP; 1-Methy1bromo-pseudo—UTP; 1-Methy1buty1-pseudo-UTP; 1-Methy1chloro-pseudo-UTP; 1-Methy1cyano-pseudo— UTP; 1-Methy1dimethylamino-pseudo-UTP; 1-Methy1ethoxy-pseudo-UTP; 1-Methyl ethylcarboxylate-pseudo-UTP; 1-Methy1ethyl-pseudo—UTP; y1?uoro—pseudo-UTP; 1-Methy1formy1-pseudo—UTP; y1hydroxyamino-pseudo-UTP; 1-Methy1hydroxy- pseudo-UTP; 1-Methy1iodo-pseudo-UTP; 1-Methy1iso-propyl-pseudo—UTP; 1-Methy1 methoxy-pseudo-UTP; 1-Methy1methy1amino-pseudo—UTP; 1-Methy1pheny1-pseudo—UTP; y1propy1-pseudo-UTP; y1tert-buty1-pseudo-UTP; 1-Methy1 romethoxy-pseudo-UTP; 1-Methy1tri?uoromethyl-pseudo-UTP; 1- Morpholinomethylpseudouridine TP; 1-Penty1-pseudo—UTP; 1-Pheny1-pseudo—UTP; 1- Pivaloylpseudouridine TP; 1-Propargy1pseudouridine TP; 1-Propy1-pseudo—UTP; 1-propyny1- pseudouridine; 1-p-toly1-pseudo—UTP; l-tert-Butyl-pseudo-UTP; 1- Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1- Tri?uoroacetylpseudouridine TP; 1-Tri?uoromethy1-pseudo-UTP; l-Vinylpseudouridine TP; 2,2'—anhydro-uridine TP; 2'—bromo-deoxyuridine TP; 2'-FMethy1-2'—deoxy-UTP; 2'—OMe Me-UTP; 2'—OMe-pseudo-UTP; 2'-a-Ethyny1uridine TP; 2'—a-Tri?uoromethy1uridine TP; 2'—b- Ethynyluridine TP; 2'-b-Tri?uoromethyluridine TP; 2'—Deoxy-2',2'—di?uorouridine TP; 2'—Deoxy- 2'—a-mercaptouridine TP; 2'—Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'—b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP; 2'—Deoxy-2'—b-bromouridine TP; xy-2'-b-chlorouridine TP; 2'—Deoxy-2'-b-?uorouridine TP; 2'—Deoxy-2'—b-iodouridine TP; 2'—Deoxy-2'-b-mercaptouridine TP; 2'—Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxythio-uridine; 2-methoxyuridine; 2'—O- Methyl-S-(l-propyny1)uridine TP; 3-A1ky1-pseudo-UTP; 4'-Azidouridine TP; 4'—Carbocyclic uridine TP; 4'—Ethynyluridine TP; ropynyl)ara—uridine TP; 5-(2-Furanyl)uridine TP; 5- Cyanouridine TP; 5-Dimethy1aminouridine TP; 5'—Homo—uridine TP; —2'—?uorodeoxyuridine TP; 5-Pheny1ethynyluridine TP; 5-Trideuteromethyldeuterouridine TP; 5- Tri?uoromethyl-Uridine TP; S-Vinylarauridine TP; 6-(2;2,2-Tri?uoroethy1)—pseudo—UTP; 6-(4- Morpholino)—pseudo—UTP; 6-(4-Thiomorpholino)—pseudo-UTP; 6-(Substituted-Pheny1)-pseudo- UTP; 6-Amino—pseudo-UTP; 6-Azido—pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Buty1-pseudo- UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo—UTP; 6-Dimethy1amino—pseudo—UTP; 6-Ethoxy- pseudo-UTP; 6-Ethy1carboxylate-pseudo-UTP; 6-Ethy1-pseudo—UTP; 6-F1uoro-pseudo-UTP; 6- Formyl-pseudo-UTP; 6-Hydroxyamino—pseudo-UTP; 6-Hydroxy-pseudo—UTP; -pseudo- UTP; 6-iso-Propy1-pseudo—UTP; 6-Methoxy-pseudo-UTP; 6-Methy1amino-pseudo-UTP; 6- Methyl-pseudo-UTP; y1-pseudo-UTP; 6-Pheny1-pseudo-UTP; 6-Propy1-pseudo-UTP; 6- tert-Butyl-pseudo—UTP; 6-Tri?uoromethoxy-pseudo—UTP; 6-Tri?uoromethyl-pseudo—UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1-(4-methy1benzenesu1fonic acid) TP; Pseudouridine 1- (4-methy1benzoic acid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy )-ethoxy]—ethoxy )-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2- (2-[2-{2(2-ethoxy )-ethoxy}-ethoxy]—ethoxy )-ethoxy}]propionic acid; Pseudouridine TP 1-[3- {2-(2-[2-ethoxy ]—ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)- ethoxy}] propionic acid; Pseudouridine TP l-methylphosphonic acid; Pseudouridine TP 1- methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3 -propionic acid; -UTP-N1 butanoic acid; Pseudo-UTP-Nlpentanoic acid; Pseudo-UTP-N1hexanoic acid; Pseudo- UTP-N1heptanoic acid; Pseudo-UTP-Nl 1-p-benzoic acid; Pseudo-UTP-Nl -p-benzoic acid; sine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodi?ed hydroxywybutosine; 4-demethy1wyosine; 2,6-(diamino)purine;1-(aza)(thio)—3-(aza)— phenoxazin-l-yl: 1,3-( diaza)( OX0 thiaziny1;1,3-(diaza)—2-(oxo)-phenoxazin yl;1,3;5-(triaza)-2;6-(dioxa)—naphtha1ene;2 (amino)purine;2;4;5-(trimethy1)pheny1;2' ; 2'amino; 2'azido; 2'?uro—cytidine;2' methyl; 2'amino; 2'azido; 2'?uro—adenine;2'methy1; 2'amino; 2'azido; 2'?uro—uridine;2'—amino—2'-deoxyribose; 2-amino—6-Chloro-purine; 2-aza-inosiny1; 2'- azido—2'—deoxyribose; 2'?uoro-2'—deoxyribose; ro-modi?ed bases; 2'—O-methy1-ribose; 2- oxoaminopyridopyrimidiny1; 2-0X0-pyridopyrimidiney1; 2-pyridinone; 3 nitropyrrole; 3- (methyl)(propynyl)isocarbostyrilyl; 3-(methy1)isocarbostyrilyl; ro)—6- (methy1)benzimidazole; 4-(methy1)benzimidazole; 4-(methy1)indoly1; 4,6-(dimethy1)indolyl; 5 nitroindole; S tuted pyrimidines; 5-(methy1)isocarbostyrilyl; 5-nitroindole; 6- (aza)pyrimidine; 6-(azo)thymine; 6-(methy1)(aza)indoly1; 6-chloro-purine; 6-pheny1-pyrrolo— pyrimidin-Z-on-3 -y1; 7-(aminoalky1hydroxy)—1-(aza)(thio )(aza)-phenthiaziny1; 7- (aminoalkylhydroxy)- l -(aza)—2-(thio)-3 -(aza)-phenoxazin- l -yl; 7-(aminoalkylhydroxy)-1,3 - (diaza)(oxo)-phenoxazin-l-yl; 7-(aminoalkylhydroxy)-l,3-( diaza)( oxo )-phenthiazin-l-yl; noalkylhydroxy)—l;3-( diaza)—2-(oxo)-phenoxazin-l-yl; 7-(aza)indolyl; 7- (guanidiniumalkylhydroxy)- l -(aza)(thio )(aza)—phenoxazinl-yl; 7- (guanidiniumalkylhydroxy)- l -(aza)(thio )(aza)—phenthiazin-l-yl; 7- (guanidiniumalkylhydroxy)- l -(aza)(thio)—3 -(aza)-phenoxazin- l -yl; 7- diniumalkylhydroxy)- l ; 3 -(diaza)(oxo)-phenoxazin- l -yl; nidiniumalkyl-hydroxy)- l;3-( diaza)—2-( oxo )-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l;3-(diaza)( oxo )- phenoxazin-l-yl; 7-(propynyl)isocarbostyrilyl; pynyl)isocarbostyrilyl; propynyl (aza)indolyl; 7-deaza-inosinyl; 7-substituted l-(aza)(thio)(aza)-phenoxazin-l-yl; 7- substituted l,3-(diaza)—2-(oxo)—phenoxazin-l-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)—6-phenyl-pyrrolo-pyrimidinon-3 -yl; bis-ortho- substitutedphenyl-pyrrolo-pyrimidinon-3 -yl; Di?uorotolyl; Hypoxanthine; opyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl amino-purine; N6-substituted purines; lated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; ndazolyl; yrazolyl; rine; O6-substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)phenyl-pyrrolo-pyrimidinon-3 -yl; ortho-sub stituted- 6-phenyl-pyrrolo-pyrimidinonyl; Oxoformycin TP; para-(aminoalkylhydroxy)phenylpyrrolo-pyrimidinon-3 -yl; para-sub stitutedphenyl-pyrrolo-pyrimidinon-3 -yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl(aza)indolyl; Pyrenyl; pyridopyrimidinyl; pyridopyrimidinyl; 7-amino-pyridopyrimidinyl; pyrrolo-pyrimidinonyl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2;4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5'-TP; 2-thio-zebularine; 5-azathio-zebularine; a amino-purine; pyridinone ribonucleoside; 2-Amino-riboside-TP; Forrnycin A TP; Formycin B TP; Pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'—OH-ara-cytidine TP; 2'-OH—ara-uridine TP; 2'- OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(l9-Aminopentaoxanonadecyl )adenosine TP.

In some embodiments; the polynucleotide (e.g.; RNA polynucleotide; such as mRNA polynucleotide) includes a combination of at least two (e.g.; 2; 3; 4 or more) of the entioned modi?ed nucleobases.

In some embodiments; the mRNA comprises at least one chemically modi?ed nucleoside.

In some embodiments; the at least one chemically modi?ed side is selected from the group consisting of pseudouridine (w); 2-thiouridine (s2U); 4'—thiouridine; 5-methylcytosine; 2-thio-l- methyl-l-deaza-pseudouridine; 2-thio-l-methyl-pseudouridine; 2-thioaza-uridine; 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxythio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, -aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyl uridine, l- methyl-pseudouridine (mlw), l-ethyl-pseudouridine (elm), 5-methoxy-uridine (moSU), 5- methyl-cytidine (mSC), d-thio-guanosine, d-thio-adenosine, 5-cyano e, o uridine 7- deaza-adenine, l-methyl-adenosine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-Diaminopurine, (I), l-methyl-inosine (mlI), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyanodeaza-guanosine (preQO), 7-aminomethyldeazaguanosine (prte), 7-methyl-guanosine (m7G), yl-guanosine (mlG), guanosine, 7- methyloxo-guanosine, 2,8-dimethyladenosine, 2-geranylthiouridine, 2-lysidine, 2- selenouridine, 3-(3 -aminocarboxypropyl)-5,6-dihydrouridine, 3-(3-amino carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O- methyluridine methyl ester, 5-aminomethylgeranylthiouridine, 5-aminomethyl selenouridine, omethyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl thiouridine, 5-carboxymethylthiouridine, 5-carboxymethylaminomethylgeranylthiouridine, -carboxymethylaminomethylselenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5- aminomethylgeranylthiouridine, 7-aminocarboxypropyl-demethylwyosine, 7- aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, dine, cyclic 2O N6-threonylcarbamoyladenosine, glutamyl-queuosine, methylated undermodif1ed hydroxywybutosine, N4,N4,2'-O-trimethylcytidine, geranylated 5-methylaminomethyl thiouridine, lated 5-carboxymethylaminomethylthiouridine, Qbase , preQObase, prtebase, and two or more combinations thereof. In some embodiments, the at least one chemically modi?ed nucleoside is selected from the group ting of pseudouridine, lpseudouridine, l-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned ed nucleobases.

In some embodiments, the mRNA is a uracil-modif1ed sequence comprising an ORF encoding one or more cancer epitope polypeptides, wherein the mRNA comprises a chemically modified nucleobase, e.g., 5-methoxyuracil. In certain aspects of the invention, when the 5- methoxyuracil base is connected to a ribose sugar, as it is in polynucleotides, the ing ed nucleoside or nucleotide is refered to as 5-methoxyuridine. In some ments, uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% 5-methoxyuracil. In one embodiment, uracil in the polynucleotide is at least 95% 5-methoxyuracil. In another embodiment, uracil in the polynucleotide is 100% 5- methoxyuracil.

In embodiments where uracil in the polynucleotide is at least 95% 5-methoxyuracil, overall uracil content can be adjusted such that an mRNA provides suitable protein sion levels while inducing little to no immune response. In some embodiments, the uracil content of the ORF is between about 105% and about 145%, about 105% and about 140%, about 110% and about 140%, about 110% and about 145%, about 115% and about 135%, about 105% and about 135%, about 110% and about 135%, about 115% and about 145%, or about 115% and about 140% of the theoretical minimum uracil content in the ponding wild-type ORF (%Utm). In other embodiments, the uracil content of the ORF is n about 117% and about 134% or between 118% and 132% of the %UTM. In some embodiments, the uracil content of the ORF encoding one or more cancer epitope polypeptides is about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% ofthe %Utm. In this context, the term "uracil" can refer to 5-methoxyuracil and/or naturally occurring uracil.

In some embodiments, the uracil content in the ORF of the mRNA encoding one or more cancer e polypeptides of the invention is less than about 50%, about 40%, about 30%, about 20%, about 15%, or about 12% of the total nucleobase content in the ORF. In some embodiments, the uracil content in the ORF is between about 12% and about 25% of the total nucleobase content in the ORF. In other embodiments, the uracil t in the ORF is between about 15% and about 17% of the total ase content in the ORF. In one embodiment, the uracil content in the ORF of the mRNA encoding one or more cancer epitope polypeptides is less than about 20% of the total nucleobase content in the open g frame. In this context, the term "uracil" can refer to 5-methoxyuracil and/or naturally occurring .

In further embodiments, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention comprises 5-methoxyuracil and has an adjusted uracil content containing less uracil pairs (UU) and/or uracil triplets (UUU) and/or uracil quadruplets (UUUU) than the corresponding wild-type nucleotide sequence encoding the one or more cancer epitope polypeptides. In some embodiments, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention ns no uracil pairs and/or uracil triplets and/or uracil quadruplets. In some embodiments, uracil pairs and/or uracil triplets and/or uracil quadruplets are reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the ORF of the mRNA encoding the one or more cancer epitope ptides. In a particular embodiment, the ORF of the mRNA encoding the one or more cancer epitope polypeptides of the invention contains less than 20, 19, 18, 17, 16, , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-phenylalanine uracil pairs and/or ts. In r embodiment, the the ORF of the mRNA ng the one or more cancer epitope polypeptides contains no non-phenylalanine uracil pairs and/or triplets.

In further embodiments, the ORF of the mRNA encoding one or more cancer epitope polypeptides of the invention comprises 5-methoxyuracil and has an adjusted uracil content containing less uracil-rich clusters than the corresponding wild-type nucleotide sequence encoding the one or more cancer e ptides. In some ments, the ORF of the mRNA encoding the one or more cancer epitope polypeptides of the invention contains uracil- rich clusters that are shorter in length than corresponding uracil-rich clusters in the corresponding wild-type nucleotide sequence encoding the one or more cancer epitope polypeptides.

In further embodiments, alternative lower frequency codons are employed. At least about %, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in the one or more cancer epitope polypeptides —encoding ORF of the 5- methoxyuracil-comprising mRNA are substituted with alternative codons, each ative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set. The ORF also has adjusted uracil content, as described above. In some embodiments, at least one codon in the ORF of the mRNA encoding the one or more cancer epitope polypeptides is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.

In some embodiments, the adjusted uracil content, of the one or more cancer epitope polypeptides-encoding ORF of the 5-methoxyuracil-comprising mRNA exhibits expression levels of the one or more cancer epitope polypeptides when administered to a mammalian cell that are higher than expression levels of the one or more cancer epitope polypeptides from the corresponding wild-type mRNA. In other embodiments, the expression levels of the one or more cancer epitope polypeptides when administered to a ian cell are increased relative to a corresponding mRNA containing at least 95% 5-methoxyuracil and having a uracil content of about 160%, about 170%, about 180%, about 190%, or about 200% of the theoretical minimum.

In yet other embodiments, the expression levels of the one or more cancer epitope polypeptides when stered to a ian cell are increased relative to a corresponding mRNA, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of s are l-methylpseudouracil or uracils. In some embodiments, the mammalian cell is a mouse cell, a rat cell, or a rabbit cell. In other embodiments, the mammalian cell is a monkey cell or a human cell. In some embodiments, the human cell is a HeLa cell, a BI ?broblast cell, or a peripheral blood mononuclear cell (PBMC).

In some embodiments, one or more cancer epitope polypeptides is expressed when the mRNA is administered to a mammalian cell in vivo. In some embodiments, the mRNA is administered to mice, rabbits, rats, monkeys, or humans. In one embodiment, mice are null mice. In some embodiments, the mRNA is administered to mice in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, or about 0.15 mg/kg. In some embodiments, the mRNA is administered intravenously or intramuscularly. In other embodiments, the one or more cancer e polypeptides is expressed when the mRNA is administered to a mammalian cell in vitro. In some embodiments, the expression is increased by at least about 2-fold, at least about 5-fold, at least about d, at least about d, at least about 500-fold, at least about 1500-fold, or at least about 3000-fold. In other embodiments, the expression is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about 100%.

In some ments, adjusted uracil content, one or more cancer epitope polypeptides - encoding ORF of the 5-methoxyuracil-comprising mRNA exhibits increased stability. In some ments, the mRNA exhibits increased stability in a cell relative to the stability of a corresponding wild-type mRNA under the same conditions. In some embodiments, the mRNA exhibits increased stability including resistance to nucleases, thermal stability, and/or increased stabilization of secondary structure. In some embodiments, increased stability ted by the mRNA is measured by determining the half-life of the mRNA (e.g., in a plasma, cell, or tissue sample) and/or determining the area under the curve (AUC) of the protein expression by the mRNA over time (e.g., in vitro or in vivo). An mRNA is identi?ed as having increased stability if the half-life and/or the AUC is greater than the half-life and/or the AUC of a corresponding wild-type mRNA under the same conditions.

In some embodiments, the mRNA of the present invention induces a detectably lower immune response (e.g., innate or acquired) ve to the immune response induced by a ponding wild-type mRNA under the same conditions. In other embodiments, the mRNA of the present disclosure induces a detectably lower immune se (e.g., innate or acquired) relative to the immune response induced by an mRNA that encodes for one or more cancer e polypeptides but does not comprise 5-methoxyuracil under the same conditions, or relative to the immune response induced by an mRNA that encodes for one or more cancer epitope polypeptides and that comprises 5-methoxyuracil but that does not have adjusted uracil content under the same conditions. The innate immune response can be manifested by increased expression of pro-in?ammatory cytokines, activation of intracellular PRRs (RIG-I, MDA5, etc), cell death, and/or termination or reduction in n ation. In some embodiments, a reduction in the innate immune response can be measured by expression or activity level of Type 1 interferons (e.g., IFN—Ot, IFN—B, IFN—K, IFN-o, IFN—s, IFN-T, IFN—oa, and IFN—C) or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8), and/or by decreased cell death following one or more administrations of the mRNA of the invention into a cell.

In some embodiments, the expression of Type-1 interferons by a mammalian cell in response to the mRNA of the present disclosure is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% relative to a corresponding wild-type mRNA, to an mRNA that encodes one or more cancer epitope polypeptides but does not comprise 5-methoxyuracil, or to an mRNA that encodes one or more cancer e polypeptides and that comprises 5-methoxyuracil but that does not have adjusted uracil content.

In some embodiments, the interferon is IFN—B. In some ments, cell death frequency cased by administration of mRNA of the present disclosure to a mammalian cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding wild-type mRNA, an mRNA that encodes for one or more cancer e polypeptides but does not comprise 5-methoxyuracil, or an mRNA that encodes for one or more cancer e polypeptides and that comprises 5-methoxyuracil but that does not have adjusted uracil content. In some embodiments, the mammalian cell is a BI ?broblast cell. In other embodiments, the ian cell is a splenocyte. In some embodiments, the mammalian cell is that of a mouse or a rat. In other embodiments, the ian cell is that of a human. In one ment, the mRNA of the present disclosure does not substantially induce an innate immune se of a mammalian cell into which the mRNA is introduced.

In some embodiments, the polynucleotide is an mRNA that comprises an ORF that encodes one or more cancer epitope polypeptides, n uracil in the mRNA is at least about 95% 5-methoxyuracil, wherein the uracil content of the ORF is n about 115% and about 135% of the theoretical minimum uracil content in the ponding wild-type ORF, and wherein the uracil content in the ORF encoding the one or more cancer epitope polypeptides is less than about 23% of the total nucleobase content in the ORF. In some embodiments, the ORF that encodes the one or more cancer epitope polypeptides is further modi?ed to decrease G/C content of the ORF (ab solute or ve) by at least about 40%, as compared to the corresponding wild-type ORF. In yet other embodiments, the ORF encoding the one or more cancer epitope polypeptides contains less than 20 non-phenylalanine uracil pairs and/or triplets.

In some embodiments, at least one codon in the ORF of the mRNA ng the one or more cancer epitope polypeptides is further tuted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.

In some embodiments, the expression of the one or more cancer e polypeptides encoded by an mRNA comprsing an ORF wherein uracil in the mRNA is at least about 95% 5- methoxyuracil, and wherein the uracil content of the ORF is n about 115% and about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, is increased by at least about 10-fold when compared to expression of the one or more cancer epitope polypeptides from the corresponding wild-type mRNA. In some embodiments, the mRNA comprises an open ORF n uracil in the mRNA is at least about 95% 5- methoxyuracil, and wherein the uracil content of the ORF is n about 115% and about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, and wherein the mRNA does not ntially induce an innate immune response of a ian cell into which the mRNA is introduced.

In certain embodiments, the chemical modi?cation is at nucleobases in the polynucleotides (e.g., RNA polynucleotide, such as mRNA polynucleotide). In some embodiments, modi?ed nucleobases in the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) are selected from the group consisting of 1-methyl-pseudouridine (mlw), 1-ethyl-pseudouridine (elw), 5-methoxy-uridine (moSU), 5-methyl-cytidine (m5C), pseudouridine (w), d-thio-guanosine and d-thio-adenosine. In some embodiments, the polynucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises uridine (w) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (mlw). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-ethyl-pseudouridine (elw). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (mlw) and 5-methyl-cytidine (m5C). In some ments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) ses 1-ethyl- pseudouridine (elw) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) ses 2-thiouridine (s2U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises methoxy- uridine . In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 5-methoxy-uridine (moSU) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2'—O-methyl uridine. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises ethyl uridine and 5- methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA cleotide, such as mRNA cleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6- methyl-adenosine (m6A) and yl-cytidine (m5C).

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) is uniformly modi?ed (e.g., fully modi?ed, modi?ed hout the entire sequence) for a particular ation. For example, a polynucleotide can be uniformly modi?ed with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). As r example, a polynucleotide can be uniformly modi?ed with l-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with yl-pseudouridine. Similarly, a polynucleotide can be uniformly modi?ed for any type of nucleoside residue present in the sequence by replacement with a modi?ed residue such as any of those set forth above.

In some embodiments, the chemically modi?ed nucleosides in the open reading frame are selected from the group consisting of uridine, adenine, cytosine, e, and any combination thereof.

In some embodiments, the modi?ed nucleobase is a modi?ed cytosine. Exemplary nucleobases and nucleosides haVing a modi?ed cytosine include N4-acetyl-cytidine (ac4C), 5- -cytidine (m5C), -cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), l-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thiomethyl-cytidine.

In some embodiments, a modi?ed nucleobase is a d uridine. Exemplary nucleobases and nucleosides haVing a modi?ed uridine include l-methyl-pseudouridine (mlw), l-ethyl-pseudouridine (elw), 5-methoxy uridine, 2-thio uridine, 5-cyano uridine, 2’-O-methyl uridine, and 4'—thio uridine.

In some embodiments, a modi?ed nucleobase is a modi?ed adenine. Exemplary nucleobases and sides having a modi?ed adenine include a-adenine, l-methyl- adenosine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6- Diaminopurine.

In some embodiments, a modi?ed nucleobase is a modi?ed guanine. e nucleobases and nucleosides having a modi?ed guanine include inosine (I), l-methyl-inosine (mlI), wyosine (imG), methylwyosine (mimG), a-guanosine, 7-cyanodeaza-guanosine (preQO), 7-aminomethyldeaza-guanosine (prte), 7-methyl-guanosine (m7G), l-methylguanosine (mlG), 8-oxo-guanosine, 7-methyloxo-guanosine.

In some embodiments, the nucleobase modi?ed nucleotides in the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) are 5-methoxyuridine.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a ation of at least two (e.g., 2, 3, 4 or more) of modi?ed nucleobases.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises oxyuridine (5moSU) and 5-methyl-cytidine (m5C).

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) is uniformly modi?ed (e.g., fully modi?ed, modi?ed throughout the entire sequence) for a particular modi?cation. For example, a polynucleotide can be uniformly modi?ed with 5-methoxyuridine, meaning that substantially all e residues in the mRNA sequence are replaced with 5-methoxyuridine. Similarly, a polynucleotide can be uniformly d for any type of side residue present in the sequence by replacement with a d residue such as any of those set forth above.

In some embodiments, the modi?ed nucleobase is a modi?ed cytosine.

In some embodiments, a modi?ed nucleobase is a modi?ed uracil. Example nucleobases and nucleosides haVing a modi?ed uracil include 5-methoxyuracil.

In some embodiments, a modi?ed nucleobase is a modi?ed adenine.

In some embodiments, a modi?ed nucleobase is a modi?ed guanine.

In some embodiments, the polynucleotides can include any useful linker between the nucleosides. Such linkers, including backbone modi?cations, that are useful in the ition of the present sure include, but are not limited to the following: 3'—alkylene phosphonates, 3'—amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)- CH2-, -CH2-NH-CH2-, chiral onates, chiral phosphorothioates, formacetyl and thioformacetyl nes, methylene limino), methylene formacetyl and thioformacetyl backbones, methyleneimino and methylenehydrazino backbones, lino linkages, )- CH2-CH2-, oligonucleosides with atom internucleoside linkage, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNA, siloxane backbones, sulfamate backbones, sul?de sulfoxide and sulfone backbones, sulfonate and amide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates.

The modi?ed sides and nucleotides (e.g., building block molecules), which can be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be modi?ed on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modi?ed or replaced with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted CH, alkyl, optionally substituted C1- 6 alkoxy, optionally substituted C640 aryloxy, optionally substituted C3-g cycloalkyl, optionally substituted C3-g lkoxy, optionally substituted C640 aryloxy, optionally substituted C640 aryl-C1-6 alkoxy, optionally substituted C142 (heterocyclyl)oxy, a sugar (e.g., ribose, pentose, or any described herein), a polyethyleneglycol (PEG), -O(CH2CH20)nCH2CH20R, where R is H or optionally substituted alkyl, and n is an integer from O to 20 (e.g., from O to 4, from O to 8, from O to 10, from O to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and 2O from 4 to 20), "locked" nucleic acids (LNA) in which the 2'-hydroxyl is connected by a CH, ne or CH, heteroalkylene bridge to the 4'-carbon of the same ribose sugar, where exemplary bridges included ene, propylene, ether, or amino bridges, aminoalkyl, as de?ned herein, lkoxy, as de?ned herein, amino as de?ned herein, and amino acid, as de?ned herein Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modi?ed nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ne), addition of a double bond (e.g., to e ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., to form a ered ring of cyclobutane or oxetane), ring expansion of ribose (e.g., to form a 6- or 7-membered ring haVing an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone), multicyclic forms (e.g., tricyclo, and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with d-L- threofuranosyl-(3 '—>2')) and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical con?guration than that of the corresponding carbon in ribose. Thus, a polynucleotide le can include nucleotides containing, e.g., arabinose, as the sugar. Such sugar modi?cations are taught ational Patent ation Nos.

W02013052523 and W02014093 924, the contents of each of which are incorporated herein by reference in their entireties.

The polynucleotides of the invention (e.g., a polynucleotide comprising a nucleotide sequence encoding one or more cancer epitope polypeptides or a functional fragment or variant thereof) can include a combination of modi?cations to the sugar, the nucleobase, and/or the intemucleoside linkage. These combinations can include any one or more modi?cations described herein.

The polynucleotides of the present disclosure may be partially or fully modi?ed along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modi?ed in a polynucleotide of the invention, or in a given predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in a cleotide of the present disclosure (or in a given sequence region thereof) are modi?ed nucleotides, wherein X may any one of nucleotides A, G, U, C, orany hecombinationsAIG, AIU, AIC, GIU, GIC,UIC, AIGIU, A+G+C, G+U+C or A+G+C.

The polynucleotide may contain from about 1% to about 100% modi?ed nucleotides (either in on to overall nucleotide content, or in on to one or more types of nucleotide, 1'. e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from % to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be tood that any remaining tage is accounted for by the presence of unmodi?ed A, G, U, or C.

The polynucleotides may contain at a m 1% and at maximum 100% modi?ed tides, or any intervening percentage, such as at least 5% modi?ed tides, at least % d tides, at least 25% modi?ed nucleotides, at least 50% modi?ed nucleotides, at least 80% modi?ed nucleotides, or at least 90% modi?ed nucleotides. For example, the cleotides may contain a modi?ed pyrimidine such as a modi?ed uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide is replaced with a modi?ed uracil (e.g., a 5-substituted uracil). The modi?ed uracil can be ed by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least %, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% ofthe cytosine in the polynucleotide is replaced with a modi?ed cytosine (e.g., a 5-substituted cytosine). The modi?ed ne can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

Thus, in some embodiments, the RNA vaccines comprise a 5'UTR element, an optionally codon optimized open reading frame, and a 3'UTR element, a ) sequence and/or a enylation signal wherein the RNA is not ally modi?ed.

In some embodiments, the modi?ed nucleobase is a modi?ed uracil. Exemplary nucleobases and nucleosides having a modi?ed uracil include pseudouridine (\y), pyridin one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thioaza-uridine, 2-thio-uridine (sZU), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (hoSU), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (moSU), uridine 5-oxyacetic acid (cmoSU), uridine cetic acid methyl ester (mcmoSU), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethylthio-uridine (mcm5s2U), 5-aminomethylthio-uridine (nm5s2U), -methylaminomethyl-uridine (mnm5U), 5-methylaminomethylthio-uridine (mnm5s2U), 5- methylaminomethylseleno-uridine (mnm5se2U), amoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnmSU), 5-carboxymethylaminomethylthio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rmSU), inomethyl-pseudouridine, 5-taurinomethylthio-uridine(rm5szU), 1-taurinomethyl thio-pseudouridine, 5-methyl-uridine (m5U, 1'. e., having the nucleobase deoxythymine), 1- methyl-pseudouridine (mlw), l-ethyl-pseudouridine (elw), 5-methylthio-uridine (m5s2U), 1 shy), 4-thi l -methylthio-pseudouridine (m o- 1 l-pseudouridine, 3 -methyl- pseudouridine (m3q1), 2-thio- l l-pseudouridine, l-methyl- l -deaza-pseudouridine, 2- thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (mSD), 2-thio-dihydrouridine, - dihydropseudouridine, 2-methoxy-uridine, 2-methoxythio-uridine, 4-methoxypseudouridine , 4-methoxythio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino carboxypropyl)uridine (acp3U), l-methyl(3 -aminocarboxypropyl)pseudouridine (acp3 w), 5-(isopentenylaminomethyl)uridine (inmSU), 5-(isopentenylaminomethyl)thio-uridine (inm5s2U), 0t-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'—o— methyl-pseudouridine (wm), -2'-O-methyl-uridine (szUm), 5-methoxycarbonylmethyl- 2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncmSUm), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), l-thio-uridine, hymidine, 2’-F-ara-uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyyinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some ments, the modi?ed nucleobase is a modi?ed cytosine. Exemplary nucleobases and sides having a modi?ed cytosine include 5-aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl- cytidine (fSC), N4-methyl-cytidine (m4C), 5-methyl-cytidine (mSC), 5-halo—cytidine (e.g., 5- iodo-cytidine), 5-hydroxymethyl-cytidine (thC), yl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (szC), 2-thiomethyl-cytidine, 4-thio- pseudoi socytidine, methyl -pseudoi socyti dine, 4-thiomethyldeaza- pseudoisocytidine, l-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-azathio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-S-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methylpseudoisocytidine , ne (sz), 0t-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O- dimethyl-cytidine (mSCm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethylcytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (fSCm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), l-thio-cytidine, 2’-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

In some embodiments, the modi?ed nucleobase is a modi?ed adenine. Exemplary bases and nucleosides having a modi?ed adenine include 2-amino-purine, 2, 6- diaminopurine, ohalo-purine (e.g., ochloro-purine), 6-halo-purine (e.g., 6- chloro-purine), 2-aminomethyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza aza-adenine, 7-deazaamino-purine, 7-deazaazaamino-purine, 7-deaza-2,6- diaminopurine, 7-deazaaza-2,6-diaminopurine, 1-methy1-adenosine (mlA), 2-methy1- adenine (mZA), N6-methy1-adenosine (m6A), ylthio-N6-methy1-adenosine (ms2m6A), N6-isopenteny1-adenosine (i6A), y1thio-N6-isopentenyl-adenosine (ms2i6A), N6-(cishydroxyisopenteny1 )adenosine (io6A), 2-methy1thio-N6-(cis-hydroxyisopenteny1)adenosine (mszio6A), N6-glyciny1carbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methy1-N6-threony1carbamoyl-adenosine (m6t6A), 2-methy1thio-N6-threony1carbamoyl- adenosine (ms2g6A), N6,N6-dimethy1-adenosine (m62A), N6-hydroxynorva1y1carbamoyl- adenosine (hn6A), 2-methylthio-N6-hydroxynorva1y1carbamoyl-adenosine (ms2hn6A), N6- acetyl-adenosine (ac6A), 7-methy1-adenine, 2-methy1thio-adenine, 2-methoxy-adenine, 0t- thio-adenosine, 2'-O-methy1-adenosine (Am), O-dimethy1-adenosine (m6Am), N6,N6,2'-O-trimethy1-adenosine ), 1,2'-O-dimethy1-adenosine (mlAm), 2'-O- ladenosine (phosphate) (Ar(p)), 2-amino-N6-methy1-purine, 1-thio-adenosine, 8-azido- adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2’-OH—ara-adenosine, and N6-(19-amino- pentaoxanonadecy1)-adenosine.

In some embodiments, the modi?ed base is a modi?ed guanine. Exemplary nucleobases and nucleosides having a modi?ed guanine include inosine (I), 1-methy1-inosine (mII), wyosine (imG), methylwyosine (mimG), 4-demethy1-wyosine (imG—14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (ozyW), hydroxywybutosine (OhyW), undermodi?ed hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano deaza-guanosine (prer), 7-aminomethy1deaza-guanosine (prte), archaeosine (G+), 7- 8-aza-guanosine, 6-thio-guanosine, 6-thiodeaza-guanosine, 6-thiodeazaaza- guanosine, 7-methy1-guanosine (m7G), 6-thiomethy1-guanosine, 7-methy1-inosine, 6- methoxy-guanosine, 1-methy1-guanosine (mlG), N2-methy1-guanosine (sz), N2,N2- dimethyl-guanosine (mzzG), imethy1-guanosine (m2’7G), N2, N2,7-dimethy1-guanosine (m2’2’7G), 8-oxo-guanosine, 7-methy1oxo-guanosine, y1thio-guanosine, N2- methy1thio-guanosine, N2,N2-dimethy1thio-guanosine, d-thio-guanosine, 2'-O-methy1- guanosine (Gm), N2-methy1-2'-O-methy1-guanosine (szm), dimethy1-2'-O-methy1- guanosine (mzsz), y1-2'-O-methy1-guanosine (mle), N2,7-dimethy1-2'-O-methy1- guanosine (m2’7Gm), 2'-O-methy1-inosine (Im), 1,2'-O-dimethy1-inosine , 2'-O- ribosylguanosine (phosphate) (Gr(p)) 2’-F-ara- , 1-thio-guanosine, O6-methy1-guanosine, uanosine and 2’-F- anosine.

In vitro Transcription ofRNA (e.g., mRNA) Cancer vaccines of the present disclosure comprise at least one RNA polynucleotide, such as a mRNA (e.g., modi?ed mRNA). mRNA, for example, is ribed in vitro from template DNA, referred to as an "in vitro transcription template." In some embodiments, an in vitro ription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides. For example, a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides).

In other aspects, the invention relates to a method for preparing an mRNA cancer vaccine by IVT methods. In vitro transcription (IVT) methods permit template-directed synthesis ofRNA molecules of almost any sequence. The size of the RNA molecules that can be synthesized using IVT methods range from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT methods permit synthesis of large quantities of RNA transcript (e.g., from microgram to milligram quantities) (Beckert et (1]., Synthesis of RNA by in vitro transcription, Methods M0] Biol. 703 :29-41(201 1), Rio et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205- 220., Cooper, Geoffery M. The Cell: A lar ch. 4th ed. Washington DC: ASM Press, 2007. 262-299). Generally, IVT utilizes a DNA template ing a promoter sequence upstream of a sequence of interest. The promoter sequence is most commonly of bacteriophage origin (ex. the T7, T3 or SP6 promoter ce) but many other promotor sequences can be tolerated including those designed de novo. Transcription of the DNA template is typically best achieved by using the RNA polymerase corresponding to the speci?c iophage promoter sequence. Exemplary RNA polymerases include, but are not limited to T7 RNA rase, T3 RNA polymerase, or SP6 RNA polymerase, among . IVT is generally ted at a dsDNA but can proceed on a single strand.

It will be appreciated that mRNA es of the present disclosure, e.g., mRNAs encoding the cancer antigen or e.g., ting oncogene mutation peptide, may be made using any appropriate synthesis method. For example, in some embodiments, mRNA vaccines of the present disclosure are made using IVT from a single bottom strand DNA as a template and complementary oligonucleotide that serves as promotor. The single bottom strand DNA may act as a DNA template for in vitro transcription of RNA, and may be obtained from, for example, a plasmid, a PCR product, or al synthesis. In some embodiments, the single bottom strand DNA is ized from a circular te. The single bottom strand DNA template generally includes a er sequence, e.g., a bacteriophage promoter sequence, to facilitate IVT. Methods of making RNA using a single bottom strand DNA and a top strand promoter complementary ucleotide are known in the art. An exemplary method includes, but is not limited to, annealing the DNA bottom strand template with the top strand promoter complementary oligonucleotide (e.g., T7 promoter complementary oligonucleotide, T3 promoter complementary oligonucleotide, or SP6 promoter complementary oligonucleotide), followed by IVT using an RNA polymerase corresponding to the promoter sequence, e.g., aT7 RNA polymerase, a T3 RNA polymerase, or an SP6 RNA polymerase.

IVT s can also be performed using a double-stranded DNA template. For example, in some embodiments, the double-stranded DNA template is made by extending a complementary oligonucleotide to generate a complementary DNA strand using strand extension techniques available in the art. In some embodiments, a single bottom strand DNA te containing a promoter sequence and sequence encoding one or more epitopes of interest is annealed to a top strand promoter complementary oligonucleotide and subjected to a PCR-like s to extend the top strand to generate a double-stranded DNA template.

Alternatively or onally, a top strand DNA containing a sequence complementary to the bottom strand promoter sequence and complementary to the sequence encoding one or more epitopes of interest is annealed to a bottom strand promoter oligonucleotide and subjected to a PCR-like process to extend the bottom strand to te a double-stranded DNA template.

In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. In some embodiments, a double-stranded DNA template is synthesized wholly or in part by chemical synthesis methods. The double-stranded DNA template can be subjected to in vitro transcription as described herein.

In another aspect, mRNA vaccines of the present sure, e.g., mRNAs encoding the cancer antigen or eptiope, may be made using two DNA strands that are complementary across an overlapping portion of their ce, leaving single-stranded overhangs (i.e., sticky ends) when the complementary portions are annealed. These -stranded overhangs can be made double-stranded by extending using the other strand as a template, thereby generating double-stranded DNA. In some cases, this primer ion method can permit larger ORFs to be incorporated into the template DNA sequence, e.g., as compared to sizes incorporated into the template DNA sequences obtained by top strand DNA synthesis methods. In the primer extension method, a n of the 3’-end of a ?rst strand (in the 5"-3’ ion) is complementary to a portion the 3'-end of a second strand (in the 3"-5’ direction).

In some such embodiments, the single ?rst strand DNA may include a sequence of a promoter (e.g., T7, T3, or SP6), ally a 5'-UTR, and some or all of an ORF (e.g., a portion of the 5'-end of the ORF). In some embodiments, the single second strand DNA may include complementary sequences for some or all of an ORF (e.g., a n complementary to the 3 '-end of the ORF), and optionally a 3 '-UTR, a stop sequence, and/or a poly(A) tail.

Methods of making RNA using two synthetic DNA strands may include annealing the two strands with overlapping complementary portions, followed by primer extension using one or more PCR-like cycles to extend the strands to generate a double-stranded DNA template. In some embodiments, the number of PCR—like cycles ranges from 1 to 20 , e.g., 3 tolO cycles. Such double-stranded DNA can be subjected to in vitro transcription as described herein.

In another aspect, mRNA vaccines of the present disclosure, e.g., mRNAs encoding the cancer antigen or eptiope, may be made using synthetic double-stranded linear DNA molecules, such as gBlocks® (Integrated DNA Technologies, Coralville, Iowa), as the double-stranded DNA template. An advantage to such synthetic double-stranded linear DNA molecules is that they provide a longer template from which to generate mRNAs. For example, gBlocks® can range in size from 45-1000 (e.g., 125-750 nucleotides). In some embodiments, a tic double-stranded linear DNA template includes a full length 5'- UTR, a full length 3'-UTR, or both. A full length 5'-UTR may be up to 100 nucleotides in length, e.g., about 40-60 nucleotides. A full length 3 '-UTR may be up to 300 nucleotides in , e.g., about 100-150 nucleotides.

To facilitate generation of longer constructs, two or more double-stranded linear DNA molecules and/or gene nts that are designed with overlapping sequences on the 3' strands may be assembled together using s known in art. For example, the Gib son AssemblyTM Method (Synthetic cs, Inc., La Jolla, CA) may be performed with the use of a mesophilic exonuclease that cleaves bases from the 5'-end of the double-stranded DNA fragments, followed by annealing of the newly formed complementary -stranded 3 '-ends, polymerase-dependent extension to ?ll in any single-stranded gaps, and ?nally, covalent joining of the DNA segments by a DNA ligase.

In another , mRNA es of the t disclosure, e.g., mRNAs encoding the cancer antigen or epitope, may be made using chemical synthesis of the RNA. Methods, for instance, e annealing a ?rst polynucleotide comprising an open reading frame encoding the polypeptide and a second polynucleotide comprising a 5'-UTR to a complementary polynucleotide conjugated to a solid support. The 3'-terminus of the second polynucleotide is then ligated to the 5'-terminus of the ?rst polynucleotide under suitable conditions. Suitable conditions include the use of a DNA Ligase. The ligation reaction produces a ?rst ligation product. The 5' terminus of a third polynucleotide comprising a 3'- UTR is then ligated to the minus of the ?rst ligation product under suitable ions.

Suitable conditions for the second ligation reaction include an RNA . A second ligation product is produced in the second ligation reaction. The second on product is released from the solid support to produce an mRNA encoding a polypeptide of interest. In some embodiments the mRNA is between 30 and 1000 nucleotides.

An mRNA encoding a polypeptide of interest may also be prepared by g a ?rst polynucleotide comprising an open g frame encoding the polypeptide to a second polynucleotide comprising 3'-UTR to a complementary polynucleotide conjugated to a solid t. The 5'-terminus of the second polynucleotide is ligated to the 3'-terminus of the ?rst polynucleotide under suitable conditions. The suitable conditions include a DNA Ligase. The method produces a ?rst ligation product. A third polynucleotide comprising a 5'-UTR is ligated to the ?rst ligation product under suitable conditions to produce a second ligation product. The suitable conditions include an RNA Ligase, such as T4 RNA. The second ligation product is released from the solid support to produce an mRNA ng a polypeptide of interest.

In some embodiments the ?rst polynucleotide features a 5'-triphosphate and a 3'-OH.

In other embodiments the second cleotide comprises a 3'-OH. In yet other embodiments, the third cleotide comprises a 5'-triphosphate and a 3 '-OH. The second polynucleotide may also e a 5'-cap structure. The method may also involve the further step of ligating a fourth polynucleotide comprising a poly-A region at the 3 '-terminus of the third polynucleotide. The fourth cleotide may comprise a 5'-triphosphate.

The method may or may not comprise e phase puri?cation. The method may also include a washing step wherein the solid support is washed to remove unreacted polynucleotides. The solid support may be, for ce, a capture resin. In some ments the method involves dT puri?cation.

In accordance with the present disclosure, template DNA encoding the mRNA vaccines of the present disclosure includes an open reading frame (ORF) encoding one or more cancer epitopes. In some embodiments, the template DNA includes an ORF of up to 1000 nucleotides, e.g., about 10-350, 30-300 nucleotides or about 50-250 nucleotides. In some embodiments, the te DNA includes an ORF of about 150 nucleotides. In some embodiments, the template DNA includes an ORF of about 200 nucleotides.

In some embodiments, IVT transcripts are d from the components of the IVT reaction mixture after the reaction takes place. For example, the crude IVT mix may be treated with RNase-free DNase to digest the original te. The mRNA can be puri?ed using s known in the art, including but not limited to, precipitation using an organic solvent or column based puri?cation method. Commercial kits are available to purify RNA, e.g., MEGACLEARTM Kit (Ambion, Austin, TX). The mRNA can be quanti?ed using methods known in the art, including but not limited to, commercially available instruments, e.g., NanoDrop. Puri?ed mRNA can be ed, for example, by e gel electrophoresis to con?rm the RNA is the proper size and/or to con?rm that no degradation of the RNA has occurred.

UntranslatedRegions (UTRs) Untranslated regions (UTRs) are nucleic acid sections of a polynucleotide before a start codon (5'UTR) and after a stop codon (3'UTR) that are not translated. In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the invention comprising an open reading frame (ORF) encoding one or more cancer antigen or epitope further comprises UTR (e.g. , a 5'UTR or functional fragment thereof, a 3 'UTR or functional fragment thereof, or a combination thereof).

A UTR can be homologous or heterologous to the coding region in a polynucleotide. In some embodiments, the UTR is gous to the ORF ng the one or more cancer epitope ptides. In some embodiments, the UTR is heterologous to the ORF ng the one or more cancer epitope polypeptides. In some embodiments, the polynucleotide comprises two or more 5'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences. In some embodiments, the polynucleotide comprises two or more 3'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences.

In some embodiments, the 5 'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any ation thereof is sequence optimized.

In some ments, the 5 'UTR or functional nt thereof, 3' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modi?ed nucleobase, e.g., 5-methoxyuracil.

UTRs can have features that e a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency. A polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory es can be measured using routine methods. In some embodiments, a functional fragment of a 5'UTR or 3'UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively.

Natural 5 'UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ ID NO: 246), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5'UTRs also have been known to form ary structures that are involved in elongation factor binding.

By engineering the features typically found in abundantly expressed genes of ic target organs, one can e the stability and n production of a cleotide. For example, introduction of 5'UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E, errin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of polynucleotides in c cell lines or liver. Likewise, use of 5'UTR from other tissue-specif1c mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, bin, Myogenin, Herculin), for endothelial cells (e.g., Tie-l, CD36), for myeloid cells (e.g., C/EBP, AMLl, G—CSF, GM-CSF, CDl lb, MSR, Fr-l, , for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).

In some embodiments, UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, an encoded polypeptide can belong to a family of proteins (1'.e., that share at least one function, structure, feature, zation, origin, or expression pattern), which are expressed in a ular cell, tissue or at some time during pment. The UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.

In some embodiments, the 5’UTR and the 3 ’UTR can be heterologous. In some embodiments, the 5'UTR can be derived from a different species than the 3'UTR. In some embodiments, the 3'UTR can be derived from a different species than the 5'UTR.

Co-owned International Patent Application No. PCT/USZOl4/021522 (Publ. No.

WO/2014/164253, incorporated herein by reference in its entirety) provides a listing of exemplary UTRs that can be utilized in the polynucleotide of the present invention as ?anking regions to an ORF.

Exemplary UTRs of the ation include, but are not limited to, one or more 5'UTR and/or 3'UTR derived from the nucleic acid sequence of: a globin, such as an OL- or B-globin (e.g., aXenopus, mouse, rabbit, or human globin), a strong Kozak translational tion signal, a CYBA (e.g., human cytochrome b-245 0t polypeptide), an albumin (e.g., human albumin7), a HSDl7B4 (hydroxysteroid (17-13) ogenase), a Virus (e.g., a tobacco etch Virus (TEV), a Venezuelan equine encephalitis Virus (VEEV), a Dengue Virus, a galovirus (CMV) (e.g., CMV immediate early 1 (ED), a hepatitis Virus (e.g., hepatitis B Virus), a sindbis Virus, or a PAV barley yellow dwarf Virus), a heat shock protein (e.g., hsp70), a ation initiation factor (e.g., elF4G), a e transporter (e.g., hGLUTl (human glucose transporter 1)), an actin (e.g., human 0L or B , a GAPDH, a tubulin, a histone, a citric acid cycle enzyme, a topoisomerase (e.g., a 5'UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract)), a ribosomal protein Large 32 (L32), a ribosomal protein (e.g., human or mouse ribosomal protein, such as, for example, rps9), an ATP synthase (e.g., ATPSAl or the [3 subunit of mitochondrial H+-ATP synthase), a growth hormone e (e.g., bovine (bGH) or human (hGH)), an elongation factor (e.g., elongation factor 1 d1 (EEF1A1)), a manganese superoxide dismutase (MnSOD), a myocyte enhancer factor 2A (MEFZA), a B-Fl-ATPase, a ne kinase, a myoglobin, a granulocyte- colony stimulating factor (G—C SF), a collagen (e.g., collagen type 1, alpha 2 (CollA2), collagen type 1, alpha 1 (CollAl), en type VI, alpha 2 (Col6A2), en type VI, alpha 1 (Col6A1)), a orin (e.g., ribophorin I (RPNI)), a low density lipoprotein receptor-related protein (e.g., LRPl), a cardiotrophin-like ne factor (e.g., Nntl), calreticulin (Calr), a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plodl), and a nucleobindin (e.g., Nucbl).

Other exemplary 5' and 3' UTRs include, but are not limited to, those described in Kariko et al, Mol. Ther. 2008 16(11):1833-1840, Kariko et (1]., Mol. Ther. 2012 20(5):948-953, Kariko et al, Nucleic Acids Res. 2011 39(21):e142, Strong et (1]., Gene Therapy 1997 4:624-627, Hansson et al., J. Biol. Chem. 2015 290(9):5661-5672, Yu et al, Vaccine 2007 25(10):1701- 1711, Cafri et (1]., Mol. Ther. 2015 23(8):1391-1400, Andries et (1]., Mol. Pharm. 2012 9(8):2136-2145, Crowley et (1]., Gene Ther. 2015 Jun 30, doi:10.1038/gt.2015.68, Ramunas et al., FASEB J. 2015 29(5):1930-1939, Wang et (1]., Curr. Gene Ther. 2015 15(4):428-435, Holtkamp et al., Blood 2006 108(13):4009-4017, Kormann et al, Nat. Biotechnol. 2011 29(2):154-157, Poleganov et (1]., Hum. Gen. Ther. 2015 26(11):751-766, Warren et al., Cell Stem Cell 2010 7(5):618-630, Mandal and Rossi, Nat. . 2013 8(3):568-582, Holcik and Liebhaber, PNAS 1997 94(6):2410-2414, Ferizi et (1]., Lab Chip. 2015 15(17):3561-3571, Thess et (1]., Mol. Ther. 2015 23(9):1456-1464, Boros et al., PLoS One 2015 e0131141, Boros et al., J. Photochem. Photobiol. B. 2013 -99, Andries et al., J. Control. Release 2015 7—344; Zinckgraf et al; Vaccine 2003 21(15): 1640-9; Gameau et al.; J. Virol. 2008 82(2):880-892; Holden and Harris; Virology 2004 329(1):119-133; Chiu et al; J. Virol. 2005 79(13):8303-8315; Wang et al; E1V?30 J. 1997 16(13):4107-4116; Al-Zoghaibi et al; Gene 2007 391(1-2):130-9; ViVinus et al; Eur. J. Biochem. 2001 268(7):1908-1917; Gan and Rhoads; J.

Biol. Chem. 1996 271(2):623-626; Boado et al; J. Neurochem. 1996 67(4): 1335-1343; Knirsch and Clerch; Biochem. Biophys. Res. . 2000 272(1): 164-168; Chung et (1].; Biochemistry 1998 37(46): 16298-16306; Izquierdo and Cuevza; Biochem. J. 2000 346 Pt 3:849-855; Dwyer et al.; J. Neurochem. 1996 66(2):449-458; Black et al; Mol. Cell. Biol. 1997 17(5):2756-2763; Izquierdo and Cuevza; Mol. Cell. Biol. 1997 17(9):5255-5268; US8278036; US8748089; US8835108; US9012219; US2010/0129877; US2011/0065103; US2011/0086904; US2012/0195936; US2014/020675; US2013/0195967; US2014/029490; US2014/0206753; WO2007/036366; WO2011/015347; WO2012/072096; WO2013/143555; WO2014/071963; WO2013/185067; WO2013/182623; WO2014/089486; WO2013/185069; WO2014/144196; WO2014/152659; 2014/152673; WO2014/152940; WO2014/152774; WO2014/153052; WO2014/152966; WO2014/152513; WO2015/101414; WO2015/101415; WO2015/062738; and W02015/024667; the contents of each of which are orated herein by reference in their entirety.

In some embodiments; the 5'UTR is selected from the group ting of a B-globin ’UTR; a 5'UTR containing a strong Kozak ational initiation ; a cytochrome b-245 0L polypeptide (CYBA) 5'UTR; a hydroxysteroid (17-13) dehydrogenase (HSD17B4) 5'UTR; a Tobacco etch Virus (TEV) 5'UTR; a Venezuelen equine encephalitis Virus (TEEV) 5'UTR; a 5' proximal open reading frame of rubella Virus (RV) RNA encoding nonstructural proteins; a Dengue Virus (DEN) 5'UTR; a heat shock protein 70 (Hsp70) 5'UTR; a e1F4G 5'UTR; a GLUT1 'UTR; functional fragments thereof and any combination thereof.

In some embodiments; the 3'UTR is selected from the group consisting of a B-globin 3’UTR; a CYBA 3'UTR; an albumin 3'UTR; a growth hormone (GH) 3'UTR; a VEEV 3'UTR; a hepatitis B Virus (HBV) 3'UTR; d-globin 3'UTR; a DEN 3'UTR; a PAV barley yellow dwarf Virus (BYDV-PAV) 3'UTR; an elongation factor 1 Otl l) 3'UTR; a ese superoxide dismutase (MnSOD) 3'UTR; a [3 subunit of mitochondrial TP se ([3- mRNA) 3'UTR; a GLUT1 3'UTR; a MEF2A 3'UTR; a B-Fl-ATPase 3'UTR; functional fragments f and combinations thereof.

Other exemplary UTRs include; but are not limited to; one or more of the UTRs; ing any ation of UTRs; disclosed in WO2014/164253; the contents of which are incorporated herein by reference in their entirety. Shown in Table 21 ofUS. Provisional Application No. 61/775,509 and in Table 22 of US. Provisional Application No. 61/829,372, the contents of each are incorporated herein by reference in their entirety, is a listing start and stop sites for 5'UTRs and . In Table 21, each 5'UTR R-005 to 5'-UTR 68511) is identi?ed by its start and stop site relative to its native or wild-type (homologous) transcript (ENST, the identi?er used in the ENSE1V?3L database).

Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the invention. In some embodiments, a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF, or by inclusion of additional tides, deletion of nucleotides, swapping or transposition of nucleotides. In some embodiments, variants of 5' or 3' UTRs can be utilized, for example, mutants of wild type UTRs, or ts wherein one or more nucleotides are added to or removed from a terminus of the UTR.

Additionally, one or more synthetic UTRs can be used in ation with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, and sequences available at www.addgene.org/Derrick_Rossi/, the ts of each are incorporated herein by reference in their entirety. UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a ' and/or 3' UTR can be inverted, shortened, lengthened, or ed with one or more other 5' UTRs or 3' UTRs.

In some embodiments, the polynucleotide comprises multiple UTRs, e.g., a , a triple or a quadruple 5’UTR or 3’UTR. For example, a double UTR comprises two copies of the same UTR either in series or substantially in series. For e, a double beta-globin 3 'UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).

In certain embodiments, the polynucleotides of the invention comprise a 5'UTR and/or a 3'UTR selected from any of the UTRs disclosed herein. In some embodiments, the 5’UTR and/or the 3’ UTR comprise: 'UTR-001 eam UTR) 247 'UTR-002 (Upstream UTR) 248 'UTR-003 (Upstream UTR) 249 'UTR-004 (Upstream UTR) 250 'UTR-005 (Upstream UTR) Name SEQ ID NO: ' 'TR—OO6 (Upstream U 252 ' 'TR—OO7 (Upstream U 253 ' 'TR—OO8 (Upstream U 254 ' 9 (Upstream U 255 ' 'TR—OlO eam U 256 ' 'TR-Oll (Upstream U 257 ' 'TR—Ol2 (Upstream U 258 ' 'TR-Ol3 (Upstream U 259 ' 'TR—Ol4 (Upstream U 260 ' 'TR-Ol5 (Upstream U 261 ' 'TR—Ol6 (Upstream U 262 ' 'TR—Ol7 (Upstream U 263 ' 'TR—Ol8 (Upstream U 264 l42-3p 5' 'TR-OOl (Upstream L'TR including miRl42-3p binding site) 265 l42-3p 5' 'TR-OO2 (Upstream L'TR including -3p binding site) 266 l42-3p 5' 3 (Upstream L'TR including miRl42-3p binding site) 267 l42-3p 5' 'TR-OO4 (Upstream L'TR including miRl42-3p binding site) 268 l42-3p 5' 'TR-OO5 (Upstream L'TR ing miRl42-3p binding site) 269 l42-3p 5' 'TR-OO6 (Upstream L'TR including miRl42-3p binding site) 270 l42-3p 5' 'TR-OO7 (Upstream L'TR including miRl42-3p binding site) 271 3' 'TR comprises: 3'UTR—OOl (Creatine Kinase UTR) 272 3' 'TR—OO2 obin UTR) 273 3' 'TR-OO3 (d-actin UTR) 274 3' 'TR-OO4 (Albumin UTR) 275 3' 'TR-005 (or-globin UTR) 276 3' 'TR-OO6 (G—CSF UTR) 277 3' 'TR—OO7 2; collagen, type 1, alpha 2 UTR) 278 3' 'TR—OO8 (Col6a2; collagen, type VI, alpha 2 UTR) 279 3' 'TR—OO9 (RPNl; ribophorin I UTR) 280 3' 'TR—OlO (LRPl; low density lipoprotein receptor-related protein 1 UTR) 3' l (Nntl; cardiotrophin-like cytokine factor 1 UTR) 3'UTR-015 (Plodl, procollagen-lysine, 2-oxoglutarate 5-dioxygenase l 286 . . 222 3'UTR with miR 142-3p binding site, P1 insertion 297 3'UTR with miR 142-3p binding site, P2 insertion 298 3'UTR with m1R 142 3p b1nd1ng s1te P3 1nsertlon. . . . . . _ 299 3’UTR w1th m1R 155 5p b1nd1ng s1te 300 3’ UTR w1th 3 m1R 155 5p b1nd1ng s1tes- 301 3’UTR w1th 2 m1R 155-5p b1nd1ng s1tes and' ' 1 m1R 142-3p binding site 302 In certain embodiments, the 5'UTR and/or 3'UTR sequence of the invention comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence ed from the group consisting of 5'UTR sequences comprising any of SEQ ID NOs: 247-271 and/or 3'UTR sequences comprises any of SEQ ID NOs: 272-3 02, and any combination thereof.

The cleotides of the invention can comprise combinations of features. For example, the ORF can be ?anked by a 5'UTR that comprises a strong Kozak ational initiation signal and/or a 3 'UTR comprising an oligo(dT) sequence for templated addition of a poly-A tail. A 5'UTR can se a ?rst polynucleotide fragment and a second polynucleotide fragment from the same and/or different UTRs (see, e.g., US2010/0293 625, herein incorporated by nce in its entirety).

It is also within the scope of the present invention to have patterned UTRs. As used herein "patterned UTRs" include a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR nucleic acid sequence.

Other non-UTR sequences can be used as s or subregions within the polynucleotides of the invention. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of the invention. Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels. In some embodiments, the polynucleotide of the invention comprises an internal me entry site (IRES) instead of or in on to a UTR (see, e.g., Yakubov et (1]., Biochem. Biophys. Res. . 2010 394(1): 189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the polynucleotide of the invention comprises 5' and/or 3' sequence associated with the 5' and/or 3' ends of rubella virus (RV) genomic RNA, respectively, or deletion derivatives thereof, including the 5' proximal open reading frame ofRV RNA encoding uctural proteins (e.g., see Pogue et al., J. Virol. 67(12):7106-7117, the contents of which are incorporated herein by reference in their ty). Viral capsid sequences can also be used as a ational enhancer, e.g., the 5' portion of a capsid sequence, (e.g., semliki forest virus and sindbis virus capsid RNAs as described in Sjoberg et al, Biotechnology (NY) 1994 12(11):1127- 1131, and Frolov and Schlesinger J. Virol. 1996 70(2): 1 182-1 190, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, the polynucleotide comprises an IRES instead of a 5’UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide ses a synthetic 5'UTR in combination with a non-synthetic 3'UTR.

In some embodiments, the UTR can also include at least one ation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, "TEE," which refers to c acid ces that increase the amount of polypeptide or protein produced from a polynucleotide. As a non-limiting example, the TEE can include those described in /0226470, incorporated herein by reference in its entirety, and others known in the art.

As a miting example, the TEE can be located between the transcription promoter and the start codon. In some embodiments, the 5'UTR ses a TEE.

In one aspect, a TEE is a conserved element in a UTR that can promote translational ty of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation. The conservation of these sequences has been shown across 14 species including humans. See, e.g., Panek et al, "An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5'UTRs and its implications for eukaryotic gene translation regulation," Nucleic Acids Research 2013, doi:10.1093/nar/gkt548, incorporated herein by reference in its entirety.

In one non-limiting example, the TEE comprises the TEE sequence in the der of the GtX homeodomain protein. See Chappell et al., PNAS 2004 101 9594, incorporated herein by reference in its entirety.

In another non-limiting example, the TEE comprises a TEE having one or more of the sequences of SEQ ID NOS: 1-35 in US2009/0226470, /0177581, and /075886, SEQ ID NOs: 1-5 and 7-645 in WO2012/009644, and SEQ ID NO: 1 WOl999/024595, US6310197, and US6849405, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the TEE is an internal ribosome entry site , ES, or an IRES element such as, but not limited to, those described in: US7468275, U82007/0048776, US2011/0124100, WO2007/025008, and WO2001/055369, the contents of each of which re incorporated herein by reference in their entirety. The IRES elements can include, but are not limited to, the GtX sequences (e.g., GtX9-nt, GtX8-nt, GtX7-nt) as described by Chappell et al, PNAS 2004 101 :9590-9594, Zhou et al., PNAS 2005 102:6273-6278, US2007/0048776, U82011/0124100, and W02007/025008, the ts of each of which are incorporated herein by reference in their ty.

"Translational enhancer polynucleotide" or lation enhancer cleotide sequence" refer to a polynucleotide that includes one or more of the TEE provided herein and/or known in the art (see. e.g., US6310197, US6849405, US7456273, US7183395, US2009/0226470, US2007/0048776, /0124100, US2009/0093049, US2013/0177581, WO2009/075886, WO2007/025008, WO2012/009644, WO2001/055371, WOl999/024595, EP2610341A1, and EP2610340A1, the contents of each of which are incorporated herein by reference in their entirety), or their variants, homologs, or functional derivatives. In some embodiments, the polynucleotide of the invention comprises one or multiple copies of a TEE.

The TEE in a translational enhancer polynucleotide can be organized in one or more sequence segments. A sequence t can harbor one or more of the TEEs ed herein, with each TEE being present in one or more copies. When multiple sequence segments are present in a translational enhancer polynucleotide, they can be homogenous or heterogeneous. Thus, the multiple sequence segments in a translational er polynucleotide can harbor identical or different types of the TEE provided herein, identical or different number of copies of each of the TEE, and/or identical or different organization of the TEE within each sequence segment. In one embodiment, the polynucleotide of the invention comprises a translational enhancer polynucleotide sequence.

In some embodiments, a 5'UTR and/or 3'UTR of a polynucleotide of the invention comprises at least one TEE or portion thereof that is disclosed in: WOl999/024595, WO2012/009644, /075886, WO2007/025008, WOl999/024595, WO2001/055371, EP2610341A1, EP2610340A1, US6310197, US6849405, 273, US7183395, US2009/0226470, /0124100, /0048776, US2009/0093049, or US2013/0177581, the ts of each are incorporated herein by reference in their entirety.

In some embodiments, a 5'UTR and/or 3'UTR of a polynucleotide of the invention comprises a TEE that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least %, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a TEE disclosed in: /0226470, US2007/0048776, US2013/0177581, US2011/0124100, WOl999/024595, WO2012/009644, WO2009/075886, WO2007/025008, 341A1, EP2610340A1, US6310197, 405, US7456273, US7183395, Chappell et al, PNAS 2004 101 :9590-9594, Zhou et al., PNAS 2005 102:6273-6278, and Supplemental Table 1 and in Supplemental Table 2 of Wellensiek et al., "Genome-wide pro?ling of human cap-independent ation-enhancing elements," Nature Methods 2013, DOI: 10.103 8/NMETH2522, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, a 5'UTR and/or 3'UTR of a polynucleotide of the invention comprises a TEE which is selected from a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, or a 5-10 nucleotide fragment (including a fragment of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 tides) of a TEE sequence disclosed in: US2009/0226470, US2007/0048776, /0177581, US2011/0124100, WOl999/024595, WO2012/009644, WO2009/075886, WO2007/025008, EP2610341A1, EP2610340A1, US6310197, US6849405, US7456273, US7183395, Chappell et al, PNAS 2004 101:9590-9594, Zhou et al, PNAS 2005 73- 6278, and Supplemental Table 1 and in Supplemental Table 2 of Wellensiek et al, "Genome- wide pro?ling of human cap-independent translation-enhancing elements," Nature Methods 2013, DOI:10.1038/NMETH.2522.

In some embodiments, a 5'UTR and/or 3'UTR of a polynucleotide of the invention comprises a TEE which is a transcription regulatory element described in any of US7456273, US7183395, US2009/0093049, and WO200l/O55371, the contents of each of which are incorporated herein by reference in their entirety. The ription regulatory elements can be identi?ed by s known in the art, such as, but not limited to, the methods described in US7456273, US7183395, US2009/0093049, and /O55371.

In some embodiments, a 5'UTR and/or 3'UTR comprising at least one TEE described herein can be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid . As non-limiting examples, the vector systems and nucleic acid vectors can include those described in US7456273, US7183395, US2007/0048776, US2009/0093049, US2011/0124100, /O25008, and /O55371.

In some ments, a 5'UTR and/or 3'UTR of a polynucleotide of the invention comprises a TEE or portion thereof described herein. In some embodiments, the TEEs in the 3'UTR can be the same and/or different from the TEE located in the 5'UTR.

In some embodiments, a 5'UTR and/or 3'UTR of a polynucleotide of the invention can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least , at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In 2O one embodiment, the 5'UTR of a polynucleotide of the ion can e 1-60, 1-55, 1-50, 1- 45, 1—40, 1—35, 1—30, 1—25, 1—20, 1—15, 1—10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 TEE sequences. The TEE sequences in the 5'UTR of the polynucleotide of the invention can be the same or different TEE sequences. A combination of different TEE sequences in the 5'UTR of the polynucleotide of the invention can include combinations in which more than one copy of any of the different TEE sequences are incorporated. The TEE sequences can be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated one, two, three, or more than three times. In these patterns, each , A, B, or C represent a different TEE nucleotide sequence.

In some embodiments, the 5'UTR and/or 3'UTR comprises a spacer to separate two TEE sequences. As a non-limiting example, the spacer can be a 15 nucleotide spacer and/or other spacers known in the art. As another non-limiting example, the 5'UTR and/or 3'UTR comprises a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more than 10 times in the 5'UTR and/or 3'UTR, respectively. In some embodiments, the 5'UTR and/or 3'UTR comprises a TEE sequence-spacer module repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

In some embodiments, the spacer separating two TEE sequences can include other sequences known in the art that can regulate the translation of the polynucleotide of the invention, e.g., miR sequences described herein (e.g., miR binding sites and miR seeds). As a non-limiting example, each spacer used to separate two TEE sequences can include a different miR sequence or component of a miR sequence (e.g., miR seed ce).

In some embodiments, a cleotide of the invention comprises a miR and/or TEE sequence. In some embodiments, the oration of a miR ce and/or a TEE sequence into a polynucleotide of the invention can change the shape of the stem loop , which can increase and/or decrease translation. See e.g., Kedde et al, Nature Cell Biology 2010 12(10): 1014-20, herein orated by nce in its entirety).

A/[icroRNA (miRNA) g Sites Polynucleotides of the invention can include regulatory elements, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, arti?cial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof. In some embodiments, cleotides including such regulatory elements are referred to as including "sensor 2O sequences". Non-limiting examples of sensor sequences are described in US. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a ger RNA (mRNA)) of the invention ses an open reading frame (ORF) encoding a polypeptide of interest and further comprises one or more miRNA binding site(s). Inclusion or incorporation of miRNA binding site(s) provides for regulation of polynucleotides of the invention, and in turn, of the ptides encoded therefrom, based on tissue-specif1c and/or cell-type specific expression of naturally-occurring miRNAs.

A miRNA, e.g., a natural-occurring miRNA, is a 19-25 tide long noncoding RNA that binds to a polynucleotide and down-regulates gene expression either by reducing stability or by inhibiting translation of the polynucleotide. A miRNA sequence comprises a "seed" region, i.e., a ce in the region of positions 2-8 of the mature miRNA. A miRNA seed can comprise positions 2-8 or 2-7 of the mature miRNA. In some embodiments, a miRNA seed can comprise 7 tides (e.g., nucleotides 2-8 of the mature miRNA), wherein the seed- complementary site in the corresponding miRNA binding site is ?anked by an adenosine (A) opposed to miRNA position 1. In some embodiments, a miRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is ?anked by an adenosine (A) opposed to miRNA position 1.

See, for example, Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP, Mol Cell. 2007 Jul 6,27(l):9l-105. miRNA pro?ling of the target cells or tissues can be conducted to determine the presence or absence of miRNA in the cells or tissues. In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the invention comprises one or more microRNA binding sites, microRNA target ces, microRNA complementary sequences, or microRNA seed complementary sequences. Such sequences can correspond to, e.g., have mentarity to, any known NA such as those taught in US Publication /0261218 and US Publication US2005/0059005, the contents of each of which are incorporated herein by reference in their entirety.

As used herein, the term "microRNA (miRNA or miR) binding site" refers to a sequence within a cleotide, e.g., within a DNA or within an RNA transcript, including in the 5'UTR and/or 3 'UTR, that has suf?cient complementarity to all or a region of a miRNA to interact with, ate with or bind to the miRNA. In some embodiments, a polynucleotide of the invention comprising an ORF encoding a polypeptide of interest and further comprises one or more miRNA binding site(s). In exemplary embodiments, a 5'UTR and/or 3'UTR of the 2O polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprises the one or more miRNA binding site(s).

A miRNA binding site having suf?cient complementarity to a miRNA refers to a degree of complementarity suf?cient to facilitate mediated regulation of a polynucleotide, e.g., miRNA-mediated translational repression or degradation of the cleotide. In exemplary aspects of the invention, a miRNA binding site having suf?cient complementarity to the miRNA refers to a degree of complementarity suf?cient to facilitate miRNA-mediated degradation of the polynucleotide, e.g., miRNA-guided duced ing complex (RISC)-mediated cleavage of mRNA. The miRNA binding site can have complementarity to, for example, a 19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA ce, or to a 22 nucleotide miRNA ce. A miRNA binding site can be mentary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA ce. Full or complete complementarity (e.g., full mentarity or complete complementarity over all or a signi?cant portion of the length of a naturally-occurring miRNA) is preferred when the desired regulation is mRNA degradation.

In some ments, a miRNA g site includes a sequence that has complementarity (e.g., l or complete complementarity) with an miRNA seed sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA sequence. In some embodiments, the miRNA binding site includes a sequence that has complete mentarity with a miRNA sequence. In some embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for l, 2, or 3 nucleotide substitutions, terminal ons, and/or truncations.

In some embodiments, the miRNA binding site is the same length as the corresponding miRNA. In other embodiments, the miRNA binding site is one, two, three, four, ?ve, siX, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5' terminus, the 3' terminus, or both. In still other embodiments, the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5' terminus, the 3' terminus, or both.

The miRNA g sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.

In some embodiments, the miRNA binding site binds the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has suf?cient complementarity to miRNA so that a RISC compleX comprising the miRNA cleaves the polynucleotide comprising the miRNA binding site. In other embodiments, the miRNA binding site has imperfect complementarity so that a RISC compleX comprising the miRNA s instability in the polynucleotide comprising the miRNA binding site. In another embodiment, the miRNA binding site has imperfect mentarity so that a RISC compleX comprising the miRNA ses ription of the polynucleotide comprising the miRNA binding site.

In some embodiments, the miRNA g site has one, two, three, four, ?ve, siX, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.

In some embodiments, the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about , at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about ?fteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about -one, tively, uous nucleotides of the corresponding miRNA.

By ering one or more miRNA binding sites into a polynucleotide of the ion, the polynucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the cleotide. For example, if a polynucleotide of the invention is not intended to be delivered to a tissue or cell but ends up is said tissue or cell, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5 'UTR and/or 3 'UTR of the polynucleotide.

Conversely, miRNA binding sites can be removed from polynucleotide sequences in which they naturally occur in order to increase protein expression in speci?c tissues. For example, a binding site for a speci?c miRNA can be d from a polynucleotide to e protein sion in tissues or cells containing the miRNA.

In one embodiment, a polynucleotide of the invention can include at least one miRNA- binding site in the 5'UTR and/or 3'UTR in order to regulate cytotoxic or cytoprotective mRNA therapeutics to speci?c cells such as, but not limited to, normal and/or cancerous cells. In another embodiment, a cleotide of the ion can include two, three, four, ?ve, six, seven, eight, nine, ten, or more miRNA-binding sites in the 5'—UTR and/or 3 '-UTR in order to regulate cytotoxic or cytoprotective mRNA therapeutics to speci?c cells such as, but not limited to, normal and/or cancerous cells.

Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites, e.g., one or more distinct miRNA binding sites.

The decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and/or their pro?lings in tissues and/or cells in development and/or disease.

Identi?cation of miRNAs, miRNA binding sites, and their expression patterns and role in biology have been reported (e.g., Bonauer et al, Curr Drug Targets 2010 11:943-949, Anand and Cheresh Curr Opin Hematol 2011 18: 171-176, Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi: 10.1038/leu.2011.356), Bartel Cell 2009 136:215-233, af et al, Cell, 2007 129: 1401-1414, Gentner and Naldini, Tissue Antigens. 2012 80:393-403 and all references therein, each of which is incorporated herein by reference in its entirety). miRNAs and miRNA binding sites can correspond to any known sequence, including non-limiting examples described in US. Publication Nos. 2014/0200261, 2005/0261218, and 2005/0059005, each of which are incorporated herein by reference in their entirety.

Examples of tissues where miRNA are known to regulate mRNA, and y protein expression, include, but are not d to, liver (miR-122), muscle (miR-133, miR-206, miR- 208), endothelial cells (miR92, miR—126), myeloid cells (miR3p, miR5p, miR-16, miR-21, miR-223, miR—24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR—149), kidney (miR—192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).

Speci?cally, miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell speci?c miRNAs are involved in immunogenicity, autoimmunity, the immune-response to ion, in?ammation, as well as unwanted immune response after gene therapy and /organ transplantation. Immune cells speci?c miRNAs also regulate many s of development, proliferation, entiation and apoptosis of hematopoietic cells (immune cells). For e, miR-142 and miR-146 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstrated that the immune response to a polynucleotide can be shut-off by adding miR-142 binding sites to the 3 '-UTR of the polynucleotide, enabling more stable gene transfer in tissues and cells. miR-142 ef?ciently es exogenous polynucleotides in antigen presenting cells and sses cytotoxic elimination of transduced cells (e.g., Annoni A et (1]., blood, 2009, 114, 5152-5161, Brown BD, et al, Nat med. 2006, 12(5), 585-591, Brown BD, et (1]., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by nce in its entirety).

An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the n presenting cells. T cells can recognize the ted antigen and induce a cytotoxic ation of cells that express the antigen.

Introducing a miR-142 binding site into the 5'UTR and/or 3'UTR of a polynucleotide of the invention can selectively repress gene expression in antigen presenting cells through miR-142 mediated degradation, limiting antigen presentation in antigen ting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the ry of the polynucleotide. The cleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.

In one embodiment, binding sites for miRNAs that are known to be expressed in immune cells, in particular, n presenting cells, can be engineered into a polynucleotide of the invention to ss the expression of the polynucleotide in antigen presenting cells through miRNA mediated RNA degradation, subduing the antigen-mediated immune se.

Expression of the polynucleotide is maintained in non-immune cells where the immune cell speci?c miRNAs are not sed. For example, in some embodiments, to prevent an immunogenic reaction against a liver speci?c protein, any miR-122 binding site can be d and a miR-142 (and/or mirR-l46) binding site can be engineered into the 5'UTR and/or 3'UTR of a polynucleotide of the invention.

In one embodiment, g sites for miRNAs that are known to be expressed in liver cells can be engineered into a polynucleotide of the invention to ss the expression of the polynucleotide in liver. For example, in some embodiments, to prevent expression of an antigen in liver, any liver speci?c miR binding site can be ered into the 5'UTR and/or 3'UTR of a polynucleotide of the invention.

To further drive the selective degradation and suppression in APCs and macrophage, a polynucleotide of the invention can include a further negative regulatory element in the 5'UTR and/or 3'UTR, either alone or in combination with miR-142 and/or miR-146 binding sites. As a non-limiting example, the further negative regulatory element is a Constitutive Decay Element (CDE).

Immune cell speci?c miRNAs include, but are not limited to, hsa-let-7a3p, hsa-let-7a- 3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i- 3p, hsa-let-7i-5p, miR-lOa-3p, miR-lOa-Sp, miR-1184, hsa-let-7f-l--3p, t-7f-5p, hsa-let- 7f—5p, miR-125b-l-3p, miR-125b3p, miR-125b-5p, miR-1279, miR-l30a-3p, miR-l30a-5p, miR-l32-3p, miR-l32-5p, miR-l42-3p, miR-l42-5p, miR-l43-3p, 3-5p, miR-l46a-3p, miR-l46a-5p, miR-l46b-3p, miR-l46b-5p, miR-147a, miR-147b, miR-l48a-5p, miR-l48a-3p, miR3p, miR- l 50-5p, miR-15 lb, miR3p, miR5p, miR-15a-3p, miR- l 5a-5p, miR- 15b-5p, miR-15b-3p, -l-3p, miR-l63p, miR-l6-5p, -5p, miR-181a-3p, miR- 181a-5p, miR-181a3p, miR3p, 2-5p, miR-l97-3p, miR-l97-5p, miR5p, miR3p, miR3p, miR5p, 3-3p, 3-5p, miR3p, miR5p, miR-23b-3p, miR-23b-5p, miRl-5p,miR2-5p, -3p, miR-26a-l-3p, miR-26a3p, miR-26a-5p, miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p,miR-27b-5p, miR3p, miR5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-l-5p, miR-29b5p, miR-29c-3p, miR-29c-5p,, miR-30e-3p, e-5p, miR-33 l-5p, miR3p, miR5p, miR3p, miR5p, miR-346, miR-34a-3p, a-5p, miR- , miR3p, miR5p, 372, miR3p, miR5p, miR3p, miR5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j, miR-548n, miR3p, miR—598, miR—718, miR—935, miR-99a-3p, miR- 99a-5p, miR—99b-3p, and miR-99b-5p. Furthermore, novel miRNAs can be identi?ed in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, 116:e118-e127, Vaz C et al., BMC Genomics, 2010, 11,288, the t of each ofwhich is incorporated herein by reference in its entirety.) miRNAs that are known to be expressed in the liver include, but are not limited to, miR- 107, miR3p, miR5p, miR3p, miR—1228-5p, 49, miR5p, miR—1303, miR-151a-3p, miR—151a-5p, miR-152, miR3p, miR5p, miR-199a-3p, miR-199a-5p, 9b-3p, miR-199b-5p, miR5p, miR-557, miR-581, miR3p, and miR5p.

MiRNA binding sites from any liver speci?c miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the liver. Liver speci?c miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention. miRNAs that are known to be expressed in the lung include, but are not limited to, let-7a- 2-3p, let-7a-3p, let-7a-5p, miR3p, miR5p, miR3p, 7-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR—133a, miR-133b, miR—134, miR—18a-3p, miR- 18a-5p, miR-18b-3p, miR-18b-5p, miR—245p, miR2-5p, miR3p, miR3p, miR- 296-5p, miR—32-3p, 7-3p, 7-5p, miR3p, and miR5p. miRNA binding 2O sites from any lung speci?c miRNA can be introduced to or removed from a polynucleotide of the invention to te expression of the polynucleotide in the lung. Lung speci?c miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention. miRNAs that are known to be expressed in the heart include, but are not limited to, miR- 1, miR-133a, miR-133b, miR3p, miR5p, miR3p, miR5p, miR-208a, miR- 208b, 0, miR3p, miR-320, 1a, miR-451b, miR-499a-3p, miR-499a-5p, miR- p, miR—499b-5p, miR3p, miR5p, miR-92b-3p, and miR-92b-5p. mMiRNA binding sites from any heart c NA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the cleotide in the heart. Heart speci?c miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA g sites in a polynucleotide of the ion. miRNAs that are known to be expressed in the s system include, but are not limited to, 4-5p, miR-125a-3p, miR-125a-5p, miR-125b3p, miR-125b3p, miR- 125b-5p,miR3p, miR—1271-5p, miR-128, miR5p, 5a-3p, miR-135a-5p, miR- 135b-3p, miR—135b-5p, miR—137, miR5p, miR3p, miR3p, miR5p, miR- 153, miR-181c-3p, miR-181c-5p, miR3p, miR5p, miR-190a, miR-190b, miR3p, miR5p, miR1-3p, miR2-3p, a-3p, miR-23a-5p,miR-30a-5p, miR-30b-3p, miR-30b-5p, c3p, miR—30c3p, miR—30c-5p, miR-30d-3p, miR-30d-5p, 9, miR3p, miR-3665, miR-3666, 0-3p, miR5p, miR—3 83, miR-410, miR3p, miR5p, miR3p, miR5p, miR-483, miR-510, miR-516a-3p, miR-548b-5p, miR- 548c-5p, miR-571, miR1-3p, miR2-3p, miR—7-5p, miR-802, miR-922, miR3p, and miR- 9-5p. miRNAs enriched in the nervous system further e those speci?cally sed in neurons, including, but not limited to, miR3p, miR—132-3p, miR-148b-3p, miR—148b-5p, 1a-3p, miR—151a-5p, miR3p, 2-5p, miR-320b, miR—320e, miR-323a-3p, miR-323a-5p, 4-5p, miR-325, miR-326, miR-328, miR—922 and those speci?cally expressed in glial cells, including, but not limited to, miR-1250, miR1-3p, 93p, miR5p, miR-23a-3p, a-5p, miR3p, miR5p, miR—30e-3p, miR-30e-5p, miR5p, miR—338-5p, and miR—657. miRNA binding sites from any CNS speci?c miRNA can be uced to or removed from a polynucleotide of the invention to regulate expression of the cleotide in the nervous system. Nervous system speci?c miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention. miRNAs that are known to be expressed in the pancreas include, but are not limited to, 5-3p, miR5p, miR-184, miR3p, miR5p, miR-196a-3p, miR-196a-5p, miR3p, miR5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR1-3p, miR2-3p, miR3p, 3-5p, and miR—944. MiRNA binding sites from any as speci?c miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the pancreas. Pancreas speci?c miRNA binding sites can be engineered alone or further in combination with immune cell (e.g.

APC) miRNA binding sites in a polynucleotide of the invention. miRNAs that are known to be expressed in the kidney e, but are not limited to, miR3p, miR5p, miR5p, miR3p, miR5p, miR3p, miR5p, miR-20a-3p, miR-20a-5p, miR3p, miR5p, 0, miR-216a-3p, miR-216a-5p, miR3p, miR-30a-3p, a-5p, miR-30b-3p, miR-30b-5p, miR—30c3p, miR—30c3p, miR30c-5p, miR3p, miR3p, miR5p, miR3p, miR5p, and miR-562. miRNA binding sites from any kidney speci?c miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the kidney.

Kidney speci?c miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention. miRNAs that are known to be expressed in the muscle include, but are not d to, let- 7g-3p, let-7g-5p, miR-l, 86, miR—l33a, miR-l33b, miR-l40-3p, miR-l43-3p, miR-l43- 5p, miR-l45-3p, miR-l45-5p, miR3p, miR5p, miR-206, miR-208a, 8b, miR- -3p, and miR5p. MiRNA binding sites from any muscle speci?c miRNA can be introduced to or removed from a cleotide of the invention to regulate sion of the polynucleotide in the muscle. Muscle speci?c miRNA binding sites can be ered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention. miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes. miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, -5p, miR-lOO-3p, miR-lOO-Sp, miR-lOl-3p, miR-lOl-Sp, miR-l26-3p, miR- l26-5p, miR—l236-3p, miR-l236-5p, miR-l30a-3p, miR-l30a-5p, miR-l7-5p, miR-l7-3p, miR- 18a-3p, miR-18a-5p, miR—l9a-3p, miR-l9a-5p, miR-l9b-l-5p, miR-l9b5p, miR-l9b-3p, miR-20a-3p, miR-20a-5p, miR-2l7, O, miR-2l-3p, miR-2l-5p, miR-22l-3p, miR-22l-5p, miR3p, miR5p, miR-23a-3p, miR-23a-5p, miR5p, miR-36l-3p, miR-36l-5p, miR-42l, miR3p, miR5p, miR-513a-5p, miR—92a-l-5p, miR—92a5p, miR-92a-3p, 2O miR-92b-3p, and miR-92b-5p. Many novel miRNAs are ered in endothelial cells from deep-sequencing analysis (e.g., Voellenkle C et (11., RNA, 2012, 18, 472-484, herein incorporated by reference in its entirety). miRNA binding sites from any endothelial cell c miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the endothelial cells. miRNAs that are known to be expressed in epithelial cells include, but are not limited to, -3p, let-7b-5p, miR-l246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR- 200c-3p, miR-200c-5p, miR3p, miR-429, miR—451a, miR—45 lb, 4, miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p speci?c in respiratory ciliated lial cells, let-7 family, miR—l33a, miR-l33b, miR-126 speci?c in lung epithelial cells, miR-3 82-3p, miR-3 82-5p c in renal lial cells, and miR-762 speci?c in corneal epithelial cells. miRNA binding sites from any epithelial cell speci?c miRNA can be introduced to or removed from a cleotide of the invention to regulate expression of the polynucleotide in the epithelial cells.

In addition, a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell enewal as well as the development and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy KT et (1]., Curr. Mol Med, 2013, 13(5), 4, Vidigal JA and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436, Goff LA et al., PLoS One, 2009, 4:e7192, Morin RD et al, Genome Res,2008,18, 610-621, Yoo JK et al, Stem Cells Dev. 2012, 21(11), 2049- 2057, each of which is herein incorporated by reference in its entirety). MiRNAs abundant in embryonic stem cells include, but are not limited to, let-7a3p, let-a-3p, let-7a-5p, let7d-3p, let- 7d-5p, miR—103a3p, miR-103a-5p, miR—106b-3p, miR-106b-5p, miR-1246, miR-1275, miR- 1383p, miR—1383p, miR5p, miR3p, miR5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR—302a-3p, miR-302a-5p, miR-302b-3p, miR-302b- 5p, miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR3p, miR 5p, miR3p, miR5p, miR-370, miR-371, miR-373, miR5p, 3-3p, miR- 423-5p, 6-5p, miR-520c-3p, miR—548e, miR—548f, miR-548g-3p, miR—548g-5p, miR- 548i, miR-548k, miR-548l, miR—548m, miR-548n, 8o-3p, miR—548o-5p, miR—548p, miR- 664a-3p, miR-664a-5p, miR-664b-3p, 4b-5p, miR3p, miR5p, miR3p, miR5p,miR3p, miR5p, miR-941,miR3p, miR5p, miR-99b-3p and miR- 99b-5p. Many predicted novel miRNAs are discovered by deep sequencing in human embryonic stem cells (e.g., Morin RD et al., Genome Res,2008,18, 610-621, Goff LA et al., PLoS One, 2009, 4:e7192, Bar M et al, Stem cells, 2008, 26, 2496-2505, the content of each of which is incorporated herein by reference in its entirety).

Many miRNA expression studies are conducted to profile the differential sion of miRNAs in various cancer cells/tissues and other diseases. Some miRNAs are abnormally over- expressed in certain cancer cells and others are under-expressed. For example, miRNAs are differentially expressed in cancer cells (WO2008/154098, US2013/0059015, US2013/0042333, W02011/157294), cancer stem cells (U82012/0053224), pancreatic cancers and diseases 9/0131348, U82011/0171646, /0286232, 210), asthma and in?ammation (US8415096), te cancer 3/0053264), hepatocellular carcinoma 2/151212, U82012/0329672, W02008/054828, US825253 8), lung cancer cells (W02011/076143, /033640, W02009/070653, U82010/0323357), cutaneous T cell lymphoma (WO2013/011378), colorectal cancer cells (WO2011/0281756, WO2011/076142), cancer positive lymph nodes (WO2009/100430, US2009/0263803), aryngeal carcinoma (EP2112235), c obstructive pulmonary disease (US2012/0264626, US2013/0053263), thyroid cancer (WO2013/066678), n cancer cells (US2012/0309645, WO2011/095623), breast cancer cells (WO2008/154098, WO2007/081740, US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974, US2012/0316081, US2012/0283310, W02010/018563, the content of each of which is incorporated herein by reference in its entirety.) As a non-limiting example, miRNA binding sites for miRNAs that are xpressed in certain cancer and/or tumor cells can be removed from the 3'UTR of a cleotide of the invention, restoring the expression suppressed by the over-expressed miRNAs in cancer cells, thus ameliorating the corresponsive biological function, for instance, transcription stimulation and/or repression, cell cycle arrest, apoptosis and cell death. Normal cells and tissues, wherein miRNAs expression is not up-regulated, will remain unaffected. miRNA can also regulate complex biological processes such as angiogenesis (e.g., miR- 132) (Anand and Cheresh Curr Opin Hematol 2011 18: 6). In the polynucleotides of the ion, miRNA g sites that are involved in such processes can be removed or uced, in order to tailor the expression of the polynucleotides to biologically relevant cell types or relevant biological ses. In this context, the polynucleotides of the invention are de?ned as ophic polynucleotides.

In some embodiments, a polynucleotide of the invention comprises a miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from TABLE 1 or described elsewhere herein, ing one or more copies of any one or more of the miRNA binding site sequences. In some embodiments, a polynucleotide of the invention further comprises at least one, two, three, four, ?ve, six, seven, eight, nine, ten, or more of the same or different miRNA binding sites selected from TABLE 1 or described elsewhere herein, including any combination f. In some embodiments, the miRNA binding site binds to miR-142 or is mentary to miR-142. In some embodiments, the miR—142 comprises SEQ ID NO: 303.

In some embodiments, the miRNA binding site binds to miR3p or 2-5p. In some embodiments, the miR3p binding site comprises SEQ ID NO: 305. In some embodiments, the miR5p binding site comprises SEQ ID NO: 307. In some embodiments, the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NOs: 305 or 307.

TABLE 1. miR-142 and alternative 2 binding sites 303 miR-l42 GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUA ACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGA UGAGUGUACUGUG miR-l42-3p UGUAGUGUUUCCUACUUUAUGGA miR-l42-3p binding site UCCAUAAAGUAGGAAACACUACA miR-l42-5p CAUAAAGUAGAAAGCACUACU miR-l42-5p binding site AGUAGUGCUUUCUACUUUAUG In some embodiments, a miRNA binding site is inserted in the polynucleotide of the invention in any on of the polynucleotide (e.g., the 5'UTR and/or 3'UTR). In some ments, the 5'UTR ses a miRNA binding site. In some embodiments, the 3'UTR comprises a miRNA binding site. In some embodiments, the 5'UTR and the 3'UTR comprise a miRNA binding site. The insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the ation of a functional ptide in the absence of the corresponding miRNA, and in the presence of the miRNA, the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.

In some embodiments, a miRNA binding site is ed in at least about 30 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention comprising the ORF. In some embodiments, a miRNA binding site is inserted in at least about 10 tides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 tides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 tides, at least about 90 2O nucleotides, at least about 95 tides, or at least about 100 tides downstream from the stop codon of an ORF in a polynucleotide of the invention. In some embodiments, a miRNA binding site is inserted in about 10 nucleotides to about 100 tides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 tides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention. miRNA gene regulation can be in?uenced by the sequence surrounding the miRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, exogenous, endogenous, or arti?cial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence. The miRNA can be in?uenced by the 5'UTR and/or 3 'UTR. As a non-limiting example, a non-human 3 'UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3 'UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elements of the 5'UTR can in?uence miRNA mediated gene regulation. One example of a regulatory element and/or structural t is a structured IRES (Internal Ribosome Entry Site) in the 5'UTR, which is necessary for the binding of translational elongation factors to initiate protein translation.

EIF4A2 binding to this secondarily ured element in the 5'-UTR is necessary for miRNA mediated gene expression (Meij er HA et al, Science, 2013, 340, 82-85, herein incorporated by reference in its entirety). The polynucleotides of the ion can further include this structured 5'UTR in order to enhance microRNA mediated gene regulation.

At least one miRNA binding site can be engineered into the 3 'UTR of a cleotide of the invention. In this context, at least two, at least three, at least four, at least ?ve, at least six, at least seven, at least eight, at least nine, at least ten, or more miRNA binding sites can be engineered into a 3'UTR of a polynucleotide of the invention. For example, 1 to 10, l to 9, l to 8, l to 7, l to 6, l to 5, l to 4, l to 3, 2, or 1 miRNA binding sites can be engineered into the 3'UTR of a polynucleotide of the ion. In one embodiment, miRNA binding sites incorporated into a polynucleotide of the invention can be the same or can be ent miRNA sites. A combination of different miRNA binding sites orated into a polynucleotide of the invention can include combinations in which more than one copy of any of the different miRNA 2O sites are incorporated. In another ment, miRNA binding sites incorporated into a polynucleotide of the invention can target the same or different tissues in the body. As a non- limiting example, through the introduction of tissue-, cell-type-, or disease-specif1c miRNA binding sites in the 3'-UTR of a polynucleotide of the invention, the degree of expression in c cell types (e.g., hepatocytes, myeloid cells, endothelial cells, cancer cells, etc.) can be reduced.

In one embodiment, a miRNA binding site can be engineered near the 5' terminus of the 3'UTR, about halfway between the 5' terminus and 3' terminus of the 3'UTR and/or near the 3' terminus of the 3 'UTR in a polynucleotide of the invention. As a non-limiting example, a miRNA binding site can be ered near the 5' terminus of the 3'UTR and about halfway between the 5' terminus and 3' us of the 3'UTR. As another non-limiting e, a miRNA binding site can be engineered near the 3' terminus of the 3'UTR and about halfway between the 5' terminus and 3' us of the 3'UTR. As yet another non-limiting example, a miRNA binding site can be engineered near the 5' terminus of the 3'UTR and near the 3' terminus of the 3'UTR.

In another embodiment, a 3'UTR can comprise l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites. The miRNA binding sites can be complementary to a miRNA, miRNA seed ce, and/or miRNA sequences ?anking the seed sequence.

In one embodiment, a polynucleotide of the invention can be engineered to include more than one miRNA site expressed in different tissues or different cell types of a subject. As a non- limiting example, a polynucleotide of the invention can be engineered to include miR-192 and miR-122 to te expression of the cleotide in the liver and kidneys of a subject. In another embodiment, a polynucleotide of the invention can be engineered to include more than one miRNA site for the same .

In some embodiments, the therapeutic window and or differential expression ated with the polypeptide encoded by a polynucleotide of the invention can be d with a miRNA binding site. For example, a polynucleotide encoding a polypeptide that provides a death signal can be designed to be more highly expressed in cancer cells by virtue of the miRNA signature of those cells. Where a cancer cell ses a lower level of a particular miRNA, the polynucleotide encoding the binding site for that miRNA (or miRNAs) would be more highly expressed. Hence, the polypeptide that provides a death signal triggers or induces cell death in the cancer cell. Neighboring noncancer cells, harboring a higher expression of the same miRNA would be less ed by the d death signal as the polynucleotide would be expressed at a lower level due to the effects of the miRNA binding to the binding site or "sensor" encoded in 2O the 3'UTR. sely, cell survival or cytoprotective signals can be delivered to tissues containing cancer and non-cancerous cells where a miRNA has a higher expression in the cancer cells—the result being a lower survival signal to the cancer cell and a larger survival signal to the normal cell. Multiple polynucleotides can be designed and administered having different signals based on the use of miRNA g sites as described herein.

In some embodiments, the expression of a polynucleotide of the invention can be controlled by incorporating at least one miR binding site or sensor sequence in the polynucleotide and formulating the polynucleotide for administration. As a non-limiting example, a polynucleotide of the invention can be ed to a tissue or cell by incorporating a miRNA binding site and formulating the polynucleotide in a lipid nanoparticle comprising a ionizable lipid (e.g., a cationic lipid), including any of the lipids described .

A cleotide of the invention can be engineered for more targeted expression in speci?c tissues, cell types, or biological conditions based on the expression patterns of miRNAs in the different tissues, cell types, or ical conditions. Through introduction of tissue- speci?c miRNA binding sites, a polynucleotide of the ion can be ed for optimal protein expression in a tissue or cell, or in the context of a ical condition.

In some embodiments, a polynucleotide of the invention can be ed to orate miRNA binding sites that either have 100% identity to known miRNA seed sequences or have less than 100% identity to miRNA seed sequences. In some embodiments, a polynucleotide of the invention can be designed to incorporate miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed sequences. The miRNA seed sequence can be partially mutated to decrease miRNA binding af?nity and as such result in reduced downmodulation of the polynucleotide. In essence, the degree of match or mis-match between the miRNA binding site and the miRNA seed can act as a rheostat to more ?nely tune the ability of the miRNA to modulate protein expression. In addition, mutation in the non-seed region of a miRNA binding site can also impact the ability of a miRNA to modulate protein expression.

In one embodiment, a miRNA sequence can be incorporated into the loop of a stem loop.

In another embodiment, a miRNA seed sequence can be incorporated in the loop of a stem loop and a miRNA binding site can be incorporated into the 5' or 3' stem of the stem loop.

In one embodiment, a translation enhancer element (TEE) can be incorporated on the 'end of the stem of a stem loop and a miRNA seed can be incorporated into the stem of the stem loop. In another ment, a TEE can be incorporated on the 5' end of the stem of a stem loop, a miRNA seed can be incorporated into the stem of the stem loop and a miRNA binding site can be incorporated into the 3' end of the stem or the sequence after the stem loop. The miRNA seed and the miRNA g site can be for the same and/or different miRNA sequences.

In one embodiment, the incorporation of a miRNA sequence and/or a TEE sequence changes the shape of the stem loop region which can increase and/or decrease translation. (see e. g, Kedde et al., "A Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221 and miR-22 accessibility." Nature Cell Biology. 2010, incorporated herein by reference in its entirety).

In one embodiment, the 5'-UTR of a polynucleotide of the invention can comprise at least one miRNA sequence. The miRNA sequence can be, but is not d to, a 19 or 22 nucleotide ce and/or a miRNA sequence without the seed.

In one embodiment the miRNA sequence in the 5'UTR can be used to stabilize a polynucleotide of the invention described herein.

In another embodiment, a miRNA sequence in the 5'UTR of a polynucleotide of the ion can be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon. See, e.g., Matsuda et al, PLoS One. 2010 11(5):e15057, orated herein by reference in its entirety, which used antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) around a start codon (-4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the ?rst start codon (AUG).

Matsuda showed that altering the ce around the start codon with an LNA or EJC affected the efficiency, length and structural stability of a cleotide. A cleotide of the invention can comprise a miRNA sequence, instead of the LNA or EJC sequence bed by Matsuda et al, near the site of translation tion in order to decrease the accessibility to the site of translation initiation. The site of translation initiation can be prior to, after or within the miRNA ce. As a non-limiting example, the site of ation initiation can be located within a miRNA sequence such as a seed sequence or binding site. As another non-limiting example, the site of translation initiation can be located within a miR-122 sequence such as the seed ce or the 2 binding site.

In some embodiments, a polynucleotide of the ion can include at least one miRNA in order to dampen the antigen presentation by antigen presenting cells. The miRNA can be the complete miRNA sequence, the miRNA seed sequence, the miRNA sequence t the seed, or a combination thereof. As a non-limiting example, a miRNA incorporated into a 2O polynucleotide of the invention can be specific to the hematopoietic . As another nonlimiting example, a miRNA incorporated into a polynucleotide of the invention to dampen antigen presentation is miR—142-3p.

In some embodiments, a polynucleotide of the invention can include at least one miRNA in order to dampen expression of the encoded polypeptide in a tissue or cell of interest. As a non-limiting example, a polynucleotide of the invention can include at least one miR—122 binding site in order to dampen expression of an encoded polypeptide of interest in the liver. As another non-limiting example a polynucleotide of the invention can include at least one miR3p binding site, miR3p seed sequence, miR3p binding site without the seed, miR5p binding site, miR5p seed sequence, miR5p g site without the seed, miR—146 binding site, miR-146 seed sequence and/or miR-146 binding site without the seed sequence.

In some embodiments, a polynucleotide of the ion can comprise at least one miRNA binding site in the 3 'UTR in order to selectively degrade mRNA therapeutics in the immune cells to subdue unwanted immunogenic reactions caused by therapeutic delivery. As a non-limiting example, the miRNA binding site can make a polynucleotide of the invention more unstable in antigen presenting cells. Non-limiting examples of these miRNAs include mir-l42- 5p, 2-3p, mir-l46a-5p, and mir-l46-3p.

In one embodiment, a polynucleotide of the invention comprises at least one miRNA sequence in a region of the polynucleotide that can interact with a RNA g protein.

In some embodiments, the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprising (i) a sequence-optimized nucleotide sequence (e.g., an ORF) encoding one or more wild type epitope antigens and (ii) a miRNA binding site (e.g., a miRNA binding site that binds to miR-142).

In some embodiments, the polynucleotide of the invention comprises a uracil-modi?ed sequence encoding one or more cancer epitope polypeptides disclosed herein and a miRNA binding site disclosed herein, e.g., a miRNA binding site that binds to miR-l42. In some embodiments, the uracil-modi?ed sequence encoding one or more cancer epitope ptides comprises at least one chemically modi?ed nucleobase, e.g., 5-methoxyuracil. In some embodiments, at least 95% of a type of nucleobase (e.g., uricil) in a uracil-modi?ed sequence encoding one or more cancer epitope polypeptides of the invention are modi?ed nucleobases. In some embodiments, at least 95% of uricil in a uracil-modi?ed sequence encoding one or more cancer epitope ptides is 5-methoxyuridine. In some embodiments, the polynucleotide comprising a nucleotide ce ng one or more cancer epitope polypeptides disclosed herein and a miRNA binding site is formulated with a delivery agent, e.g., a LNP comprising, for ce, a lipid having the Formula (I), (IA), (II), (IIa), (IIb), (11c), (11d) or (IIe), e.g., any of Compounds 1-232. 3 ' UTR and the A URich Elements In certain embodiments, a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a cancer antigen epitope of the ion) further ses a 3' UTR. In certain embodiments, a polynucleotide of the present invention (e.g., a polynucleotide comprising a tide sequence encoding an ting oncogene mutation peptide of the invention) further comprises a 3' UTR. 3'—UTR is the n of mRNA that immediately follows the translation termination codon and often contains regulatory regions that post-transcriptionally in?uence gene expression. Regulatory regions within the 3'—UTR can in?uence polyadenylation, translation ef?ciency, localization, and ity of the mRNA. In one embodiment, the 3'—UTR useful for the invention comprises a g site for regulatory proteins or microRNAs. In some embodiments, the 3'—UTR has a silencer , which binds to repressor proteins and inhibits the expression of the mRNA. In other embodiments, the 3'—UTR comprises an AU-rich element. ns bind AREs to affect the stability or decay rate of transcripts in a localized manner or affect translation initiation. In other embodiments, the 3'—UTR ses the sequence AAUAAA that directs addition of several hundred adenine residues called the ) tail to the end of the mRNA transcript.

Natural or wild type 3' UTRs are known to have stretches of Adenosines and es embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain l sed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class IAREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. les containing this type of AREs include GM- CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, s members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR speci?c binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo. 2O Introduction, removal or modi?cation of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides of the invention. When engineering specif1c polynucleotides, one or more copies of an ARE can be introduced to make polynucleotides of the invention less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and tion of the resultant protein. ection experiments can be conducted in relevant cell lines, using polynucleotides of the invention and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different gineering les and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

Regions having a 5 ' Cap The invention also includes a polynucleotide that comprises both a 5' Cap and a polynucleotide of the t invention (e.g., a polynucleotide comprising a nucleotide ce ng a cancer antigen epitope such as an activating oncogene mutation peptide).

The 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding n (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns during mRNA splicing.

Endogenous mRNA molecules can be 5'-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense tide of the mRNA le. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the al and/or anteterminal transcribed nucleotides of the 5' end of the mRNA can optionally also be 2 methylated. 5'-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.

In some embodiments, the polynucleotides of the t invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a cancer antigen epitope) incorporate a cap moiety.

In some embodiments, polynucleotides of the present invention (e.g., a cleotide comprising a nucleotide sequence encoding a cancer antigen epitope such as an ting oncogene mutation peptide) comprise a non-hydrolyzable cap structure ting decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'- ppp-5' phosphorodiester linkages, modi?ed tides can be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs ch, MA) can be used with d-thio-guanosine nucleotides according to the cturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modi?ed guanosine nucleotides can be used such as d-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modi?cations include, but are not limited to, ethylation of the ribose sugars of 5'-terminal and/or 5'-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap s, or structural or onal cap analogs, differ from natural (1'.e., endogenous, wild-type or physiological) 5'- caps in their chemical structure, while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the invention.

For example, the Anti-Reverse Cap Analog (ARCA) cap ns two guanines linked by a triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G- 3'mppp-G, which can equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other, unmodi?ed, guanine becomes linked to the 5'-terminal nucleotide of the capped polynucleotide. The N7- and 3 '-O-methlyated guanine provides the terminal moiety of the capped cleotide.

Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine (1'. e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm- ppp-G) In some embodiments, the cap is a eotide cap analog. As a non-limiting e, the dinucleotide cap analog can be modi?ed at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US. Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.

In another embodiment, the cap is a cap analog is a N7-(4-chlorophenoxyethyl) 2O substituted dicucleotide form of a cap analog known in the art and/or described herein. Non- limiting examples of a N7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and a N7-(4- phenoxyethyl)-m3'-OG(5')ppp(5')G cap analog (See, e.g., the various cap s and the methods of synthesizing cap analogs described in Kore et al. anic & Medicinal Chemistry 2013 21 :4570-4574, the contents of which are herein incorporated by reference in its entirety). In r embodiment, a cap analog of the present invention is a 4- /bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotide or a region thereof, in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped.

This, as well as the structural ences of a cap analog from an endogenous 5'-cap structures of nucleic acids produced by the endogenous, cellular ription machinery, can lead to reduced translational competency and reduced cellular ity.

Polynucleotides of the invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a cancer antigen epitope) can also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes, in order to te more authentic 5'-cap ures. As used , the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding nous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 'cap structures of the present invention are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5'decapping, as compared to synthetic 5'cap structures known in the art (or to a wild-type, natural or physiological 5'cap structure). For example, recombinant Vaccinia Virus g Enzyme and recombinant ethyltransferase enzyme can create a canonical 5'-5'-triphosphate e between the minal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 ation and the 5'- terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-in?ammatory cytokines, as compared, e.g., to other 5'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')Nlmpr (cap 1), and 7mG(5')- ppp(5')NlmpN2mp (cap 2).

As a non-limiting example, capping chimeric polynucleotides post-manufacture can be more efficient as nearly 100% of the chimeric polynucleotides can be . This is in contrast to ~80% when a cap analog is linked to a ic polynucleotide in the course of an in vitro transcription reaction.

According to the t invention, 5' terminal caps can e endogenous caps or cap analogs. According to the present invention, a 5' terminal cap can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'?uoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine.

Poly-A Tails In some embodiments, the polynucleotides of the present disclosure (e.g., a polynucleotide sing a nucleotide sequence encoding a cancer antigen epitope such as an activating oncogene mutation peptide) further comprise a poly-A tail. In further embodiments, terminal groups on the poly-A tail can be incorporated for ization. In other embodiments, a poly-A tail comprises des-3' hydroxyl tails.

During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to a polynucleotide such as an mRNA molecule in order to se stability. ately after transcription, the 3' end of the transcript can be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.

PolyA tails can also be added after the construct is exported from the nucleus.

According to the present invention, terminal groups on the poly A tail can be incorporated for stabilization. Polynucleotides of the present invention can include des-3' hydroxyl tails. They can also e structural moieties or 2'—Omethyl modi?cations as taught by Junjie Li, et al (Current Biology, Vol. 15, 1501—1507, August 23, 2005, the contents of which are orated herein by reference in its entirety).

The cleotides of the present invention can be designed to encode transcripts with alternative polyA tail structures including histone mRNA. According to Norbury, "Terminal uridylation has also been detected on human replication-dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the tion or inhibition of chromosomal DNA replication. These mRNAs are distinguished by their lack of a 3' poly(A) tail, the function of which is instead assumed by a stable stem—loop structure and its cognate stem—loop binding protein (SLBP), the latter carries out the same functions as those of PABP on polyadenylated mRNAs" (Norbury, "Cytoplasmic RNA: a case of the tail wagging the dog," Nature Reviews Molecular Cell Biology, AOP, published online 29 August 2013, doi: 10. 103 8/nrm3 645) the contents of which are incorporated herein by nce in its entirety.

Unique poly-A tail s provide certain ages to the polynucleotides of the present invention. Generally, the length of a poly-A tail, when t, is greater than 30 nucleotides in . In another embodiment, the poly-A tail is greater than 35 tides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).

In some embodiments, the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

In some embodiments, the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate t expressed from the polynucleotides.

In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or e thereof. The poly-A tail can also be designed as a fraction of the polynucleotides to which it s. In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered g sites and conjugation of polynucleotides for Poly-A binding protein can enhance expression.

Additionally, multiple ct polynucleotides can be linked together via the PABP (Poly-A binding protein) through the 3'-end using modi?ed nucleotides at the 3'-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr and day 7 post- transfection.

In some embodiments, the polynucleotides of the present ion are designed to include a polyA-G Quartet region. The G—quartet is a cyclic en bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G—quartet is incorporated at the end of the poly-A tail. The resultant cleotide is assayed for stability, protein tion and other ters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.

Start codon region The invention also includes a polynucleotide that comprises both a start codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a cancer antigen epitope such as an activating oncogene mutation peptide). In some embodiments, the polynucleotides of the t invention can have regions that are analogous to or function like a start codon region.

In some embodiments, the translation of a polynucleotide can initiate on a codon that is not the start codon AUG. Translation of the polynucleotide can initiate on an ative start codon such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG (see Touriol et al Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 2010 5:11, the contents of each of which are herein incorporated by reference in its entirety).

As a non-limiting example, the translation of a polynucleotide begins on the alternative start codon ACG. As another non-limiting example, polynucleotide translation begins on the alternative start codon CTG or CUG. As yet another non-limiting example, the translation of a polynucleotide begins on the alternative start codon GTG or GUG.

Nucleotides ?anking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See, e.g., a and Mauro PLoS ONE, 2010 5:11, the ts of which are herein incorporated by reference in its entirety). g any of the nucleotides ?anking a codon that initiates translation can be used to alter the position of translation initiation, translation ef?ciency, length and/or ure of a cleotide.

In some embodiments, a g agent can be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon. Non-limiting es of masking agents include antisense locked c acids (LNA) polynucleotides and exon- junction complexes (EJCs) (See, e.g., Matsuda and Mauro bing masking agents LNA polynucleotides and EJCs (PLoS ONE, 2010 5:11), the contents of which are herein incorporated by reference in its entirety).

In another embodiment, a masking agent can be used to mask a start codon of a polynucleotide in order to increase the likelihood that translation will initiate on an alternative start codon. In some embodiments, a masking agent can be used to mask a first start codon or ative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.

In some embodiments, a start codon or alternative start codon can be d within a perfect complement for a miR binding site. The perfect complement of a miR binding site can help control the translation, length and/or structure of the polynucleotide similar to a masking agent. As a non-limiting example, the start codon or alternative start codon can be located in the middle of a perfect complement for a miRNA binding site. The start codon or alternative start codon can be located after the ?rst nucleotide, second nucleotide, third nucleotide, fourth nucleotide, ?fth nucleotide, sixth nucleotide, h nucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth nucleotide, ?fteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth tide, twentieth tide or twenty-?rst nucleotide.

In another ment, the start codon of a polynucleotide can be removed from the cleotide sequence in order to have the translation of the polynucleotide begin on a codon that is not the start codon. Translation of the polynucleotide can begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon. In a miting example, the start codon ATG or AUG is removed as the ?rst 3 tides of the polynucleotide sequence in order to have translation initiate on a downstream start codon or alternative start codon. The cleotide sequence where the start codon was removed can further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the ure of the polynucleotide.

Stop Codon Region The invention also includes a polynucleotide that comprises both a stop codon region and the polynucleotide described herein (e.g., a cleotide comprising a nucleotide sequence encoding a cancer antigen epitope such as an activating oncogene mutation peptide). In some embodiments, the cleotides of the present invention can include at least two stop codons before the 3' untranslated region (UTR). The stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA. In some ments, the polynucleotides of the present invention include the stop codon TGA in the case or DNA, or the stop codon UGA in the case of RNA, and one additional stop codon. In a further embodiment the addition stop codon can be TAA or UAA.

In another ment, the polynucleotides of the present invention include three consecutive stop codons, four stop codons, or more.

Insertions and Substitutions The invention also includes a polynucleotide of the present disclosure that further ses ions and/or substitutions.

In some embodiments, the 5'UTR of the polynucleotide can be replaced by the insertion of at least one region and/or string of nucleosides of the same base. The region and/or string of nucleotides can e, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 nucleotides and the nucleotides can be natural and/or ral.

As a miting example, the group of nucleotides can include 5-8 e, cytosine, thymine, a string of any of the other nucleotides disclosed herein and/or combinations thereof.

In some ments, the 5'UTR of the polynucleotide can be replaced by the insertion of at least two regions and/or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein and/or combinations f. For example, the 5'UTR can be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases. In another example, the 5'UTR 2O can be replaced by ing 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.

In some ments, the cleotide can include at least one substitution and/or insertion downstream of the transcription start site that can be recognized by an RNA polymerase. As a non-limiting example, at least one substitution and/or insertion can occur downstream of the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6).

Changes to region of nucleotides just downstream of the transcription start site can affect initiation rates, increase apparent nucleotide triphosphate (NTP) reaction constant values, and increase the dissociation of short transcripts from the transcription x curing initial transcription (Brieba et al, Biochemistry (2002) 41: 5144-5149, herein incorporated by reference in its entirety). The modification, substitution and/or insertion of at least one nucleoside can cause a silent mutation of the sequence or can cause a mutation in the amino acid sequence.

In some embodiments, the polynucleotide can include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 guanine bases downstream of the transcription start site.

In some embodiments, the polynucleotide can e the substitution of at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine bases in the region just downstream of the transcription start site. As a non-limiting example, if the tides in the region are GGGAGA, the guanine bases can be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides. In r non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases can be substituted by at least 1, at least 2, at least 3 or at least 4 cytosine bases. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases can be substituted by at least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the nucleotides described herein.

In some embodiments, the polynucleotide can include at least one substitution and/or insertion upstream of the start codon. For the purpose of y, one of skill in the art would appreciate that the start codon is the first codon of the protein coding region whereas the transcription start site is the site where transcription begins. The polynucleotide can e, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or insertions of nucleotide bases. The tide bases can be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4 or at least 5 locations 2O upstream of the start codon. The nucleotides inserted and/or substituted can be the same base (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T or A, C and T) or at least four ent bases.

As a non-limiting example, the guanine base upstream of the coding region in the polynucleotide can be tuted with adenine, cytosine, thymine, or any of the nucleotides described . In another non-limiting example, the substitution of e bases in the polynucleotide can be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472(7344):499-503, the contents of which is herein incorporated by reference in its entirety).

As a non-limiting example, at least 5 nucleotides can be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 tides can be the same base type.

According to the present disclosure, two regions or parts of a chimeric polynucleotide may be joined or ligated, for example, using triphosphate chemistry. In some embodiments, a ?rst region or part of 100 nucleotides or less is chemically synthesized with a 5'- monophosphate and terminal 3 '-desOH or d OH. If the region is longer than 80 nucleotides, it may be synthesized as two or more strands that will subsequently be chemically linked by ligation. If the ?rst region or part is synthesized as a non-positionally d region or part using IVT, conversion to the 5'-monophosphate with sub sequent capping of the 3 '-terminus may follow. osphate protecting groups may be selected from any of those known in the art. A second region or part of the chimeric polynucleotide may be synthesized using either chemical synthesis or IVT methods, e.g., as described herein.

IVT methods may include use of an RNA polymerase that can utilize a primer with a modi?ed cap. Alternatively, a cap may be chemically synthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase followed by DNAse ent (to eliminate the DNA splint required for DNA T4 Ligase activity) should readily prevent the undesirable formation of concatenation products.

The entire chimeric polynucleotide need not be manufactured with a phosphate-sugar backbone. If one of the regions or parts encodes a polypeptide, then it is preferable that such region or part comprise a ate-sugar backbone.

Ligation may be performed using any appropriate technique, such as enzymatic ligation, click chemistry, lick chemistry, solulink, or other bioconjugate tries known to those in the art. In some embodiments, the on is ed by a mentary oligonucleotide splint. In some embodiments, the ligation is performed without a complementary oligonucleotide splint.

In other aspects, the ion relates to kits for preparing an mRNA cancer vaccine by IVT methods. In personalized cancer vaccines, it is important to identify patient c mutations and vaccinate the patient with one or more neoepitopes. In such vaccines, the antigen(s) encoded by the ORFs of an mRNA will be speci?c to the patient. The 5'- and 3'- ends of RNAs encoding the antigen(s) may be more broadly applicable, as they include untranslated regions and stabilizing regions that are common to many RNAs. Among other things, the present disclosure provides kits that include one or parts of a chimeric polynucleotide, such as one or more 5'- and/or 3'-regions of RNA, which may be combined with an ORF encoding a patient-speci?c epitope. For example, a kit may include a polynucleotide containing one or more of a 5'-ORF, a 3'-ORF, and a poly(A) tail. In some embodiments, each polynucleotide component is in an individual ner. In other embodiments, more than one polynucleotide component is present together in a single container. In some embodiments, the kit includes a ligase enzyme. In some embodiments, provided kits e instructions for use. In some embodiments, the ctions include an instruction to ligate the epitope encoding ORF to one or more other ents from the kit, e.g., 5'-ORF, a 3 '-ORF, and/or a poly(A) tail.

Methods for generating personalized cancer vaccines according to the invention involve identi?cation of mutations using techniques such as deep nucleic acid or protein sequencing methods as described herein of tissue samples. In some embodiments an initial identi?cation of mutations in a patient’s transcriptome is performed. The data from the patient’s transcriptome is compared with sequence information from the patients exome in order to identify patient speci?c and tumor speci?c mutations that are expressed. The ison produces a dataset of putative neoepitopes, referred to as a mutanome. The mutanome may include imately lOO-l0,000 ate mutations per patients. The mutanome is subject to a data probing analysis using a set of inquiries or algorithms to identify an optimal mutation set for generation of a neoantigen vaccine. In some embodiments an mRNA neoantigen vaccine is ed and manufactured. The patient is then treated with the vaccine.

The igen vaccine may be a polycistronic vaccine including multiple neoepitopes or one or more single RNA vaccines or a combination thereof.

In some embodiments the entire method from the initiation of the mutation identi?cation process to the start of patient ent is achieved in less than 2 months. In 2O other embodiments the whole process is achieved in 7 weeks or less, 6 weeks or less, 5 weeks or less, 4 weeks or less, 3 weeks or less, 2 weeks or less or less than 1 week. In some embodiments the whole method is performed in less than 30 days.

The on identi?cation process may involve both transcriptome and exome analysis or only transcriptome or exome analysis. In some embodiments transcriptome analysis is performed ?rst and exome is is performed second. The analysis is performed on a biological or tissue sample. In some embodiments a biological or tissue sample is a blood or serum sample. In other embodiments the sample is a tissue bank sample or EBV transformation of B-cells.

It has been recognized and appreciated that, by analyzing certain properties of cancer associated mutations, l neoepitopes may be assessed and/or ed for inclusion in an mRNA e. For example, at a given time, one or more of several properties may be assessed and weighted in order to select a set of neoepitopes for inclusion in a vaccine. A property of a neoepitope or set of topes may include, for instance, an assessment of gene or transcript-level expression in patient RNA-seq or other nucleic acid analysis, - speci?c expression in available databases, known oncogenes/tumor suppressors, variant call con?dence score, RNA-seq allele-specif1c expression, conservative vs. non-conservative AA substitution, position of point mutation (Centering Score for increased TCR engagement), position of point on (Anchoring Score for differential HLA binding), Selfness: core epitope homology with patient WES data, HLA-A and —B IC50 for 8mers-l lmers, HLA-DRBl IC50 for lSmers-20mers, promiscuity Score (1'. e. number of patient HLAs predicted to bind), HLA-C IC50 for l lmers, HLA-DRB3-5 IC50 for 15mers-20mers, Bl/Al IC50 for 15mers-20mers, HLA-DPBl/Al IC50 for 15mers-20mers, Class I vs Class II proportion, ity of patient HLA-A, -B and DRBl allotypes covered, proportion of point mutation vs complex epitopes (e.g. hifts), and /or pseudo-epitope HLA g scores.

In some embodiments, the properties of cancer associated mutations used to identify optimal neoepitopes are properties related to the type of mutation, abundance of mutation in patient sample, immunogenicity, lack of self-reactivity, and nature of peptide composition.

The type of mutation should be determined and ered as a factor in determining whether a putative e should be included in a vaccine. The type of mutation may vary.

In some instances it may be desirable to include multiple different types of mutations in a single vaccine. In other instances a single type of on may be more desirable. A value for particular mutation can be weighted and calculated. In some embodiments, a particular mutation is a single nucleotide polymorphism (SNP). In some embodiments, a particular mutation is a complex variant, for example, a e sequence resulting from intron retention, complex splicing events, or insertion / deletion mutations changing the reading frame of a sequence.

The abundance of the mutation in patient sample may also be scored and ed into the decision of whether a putative epitope should be included in a vaccine. Highly nt mutations may promote a more robust immune response.

The consideration of the immunogenicity is an important component in the selection of optimal neoepitopes for inclusion in a vaccine. genicity may be assessed for instance, by analyzing the MHC binding capacity of a neoepitope, HLA promiscuity, mutation position, ted T cell reactivity, actual T cell reactivity, structure leading to ular conformations and resultant solvent exposure, and representation of speci?c amino acids. Known algorithms such as the NetMHC prediction algorithm can be used to predict capacity of a peptide to bind to common HLA-A and -B s. Structural assessment of a MHC bound peptide may also be conducted by in silico nsional analysis and/or protein docking programs. Use of a predicted epitope structure when bound to a MHC molecule, such as acquired from a Rosetta thm, may be used to evaluate the degree of t exposure of an amino acid residues of an epitope when the epitope is bound to a MHC molecule. T cell reactivity may be assessed experimentally with es and T cells in vitro. Alternatively T cell reactivity may be assessed using T cell response/ sequence datasets.

An important component of a neoepitope included in a vaccine, is a lack of self- reactivity. The putative neoepitopes may be screened to con?rm that the epitope is restricted to tumor tissue, for instance, arising as a result of genetic change within malignant cells.

Ideally, the epitope should not be present in normal tissue of the patient and thus, self-similar epitopes are ?ltered out of the dataset. A personalized coding genome may be used as a reference for comparison of neoantigen candidates to determine lack of eactivity. In some embodiments, a personalized coding genome is generated from an individualized transcriptome and/or exome.

The nature of peptide composition may also be considered in the epitope design. For instance a score can be provided for each putative epitope on the value of conserved versus non-conserved amino acids found in the epitope.

In some ments, the analysis performed by the tools described herein may include comparing ent sets of properties ed at different times from a patient, i.e. prior to and following a therapeutic intervention, from different tissue samples, from different patients having similar tumors, etc. In some embodiments, an average of peak values from one set of properties may be compared with an average of peak values from another set of properties. For example, an average value for HLA binding may be compared between two different sets of distributions. The two sets of distributions may be ined for time ons separated by days, months, or years, for instance.

Moreover, the inventors have recognized and appreciated that such data on ties of cancer mutations may be collected and analyzed using the algorithms described herein.

The data is useful for fying neoepitopes and sets of neoepitopes for the development of personalized cancer vaccines.

In some embodiments, all annotated transcripts of a tumor variant e are ed in a vaccine in ance with the invention. In some embodiments, translations of RNA identified in RNAseq are included in a vaccine in accordance with the present invention.

It will be appreciated that a concatamer of 2 or more peptides, e.g., 2 or more neoantigens, may create unintended new epitopes (pseudoepitopes) at peptide boundaries. To prevent or eliminate such pseudoepitopes, class I s may be scanned for hits across peptide boundaries in a concatamer. In some embodiments, the peptide order within the concatamer is d to reduce or eliminate pseudoepitope ion. In some embodiments, a linker is used between peptides, e.g., a single amino acid linker such as glycine, to reduce or eliminate pseudoepitope formation. In some embodiments, anchor amino acids can be replaced with other amino acids which will reduce or eliminate pseudoepitope formation. In some embodiments, peptides are trimmed at the peptide boundary within the concatamer to reduce or eliminate pseudoepitope ion.

In some ments the multiple peptide epitope antigens are arranged and ordered to minimize pseudoepitopes. In other embodiments the multiple peptide epitope antigens are a polypeptide that is free of pseudoepitopes. When the cancer antigen epitopes are arranged in a concatemeric structure in a head to tail formation a junction is formed between each of the cancer antigen epitopes. That includes several, i.e. l-lO, amino acids from an epitope on a N—terminus of the peptide and several, i.e. l-lO, amino acids on a C-terminus of an adjacent directly linked epitope. It is important that the junction not be an immunogenic peptide that may produce an immune response. In some embodiments the junction forms a peptide sequence that binds to an HLA protein of a t for which the personalized cancer e 2O is designed with an ICSO greater than about 50 nM. In other embodiments the on peptide sequence binds to an HLA protein of a t with an ICSO greater than about 10 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nm, or 500 nM.

A neoepitope characterization system in accordance with the techniques described herein may take any suitable form, as embodiments are not limited in this t. An illustrative implementation of a computer system 900 that may be used in connection with some ments is shown in One or more computer systems such as computer system 900 may be used to implement any of the functionality described above. The computer system 900 may include one or more processors 910 and one or more computer- readable storage media (1'.e., tangible, non-transitory er-readable media), e.g., volatile e 920 and one or more non-volatile storage media 930, which may be formed of any suitable data storage media. The processor 910 may control writing data to and reading data from the volatile storage 920 and the non-volatile storage device 930 in any le manner, as embodiments are not limited in this respect. To perform any of the functionality described herein, the processor 910 may execute one or more instructions stored in one or more computer-readable storage media (e.g., le storage 920 and/or non-volatile storage 930), which may serve as tangible, non-transitory computer-readable media storing instructions for execution by the processor 910.

The above-described embodiments can be implemented in any of numerous ways.

For example, the ments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or buted among multiple computers. It should be appreciated that any component or collection of components that perform the functions described above can be generically ered as one or more controllers that control the discussed functions. The one or more controllers can be ented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above.

In this respect, it should be appreciated that one implementation comprises at least one computer-readable storage medium (1'.e., at least one tangible, non-transitory computer- readable medium), such as a er memory (e.g., hard drive, ?ash memory, processor working memory, etc.), a ?oppy disk, an optical disk, a magnetic tape, or other tangible, non- transitory computer-readable medium, encoded with a computer program (i.e., a plurality of instructions), which, when executed on one or more processors, performs above-discussed functions. The computer-readable storage medium can be ortable such that the program stored thereon can be loaded onto any er resource to implement techniques discussed herein. In on, it should be appreciated that the reference to a computer program which, when executed, performs discussed functions, is not limited to an application program running on a host er. , the term "computer program" is used herein in a generic sense to reference any type of computer code (e.g., software or microcode) that can be ed to program one or more processors to implement abovetechniques.

GC—Rich Domains ions GC—rich: As used herein, the term "GC-rich" refers to the nucleobase composition of a polynucleotide (e.g., mRNA), or any portion thereof (e.g., an RNA element), comprising guanine (G) and/or cytosine (C) nucleobases, or derivatives or analogs thereof, wherein the GC-content is greater than about 50%. The term "GC-rich" refers to all, or to a portion, of a polynucleotide, including, but not d to, a gene, a non-coding region, a 5’ UTR, a 3’ UTR, an open reading frame, an RNA element, a ce motif, or any discrete sequence, fragment, or segment thereof which comprises about 50% GC-content. In some embodiments of the disclosure, GC-rich polynucleotides, or any portions thereof, are ively comprised of guanine (G) and/or cytosine (C) nucleobases.

GC—conieni: As used herein, the term "GC-content" refers to the percentage of nucleobases in a cleotide (e.g., mRNA), or a portion thereof (e.g., an RNA element), that are either e (G) and cytosine (C) nucleobases, or derivatives or analogs thereof, (from a total number of le nucleobases, including adenine (A) and thymine (T) or uracil (U), and derivatives or analogs thereof, in DNA and in RNA). The term "GC-content" refers to all, or to a n, of a polynucleotide, including, but not limited to, a gene, a non- coding region, a 5’ or 3’ UTR, an open reading frame, an RNA element, a sequence motif, or any discrete sequence, fragment, or segment thereof. tion Codon: As used herein, the term "initiation codon", used hangeably with the term "start codon", refers to the first codon of an open reading frame that is translated by the ribosome and is comprised of a triplet of linked adenine-uracil-guanine nucleobases. The initiation codon is depicted by the ?rst letter codes of adenine (A), uracil (U), and guanine (G) and is often written simply as "AUG". Although natural mRNAs may use codons other than AUG as the initiation codon, which are referred to herein as "alternative initiation codons", the initiation codons of polynucleotides described herein use the AUG codon. During the process of translation initiation, the sequence sing the initiation codon is recognized via complementary base-pairing to the anticodon of an initiator tRNA (Met-tRNAiMet) bound by the ribosome. Open reading frames may contain more than one AUG initiation codon, which are referred to herein as "alternate initiation codons".

The initiation codon plays a critical role in ation initiation. The initiation codon is the ?rst codon of an open reading frame that is translated by the ribosome. Typically, the initiation codon comprises the nucleotide triplet AUG, however, in some instances translation tion can occur at other codons comprised of distinct nucleotides. The initiation of ation in otes is a multistep biochemical process that involves numerous protein- protein, protein-RNA, and RNA-RNA interactions between messenger RNA molecules (mRNAs), the 408 mal subunit, other components of the translation machinery (e.g., eukaryotic initiation factors, elFs). The current model of mRNA translation initiation postulates that the pre-initiation compleX natively "43S itiation complex", abbreviated as "PIC") translocates from the site of recruitment on the mRNA (typically the 5’ cap) to the initiation codon by ng nucleotides in a 5' to 3' direction until the ?rst AUG codon that resides within a speci?c translation-promotive nucleotide context (the Kozak sequence) is encountered (Kozak (1989) J Cell Biol 9-241). Scanning by the PIC ends upon complementary base-pairing between nucleotides comprising the anticodon of the initiator Met-tRNAiMet transfer RNA and nucleotides comprising the initiation codon of the mRNA. tive base-pairing between the AUG codon and the Met-tRNAiMet anticodon elicits a series of structural and biochemical events that ate in the joining of the large 60S ribosomal subunit to the PIC to form an active ribosome that is competent for translation elongation.

Kozak Sequence: The term "Kozak sequence" (also referred to as "Kozak consensus sequence") refers to a translation initiation enhancer t to enhance expression of a gene or open g frame, and which in eukaryotes, is located in the 5’ UTR. The Kozak consensus sequence was originally de?ned as the sequence GCCRCC where R = a purine, following an analysis of the effects of single mutations surrounding the initiation codon (AUG) on translation of the preproinsulin gene (Kozak (1986) Cell 44:283-292).

Polynucleotides disclosed herein comprise a Kozak sus sequence, or a derivative or modi?cation thereof. (Examples of translational enhancer compositions and methods of use thereof, see US. Pat. No. 5,807,707 to Andrews et al., incorporated herein by reference in its entirety, US. Pat. No. 5,723,332 to Chemajovsky, incorporated herein by reference in its entirety, US. Pat. No. 665 to Wilson, incorporated herein by reference in its ty.) Leaky scanning: A phenomenon known as "leaky scanning" can occur whereby the PIC bypasses the initiation codon and d continues scanning downstream until an alternate or alternative initiation codon is recognized. Depending on the frequency of occurrence, the bypass of the initiation codon by the PIC can result in a decrease in translation ef?ciency. Furthermore, translation from this ream AUG codon can occur, which will result in the production of an undesired, aberrant translation product that may not be capable of eliciting the desired therapeutic response. In some cases, the nt translation product may in fact cause a deleterious response t et al, (2017) Nat Med 23(4):501—507).

Modified: As used herein "modi?ed" or "modi?cation" refers to a changed state or a change in composition or structure of a polynucleotide (e.g., mRNA). cleotides may be modi?ed in various ways including chemically, structurally, and/or functionally. For example, cleotides may be structurally modi?ed by the incorporation of one or more RNA elements, wherein the RNA element comprises a sequence and/or an RNA secondary structure(s) that provides one or more functions (e.g., translational regulatory activity).

Accordingly, polynucleotides of the disclosure may be comprised of one or more modi?cations (e.g., may include one or more al, structural, or functional modi?cations, including any combination thereof).

Nucleobase: As used herein, the term "nucleobase" (alternatively "nucleotide base" or "nitrogenous base") refers to a purine or dine heterocyclic compound found in nucleic acids, including any derivatives or analogs of the naturally occurring purines and pyrimidines that confer improved properties (e.g., binding y, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the nucleobases predominately found in natural nucleic acids. Other natural, non- natural, and/or synthetic nucleobases, as known in the art and/or described herein, can be incorporated into nucleic acids. side/Nucleolide: As used herein, the term oside" refers to a compound containing a sugar molecule (e.g., a ribose in RNA or a deoxyribose in DNA), or tive or analog f, ntly linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as "nucleobase"), but g an internucleoside linking group (e.g., a phosphate group). As used herein, the term otide" refers to a nucleoside covalently bonded to an ucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modi?cation thereof that confers improved chemical and/or functional properties (e.g., binding af?nity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

Nucleic acid: As used herein, the term "nucleic acid" is used in its broadest sense and encompasses any compound and/or sub stance that includes a polymer of nucleotides, or derivatives or analogs thereof. These polymers are often referred to as "polynucleotides".

Accordingly, as used herein the terms "nucleic acid" and "polynucleotide" are equivalent and are used interchangeably. Exemplary nucleic acids or polynucleotides of the disclosure e, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, mRNAs, modi?ed mRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, ing LNA having a bo con?guration, d-LNA having an d-L-ribo con?guration (a diastereomer of LNA), 2'—amino- LNA having a no functionalization, and 2'—amino-0t-LNA having a 2'-amino functionalization) or hybrids thereof.

Nucleic Acid Structure: As used herein, the term "nucleic acid structure" (used hangeably with "polynucleotide structure") refers to the arrangement or organization of atoms, chemical constituents, elements, motifs, and/or sequence of linked nucleotides, or tives or analogs thereof, that comprise a nucleic acid (e.g., an mRNA). The term also refers to the two-dimensional or three-dimensional state of a nucleic acid. Accordingly, the term "RNA structure" refers to the arrangement or organization of atoms, chemical tuents, elements, motifs, and/or sequence of linked tides, or derivatives or analogs thereof, comprising an RNA molecule (e.g., an mRNA) and/or refers to a two- dimensional and/or three dimensional state of an RNA molecule. Nucleic acid structure can be further demarcated into four organizational categories ed to herein as "molecular structure77 (L (4 , primary structure77 , secondary ure", and "tertiary structure" based on increasing organizational complexity.

Open Reading Frame: As used herein, the term "open reading frame", abbreviated as "ORF", refers to a segment or region of an mRNA molecule that encodes a polypeptide. The ORF ses a continuous stretch of erlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

[Dre-Initiation Complex (PIC): As used herein, the term "pre-initiation complex" (alternatively "43S pre-initiation complex", abbreviated as "PIC") refers to a ribonucleoprotein complex comprising a 408 ribosomal subunit, eukaryotic initiation factors (elFl, elFlA, e1F3, eIFS), and the TP-Met-tRNAiMet ternary complex, that is intrinsically capable of ment to the 5’ cap of an mRNA molecule and, after attachment, of performing ribosome scanning of the 5’ UTR.

RNA element: As used herein, the term "RNA element" refers to a n, fragment, or segment of an RNA molecule that provides a biological function and/or has biological activity (e.g., translational regulatory activity). Modification of a polynucleotide by the incorporation of one or more RNA elements, such as those described herein, provides one or more desirable onal properties to the modi?ed polynucleotide. RNA elements, as described herein, can be naturally-occurring, non-naturally occurring, synthetic, engineered, or any combination thereof. For e, naturally-occurring RNA elements that provide a regulatory activity include ts found throughout the transcriptomes of viruses, prokaryotic and eukaryotic organisms (e.g., humans). RNA elements in particular eukaryotic mRNAs and translated viral RNAs have been shown to be ed in mediating many functions in cells. ary natural RNA elements include, but are not limited to, translation initiation elements (e.g., internal ribosome entry site (IRES), see Kieft et al., (2001) RNA 7(2): 194-206), translation enhancer elements (e.g., the APP mRNA translation enhancer element, see Rogers et al., (1999) J Biol Chem ):6421-6431), mRNA stability elements (e.g., AU-rich elements (AREs), see Garneau et al, (2007) Nat Rev Mol Cell Biol 8(2): 1 13-126), ational repression element (see e.g., Blumer et al., (2002) Mech Dev 110(1-2):97-112), protein-binding RNA elements (e.g., iron-responsive element, see Selezneva et al, (2013) J Mol Biol 425(18):3301-3310), asmic polyadenylation elements (Villalba et al, (2011) Curr Opin Genet Dev 21(4):452-457), and catalytic RNA elements (e.g., ribozymes, see Scott et al, (2009) Biochim s Acta 1789(9-10):634- 641).

Residence time: As used herein, the term "residence time" refers to the time of occupancy of a pre-initiation complex (PIC) or a ribosome at a discrete position or location along an mRNA molecule.

Translational Regulatory Activity: As used herein, the term "translational regulatory activity" (used interchangeably with "translational regulatory function") refers to a biological function, mechanism, or process that modulates (e.g., regulates, in?uences, controls, varies) the activity of the translational apparatus, including the activity of the PIC and/or ribosome.

In some aspects, the d translation regulatory activity promotes and/or enhances the translational ?delity of mRNA translation. In some s, the desired translational regulatory activity reduces and/or inhibits leaky scanning.

Translation of a polynucleotide comprising an open reading frame encoding a polypeptide can be controlled and ted by a variety of mechanisms that are provided by various cis-acting c acid structures. For example, naturally-occurring, cis-acting RNA elements that form hairpins or other higher-order (e.g., pseudoknot) intramolecular mRNA secondary structures can provide a translational regulatory activity to a polynucleotide, wherein the RNA element in?uences or modulates the initiation of polynucleotide translation, particularly when the RNA t is positioned in the 5' UTR close to the 5’-cap structure (Pelletier and Sonenberg (1985) Cell 40(3):515-526, Kozak (1986) Proc Natl Acad Sci 83:2850-2854). Cis-acting RNA ts can also affect translation tion, being involved in numerous frameshifting events (Namy et al, (2004) Mol Cell 13(2): 157-168). al ribosome entry sequences (IRES) represent r type of cis-acting RNA element that are typically located in 5' UTRs, but have also been ed to be found within the coding region of naturally-occurring mRNAs (Holcik et al (2000) Trends Genet 16(10):469- 473). In cellular mRNAs, IRES often coexist with the 5'-cap structure and provide mRNAs with the functional capacity to be translated under conditions in which cap-dependent translation is compromised (Gebauer et al., (2012) Cold Spring Harb Perspect Biol 4(7):a012245). Another type of naturally-occurring cis-acting RNA element comprises upstream open reading frames (uORFs). lly-occurring uORFs occur singularly or multiply within the 5' UTRs of numerous mRNAs and in?uence the translation of the downstream major ORF, usually negatively (with the notable exception of GCN4 mRNA in yeast and ATF4 mRNA in mammals, where uORFs serve to promote the translation of the downstream major ORF under ions of increased eIF2 phosphorylation busch (2005) Annu Rev Microbiol 59:407-450)). onal exemplary translational regulatory activities provided by components, structures, elements, motifs, and/or speci?c sequences comprising cleotides (e.g., mRNA) include, but are not limited to, mRNA stabilization or destabilization (Baker & Parker (2004) Curr Opin Cell Biol l6(3):293-299), translational activation (Villalba et al., (201 1) Curr Opin Genet Dev 2l(4):452-457), and translational repression (Blumer et al, (2002) Mech Dev llO(l-2):97-l 12). Studies have shown that lly-occurring, ting RNA elements can confer their tive functions when used to modify, by incorporation into, heterologous polynucleotides (Goldberg-Cohen et al, (2002) J Biol Chem 277(16): 13635-13640). d Polynucleotides Comprising Functional RNA Elements The present disclosure provides synthetic polynucleotides comprising a modi?cation (e.g., an RNA element), wherein the modi?cation provides a desired translational regulatory activity. In some embodiments, the disclosure provides a polynucleotide comprising a 5’ untranslated region (UTR), an initiation codon, a full open reading frame encoding a polypeptide, a 3’ UTR, and at least one modi?cation, wherein the at least one modi?cation provides a desired translational regulatory activity, for example, a modi?cation that promotes and/or enhances the translational ?delity of mRNA translation. In some embodiments, the d ational regulatory activity is a cis-acting regulatory activity. In some embodiments, the d translational regulatory activity is an increase in the residence time of the 43S pre-initiation complex (PIC) or ribosome at, or al to, the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some embodiments, the desired ational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some ments, the desired translational regulatory activity is an increase in the y of initiation codon decoding by the PIC or me. In some embodiments, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or me. In some embodiments, the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome. In some embodiments, the desired translational tory activity is inhibition or reduction in the initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon. In some embodiments, the desired ational regulatory ty is inhibition or reduction of the amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame. In some embodiments, the desired translational tory activity is inhibition or reduction in the production of nt translation products. In some embodiments, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.

Accordingly, the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA t that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity as bed herein. In some aspects, the mRNA comprises an RNA element that comprises a sequence and/or an RNA ary ure(s) that es and/or enhances the translational ?delity of mRNA translation. In some aspects, the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a d translational regulatory activity, such as inhibiting and/or reducing leaky scanning. In some aspects, the disclosure provides an mRNA that comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that inhibits and/or reduces leaky scanning thereby promoting the translational ?delity of the mRNA.

In some embodiments, the RNA element comprises natural and/or modi?ed nucleotides. In some embodiments, the RNA element comprises of a ce of linked nucleotides, or derivatives or analogs thereof, that provides a desired translational regulatory activity as described herein. In some embodiments, the RNA element comprises a sequence of linked nucleotides, or derivatives or analogs thereof, that forms or folds into a stable RNA secondary structure, wherein the RNA secondary structure provides a d translational regulatory activity as described herein. RNA elements can be ?ed and/or characterized based on the primary sequence of the element (e.g., GC-rich element), by RNA secondary structure formed by the element (e.g. stem-loop), by the location of the element within the RNA molecule (e.g., located within the 5’ UTR of an mRNA), by the biological on and/or activity of the element (e.g., "translational enhancer element"), and any combination thereof.

In some aspects, the disclosure es an mRNA having one or more structural modi?cations that ts leaky scanning and/or promotes the translational ?delity of mRNA translation, wherein at least one of the structural modi?cations is a GC-rich RNA element. In some aspects, the disclosure provides a modi?ed mRNA comprising at least one modi?cation, n at least one modi?cation is a GC-rich RNA element comprising a ce of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA. In one embodiment, the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus ce in the 5’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located 15-30, 15-20, 15- , 10-15, or 5-10 tides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus ce in the 5’ UTR of the mRNA.

In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 3-3 0, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or s thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%- 60% cytosine, 40-50% cytosine, 30-40% cytosine bases. In any of the foregoing or related aspects, the disclosure es a GC-rich RNA element which comprises a sequence of 3- 30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

In any of the foregoing or related aspects, the sure provides a GC-rich RNA element which comprises a sequence of20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or s thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, or 30-40% cytosine. In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

In some embodiments, the disclosure provides a modi?ed mRNA sing at least one modi?cation, wherein at least one modi?cation is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is >50% cytosine. In some embodiments, the ce composition is >55% cytosine, >60% ne, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.

In other aspects, the disclosure provides a modi?ed mRNA comprising at least one modi?cation, wherein at least one modi?cation is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus ce in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of about 3-30, 5-25, 10-20, 15-20 or about 20, about 15, about 12, about 10, about 6 or about 3 nucleotides, or tives or analogues thereof, wherein the ce comprises a repeating GC-motif, wherein the repeating GC-motifis [CCG]n, wherein n = 1 to 10, 11: 2 to 8, n= 3 to 6, or 11: 4 to 5. In 2O some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1, 2, 3, 4 or 5. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1, 2, or 3. In some embodiments, the sequence comprises a repeating GC-motif , wherein n = 1. In some ments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 2. In some embodiments, the sequence ses a repeating GC-motif , wherein n = 3. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 4 (SEQ ID NO: 308). In some ments, the sequence ses a repeating GC-motif [CCG]n, wherein n = 5 (SEQ ID NO: 309).

In another aspect, the disclosure provides a modi?ed mRNA sing at least one modi?cation, wherein at least one modi?cation is a GC-rich RNA element comprising a sequence of linked tides, or tives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element comprises any one of the ces set forth in TABLE 2. In one embodiment, the GC-rich RNA t is d about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is d about 15-30, 15- , 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.

In other aspects, the disclosure provides a modi?ed mRNA comprising at least one modi?cation, wherein at least one modi?cation is a GC-rich RNA element comprising the sequence Vl [CCCCGGCGCC] (SEQ ID NO: 310) as set forth in TABLE 2, or tives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence Vl as set forth in TABLE 2 located immediately adjacent to and upstream of the Kozak sus sequence in the 5’ UTR of the mRNA. In some ments, the GC-rich element ses the sequence Vl as set forth in TABLE 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream ofthe Kozak consensus sequence in the 5’ UTR of the mRNA. In other ments, the GC-rich element comprises the sequence Vl as set forth in TABLE 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

In other aspects, the sure provides a modi?ed mRNA comprising at least one ation, wherein at least one modi?cation is a GC-rich RNA element comprising the sequence V2 [CCCCGGC] as set forth in TABLE 2, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, 2O the h element comprises the sequence V2 as set forth in TABLE 2 located immediately adjacent to and upstream of the Kozak sus sequence in the 5’ UTR of the mRNA. In some ments, the GC-rich element comprises the ce V2 as set forth in TABLE 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the ’ UTR of the mRNA. In other embodiments, the GC-rich element ses the sequence V2 as set forth in TABLE 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak sus sequence in the 5’ UTR of the mRNA.

In other aspects, the disclosure provides a modi?ed mRNA comprising at least one modi?cation, wherein at least one modi?cation is a GC-rich RNA element comprising the sequence EK [GCCGCC] as set forth in TABLE 2, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence EK as set forth in TABLE 2 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence EK as set forth in TABLE 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence EK as set forth in TABLE 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

In yet other aspects, the disclosure provides a modi?ed mRNA comprising at least one modi?cation, wherein at least one modi?cation is a GC-rich RNA element sing the sequence V1 [CCCCGGCGCC] (SEQ ID NO: 310) as set forth in TABLE 2, or derivatives or analogs thereof, preceding a Kozak sus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR ses the following sequence shown in TABLE 2: TAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA (SEQ ID NO: 311).

In some embodiments, the GC-rich element comprises the sequence V1 as set forth in TABLE 2 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR sequence shown in TABLE 2. In some embodiments, the GC-rich element comprises the sequence V1 as set forth in TABLE 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases am of the Kozak sus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in TABLE 2: GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA (SEQ ID NO: 312).

In other ments, the GC-rich element comprises the sequence V1 as set forth in Table 1 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream ofthe Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence 2O shown in TABLE 2: GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA (SEQ ID NO: 312).

In some embodiments, the 5’ UTR comprises the following sequence set forth in TABLE 2: GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGC CACC (SEQ ID NO: 313) TABLE 2 SEQ ID GGGAAATAAGAGAGAAAAGAAGAGTAAG 3 14 Standard AAGAAATATAAGAGCCACC GGGAAATAAGAGAGAAAAGAAGAGTAAG 3 13 AAGAAATATAAGACCCCGGCGCCGCCACC SEQ ID GGGAAATAAGAGAGAAAAGAAGAGTAAG 3 l5 V2-UTR AAGAAATATAAGACCCCGGCGCCACC KO (Traditional Kozak [GCCA/GCC] consensus) In another aspect, the disclosure provides a modi?ed mRNA comprising at least one modi?cation, wherein at least one modi?cation is a GC-rich RNA element comprising a stable RNA secondary structure sing a sequence of nucleotides, or derivatives or analogs thereof, linked in an order which forms a hairpin or a stem-loop. In one ment, the stable RNA secondary structure is upstream of the Kozak consensus ce. In another embodiment, the stable RNA secondary structure is located about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 20, about 15, about 10 or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 5, about 4, about 3, about 2, about 1 nucleotides upstream of the Kozak consensus sequence. In another ment, the stable RNA secondary structure is d about l5-30, about l5-20, about l5-25, about -15, or about 5-10 nucleotides upstream of the Kozak consensus sequence. In another ment, the stable RNA secondary structure is located 12-15 tides upstream of the Kozak consensus sequence. In another ment, the stable RNA secondary structure has a deltaG of about -30 kcal/mol, about -20 to -30 kcal/mol, about -20 kcal/mol, about -10 to -20 kcal/mol, about -10 kcal/mol, about -5 to -10 kcal/mol.

In another embodiment, the modi?cation is operably linked to an open reading frame encoding a polypeptide and wherein the modi?cation and the open reading frame are heterologous.

In another embodiment, the sequence of the GC-rich RNA element is comprised ively of e (G) and cytosine (C) nucleobases.

RNA elements that provide a desired translational regulatory activity as described herein can be identi?ed and characterized using known techniques, such as ribosome pro?ling . Ribosome pro?ling is a que that allows the determination of the positions of PICs and/or ribosomes bound to mRNAs (see e.g., a et al., (2009) Science 324(5924):218-23, incorporated herein by reference). The technique is based on protecting a region or segment of mRNA, by the PIC and/or ribosome, from nuclease digestion.

Protection results in the generation of a 30-bp fragment ofRNA termed a ‘footprint’. The sequence and frequency ofRNA footprints can be analyzed by methods known in the art (e.g., RNA-seq). The footprint is roughly ed on the A-site of the ribosome. If the PIC or ribosome dwells at a particular position or location along an mRNA, footprints generated at these position would be relatively common. Studies have shown that more footprints are generated at ons where the PIC and/or ribosome eXhibits decreased processivity and fewer footprints where the PIC and/or me eXhibits sed processivity (Gardin et al, (2014) eLife 3:e03735). In some embodiments, residence time or the time of occupancy of a the PIC or ribosome at a discrete position or on along an polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome pro?ling.

Methods of Treatment Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention and/or treatment of cancer in humans and other mammals. Cancer RNA vaccines can be used as eutic or prophylactic agents. They may be used in medicine to prevent and/or treat cancer. In exemplary aspects, the cancer RNA vaccines of the present disclosure are used to provide prophylactic tion from cancer. Prophylactic protection from cancer can be achieved following administration of a cancer RNA vaccine of the present disclosure. es can be administered once, twice, three times, four times or more but it is likely suf?cient to administer the vaccine once (optionally followed by a single booster). It is more desirable, to administer the vaccine to an individual having cancer to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

Once an mRNA vaccine is sized, it is administered to the t. In some embodiments the vaccine is stered on a schedule for up to two months, up to three months, up to four month, up to ?ve months, up to siX months, up to seven months, up to eight months, up to nine months, up to ten months, up to eleven months, up to 1 year, up to l and 1/2 years, up to two years, up to three years, or up to four years. The schedule may be the same or varied. In some embodiments the schedule is weekly for the ?rst 3 weeks and then monthly thereafter.

The vaccine may be administered by any route. In some ments the vaccine is administered by an HVI or IV route.

At any point in the treatment the patient may be examined to determine whether the mutations in the vaccine are still appropriate. Based on that analysis the vaccine may be adjusted or recon?gured to include one or more different mutations or to remove one or more mutations.

Therapeutic phylactic Compositions Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention, treatment or diagnosis of cancer in humans and other mammals, For example, cancer RNA vaccines can be used as therapeutic or lactic agents. They may be used in medicine to prevent and/or treat cancer. In some embodiments, the cancer es of the invention can be envisioned for use in the priming of immune effector cells, for example, to te peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

In exemplary ments, a cancer vaccine containing RNA polynucleotides as described herein can be administered to a subject (e.g., a mammalian t, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide.

The cancer RNA vaccines may be d for ation of a polypeptide (e.g., antigen or immunogen) in a cell, tissue or organism. In exemplary embodiments, such translation occurs in vivo, gh there can be envisioned embodiments where such translation occurs ex vivo, in culture or in vitro. In exemplary embodiments, the cell, tissue or sm is contacted with an effective amount of a composition containing a cancer RNA vaccine that contains a polynucleotide that has at least one a translatable region encoding an antigenic polypeptide.

An "effective " of a cancer RNA vaccine is provided based, at least in part, on the target , target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the cancer RNA vaccine, and other determinants. In general, an effective amount of the cancer RNA vaccine composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more ef?cient than a composition containing a ponding unmodi?ed cleotide encoding the same antigen or a peptide antigen.

Increased n production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation from the polynucleotide, sed nucleic acid degradation (as demonstrated, for e, by increased duration of protein translation from a modi?ed polynucleotide), or altered antigen speci?c immune response of the host cell.

In some embodiments, RNA vaccines (including polynucleotides their encoded ptides) in accordance with the present disclosure may be used for treatment of cancer.

Cancer RNA vaccines may be stered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in cancer or during active cancer after onset of symptoms. In some embodiments, the amount ofRNA vaccines of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

Cancer RNA es may be stered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an immune potentiator, adjuvant, or booster. As used herein, when referring to a composition, such as a vaccine, the term er" refers to an extra administration of the lactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier 2O administration of the prophylactic ition. The time of administration between the initial administration of the lactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 s, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years. In exemplary embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In one embodiment, the polynucleotides may be administered intramuscularly or intradermally rly to the administration of vaccines known in the art.

The mRNA cancer vaccines may be utilized in various settings depending on the severity of the cancer or the degree or level of unmet medical need. As a miting example, the mRNA cancer vaccines may be ed to treat any stage of cancer. The mRNA cancer vaccines have superior properties in that they produce much larger antibody titers, T cell responses and e responses early than commercially available anti-cancer vaccines.

While not wishing to be bound by theory, the inventors hypothesize that the mRNA cancer vaccines, as mRNAs, are better designed to e the appropriate protein conformation on translation as the mRNA cancer vaccines co-opt natural cellular machinery. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the mRNA cancer vaccines are presented to the ar system in a more native fashion.

A non-limiting list of cancers that the mRNA cancer vaccines may treat is presented below. Peptide epitopes or antigens may be derived from any antigen of these cancers or tumors. Such epitopes are referred to as cancer or tumor antigens. Cancer cells may differentially express cell surface les during different phases of tumor progression. For 2O example, a cancer cell may express a cell e antigen in a benign state, yet down-regulate that particular cell surface antigen upon metastasis. As such, it is envisioned that the tumor or cancer antigen may encompass antigens ed during any stage of cancer progression.

The methods of the invention may be ed to accommodate for these changes. For instance, several ent mRNA vaccines may be generated for a particular patient. For instance a ?rst vaccine may be used at the start of the treatment. At a later time point, a new mRNA e may be generated and administered to the patient to t for different ns being expressed.

In some embodiments, the tumor antigen is one of the following antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4— BB, 5T4, AGS-S , AGS-16, Angiopoietin 2, B71, B72, B7DC, B7H1, B7H2, B7H3, BT-O62, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGFlR, Integrin av, Integrin owl} , LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUCl, MUCl6, Nectin-4, NKGDZ, NOTCH, 0X40, OX4OL, PD-l, PDLl, PSCA, PSMA, RANKL, RORl, RORZ, SLC44A4, Syndecan-l, TACI, TAG-72, Tenascin, TlM3, TRAILRl 1 , 2,VEGFR- , VEGFR—Z, VEGFR-3, and variants thereof.

Cancers or tumors include but are not limited to neoplasms, malignant tumors, metastases, or any e or disorder characterized by uncontrolled cell growth such that it would be considered cancerous. The cancer may be a primary or metastatic cancer. Speci?c cancers that can be treated according to the present invention include, but are not limited to, those listed below (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed, J.B. Lippincott Co., Philadelphia). Cancers include, but are not limited to, biliary tract cancer, bladder cancer, brain cancer including glioblastomas and medulloblastomas, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms including acute cytic and myelogenous leukemia, multiple myeloma, AIDS-associated leukemias and adult T-cell leukemia lymphoma, intraepithelial neoplasms including Bowen’s disease and Paget’s disease, liver cancer, lung cancer, lymphomas including Hodgkin’s disease and cytic mas, neuroblastomas, oral cancer including squamous cell carcinoma, ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, as including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, ?brosarcoma, and osteosarcoma, skin cancer including melanoma, Kaposi’s sarcoma, basocellular cancer, and squamous cell cancer, testicular cancer ing germinal tumors such as seminoma, non-seminoma, teratomas, choriocarcinomas, stromal tumors and germ cell tumors, d cancer including thyroid adenocarcinoma and medullar carcinoma, and renal cancer including adenocarcinoma and Wilms’ tumor. Commonly encountered cancers include , prostate, lung, ovarian, colorectal, and brain cancer.

In some embodiments, the cancer is selected from the group consisting of all cell lung cancer (NSCLC), small cell lung cancer, ma, bladder urothelial carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), and a solid ancy that is microsatellite high (MSI H) / mismatch repair (MMR) de?cient. In some embodiments, the NSCLC lacks an EGFR sensitizing mutation and/or an ALK translocation.

In some ments, the solid ancy that is microsatellite high (MSI H) / mismatch repair (MIVIR) nt is ed from the group consisting of ctal cancer, stomach adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from cancer of the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, genital tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues. In some embodiments, the cancer is colorectal cancer.

Provided herein are pharmaceutical compositions including cancer RNA vaccines and RNA vaccine compositions and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

Cancer RNA vaccines may be ated or administered alone or in conjunction with one or more other ents. For instance, cancer RNA vaccines (vaccine itions) may comprise other components ing, but not limited to, immune potentiators (e.g., adjuvants). In some ments, cancer RNA vaccines do not include an immune potentiator or adjuvant (1'.e., they are immune potentiator or adjuvant free).

In other embodiments the mRNA cancer vaccines described herein may be combined with any other therapy useful for treating the patient. For instance a patient may be treated with the mRNA cancer vaccine and an anti-cancer agent. Thus, in one embodiment, the methods of the invention can be used in conjunction with one or more cancer therapeutics, for example, in conjunction with an anti-cancer agent, a traditional cancer vaccine, chemotherapy, radiotherapy, etc. (e.g., simultaneously, or as part of an overall treatment procedure). Parameters of cancer treatment that may vary e, but are not limited to, dosages, timing of administration or duration or therapy, and the cancer treatment can vary in , timing, or duration. Another treatment for cancer is y, which can be utilized either alone or in combination with any of the us treatment methods. Any agent or therapy (e.g., traditional cancer vaccines, herapies, radiation therapies, surgery, hormonal ies, and/or ical therapies/immunotherapies) which is known to be useful, or which has been used or is currently being used for the prevention or treatment of cancer can be used in ation with a composition of the invention in accordance with the invention described herein. One of ordinary skill in the medical arts can determine an appropriate treatment for a t.

Examples of such agents (i.e., anti-cancer agents) include, but are not limited to, DNA-interactive agents including, but not limited to, the alkylating agents (e.g., nitrogen mustards, e.g. Chlorambucil, Cyclophosphamide, Isofamide, Mechlorethamine, Melphalan, Uracil mustard, Aziridine such as pa, methanesulphonate esters such as Busulfan, nitroso ureas, such as Carmustine, Lomustine, Streptozocin, platinum complexes, such as Cisplatin, Carboplatin, bioreductive alkylator, such as Mitomycin, and Procarbazine, Dacarbazine and Altretamine), the DNA strand-breakage agents, e.g., Bleomycin, the intercalating omerase II tors, e.g., Intercalators, such as Amsacrine, Dactinomycin, ubicin, Doxorubicin, Idarubicin, Mitoxantrone, and nonintercalators, such as Etoposide and Teniposide, the nonintercalating topoisomerase II inhibitors, e.g., Etoposide and sde, and the DNA minor groove binder, e.g., Plicamydin, the antimetabolites including, but not limited to, folate nists such as Methotrexate and trimetrexate, pyrimidine antagonists, such as Fluorouracil, Fluorodeoxyuridine, CB3717, Azacitidine and Floxuridine, purine antagonists such as Mercaptopurine, guanine, Pentostatin, sugar modi?ed analogs such as Cytarabine and Fludarabine, and ribonucleotide reductase inhibitors such as hydroxyurea, tubulin Interactive agents including, but not limited to, cine, Vincristine and Vinblastine, both alkaloids and axel and cytoxan, hormonal agents including, but not limited to, estrogens, conjugated estrogens and Ethinyl Estradiol and Diethylstilbesterol, Chlortrianisen and Idenestrol, tins such as Hydroxyprogesterone caproate, Medroxyprogesterone, and rol, and androgens such as testosterone, testosterone propionate, ?uoxymesterone, methyltestosterone, adrenal corticosteroid, e.g., Prednisone, Dexamethasone, prednisolone, and Prednisolone, leutinizing hormone releasing hormone agents or gonadotropin-releasing e antagonists, e.g., leuprolide acetate and goserelin acetate, antihormonal antigens including, but not limited to, antiestrogenic agents such as Tamoxifen, antiandrogen agents such as Flutamide, and antiadrenal agents such as Mitotane and lutethimide, cytokines including, but not limited to, IL-lalpha, IL-1 [3, IL-2, 1L-3, 1L-4, IL-5, IL-6, 1L-7, 1L-8, 1L- 9, 1L-10,1L-11, 1L-12, IL-13, IL-18, TGF-B, GM-CSF, M-CSF, G—CSF, TNF-d, TNF-B, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, lFN-d, lFN-B, lFN-.y, and Uteroglobins (US. Pat. No. 5,696,092), anti-angiogenics including, but not limited to, agents that inhibit VEGF (e.g., other neutralizing antibodies), soluble receptor constructs, tyrosine kinase inhibitors, antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptors, Immunotoxins and coaguligands, tumor vaccines, and antibodies.

Speci?c es of anti-cancer agents which can be used in accordance with the methods of the invention include, but not limited to: acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, tamide, bisantrene hydrochloride, bisna?de dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, omycin, calusterone, caracemide, carbetimer, carboplatin, carmustine; carubicin hydrochloride; carzelesin; cedeflngol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; xel; bicin; doxorubicin hydrochloride; ifene; ifene citrate; dromostanolone propionate; duazomycin; xate; e?omithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hloride; fazarabine; fenretinide; dine; bine phosphate; ?uorouracil; ?urocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin 11 (including recombinant interieukin II; or r1LZ), interferon alpha-2a; interferon alpha-2b; interferon nl; interferon alpha-n3; interferon beta-I a; interferon I b; iproplatin; irinotecan hloride; lanreotide e; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; rethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; cin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porf1mer sodium; omycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; ol; saflngol hydrochloride; semustine; zene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporf1n; side; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; zamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate onate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfm; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; dine sulfate; Vinglycinate sulfate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrosidine sulfate; Vinzolidine sulfate; vorozole; zeniplatin; atin; and cin hydrochloride.

Other anti-cancer drugs include; but are not limited to: 20-epi-l;25 dihydroxyyitamin D3; 5-ethynyluracil; angiogenesis inhibitors; anti-dorsalizing morphogenetic protein-1; ara- CDP-DL-PTBA; BCR/ABL antagonists; CaRest M3; CARN 700; casein kinase inhibitors (ICOS); clotrimazole; collismycin A; collismycin B; combretastatin A4; crambescidin 816; cryptophycin 8; curacin A; odidemnin B; didemnin B; dihydroazacytidine; dihydrotaxol; duocarmycin SA; kahalalide F; lamellarin-N triacetate; leuprolide+estrogen+progesterone; lissoclinamide 7; monophosphoryl lipid A+myobacterium cell wall sk; N—acetyldinaline; N—substituted benzamides; O6-benzylguanine; placetin A; placetin B; um complex; platinum compounds; platinum-triamine complex; rhenium Re 186 etidronate; RII retinamide; rubiginone B 1; SarCNU; sarcophytol A; sargramostim; senescence derived inhibitor 1; spicamycin D; tallimustine; 5-?uorouracil; thrombopoietin; thymotrinan; thyroid ating hormone; variolin B; thalidomide; velaresol; veramine; verdins; orf1n; vinorelbine; vinxaltine; vitaxin; zanoterone; zeniplatin; and zilascorb.

The invention also encompasses administration of a composition comprising a mRNA cancer e in combination with radiation therapy comprising the use of x-rays; gamma rays and other sources of radiation to destroy the cancer cells. In preferred embodiments; the radiation treatment is administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In other preferred embodiments; the radiation treatment is administered as internal y or brachytherapy n a radioactive source is placed inside the body close to cancer cells or a tumor mass.

In speci?c embodiments; an appropriate anti-cancer regimen is selected depending on 2O the type of cancer. For instance; a patient with ovarian cancer may be administered a prophylactically or therapeutically effective amount of a composition comprising a mRNA cancer vaccine in combination with a prophylactically or therapeutically ive amount of one or more other agents useful for ovarian cancer therapy; including but not limited to; intraperitoneal radiation therapy; such as P32 therapy; total abdominal and pelvic radiation therapy; cisplatin; the combination of paclitaxel (Taxol) or docetaxel (Taxotere) and cisplatin or carboplatin; the ation of cyclophosphamide and cisplatin; the combination of cyclophosphamide and carboplatin; the ation of 5-FU and leucovorin; etoposide; liposomal doxorubicin; gemcitabine or can. Cancer therapies and their s; routes of administration and recommended usage are known in the art and have been described in such literature as the Physician's Desk Reference (56th ed.; 2002).

In some preferred ments of the invention the mRNA cancer vaccines are stered with a T cell activator such as be an immune checkpoint modulator. Immune checkpoint modulators include both stimulatory checkpoint molecules and inhibitory checkpoint molecules 1'.e.; an anti-CTLA4 and anti-PDl antibody.

Stimulatory checkpoint inhibitors function by promoting the checkpoint process. l stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily - CD27, CD40, 0X40, GITR and CD137, while others belong to the B7-CD28 superfamily - CD28 and ICOS. 0X40 (CD134), is involved in the expansion of effector and memory T cells. Anti-0X40 monoclonal antibodies have been shown to be effective in treating advanced cancer. MED10562 is a humanized 0X40 agonist. GITR, Glucocorticoid-Induced TNFR family Related gene, is involved in T cell expansion Several antibodies to GITR have been shown to promote an anti-tumor responses. ICOS, Inducible T- cell costimulator, is important in T cell effector function. CD27 supports antigen-speci?c expansion of naive T cells and is involved in the generation of T and B cell memory. l agonistic anti-CD27 dies are in development. CD122 is the Interleukin-2 receptor beta sub-unit. NKTR-2l4 is a CD122-biased immune-stimulatory ne.

Inhibitory oint molecules include but are not limited to PD-l, T11V1-3, VISTA, AZAR, B7-H3, B7-H4, BTLA, , IDO, KIR and LAG3. CTLA-4, PD-l and its ligands are members of the CD28-B7 family of co-signaling molecules that play ant roles throughout all stages of T-cell on and other cell functions. CTLA-4, Cytotoxic T- Lymphocyte-Associated n 4 (CD152) is involved in controlling T cell proliferation.

The PD-l or is expressed on the surface of activated T cells (and B cells) and, under normal circumstances, binds to its ligands (PD-L1 and PD-L2) that are expressed on 2O the surface of antigen-presenting cells, such as dendritic cells or macrophages. This interaction sends a signal into the T cell and inhibits it. Cancer cells take advantage of this system by driving high levels of sion of PD-Ll on their surface. This allows them to gain control of the PD-l pathway and switch off T cells expressing PD-l that may enter the tumor microenvironment, thus suppressing the anticancer immune response. Pembrolizumab (formerly MK-3475 and lambrolizumab, trade name Keytruda) is a human antibody used in cancer immunotherapy. It targets the PD-l receptor.

IDO, amine 2,3-dioxygenase, is a tryptophan catabolic enzyme, which suppresses T and NK cells, generates and activates Tregs and myeloid-derived suppressor cells, and promotes tumor angiogenesis. T11V1-3, T-cell Immunoglobulin domain and Mucin domain 3, acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, V-domain Ig suppressor of T cell activation.

The oint inhibitor is a molecule such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein or a combination thereof or a small molecule. For instance, the checkpoint inhibitor inhibits a oint protein which may be CTLA-4,PDL1,PDL2, PD1,B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN—15049, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof. Ligands of oint proteins include but are not limited to CTLA-4, PDLI,PDL2,PD1,B7-H3,B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN—15049, CHK 1, CHK2, A2aR, and B-7 family ligands. In some embodiments the anti-PD-1 antibody is BMS-93 6558 (nivolumab). In other embodiments the anti-CTLA-4 antibody is ipilimumab (trade name Yervoy, ly known as MDX-010 and MDX-101).

In some preferred embodiments the cancer therapeutic , including the checkpoint modulators, are delivered in the form ofmRNA encoding the cancer therapeutic agents, e.g., anti-PDl, cytokines, chemokines or stimulatory receptors/ligands (e.g., 0X40.

In some embodiments the cancer therapeutic agent is a targeted y. The targeted therapy may be a BRAF inhibitor such as fenib (PLX4032) or dabrafenib. The BRAF inhibitor may be PLX 4032, PLX 4720, PLX 4734, GDC-0879, PLX 4032, PLX-4720, PLX 4734 and Sorafenib Tosylate. BRAF is a human gene that makes a protein called B-Raf, also referred to as oncogene B-Raf and v-Raf murine sarcoma viral oncogene g B1.

The B-Raf protein is involved in sending signals inside cells, which are involved in ing cell growth. Vemurafenib, a BRAF inhibitor, was approved by FDA for treatment of late- stage melanoma.

The T-cell therapeutic agent in other embodiments is OX40L. 0X40 is a member of the tumor is factor/nerve growth factor receptor (TNFR/NGFR) family. 0X40 may play a role in T-cell activation as well as regulation of differentiation, proliferation or apoptosis of normal and malignant lymphoid cells.

In one aspect, the methods of the invention further comprise administering a PD-1 antagonist to the subject. In some aspects, the PD-l antagonist is an antibody or an antigen- binding portion thereof that speci?cally binds to PD-l. In a particular aspect, the PD-l antagonist is a monoclonal dy. In some aspects, the PD-l antagonist is selected from the group consisting of Nivolumab, Pembrolizumab, Pidilizumab, and any combination thereof.

In another aspect, the methods of the ion r comprise administering a PDL- 1 antagonist to the subject. In some aspects, the PD-Ll antagonist is an antibody or an n-binding portion thereof that speci?cally binds to PD-Ll. In a particular aspect, the PD-Ll antagonist is a monoclonal antibody. In some aspects, the PD-Ll antagonist is selected from the group consisting of Durvalumab, ab, MEDI473, BMS-93 6559, Atezolizumab, and any combination thereof.

In r aspect, the methods of the invention further comprise administering a CTLA-4 antagonist to the subject. In some aspects, the CTLA-4 antagonist is an antibody or an antigen-binding portion thereof that speci?cally binds to CTLA-4. In a particular , the CTLA-4 antagonist is a monoclonal antibody. In some aspects, the CTLA-4 antagonist is selected from the group consisting of Ipilimumab, imumab, and any combination thereof. n embodiments of the invention provide for a method of treating cancer in a subject in need thereof comprising administering a polynucleotide, in particular, a mRNA encoding a KRAS vaccine peptide with one or more anti-cancer agents to the subject. In some embodiments, the one or more anti-cancer agents is a checkpoint inhibitor antibody or antibodies. In some embodiments, the one or more anti-cancer agents are an mRNA encoding a checkpoint inhibitor antibody or antibodies.

In one aspect, the subject has been previously treated with a PD-l antagonist prior to the polynucleotide of the present disclosure. In another aspect, the subject has been treated with a onal antibody that binds to PD-l prior to the polynucleotide of the present disclosure. In another aspect, the subject has been treated with an anti-PD-l monoclonal antibody therapy prior to the polynucleotide of the present methods. In other aspects, the anti- PD-l monoclonal antibody y ses Nivolumab, Pembrolizumab, Pidilizumab, or any combination thereof.

In another aspect, the subject has been treated with a monoclonal antibody that binds to PDL-l prior to the polynucleotide of the present disclosure. In another aspect, the subject has been treated with an anti-PDL-l monoclonal antibody therapy prior to the polynucleotide of the present methods. In other aspects, the anti-PDL-l onal antibody therapy comprises Durvalumab, ab, 3, BMS-93 6559, Atezolizumab, or any combination thereof.

In some s, the subject has been treated with a CTLA-4 antagonist prior to the polynucleotide of the present disclosure. In r , the subject has been previously treated with a monoclonal antibody that binds to CTLA-4 prior to the polynucleotide of the present disclosure. In another aspect, the subject has been treated with an anti-CTLA-4 monoclonal antibody prior to the polynucleotide of the present invention. In other aspects, the anti-CTLA-4 dy therapy comprises Ipilimumab or Tremelimumab.

In one embodiment, the anti-PD-l antibody (or an n-binding portion thereof) useful for the disclosure is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®", lizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against human cell surface or PD-l (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in US. Patent No. 8,900,587, see also http://www.cancer.gov/drugdictionary?cdrid=695789 (last ed: December 14, 2014).

Pembrolizumab has been ed by the FDA for the treatment of relapsed or refractory melanoma and advanced NSCLC.

In r embodiment, the anti-PD-l dy useful for the disclosure is nivolumab.

Nivolumab (also known as "OPDIVO®", formerly designated 5C4, BMS-936558, MDX- 1106, or ONO-453 8) is a fully human IgG4 (S228P) PD-l immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 s (PD-L1 and PD-L2), thereby blocking the down-regulation of antitumor T-cell functions (US. Patent No. 8,008,449, Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). Nivolumab has shown activity in a variety of advanced solid tumors including renal cell carcinoma (renal adenocarcinoma, or phroma), ma, and non-small cell lung cancer (NSCLC) (Topalian et al., 2012a, Topalian et al., 2014, Drake et al., 2013, In other ments, the anti-PD-l antibody is MEDIO680 (formerly AMP-514), which is a monoclonal antibody against the PD-l receptor. MEDIO680 is bed, for example, in US. Patent No. 8,609,089B2 or in http://www.cancer.gov/drugdictionary?cdrid=756047 (last accessed December 14, 2014).

In certain embodiments, the anti-PD-l antibody is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U. S. Publ. No. 2015/0079109.

In certain embodiments, a PD-l antagonist is AMP-224, which is a B7-DC Fc fusion protein. AMP-224 is discussed in U. S. Publ. No. 2013/0017199 or in http://www.cancer.gov/publications/dictionaries/cancer-drug?cdrid=700595 (last accessed July 8, 2015).

In certain embodiments, the anti-PD-Ll antibody useful for the disclosure is MSB0010718C (also called Avelumab, See US 2014/0341917) or BMS-93 6559 (formerly 12A4 or MDX-1105) (see, e.g., US. Patent No. 7,943,743, embodiments, the anti-PD-Ll antibody is MPDL3280A (also known as RG7446) (see, e.g., Herbst et al. (2013) J Clin Oncol 31(suppl):3000. Abstract, U. S. Patent No. 149), 36 (also called Durvalumab, Khleif (2013) In: Proceedings from the European Cancer Congress 2013, September 27-October 1, 2013, Amsterdam, The lands.

An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (now known as ipilimumab and marketed as YERVOY®) as disclosed in US. Patent No. 6,984,720. Another anti-CTLA-4 antibody useful for the present methods is tremelimumab (also known as CP- 675,206). Tremelimumab is human IgG2 monoclonal anti-CTLA-4 antibody. imumab is described in WO/2012/122444, U.S. Publ. No. 2012/263677, or WO Publ. No. 2007/113648 A2.

The following Table (Table 10) provides examples of KRAS ons in speci?c tumor types and types of therapies in use and testing. The compositions of the invention are useful in combination with any of these therapies.

TABLE 10 Colorectal Pancreatic Lung Uterine endometrioid carcinoma #US KRAS* 57,712 49,257 26,695 10,281 Patients (mKRAS Incidence) % KRAS mutation 45.0% 97.0% 31.0% 21.4% (vs. Total) PD-Ll Inhibitors Atezolizumab Durvalumab (P2- ab (P3-R) tested ) R) Atezolizumab Durvalumab (P2- (P3 -R) NR) Durvalumab (P2- PD-l Inhibitors Nivolumab (P2-R) Nivolumab (P2-R) Nivolumab (P2-R) mab (P2-R) tested Pembrolizumab Pembrolizumab Pembrolizumab Pembrolizumab (P2-R) (P2-R) (P2-R) (P2-R) Cancer Vaccine No No GI-4000 (P2-C) No tested DPV-001 (P2-R) KRAS Vaccine No No 0 (P2-C) No tested DPV-001 (P2-R) Bull Case for KRAS 45% w/ mutant 97% w/ mutant 31% w/ mutant 21% w/ mutant KRAS Vaccine KRAS KRAS KRAS t pt pool Defines this tumor 39% G12C allele 36% G12D allele 39% G12D Allele 21% G12V allele 21% G12V allele 30% G12V Allele Priority for KRAS Vaccine (H/M/L) In other embodiments the cancer therapeutic agent is a ne. In yet other embodiments the cancer therapeutic agent is a vaccine comprising a population based tumor speci?c antigen.

In other embodiments, the cancer therapeutic agent is vaccine containing one or more traditional antigens expressed by cancer-germline genes (antigens common to tumors found in multiple patients, also referred to as "shared cancer antigens"). In some embodiments, a traditional antigen is one that is known to be found in cancers or tumors generally or in a speci?c type of cancer or tumor. In some embodiments, a ional cancer antigen is a non- mutated tumor n. In some embodiments, a ional cancer antigen is a mutated tumor antigen.

The p53 gene (of?cial symbol TP53) is d more frequently than any other gene in human cancers. Large cohort studies have shown that, for most p53 mutations, the c position is unique to one or only a few patients and the mutation cannot be used as recurrent neoantigens for therapeutic vaccines designed for a c population of patients.

A small subset of p53 loci do, however, exhibit a "hotspot" pattern, in which several positions in the gene are mutated with relatively high frequency. Strikingly, a large portion of these recurrently mutated regions occur near exon-intron boundaries, disrupting the canonical nucleotide ce motifs recognized by the mRNA splicing machinery. on of a splicing motif can alter the final mRNA sequence even if no change to the local amino acid sequence is predicted (i.e. for synonymous or intronic mutations).

Therefore, these ons are often annotated as "noncoding" by common annotation tools and neglected for further analysis, even though they may alter mRNA splicing in ictable ways and exert severe functional impact on the translated protein. If an alternatively spliced isoform produces an in-frame sequence change (1'.e., no pretermination codon (PTC) is ed), it can escape depletion by nonsense-mediated mRNA decay (NMD) and be readily sed, processed, and presented on the cell surface by the HLA system. Further, mutation-derived alternative splicing is usually "cryptic", i.e., not expressed 2O in normal tissues, and therefore may be recognized by T-cells as non-self neoantigens.

In some instances, the the cancer therapeutic agent is a vaccine which includes one or more neoantigens which are recurrent polymorphisms ("hot spot mutations"). For example, among other things, the t invention provides neoantigen peptide sequences resulting from certain recurrent somatic cancer mutations in p53. Exemplary mutations and mRNA splicing events resulting neoantigen peptides and HLA-restricted es include, but are not limited to the following: (1) mutations at the canonical 5’ splice site neighboring codon p.T125, inducing a retained intron having peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) that contains es AVSPCISFVW (SEQ ID NO: 233) (HLA-B*57:Ol, HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) (HLA-A*O2:Ol, O2:O6, HLA-B*35:Ol), (2) mutations at the canonical 5’ splice site neighboring codon p.331, inducing a retained intron having peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) that ns epitopes LQVLSLGTSY (SEQ ID NO: 237) (HLA-B*15:Ol), FQSNTQNAVF (SEQ ID NO: 238) (HLA-B*15:01), (3) mutations at the canonical 3’ splice site neighboring codon p. 126, inducing a cryptic alternative exonic 3’ splice site ing the novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 239) that contains epitopes CTMFCQLAK (SEQ ID NO: 240) (HLA-A*11:01), KSVTCTMF (SEQ ID NO: 241) (HLA-B*58:Ol), and/or (4) mutations at the canonical 5’ splice site neighboring codon p.224, inducing a cryptic alternative intronic 5’ splice site producing the novel spanning e sequence IO VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) that ns epitopes VPYEPPEVW (SEQ ID NO: 243) (HLA-B*53:Ol, HLA-B*5l:Ol), LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:Ol, HLA-B*57:Ol), wherein the transcript codon positions refer to the canonical full-length p53 ript ENST00000269305 (SEQ ID NO: 245) from the l V83 human genome annotation.

In one embodiment, the invention es a cancer therapeutic vaccine comprising mRNA encoding an open reading frame (ORF) coding for one or more of neoantigen peptides (1) through (4). In one embodiment, the invention provides the ive administration of a vaccine containing or coding for one or more of peptides (l)-(4), based on the patient’s tumor containing any of the above mutations. In one embodiment, the invention 2O provides the selective administration of the vaccine based on the dual criteria of the subject’s tumor containing any of the above mutations and the subject’s normal HLA type containing the corresponding HLA allele predicted to bind to the resulting neoantigen.

In some embodiments, the cancer therapeutic vaccine comprises one or more mRNAs encoding one or more recurrent polymorphisms. In some embodiments, the cancer therapeutic vaccine comprises one or more mRNAs encoding one or more t c igens. In some embodiments, the cancer therapeutic vaccine comprises one or more mRNAs encoding an immune oint modulator. The one or more recurrent polymorphisms, the one or more patient speci?c neoantigens, and/or the one or more immune checkpoint modulator can be combined in any manner. For example, it may desirable for one or more concatameric constructs to encode one the one or more recurrent polymorphisms, the one or more t speci?c neoantigens, and/or the one or more immune checkpoint modulator. In other instances, it may be desirable for the one or more recurrent polymorphisms, the one or more patient speci?c neoantigens, and/or the one or more immune checkpoint modulator to be d by separate mRNA constructs. It will be appreciated that the one or more recurrent rphisms, the one or more patient speci?c neoantigens, and/or the one or more immune checkpoint modulator can be administered concurrently, or can be administered sequentially.

The mRNA cancer vaccine and anti-cancer therapeutic can be combined to enhance immune therapeutic responses even r. The mRNA cancer vaccine and other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially with one another and with the mRNA cancer vaccine, when the administration of the other therapeutic agents and the mRNA cancer vaccine is temporally separated. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer, e.g. hours, days, weeks, months. For example, in some embodiments, the separation in time between the administration of these nds is 1 hour, 2 hours, 3 hours 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours or more. In some embodiments, the separation in time between the stration of these compounds is 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the mRNA cancer vaccine is administered before the anti-cancer therapeutic. In some ments, the mRNA cancer e is stered after the anti-cancer therapeutic.

Other therapeutic agents include but are not limited to anti-cancer therapeutic, 2O adjuvants, cytokines, antibodies, antigens, etc.

In some aspects, provided methods include administering an mRNA cancer vaccine in combination with an immune checkpoint modulator. In some embodiments, an immune checkpoint modulator, e.g., checkpoint inhibitor such as an anti-PD-l antibody, is administered at a dosage level suf?cient to deliver 100-300 mg to the subject. In some embodiments, an immune oint modulator, e.g., checkpoint inhibitor such as an anti- PD-l antibody, is administered at a dosage level ent to deliver 200 mg to the subject.

In some embodiments, an immune oint modulator, e.g., checkpoint inhibitor such as an anti-PD-l antibody, is administered by intravenous infusion. In some embodiments, thee immune checkpoint tor is administered to the subject twice, three times, four times or more. In some embodiments, the immune checkpoint modulator is administered to the subject on the same day as the mRNA vaccine administration.

RNA vaccines may be formulated or administered in combination with one or more pharrnaceutically-acceptable excipients. In some embodiments, vaccine itions se at least one additional active substances, such as, for example, a therapeutically- active substance, a prophylactically-active nce, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., cott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, cancer RNA vaccines are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to the RNA vaccines or the polynucleotides contained n, for example, RNA polynucleotides (e.g., mRNA cleotides) encoding antigenic polypeptides.

Formulations of the vaccine compositions bed herein may be prepared by any method known or ter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if ary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

Cancer RNA vaccines can be formulated using one or more excipients to: (1) increase stability, (2) increase cell transfection, (3) permit the sustained or delayed release (e.g., from a depot formulation), (4) alter the biodistribution (e.g., target to specific tissues or 2O cell types), (5) increase the translation of encoded protein in viva, and/or (6) alter the release pro?le of encoded n (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, sion or suspension aids, e active agents, isotonic agents, thickening or fying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, es, proteins, cells transfected with cancer RNA vaccines (e.g., for transplantation into a t), onidase, nanoparticle mimics and combinations thereof.

AcceleratedBlood Clearance The invention provides compounds, compositions and methods of use thereof for reducing the effect of ABC on a edly administered active agent such as a biologically active agent. As will be readily apparent, reducing or eliminating altogether the effect of ABC on an stered active agent effectively increases its half-life and thus its efficacy.

In some embodiments the term reducing ABC refers to any reduction in ABC in comparison to a ve reference control ABC inducing LNP such as an MC3 LNP. ABC inducing LNPs cause a reduction in circulating levels of an active agent upon a second or subsequent administration within a given time frame. Thus a ion in ABC refers to less clearance of circulating agent upon a second or subsequent dose of agent, relative to a standard LNP. The reduction may be, for instance, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%. In some embodiments the reduction is %, lO-50%, 20-100%, 20-50%, 30-100%, , 40%-100%, 40-80%, 50-90%, or 50-100%. atively the reduction in ABC may be characterized as at least a detectable level of circulating agent following a second or subsequent administration or at least a 2 fold, 3 fold, 4 fold, 5 fold increase in circulating agent relative to circulating agent ing administration of a standard LNP. In some ments the reduction is a 2-100 fold, 2-50 fold, 3-100 fold, 3-50 fold, 3-20 fold, 4-100 fold, 4-50 fold, 4-40 fold, 4-30 fold, 4-25 fold, 4-20 fold, 4-15 fold, 4-10 fold, 4-5 fold, 5 - 100 fold, 5—50 fold, 5—40 fold, 5—30 fold, 5—25 fold, 5—20 fold, 5—15 fold, 5—10 fold, 6-100 fold, 6-50 fold, 6-40 fold, 6-30 fold, 6-25 fold, 6-20 fold, 6-15 fold, 6-10 fold, 8-100 fold, 8- 50 fold, 8-40 fold, 8-30 fold, 8-25 fold, 8-20 fold, 8-15 fold, 8-10 fold, 10—100 fold, 1050 fold, 1040 fold, 1030 fold, 1025 fold, 1020 fold, 1015 fold, 20—100 fold, 2050 fold, 20— 40 fold, 20-30 fold, or 20-25 fold.

The disclosure provides lipid-comprising compounds and compositions that are less susceptible to clearance and thus have a longer half-life in vivo. This is ularly the case where the itions are intended for repeated including chronic administration, and even more particularly where such repeated administration occurs within days or weeks.

Signi?cantly, these compositions are less susceptible or altogether circumvent the observed phenomenon of accelerated blood nce (ABC). ABC is a phenomenon in which n exogenously stered agents are rapidly cleared from the blood upon second and subsequent administrations. This phenomenon has been observed, in part, for a variety of lipid-containing compositions including but not limited to lipidated agents, liposomes or other lipid-based delivery vehicles, and lipid-encapsulated agents. Heretofore, the basis of ABC has been poorly understood and in some cases attributed to a humoral immune response and accordingly strategies for limiting its impact in vivo particularly in a clinical setting have remained elusive.

This disclosure provides compounds and compositions that are less susceptible, if at all susceptible, to ABC. In some important aspects, such compounds and compositions are lipid-comprising compounds or compositions. The lipid-containing compounds or compositions of this disclosure, singly, do not experience ABC upon second and subsequent administration in vivo. This resistance to ABC renders these compounds and compositions particularly suitable for repeated use in vivo, including for repeated use within short periods of time, including days or l-2 weeks. This enhanced stability and/or half-life is due, in part, to the inability of these compositions to activate Bla and/or Blb cells and/or tional B cells, pDCs and/or platelets.

This disclosure therefore provides an elucidation of the mechanism underlying rated blood clearance (ABC). It has been found, in accordance with this disclosure and the inventions provided herein, that the ABC phenomenon at least as it relates to lipids and lipid nanoparticles is mediated, at least in part an innate immune response involving Bla and/or Blb cells, pDC and/or platelets. Bla cells are normally responsible for ing natural antibody, in the form of circulating IgM. This IgM is poly-reactive, meaning that it is able to bind to a variety of antigens, albeit with a relatively low af?nity for each.

It has been found in accordance with the invention that some lipidated agents or lipid- sing formulations such as lipid nanoparticles stered in vivo trigger and are subject to ABC. It has now been found in accordance with the invention that upon administration of a first dose of the LNP, one or more cells involved in generating an innate immune response (referred to herein as sensors) bind such agent, are activated, and then initiate a cascade of immune factors (referred to herein as effectors) that promote ABC and toxicity. For instance, Bla and Blb cells may bind to LNP, become ted (alone or in the presence of other sensors such as pDC and/or effectors such as 1L6) and secrete natural IgM that binds to the LNP. Pre-existing natural IgM in the subject may also recognize and bind to the LNP, thereby ring complement ?xation. After stration of the ?rst dose, the production of natural IgM begins within 1-2 hours of administration of the LNP. Typically by about 2-3 weeks the l IgM is cleared from the system due to the natural half-life of IgM. Natural IgG is produced beginning around 96 hours after administration of the LNP.

The agent, when administered in a naive setting, can exert its biological effects relatively unencumbered by the natural IgM produced post-activation of the Bla cells or Blb cells or natural IgG. The l IgM and natural IgG are non-speci?c and thus are distinct from anti-PEG IgM and anti-PEG IgG.

Although Applicant is not bound by mechanism, it is ed that LNPs trigger ABC and/or toxicity through the ing mechanisms. It is ed that when an LNP is administered to a subject the LNP is rapidly transported through the blood to the spleen. The LNPs may encounter immune cells in the blood and/or the spleen. A rapid innate immune response is triggered in response to the presence of the LNP within the blood and/or spleen.

Applicant has shown herein that within hours of stration of an LNP several immune sensors have reacted to the presence of the LNP. These sensors include but are not limited to immune cells involved in generating an immune response, such as B cells, pDC, and platelets. The sensors may be present in the spleen, such as in the marginal zone of the spleen and/or in the blood. The LNP may physically interact with one or more sensors, which may interact with other sensors. In such a case the LNP is ly or indirectly interacting with the sensors. The sensors may interact directly with one another in response to recognition of the LNP. For instance many sensors are located in the spleen and can easily interact with one r. Alternatively one or more of the sensors may interact with LNP in the blood and become activated. The ted sensor may then interact directly with other sensors or indirectly (e.g., through the stimulation or production of a messenger such as a cytokine e.g., 1L6).

In some embodiments the LNP may interact ly with and activate each of the ing sensors: pDC, Bla cells, Blb cells, and platelets. These cells may then interact directly or indirectly with one another to initiate the production of effectors which ultimately lead to the ABC and/or toxicity associated with repeated doses of LNP. For instance, Applicant has shown that LNP administration leads to pDC activation, platelet aggregation and activation and B cell tion. In response to LNP platelets also aggregate and are activated and aggregate with B cells. pDC cells are activated. LNP has been found to interact with the surface of platelets and B cells relatively quickly. Blocking the activation of any one or combination of these sensors in response to LNP is useful for dampening the immune response that would ordinarily occur. This dampening of the immune response results in the nce of ABC and/or toxicity.

The sensors once activated produce ors. An effector, as used herein, is an immune molecule ed by an immune cell, such as a B cell. Effectors include but are not d to immunoglobulin such as natural IgM and natural IgG and cytokines such as IL6.

Bla and Blb cells stimulate the production of natural Ing within 2-6 hours following administration of an LNP. Natural IgG can be detected within 96 hours. 1L6 levels are sed within several hours. The natural IgM and IgG circulate in the body for several days to several weeks. During this time the circulating effectors can interact with newly administered LNPs, triggering those LNPs for clearance by the body. For instance, an effector may recognize and bind to an LNP. The Fc region of the or may be recognized by and trigger uptake of the decorated LNP by macrophage. The macrophage are then transported to the spleen. The production of effectors by immune sensors is a transient response that correlates with the timing observed for ABC.

If the administered dose is the second or subsequent administered dose, and if such second or subsequent dose is administered before the previously induced l IgM and/or IgG is cleared from the system (e.g., before the 2-3 window time period), then such second or subsequent dose is targeted by the circulating natural IgM and/or natural IgG or Fc which r alternative complement pathway activation and is itself rapidly d. When LNP are stered after the effectors have cleared from the body or are reduced in number, ABC is not observed.

Thus, it is useful according to aspects of the invention to inhibit the interaction between LNP and one or more sensors, to inhibit the activation of one or more sensors by LNP (direct or ct), to inhibit the production of one or more ors, and/or to inhibit the activity of one or more effectors. In some embodiments the LNP is designed to limit or block interaction of the LNP with a sensor. For instance the LNP may have an altered PC and/or PEG to prevent interactions with sensors. Alternatively or onally an agent that inhibits immune responses induced by LNPs may be used to achieve any one or more of these effects.

It has also been determined that tional B cells are also implicated in ABC. cally, upon first administration of an agent, conventional B cells, referred to herein as CDl9(+), bind to and react against the agent. Unlike Bla and Blb cells though, conventional B cells are able to mount first an IgM response (beginning around 96 hours after administration of the LNPs) followed by an IgG response (beginning around 14 days after administration of the LNPs) concomitant with a memory response. Thus conventional B cells react against the administered agent and contribute to IgM (and eventually IgG) that mediates ABC. The IgM and IgG are lly anti-PEG IgM and anti-PEG IgG.

It is contemplated that in some instances, the majority of the ABC response is mediated through Bla cells and Bla-mediated immune responses. It is further contemplated that in some instances, the ABC response is ed by both IgM and IgG, with both conventional B cells and Bla cells ing such effects. In yet still other instances, the ABC response is mediated by natural IgM molecules, some of which are capable of binding to l IgM, which may be produced by activated Bla cells. The natural Ing may bind to one or more components of the LNPs, e.g., binding to a phospholipid component of the LNPs (such as binding to the PC moiety of the phospholipid) and/or binding to a PEG-lipid component of the LNPs (such as g to PEG-DMG, in particular, binding to the PEG moiety of PEG-DMG). Since Bla expresses CD3 6, to which phosphatidylcholine is a ligand, it is contemplated that the CD36 receptor may mediate the activation of Bla cells and thus production of natural IgM. In yet still other instances, the ABC response is mediated primarily by conventional B cells.

It has been found in accordance with the invention that the ABC phenomenon can be reduced or abrogated, at least in part, through the use of compounds and compositions (such as agents, delivery vehicles, and formulations) that do not activate Bla cells. Compounds and compositions that do not activate Bla cells may be referred to herein as Bla inert compounds and compositions. It has been further found in accordance with the invention that the ABC phenomenon can be reduced or ted, at least in part, through the use of compounds and compositions that do not activate conventional B cells. Compounds and compositions that do not te conventional B cells may in some embodiments be referred to herein as CDl9-inert compounds and compositions. Thus, in some embodiments provided herein, the compounds and compositions do not activate Bla cells and they do not te conventional B cells. Compounds and compositions that do not activate Bla cells and conventional B cells may in some embodiments be ed to herein as Bla/CDl9-inert compounds and itions.

These underlying mechanisms were not heretofore understood, and the role of B l a and Blb cells and their interplay with conventional B cells in this phenomenon was also not iated. ingly, this sure provides compounds and compositions that do not promote ABC. These may be further characterized as not capable of activating Bla and/or Blb cells, platelets and/or pDC, and ally conventional B cells also. These compounds (e.g., agents, including biologically active agents such as prophylactic agents, therapeutic agents and diagnostic agents, delivery vehicles, including mes, lipid nanoparticles, and other lipid-based encapsulating structures, etc.) and compositions (e.g., formulations, etc.) are particularly ble for ations requiring repeated administration, and in particular repeated administrations that occur within with short periods of time (e.g., within 1-2 weeks).

This is the case, for example, if the agent is a c acid based therapeutic that is provided to a subject at regular, closely-spaced intervals. The findings provided herein may be applied to these and other agents that are similarly stered and/or that are subject to ABC.

Of particular interest are lipid-comprising compounds, lipid-comprising particles, and lipid-comprising compositions as these are known to be susceptible to ABC. Such lipid- comprising compounds les, and compositions have been used extensively as biologically active agents or as delivery es for such agents. Thus, the y to improve their ef?cacy of such agents, whether by reducing the effect of ABC on the agent itself or on its delivery vehicle, is bene?cial for a wide variety of active agents.

Also provided herein are compositions that do not ate or boost an acute phase response (ARP) associated with repeat dose administration of one or more biologically active agents.

The composition, in some ces, may not bind to IgM, including but not limited to natural IgM.

The composition, in some instances, may not bind to an acute phase protein such as but not limited to C-reactive protein.

The composition, in some instances, may not trigger a CD5(+) mediated immune response. As used herein, a CD5(+) mediated immune response is an immune response that is ed by Bla and/or Blb cells. Such a response may include an ABC response, an acute phase response, induction of natural IgM and/or IgG, and the like.

The composition, in some instances, may not trigger a CDl9(+) ed immune response. As used , a CDl9(+) mediated immune response is an immune response that is mediated by conventional CDl9(+), CD5(-) B cells. Such a response may include ion of IgM, induction of IgG, induction of memory B cells, an ABC response, an anti- drug antibody (ADA) response including an anti-protein se where the protein may be encapsulated within an LNP, and the like.

Bla cells are a subset of B cells involved in innate immunity. These cells are the source of circulating IgM, referred to as natural dy or natural serum antibody. Natural IgM antibodies are characterized as having weak af?nity for a number of antigens, and therefore they are referred to as "poly-speci?c" or "poly-reactive", indicating their ability to bind to more than one antigen. Bla cells are not able to produce IgG. Additionally, they do not develop into memory cells and thus do not contribute to an adaptive immune response.

However, they are able to secrete IgM upon activation. The ed IgM is typically cleared within about 2-3 weeks, at which point the immune system is rendered relatively naive to the previously administered antigen. If the same antigen is presented after this time period (e.g., at about 3 weeks after the initial exposure), the antigen is not rapidly cleared. However, cantly, if the antigen is presented within that time period (e.g., within 2 weeks, including within 1 week, or within days), then the antigen is rapidly cleared. This delay between consecutive doses has rendered certain lipid-containing therapeutic or diagnostic agents unsuitable for use.

In humans, Bla cells are CDl9(--), CD20(+), CD27(+), CD43(+), CD70(-) and CD5(+). In mice, Bla cells are CDl9(--), , and CD45 B cell m B220(+). It is the expression of CD5 which lly distinguishes Bla cells from other convention B cells.

Bla cells may express high levels of CD5, and on this basis may be guished from other B-l cells such as B-lb cells which express low or undetectable levels of CD5. CD5 is a pan- T cell surface glycoprotein. Bla cells also express CD3 6, also known as fatty acid translocase. CD36 is a member of the class B scavenger receptor family. CD36 can bind many ligands, including oxidized low density lipoproteins, native lipoproteins, oxidized phospholipids, and long-chain fatty acids.

Blb cells are another subset of B cells involved in innate ty. These cells are r source of circulating natural IgM. Several antigens, including PS, are e of inducing T cell independent immunity through Blb activation. CD27 is lly upregulated on Blb cells in se to antigen activation. Similar to Bla cells, the Blb cells are typically located in speci?c body locations such as the spleen and peritoneal cavity and are in very low abundance in the blood. The Blb secreted natural IgM is typically cleared within about 2-3 weeks, at which point the immune system is rendered relatively naive to the previously administered antigen. If the same antigen is presented after this time period (e.g., at about 3 2O weeks after the initial exposure), the antigen is not rapidly cleared. However, signi?cantly, if the antigen is presented within that time period (e.g., within 2 weeks, including within 1 week, or within days), then the antigen is rapidly cleared. This delay between consecutive doses has rendered certain lipid-containing therapeutic or diagnostic agents unsuitable for In some embodiments it is desirable to block Bla and/or Blb cell tion. One strategy for ng Bla and/or B lb cell activation involves determining which components of a lipid nanoparticle promote B cell activation and neutralizing those components. It has been discovered herein that at least PEG and phosphatidylcholine (PC) contribute to Bla and Blb cell interaction with other cells and/or activation. PEG may play a role in promoting aggregation between Bl cells and platelets, which may lead to activation. PC (a helper lipid in LNPs) is also involved in activating the B1 cells, likely through interaction with the CD36 receptor on the B cell surface. Numerous particles have PEG-lipid alternatives, PEG-less, and/or PC replacement lipids (e.g. oleic acid or analogs thereof) have been designed and tested. Applicant has established that replacement of one or more of these components within an LNP that otherwise would promote ABC upon repeat administration, is useful in preventing ABC by reducing the production of natural IgM and/or B cell activation. Thus, the invention encompasses LNPs that have d ABC as a result of a design which eliminates the ion of B cell triggers.

Another strategy for blocking Bla and/or Blb cell activation involves using an agent that ts immune responses induced by LNPs. These types of agents are discussed in more detail below. In some embodiments these agents block the interaction between Bla/Blb cells and the LNP or platelets or pDC. For instance the agent may be an antibody or other binding agent that physically blocks the interaction. An example of this is an antibody that binds to CD36 or CD6. The agent may also be a compound that prevents or disables the Bla/Blb cell from signaling once activated or prior to activation. For instance, it is possible to block one or more components in the Bla/Blb ing cascade the results from B cell interaction with LNP or other immune cells. In other embodiments the agent may act one or more effectors produced by the Bla/Blb cells following tion. These effectors include for instance, natural IgM and cytokines.

It has been demonstrated according to aspects of the invention that when activation of pDC cells is blocked, B cell activation in response to LNP is decreased. Thus, in order to avoid ABC and/or toxicity, it may be desirable to prevent pDC activation. Similar to the strategies discussed above, pDC cell activation may be blocked by agents that interfere with the interaction between pDC and LNP and/or B cells/platelets. Alternatively agents that act on the pDC to block its ability to get activated or on its effectors can be used together with the LNP to avoid ABC.

Platelets may also play an important role in ABC and toxicity. Very quickly after a first dose of LNP is administered to a subject ets associate with the LNP, aggregate and are activated. In some embodiments it is desirable to block platelet aggregation and/or activation. One strategy for blocking et aggregation and/or tion involves determining which components of a lipid nanoparticle promote platelet ation and/or activation and neutralizing those components. It has been discovered herein that at least PEG bute to platelet aggregation, tion and/or interaction with other cells. us particles have PEG-lipid alternatives and PEG-less have been designed and tested. Applicant has established that replacement of one or more of these components within an LNP that ise would promote ABC upon repeat administration, is useful in preventing ABC by reducing the production of natural IgM and/or platelet aggregation. Thus, the invention encompasses LNPs that have reduced ABC as a result of a design which eliminates the inclusion of platelet triggers. Alternatively agents that act on the ets to block its activity once it is activated or on its effectors can be used together with the LNP to avoid ABC.

Measuring ABC activity and related activities Various nds and compositions provided herein, including LNPs, do not promote ABC activity upon administration in vivo. These LNPs may be characterized and/or identi?ed through any of a number of assays, such as but not limited to those described below.

In some embodiments the methods involve administering an LNP without producing an immune se that promotes ABC. An immune se that promotes ABC involves activation of one or more sensors, such as Bl cells, pDC, or platelets, and one or more effectors, such as natural IgM, natural IgG or cytokines such as 1L6. Thus administration of an LNP without producing an immune response that promotes ABC, at a minimum involves administration of an LNP without signi?cant activation of one or more sensors and significant production of one or more effectors. Signif1cant used in this t refers to an amount that would lead to the physiological consequence of accelerated blood clearance of all or part of a second dose with respect to the level of blood clearance expected for a second dose of an ABC triggering LNP. For instance, the immune response should be dampened such that the ABC ed after the second dose is lower than would have been expected for an ABC triggering LNP.

Bla or B lb activation assay Certain compositions provided in this disclosure do not activate B cells, such as Bla or Blb cells (CDl9+ CD5+) and/or tional B cells (CDl9+ CD5-). Activation of Bla cells, Blb cells, or conventional B cells may be ined in a number of ways, some of which are ed below. B cell population may be provided as fractionated B cell populations or unfractionated populations of splenocytes or peripheral blood mononuclear cells (PBMC). If the latter, the cell tion may be incubated with the LNP of choice for a period of time, and then ted for further analysis. Alternatively, the supernatant may be harvested and analyzed.

Upregulation of activation marker cell surface expression Activation of Bla cells, Blb cells, or conventional B cells may be demonstrated as increased expression of B cell tion markers including late activation markers such as CD86. In an exemplary non-limiting assay, unfractionated B cells are ed as a splenocyte population or as a PBMC population, incubated with an LNP of choice for a particular period of time, and then stained for a standard B cell marker such as CD19 and for an activation marker such as CD86, and analyzed using for example ?ow try. A suitable negative control involves incubating the same population with medium, and then performing the same staining and ization steps. An increase in CD86 sion in the test population compared to the negative control indicates B cell activation.

Pro-in?ammatom cytokine release B cell activation may also be assessed by cytokine release assay. For example, activation may be assessed through the production and/or secretion of cytokines such as 1L-6 and/or INF-alpha upon exposure with LNPs of interest.

Such assays may be performed using routine cytokine secretion assays well known in the art. An increase in cytokine secretion is tive of B cell activation.

LNP bindin association to and/or u take b B cells LNP association or binding to B cells may also be used to assess an LNP of interest and to further characterize such LNP. Association/binding and/or /intemalization may be assessed using a detectably labeled, such as ?uorescently labeled, LNP and tracking the location of such LNP in or on B cells following various periods of incubation.

The invention further contemplates that the compositions provided herein may be capable of g recognition or detection and optionally binding by downstream mediators of ABC such as circulating IgM and/or acute phase response mediators such as acute phase proteins (e.g., C-reactive protein (CRP).

Methods of use for reducing ABC Also provided herein are s for ring LNPs, which may ulate an agent such as a therapeutic agent, to a subject without promoting ABC.

In some embodiments, the method comprises administering any of the LNPs described , which do not promote ABC, for example, do not induce production of natural IgM binding to the LNPs, do not activate Bla and/or Blb cells. As used herein, an LNP that "does not promote ABC" refers to an LNP that induces no immune responses that would lead to substantial ABC or a substantially low level of immune responses that is not ent to lead to substantial ABC. An LNP that does not induce the production of natural Ing binding to the LNP refers to LNPs that induce either no natural IgM binding to the LNPs or a substantially low level of the natural IgM molecules, which is insufficient to lead to substantial ABC. An LNP that does not activate Bla and/or Blb cells refer to LNPs that induce no response of Bla and/or Blb cells to produce natural IgM binding to the LNPs or a substantially low level of Bla and/or B lb responses, which is icient to lead to substantial ABC.

In some embodiments the terms do not activate and do not induce production are a relative reduction to a reference value or condition. In some embodiments the reference value or condition is the amount of activation or induction of production of a molecule such as IgM by a standard LNP such as an MC3 LNP. In some embodiments the relative reduction is a reduction of at least 30%, for e at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments the terms do not activate cells such as B cells and do not induce production of a protein such as IgM may refer to an undetectable amount of the active cells or the specific protein.

Platelet effects and ty The invention is further premised in part on the elucidation of the mechanism ying dose-limiting toxicity ated with LNP administration. Such toxicity may involve coagulopathy, inated intravascular coagulation (DIC, also referred to as consumptive opathy), whether acute or chronic, and/or vascular thrombosis. In some instances, the dose-limiting toxicity associated with LNPs is acute phase response (APR) or complement activation-related psudoallergy (CARPA).

As used , coagulopathy refers to increased coagulation (blood clotting) in vivo.

The s reported in this disclosure are consistent with such increased coagulation and signi?cantly provide insight on the ying mechanism. Coagulation is a s that involves a number of different factors and cell types, and heretofore the relationship between and interaction of LNPs and platelets has not been understood in this regard. This disclosure provides evidence of such interaction and also provides compounds and compositions that are modified to have reduced platelet effect, including reduced platelet association, reduced platelet aggregation, and/or reduced et aggregation. The ability to modulate, including preferably down-modulate, such platelet effects can reduce the nce and/or severity of coagulopathy post-LNP administration. This in turn will reduce toxicity relating to such LNP, thereby allowing higher doses of LNPs and antly their cargo to be administered to patients in need thereof.

CARPA is a class of acute immune toxicity manifested in hypersensitivity reactions (HSRs), which may be triggered by nanomedicines and biologicals. Unlike allergic reactions, CARPA typically does not involve IgE but arises as a consequence of activation of the complement system, which is part of the innate immune system that enhances the body’s abilities to clear pathogens. One or more of the following pathways, the classical complement pathway (CP), the alternative pathway (AP), and the lectin pathway (LP), may be involved in CARPA. Szebeni, Molecular Immunology, 61 : 163-173 (2014).

The classical pathway is triggered by activation of the Cl-compleX, which contains.

Clq, Clr, Cls, or C1qr2s2. Activation of the Cl-compleX occurs when Clq binds to IgM or IgG complexed with antigens, or when Clq binds directly to the surface of the pathogen.

Such binding leads to conformational changes in the Clq molecule, which leads to the tion of Clr, which in turn, cleave Cls. The C1r2s2 component now splits C4 and then C2, ing C4a, C4b, C2a, and C2b. C4b and C2b bind to form the classical pathway C3- convertase (C4b2b compleX), which promotes cleavage of C3 into C3a and C3b. C3b then binds the C3 convertase to from the C5 convertase (C4b2b3b compleX). The alternative pathway is continuously activated as a result of spontaneous C3 hydrolysis. Factor P (properdin) is a positive regulator of the alternative pathway. Oligomerization of properdin 2O stabilizes the C3 convertase, which can then cleave much more C3. The C3 molecules can bind to surfaces and recruit more B, D, and P activity, leading to ication of the complement activation.

Acute phase se (APR) is a compleX ic innate immune responses for preventing infection and clearing potential pathogens. Numerous ns are involved in APR and C-reactive protein is a well-characterized one.

It has been found, in ance with the ion, that certain LNP are able to associate physically with platelets almost immediately after administration in viva, while other LNP do not associate with platelets at all or only at background levels. Signi?cantly, those LNPs that associate with platelets also apparently stabilize the platelet aggregates that are formed thereafter. al contact of the platelets with certain LNPs correlates with the ability of such ets to remain aggregated or to form aggregates continuously for an extended period of time after administration. Such aggregates comprise ted platelets and also innate immune cells such as macrophages and B cells.

LipidNanoparticles (LNPS) In one set of ments, lipid nanoparticles (LNPs) are provided. In one ment, a lipid nanoparticle comprises lipids including an ionizable lipid, a structural lipid, a phospholipid, and mRNA. Each of the LNPs described herein may be used as a formulation for the mRNA described herein. In one embodiment, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modi?ed lipid, a phospholipid and a structural lipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25-55% structural lipid, and about 05-15% PEG-modi?ed lipid. In some ments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modi?ed lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ble lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino or cationic lipid and the phospholipid is a neutral lipid, and the structural lipid is a terol. In some embodiments, the LNP has a molar ratio of 50:38.5: 10:1.5 ofionizable lipid: cholesterolzDSPC: PEG2000-DMG.

Ionizable Amino Lipids The present disclosure provides ceutical compositions with advantageous properties. For e, the lipids described herein (e.g. those having any of a (I), (IA), (II), (IIa), (IIb), (11c), (11d), (IIe), (III), (IV), (V), or (VI) may be advantageously used in lipid nanoparticle itions for the delivery of therapeutic and/or prophylactic agents to mammalian cells or organs. For example, the lipids described herein have little or no immunogenicity. For example, the lipid compounds disclosed hereinhave a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index as compared to a ponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent. In ular, the present application provides pharmaceutical compositions comprising: (a) a polynucleotide comprising a nucleotide sequence encoding one or more cancer epitope polypeptides, and (b) a ry agent.

In some ments, the delivery agent comprises a lipid compound having the Formula (I) R4\N/R wherein R1 is selected from the group consisting of C5_30 alkyl, C5_20 l, -R*YR", -YR", and -R"M’R’, R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle, R4 is selected from the group consisting of a C3-6 ycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted CH, alkyl, where Q is selected from a carbocycle, heterocycle, -OR, )nN(R)2, R, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(0)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -N(R)Rs, -0(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHRg)N(R)2, -0C(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)2C(O)OR, and each n is independently ed from 1, 2, 3, 4, and 5, each R51s ndently selected from the group consisting of C1-3 alkyl, C23 l, and H, each R6 is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, M and M’ are independently selected from -C(O)O-, -OC(O)—, -C(O)N(R’)-, -N(R’)C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group, R7 is selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, R8 is selected from the group consisting of C3-6 carbocycle and heterocycle, R9 is selected from the group consisting of H, CN, N02, CH, alkyl, -OR, -S(O)2R, -S(O)2N(R)2, CN, alkenyl, C3-6 carbocycle and heterocycle, each R is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R’ is independently selected from the group consisting of C148 alkyl, C248 alkenyl, , -YR", and H, each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl and C242 alkenyl, each Y is independently a C3-6 carbocycle, each X is ndently selected from the group consisting of F, Cl, Br, and I, and m is selected from 5,6,7, 8,9, 10, ll, 12, and 13, or salts or isomers thereof,.

In some embodiments, a subset of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5_20 alkyl, C5_20 alkenyl, -R*YR", -YR", and -R"M’R’, R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle, R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, 2O -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted CH, alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, (R)2, (0)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, and -C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5, each R5 is ndently selected from the group consisting of C1-3 alkyl, C23 alkenyl, and H, each R6 is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, M and M’ are independently selected from -, -OC(O)—, (R’)-, -N(R’)C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group, R7 is ed from the group consisting of C1-3 alkyl, C23 alkenyl, and H, each R is independently selected from the group consisting of C1.3 alkyl, C23 alkenyl, and H, each R’ is independently ed from the group consisting of C148 alkyl, C248 alkenyl, , -YR", and H, each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl and C242 alkenyl, each Y is independently a C3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I, and m is selected from 5 6 7 8 9 7 7 7 7 7 10, ll, 12, and 13, or salts or stereoisomers thereof, wherein alkyl and alkenyl groups may be linear or branched.

In some ments, a subset of compounds of Formula (1) includes those in which when R4 is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) Q is not -N(R)2 when n is l, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is l or 2.

In some embodiments, r sub set of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5_30 alkyl, C5_20 alkenyl, -R*YR", -YR", and -R"M’R’, R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, C244 2O alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, er with the atom to which they are attached, form a cycle or carbocycle, R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, 2, and unsubstituted CH, alkyl, where Q is selected from a C3-6 carbocycle, a 5- to l4-membered heteroaryl haVing one or more atoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(0)N(R)2, -N(R)C(0)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, )2C(O)OR, -N(R)Rg, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(0R)C(0)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHRg)N(R)2, 9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and a 5- to l4-membered heterocycloalkyl haVing one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, and C1-3 alkyl, and each n is independently selected from 1 2 3 4 and 5, 7 7 7 7 each R5 is independently selected from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R6 is independently selected from the group consisting of C1-3 alkyl; C23 alkenyl; and H; M and M’ are independently selected from -C(O)O-; -OC(O)-; -C(O)N(R’)-; -N(R’)C(O)-; -C(O)-; -C(S)-; -C(S)S-; -SC(S)-; -CH(OH)-; -P(O)(OR’)O-; -S(O)2-; -S-S-; an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl; C23 alkenyl; and H; R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H; CN; N02; C14, alkyl; -OR; -S(O)2R; -S(O)2N(R)2; C2-6 alkenyl; C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1.3 alkyl; C23 alkenyl; and H; each R’ is ndently selected from the group consisting of C148 alkyl; C248 alkenyl; -R*YR"; -YR"; and H; each R" is independently selected from the group ting of C344 alkyl and C344 each R* is independently selected from the group consisting of C142 alkyl and C242 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F; Cl; Br; and I; and m is selected from 5; 6; 7 8 9 7 7 7 10, 11, 12, and 13, or salts or stereoisomers thereof.

In some embodiments; r sub set of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5_30 alkyl; C5_20 alkenyl; -R*YR"; -YR"; and -R"M’R’; R2 and R3 are independently selected from the group consisting of H; CH4 alkyl; C244 alkenyl; -R*YR"; -YR"; and -R*OR"; or R2 and R3; together with the atom to which they are attached; form a cycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle; nQ; -(CH2)nCHQR; -CHQR; 2; and unsubstituted CH, alkyl; where Q is selected from a C3-6 carbocycle; a 5- to l4-membered cycle haVing one or more heteroatoms selected from N; O; and 8, -OR; -O(CH2)nN(R)2, -C(O)OR; -OC(O)R; -CX3; -CX2H, -CXHZ, -CN, -C(O)N(R)2, (O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)0R, -N(R)Rs, -0(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, (O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4, and 5, and when Q is a 5- to l4-membered heterocycle and (i) R4 is -(CH2)nQ in which n is l or 2, or (ii) R4 is -(CH2)nCHQR in which n is l, or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to l4-membered aryl or 8- to 14- membered heterocycloalkyl, each R5 is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R6 is ndently ed from the group consisting of C1-3 alkyl, C23 alkenyl, and H, M and M’ are independently selected from -C(O)O-, —, -C(O)N(R’)-, C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group, R7 is selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, R8 is selected from the group consisting of C3-6 carbocycle and heterocycle, R9 is ed from the group consisting of H, CN, N02, CH, alkyl, -OR, -S(O)2R, 2O -S(O)2N(R)2, CN, alkenyl, C3-6 carbocycle and heterocycle, each R is ndently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R’ is independently selected from the group consisting of C148 alkyl, C248 alkenyl, -R*YR", -YR", and H, each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl and C242 each Y is independently a C3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I, and mis selected from5 6 7 8 9 7 7 7 7 7 10, ll, 12, and 13, or salts or isomers thereof.

In some embodiments, another sub set of compounds of Formula (1) includes those in which R1 is ed from the group ting of C5_30 alkyl, C5_20 alkenyl, , -YR", and -R"M’R’, R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle, R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted CH, alkyl, where Q is selected from a C3-6 carbocycle, a 5- to l4-membered aryl haVing one or more heteroatoms ed from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(0)N(R)2, -N(R)C(0)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)Rg, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(0R)C(O)N(R)2, -N(0R)C(S)N(R)2, C(=NR9)N(R)2, -N(OR)C(=CHRg)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and a 5- to l4-membered heterocycloalkyl haVing one or more atoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, and C1-3 alkyl, and each n is independently selected from 1 2 3 4 and 5, each R5 is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, 2O each R6 is independently selected from the group consisting of C1-3 alkyl, C23 alkenyl, and H, M and M’ are ndently selected from -C(O)O-, -OC(O)—, (R’)-, -N(R’)C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group, R7 is selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, R8 is selected from the group consisting of C3-6 carbocycle and heterocycle, R9 is selected from the group consisting of H, CN, N02, CH, alkyl, -OR, -S(O)2R, -S(O)2N(R)2, CN, alkenyl, C3-6 carbocycle and heterocycle, each R is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R’ is independently selected from the group consisting of C148 alkyl, C248 alkenyl, -R*YR", -YR", and H, each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl and C242 alkenyl, each Y is independently a C3-6 ycle, each X is independently selected from the group consisting of F, Cl, Br, and I, and mis selected from5 6 7 8 9 10, ll, 12, and 13, or salts or isomers thereof.

In some embodiments, another sub set of nds of Formula (1) includes those in which R1 is selected from the group consisting of C5_20 alkyl, C5_20 alkenyl, , -YR", and -R"M’R’, R2 and R3 are independently ed from the group consisting of H, CH4 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle, R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted CH, alkyl, where Q is selected from a C3-6 carbocycle, a 5- to l4-membered heterocycle haVing one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXHZ, -CN, -C(0)N(R)2, -N(R)C(0)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)Rg, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, 2O -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, C(O)OR, -N(0R)C(O)N(R)2, C(S)N(R)2, C(=NR9)N(R)2, -N(OR)C(=CHRg)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -N(R)2 and -C(=NR9)N(R)2, and each n is ndently selected from 1, 2, 3, 4, and 5, and when Q is a 5- to l4-membered heterocycle and (i) R4 is -(CH2)nQ in which n is l or 2, or (ii) R4 is -(CH2)nCHQR in which n is l, or (iii) R4 is -CHQR, and 2, then Q is either a 5- to l4-membered aryl or 8- to l4-membered heterocycloalkyl, each R5 is independently selected from the group consisting of C1-3 alkyl, C23 alkenyl, and H, each R6 is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, M and M’ are independently selected from -C(O)O-, -OC(O)—, -C(O)N(R’)-, -N(R’)C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group, R7 is selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, R8 is selected from the group ting of C3-6 carbocycle and heterocycle, R9 is selected from the group consisting of H, CN, N02, C14, alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle, each R is independently selected from the group consisting of C1.3 alkyl, C23 alkenyl, and H, each R’ is independently selected from the group consisting of C148 alkyl, C248 alkenyl, -R*YR", -YR, -YR", and H, each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl, C142 l, and C242 l, each Y is independently a C3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I, and m is selected from 5, 6, 7 8 9 7 7 7 10, 11, 12, and 13, or salts or stereoisomers thereof.

In yet some embodiments, another subset of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5_20 alkyl, C5_20 alkenyl, -R*YR", -YR", and -R"M’R’, 2O R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle, R4 is selected from the group ting of a C3-6 carbocycle, nQ, -(CH2)nCHQR, -CHQR, 2, and unsubstituted CH, alkyl, where Q is selected from -N(R)2, a C3-6 carbocycle, a 5- to l4-membered heterocycle haVing one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, (S)N(R)2, -CRN(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5, and when Q is a 5- to l4-membered heterocycle and (i) R4 is -(CH2)nQ in which n is l or 2, or (ii) R4 is -(CH2)nCHQR in which n is l, or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to l4-membered heteroaryl or 8- to l4-membered heterocycloalkyl, each R5 is independently selected from the group consisting of C1-3 alkyl, C23 alkenyl, and H, each R6 is independently ed from the group consisting of C1_3 alkyl; C23 alkenyl; and H; M and M’ are independently selected from -C(O)O-; -; (R’)-; -N(R’)C(O)-; -C(O)-; -C(S)-; -C(S)S-; -SC(S)-; )-; -P(O)(OR’)O-; -S(O)2-; an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R is independently selected from the group consisting of C1.3 alkyl; C23 alkenyl; and H; each R’ is independently selected from the group consisting of C148 alkyl; C248 alkenyl; -R*YR"; -YR"; and H; each R" is independently selected from the group ting of C344 alkyl and C344 alkenyl; each R* is independently selected from the group consisting of C142 alkyl and C242 alkenyl; each Y is ndently a C3-6 carbocycle; each X is independently selected from the group consisting of F; Cl; Br; and I; and mis selected from5 6 7 8 9 10,11,12;andl3; or salts or stereoisomers thereof.

In still some embodiments; another subset of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5_30 alkyl; C5_20 alkenyl; -R*YR"; -YR"; and -R"M’R’; R2 and R3 are independently selected from the group consisting of H; CH4 alkyl; C244 l; -R*YR"; -YR"; and -R*OR"; or R2 and R3; together with the atom to which they are attached; form a heterocycle or ycle; R4 is selected from the group ting of a C3-6 carbocycle; -(CH2)nQ; -(CH2)nCHQR; -CHQR; -CQ(R)2; and unsubstituted CH, alkyl; where Q is selected from a C3-6 carbocycle; a 5- to l4-membered heteroaryl haVing one or more heteroatoms selected from N; O; and 8, -OR; -O(CH2)nN(R)2; -C(O)OR; -OC(O)R; -CX3; -CX2H; -CXH2; -CN; -C(0)N(R)2, -N(R)C(0)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR; -N(R)Rg; -O(CH2)nOR; -N(R)C(=NR9)N(R)2; -N(R)C(=CHR9)N(R)2; -OC(O)N(R)2; (O)OR; -N(OR)C(O)R; -N(OR)S(O)2R; -N(OR)C(O)OR; -N(0R)C(O)N(R)2, -N(0R)C(S)N(R)2, -N(0R)C(=NR9)N(R)2, -N(OR)C(=CHRg)N(R)2, -C(=NR9)R; -C(O)N(R)OR; and -C(=NR9)N(R)2; and each n is independently selected from 1 2 3 4 and 5; 7 7 7 7 each R5 is independently selected from the group consisting of C1-3 alkyl; C23 alkenyl and H; each R6 is independently selected from the group consisting of C1_3 alkyl; C23 alkenyl and H; M and M’ are independently selected from -C(O)O-; -OC(O)-; -C(O)N(R’)-; -N(R’)C(O)-; -C(O)-; -C(S)-; -C(S)S-; -SC(S)-; -CH(OH)-; -P(O)(OR’)O-; -S(O)2-; -S-S-; an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1_3 alkyl; C23 alkenyl; and H; R8 is ed from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H; CN; N02; CH, alkyl; -OR; -S(O)2R; - (R)2; CH, l; C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R’ is independently selected from the group consisting of C148 alkyl; C248 alkenyl; -R*YR"; -YR"; and H; each R" is independently selected from the group consisting of C314 alkyl and C344 alkenyl; each R* is independently selected from the group ting of C142 alkyl and C242 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F; Cl; Br; and I; and m is selected from 5; 6; 7 8 9 7 7 7 10, 11, 12, and 13, or salts or stereoisomers thereof.

In some embodiments; another sub set of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5-20 alkyl; C5-20 alkenyl; -R*YR"; -YR"; and -R"M’R’; R2 and R3 are independently selected from the group consisting of H; CH4 alkyl; C244 alkenyl; ; -YR"; and -R*OR"; or R2 and R3; er with the atom to which they are attached; form a cycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle; -(CH2)nQ; -(CH2)nCHQR; -CHQR; -CQ(R)2; and unsubstituted CH, alkyl; where Q is selected from a C3-6 carbocycle, a 5- to l4-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXHZ, -CN, -C(0)N(R)2, -N(R)C(0)R, (O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, )2C(O)OR, and each n is ndently selected from 1, 2, 3, 4, and 5, each R5 is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R6 is independently selected from the group consisting of C1-3 alkyl, C23 alkenyl, and H, M and M’ are independently selected from -C(O)O-, -OC(O)—, -C(O)N(R’)-, -N(R’)C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group, R7 is ed from the group consisting of C1-3 alkyl, C23 l, and H, each R is independently ed from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R’ is ndently selected from the group consisting of C148 alkyl, C248 alkenyl, -R*YR", -YR", and H, each R" is independently selected from the group ting of C314 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl and C242 alkenyl, each Y is independently a C3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I, and m is selected from 5, 6, 7 8 9 7 7 7 10, 11, 12, and 13, or salts or stereoisomers thereof.

In yet some embodiments, another subset of compounds of Formula (1) includes those in which R1 is ed from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR", and -R"M’R’, R2 and R3 are independently selected from the group consisting of H, C244 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle, R4 is -(CH2)nQ or _(CH2)nCHQR, where Q is -N(R)2, and n is selected from 3, 4, and each R5 is independently ed from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R6 is independently selected from the group consisting of C1-3 alkyl; C23 alkenyl; and H; M and M’ are independently selected from -C(O)O-; -OC(O)-; -C(O)N(R’)-; -N(R’)C(O)-; -C(O)-; -C(S)-; -C(S)S-; -; -CH(OH)-; -P(O)(OR’)O-; -S(O)2-; -S-S-; an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl; C23 alkenyl; and H; each R is independently ed from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R’ is independently selected from the group ting of C148 alkyl; C248 l; -R*YR"; -YR"; and H; each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl; each R* is independently selected from the group consisting of C142 alkyl and C142 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F; Cl; Br; and I; and m is selected from 5; 6; 7 8 9 7 7 7 10, 11, 12, and 13, or salts or stereoisomers thereof.

In yet some embodiments; another subset of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5-20 alkyl; C5-20 alkenyl; -R*YR"; -YR"; and -R"M’R’; R2 and R3 are independently selected from the group consisting of H; C244 alkyl; C244 alkenyl; ; -YR"; and -R*OR"; or R2 and R3; together with the atom to which they are attached; form a cycle or carbocycle; R4 is -(CH2)nQ or _(CH2)nCHQR, where Q is -N(R)2; and n is selected from 3; 4; and each R5 is independently ed from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R6 is independently selected from the group consisting of C1-3 alkyl; C23 alkenyl; and H; M and M’ are independently selected from -C(O)O-; -OC(O)-; -C(O)N(R’)-; -N(R’)C(O)-; -C(O)-; -C(S)-; -C(S)S-; -; -CH(OH)-; -P(O)(OR’)O-; -S(O)2-; an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl; C23 alkenyl; and H; each R is independently selected from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R’ is independently selected from the group consisting of C148 alkyl; C248 alkenyl; -R*YR"; -YR"; and H; each R" is independently ed from the group consisting of C344 alkyl and C344 alkenyl; each R* is independently selected from the group ting of C142 alkyl and C142 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F; Cl; Br; and I; and m is selected from 5; 6; 7 8 9 7 7 7 10, 11, 12, and 13, or salts or stereoisomers thereof.

In still other embodiments; another subset of compounds of a (1) includes those in which R1 is selected from the group consisting of C5-30 alkyl; C5-20 alkenyl; -R*YR"; -YR"; and -R"M’R’; R2 and R3 are independently selected from the group consisting ofC1-14 alkyl; C244 l; -R*YR"; -YR"; and -R*OR"; or R2 and R3; together with the atom to which they are attached; form a heterocycle or carbocycle; R4 is selected from the group consisting of -(CH2)nQ; -(CH2)nCHQR; -CHQR; and -CQ(R)2; where Q is -N(R)2; and n is selected from 1; 2; 3; 4; and 5; each R5 is independently selected from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R6 is independently selected from the group consisting of C1-3 alkyl; C23 alkenyl; and H; M and M’ are independently selected from -; -OC(O)-; -C(O)N(R’)-; -N(R’)C(O)-; -C(O)-; -C(S)-; -; -SC(S)-; -CH(OH)-; -P(O)(OR’)O-; -; -S-S-; an aryl group; and a heteroaryl group; R7 is selected from the group consisting of C1_3 alkyl; C23 alkenyl; and H; each R is independently selected from the group ting of C1_3 alkyl, C23 l, and H, each R’ is independently selected from the group consisting of C148 alkyl, C248 alkenyl, -R*YR", -YR", and H, each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl and C142 alkenyl, each Y is independently a C3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I, and m is selected from 5, 6, 7 8 9 7 7 7 10, 11, 12, and 13, or salts or stereoisomers thereof.

In still other embodiments, another subset of compounds of Formula (1) includes those in which R1 is selected from the group consisting of C5-20 alkyl, C5-20 l, -R*YR", -YR", and ’, R2 and R3 are independently selected from the group consisting ofC1-14 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle, R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2, and n is selected from 1, 2, 3, 4, and 5, each R5 is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R6 is independently selected from the group consisting of C1-3 alkyl, C23 alkenyl, and H, M and M’ are independently selected from -C(O)O-, -OC(O)—, -C(O)N(R’)-, -N(R’)C(O)—, , -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)-, -P(O)(OR’)O-, -, an aryl group, and a heteroaryl group, R7 is selected from the group consisting of C1_3 alkyl, C23 l, and H, each R is independently selected from the group consisting of C1_3 alkyl, C23 alkenyl, and H, each R’ is independently selected from the group ting of C118 alkyl, C218 alkenyl, -R*YR", -YR", and H, each R" is independently selected from the group consisting of C344 alkyl and C344 alkenyl, each R* is independently selected from the group consisting of C142 alkyl and C142 each Y is independently a C3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I, and m is selected from 5,6,7, 8,9, 10, ll, 12, and 13, or salts or stereoisomers thereof.

In certain ments, a subset of compounds of Formula (I) includes those of Formula (IA): M1\RI (\97 R IN 2 R3 (1A)7 or a salt or stereoisomer thereof, wherein l is selected from 1, 2, 3, 4, and 5, m is selected from 5, 6, 7, 8, and 9, M1 is a bond or M’, R4 is unsubstituted C1-3 alkyl, or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, )N(R)2, -N(R)C(O)R, -N(R)S(O)2R, g, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl, or heterocycloalkyl, M and M’ are independently selected from -C(O)O-, -OC(O)—, -C(O)N(R’)—, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group, and R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, and C244 alkenyl. 2O In some embodiments, a subset of nds of Formula (I) includes those of Formula (IA),, a (II), or a salt or stereoisomer thereof, wherein l is selected from 1, 2, 3, 4, and 5, m is selected from 5, 6, 7, 8, and 9, M1 is a bond or M’, R4 is unsubstituted C1-3 alkyl, or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2; M and M’ are independently selected from -C(O)O-, -OC(O)—, -C(O)N(R’)—, -P(O)(OR’)O-, an aryl group, and a heteroaryl group, and R2 and R3 are ndently selected from the group consisting of H, CH4 alkyl, and C244 alkenyl.

In certain embodiments, a subset of compounds of Formula (I) es those of Formula (II): M1\RI R3 (II) or a salt or stereoisomer thereof, wherein l is selected from 1 ,2, 3, 4, and 5, M1 is a bond or M’, R4 is unsubstituted C1-3 alkyl, or -(CH2)nQ, in which n is 2, 3, or 4, and Q is OH, - ’HC(S)N(R)2, -NHC(0)N(R)2, (0)R, -N(R)S(O)2R, -N(R)Rs, -NHC(=NR9)N(R)2, - ’HC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl, or heterocycloalkyl, M and M’ are independently selected from -, —, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group, and R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, and C244 alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula (II), or a salt or stereoisomer thereof, wherein l is selected from 1, 2, 3, 4, and 5, M1 is a bond or M’, R4 is unsubstituted C1-3 alkyl, or -(CH2)nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2, M and M’ are independently selected from -C(O)O-, -OC(O)—, -C(O)N(R’)-, -P(O)(OR’)O-, an aryl group, and a aryl group, and 2O R2 and R3 are independently selected from the group consisting of H, CH4 alkyl, and C244 alkenyl.

In some embodiments, the compound of formula (I) is of the formula (11a), (1121), or a salt thereof, wherein R4 is as described above.

In some embodiments, the nd of formula (I) is of the formula (IIb), O J\/\/\/\/ Rf M1m 0 O (IIb), or a salt thereof, wherein R4 is as described above.

In some embodiments, the compound of formula (I) is of the formula (IIc), (110), or a salt thereof, wherein R4 is as described above.

In some embodiments, the nd of formula (I) is of the formula (He): (116), or a salt thereof, wherein R4 is as described above.

In some embodiments, the compound of formula (IIa), (IIb), (IIc), or (IIe) comprises an R4 which is selected from -(CH2)nQ and -(CH2)nCHQR, wherein Q, R and n are as de?ned above.

In some embodiments, Q is selected from the group consisting of -OR, -OH, -O(CH2)nN(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, (0)2R, -N(R)C(0)N(R)2, -N(H)C(0)N(R)2, (0)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, (S)N(H)(R), and a heterocycle, wherein R is as d above. In some aspects, n is l or 2. In some embodiments, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2.

In some embodiments, the compound of formula (I) is of the formula (IId), HO/HFN/ Rll (R5 0 R3 R6 m Y 0 R2 (11d), or a salt or isomer thereof, wherein n is 2, 3, or 4, and m, R’, R", and R2 h R6 are as described . For example, each of R2 and R3 may be independently selected from the group consisting of C544 alkyl and C544 alkenyl, n is selected from 2, 3, and 4, and R’, R", R5, R6 and m are as de?ned above.

In some s of the compound of formula (IId), R2 is Cg alkyl. In some aspects of the compound of formula (IId), R3 is C5-C9 alkyl. In some aspects of the compound of formula (IId), m is 5, 7, or 9. In some aspects of the compound of formula (IId), each R5 is H.

In some aspects of the compound of formula (IId), each R6 is H.

In another aspect, the present application provides a lipid composition (e.g., a lipid nanoparticle (LNP)) sing: (1) a compound having the formula (I), (2) ally a helper lipid (e.g. a phospholipid), (3) optionally a structural lipid (e.g. a sterol), and (4) optionally a lipid conjugate (e.g. a pid). In exemplary embodiments, the lipid composition (e.g., LNP) further comprises a polynucleotide encoding one or more cancer epitope polypeptides, e.g., a polynucleotide encapsulated therein.

As used herein, the term "alkyl" or "alkyl group" means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, ?ve, six, seven, eight, nine, ten, , twelve, thirteen, en, ?fteen, sixteen, seventeen, eighteen, nineteen, , or more carbon atoms).

The notation "CH4 alkyl" means a linear or branched, saturated hydrocarbon 2O including l-l4 carbon atoms. An alkyl group can be ally substituted.

As used herein, the term yl" or "alkenyl group" means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, ?ve, six, seven, eight, nine, ten, , twelve, thirteen, fourteen, ?fteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond.

The notation "C244 alkenyl" means a linear or branched hydrocarbon including 2-14 carbon atoms and at least one double bond. An alkenyl group can include one, two, three, four, or more double bonds. For example, C18 alkenyl can include one or more double bonds.

A C18 alkenyl group including two double bonds can be a linoleyl group. An alkenyl group can be optionally substituted.

As used herein, the term "carbocycle" or "carbocyclic group" means a mono- or multi-cyclic system including one or more rings of carbon atoms. Rings can be three, four, ?ve, six, seven, eight, nine, ten, eleven, twelve, en, fourteen, or ?fteen membered rings.

The notation "C3-6 carbocycle" means a carbocycle including a single ring having 3-6 carbon atoms. Carbocycles can include one or more double bonds and can be aromatic (e.g., aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and l,2-dihydronaphthyl groups. Carbocycles can be optionally substituted.

As used , the term "heterocycle" or "heterocyclic group" means a mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms can be, for example, nitrogen, oxygen, or sulfur atoms. Rings can be three, four, ?ve, six, seven, eight, nine, ten, , or twelve ed rings.

Heterocycles can include one or more double bonds and can be aromatic (e.g., heteroaryl groups). Examples of heterocycles include imidazolyl, olidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, idinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. Heterocycles can be optionally substituted.

As used herein, a "biodegradable group" is a group that can facilitate faster lism of a lipid in a subject. A biodegradable group can be, but is not limited to, -C(O)O-, -OC(O)—, -C(O)N(R’)—, -N(R’)C(O)—, , -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)—, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a aryl group.

As used herein, an "aryl group" is a carbocyclic group including one or more aromatic rings. es of aryl groups e phenyl and naphthyl groups.

As used herein, a "heteroaryl group" is a heterocyclic group ing one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted. For example, M and M’ can be selected from the non-limiting group consisting of optionally substituted , oxazole, and le. In the formulas herein, M and M’ can be independently selected from the list of radable groups above.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups can be optionally substituted unless otherwise speci?ed. Optional substituents can be selected from the group consisting of, but are not limited to, a n atom (e.g., a chloride, bromide, e, or iodide group), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl, -OH), an ester (e.g., -C(O)OR or -OC(O)R), an aldehyde (e.g., -C(O)H), a carbonyl (e.g., -C(O)R, alternatively represented by C=O), an acyl halide (e.g., -C(O)X, in which X is a halide selected from bromide, ?uoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy (e.g., -OR), an acetal (e.g., -C(OR)2R"", in which each OR are alkoxy groups that can be the same or different and R"" is an alkyl or alkenyl group), a ate (e.g., P(O)43') a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulf1nic acid (e.g., -S(O)OH), a sulfonic acid (e.g., -S(O)20H), a thial (e.g., -C(S)H), a sulfate (e.g., S(O)42'), a sulfonyl (e.g., -S(O)2-), an amide (e.g., -C(O)NR2, or -N(R)C(O)R), an azido (e.g., -N3), a nitro (e.g., -N02), a cyano (e.g., -CN), an isocyano (e.g., -NC), an acyloxy (e.g., -OC(O)R), an amino (e.g., -NR2, -NRH, or -NH2), a carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), a sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, or -N(H)S(O)2H), an alkyl group, an alkenyl group, and a cyclyl (e.g., carbocyclyl or cyclyl) group.

In any of the preceding, R is an alkyl or alkenyl group, as de?ned herein. In some embodiments, the sub stituent groups themselves can be further tuted with, for example, one, two, three, four, ?ve, or siX substituents as de?ned herein. For example, a CH, alkyl group can be further substituted with one, two, three, four, ?ve, or siX substituents as described herein.

The compounds of any one of ae (I), (IA), (II), (IIa), (IIb), (11c), (11d), and (He) include one or more of the following features when able.

In some embodiments, R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is selected from a C3-6 ycle, - to 14- membered aromatic or non-aromatic heterocycle having one or more atoms selected from N, O, S, and P, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXHz, -CN, -N(R)2, -C(0)N(R)2, -N(R)C(0)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, (S)N(R)2, and -C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 2O 4, and 5.

In another ment, R4 is selected from the group ting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is selected from a C3-6 carbocycle, a 5- to l4-membered aryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -C(R)N(R)2C(O)OR, and a - to l4-membered heterocycloalkyl having one or more heteroatoms ed from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, and C1-3 alkyl, and each n is independently selected from 1 2 3 4 and 5. 7 7 7 7 In another embodiment, R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is selected from a C3-6 carbocycle, a 5- to l4-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, R, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -C(R)N(R)2C(O)OR, and each n is independently ed from 1, 2, 3, 4, and 5, and when Q is a 5- to l4-membered heterocycle and (i) R4 is -(CH2)nQ in which n is l or 2, or (ii) R4 is -(CH2)nCHQR in which n is l, or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to bered heteroaryl or 8- to l4-membered heterocycloalkyl.

In r ment, R4 is selected from the group consisting of a C34; carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is selected from a C34; carbocycle, a 5- to l4-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5.

In another embodiment, R4 is unsubstituted C1-4 alkyl, e.g., unsubstituted methyl.

In certain embodiments, the disclosure provides a compound having the Formula (I), wherein R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2, and n is selected from 3, 4, and In certain embodiments, the disclosure provides a compound having the a (I), wherein R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2, and n is selected from 1, 2, 3, 4, and 5.

In certain embodiments, the disclosure es a compound having the Formula (I), wherein R2 and R3 are independently selected from the group consisting of C244 alkyl, C244 alkenyl, , -YR", and -R*OR", or R2 and R3, together with the atom to which they are 2O attached, form a heterocycle or carbocycle, and R4 is -(CH2)nQ or nCHQR, where Q is -N(R)2, and n is selected from 3, 4, and 5.

In n embodiments, R2 and R3 are independently selected from the group consisting of C244 alkyl, C244 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle.

In some embodiments, R1 is ed from the group consisting of C5-20 alkyl and C5-20 alkenyl.

In other embodiments, R1 is selected from the group consisting of -R*YR", -YR", and -R"M’R’.

In certain ments, R1 is selected from -R*YR" and -YR". In some embodiments, Y is a cyclopropyl group. In some embodiments, R* is Cg alkyl or C8 alkenyl.

In certain embodiments, R" is C342 alkyl. For example, R" can be C3 alkyl. For e, R" can be C4-g alkyl (e.g., C4, C5, C6, C7, or Cg alkyl).

In some embodiments, R1 is C5-20 alkyl. In some embodiments, R1 is C6 alkyl. In some embodiments, R1 is Cg alkyl. In other embodiments, R1 is C9 alkyl. In certain embodiments, R1 is C14 alkyl. In other embodiments, R1 is C18 alkyl.

In some embodiments, R1 is C5-20 alkenyl. In certain embodiments, R1 is C18 alkenyl.

In some embodiments, R1 is linoleyl.

In certain embodiments, R1 is branched (e.g., decanyl, undecanyl, dodecanyl, tridecanyl, tetradecanyl, 2-methylundecanyl, 2-methyldecanyl, 3-methylundecan- 3-yl, 4-methyldodecanyl, or heptadecayl). In n embodiments, R1 is In certain embodiments, R1 is unsubstituted C5.20 alkyl or C5-20 alkenyl. In certain embodiments, R’ is substituted C5-20 alkyl or C5-20 alkenyl (e.g., substituted with a C3-6 carbocycle such as l-cyclopropylnonyl).

In other embodiments, R1 is -R"M’R’.

In some ments, R’ is selected from -R*YR" and -YR". In some embodiments, Y is C3-g cycloalkyl. In some embodiments, Y is C640 aryl. In some ments, Y is a cyclopropyl group. In some embodiments, Y is a cyclohexyl group. In certain embodiments, R* is C1 alkyl.

In some ments, R" is selected from the group consisting of C342 alkyl and C342 alkenyl. In some embodiments, R" adjacent to Y is C1 alkyl. In some embodiments, R" adjacent to Y is C4-9 alkyl (e.g., C4, C5, C6, C7 or C8 or C9 alkyl).

In some ments, R’ is selected from C4 alkyl and C4 alkenyl. In certain embodiments, R’ is selected from C5 alkyl and C5 l. In some embodiments, R’ is selected from C6 alkyl and C6 alkenyl. In some embodiments, R’ is selected from C7 alkyl and C7 alkenyl. In some ments, R’ is ed from C9 alkyl and C9 alkenyl.

In other embodiments, R’ is selected from C11 alkyl and C11 alkenyl. In other embodiments, R’ is selected from C12 alkyl, C12 l, C13 alkyl, C13 alkenyl, C14 alkyl, C14 l, C15 alkyl, C15 alkenyl, C16 alkyl, C16 alkenyl, C17 alkyl, C17 alkenyl, C18 alkyl, and C18 alkenyl. In certain embodiments, R’ is branched (e.g., decanyl, undecanyl, dodecan- 4-yl, tridecanyl, tetradecanyl, 2-methylundecanyl, 2-methyldecanyl, 3- methylundecanyl, 4-methyldodecanyl or heptadecayl). In certain embodiments, R’ is In certain embodiments, R’ is unsubstituted C1.1g alkyl. In certain embodiments, R’ is substituted C1-1g alkyl (e. g., C145 alkyl tuted with a C3-6 carbocycle such as l- cyclopropylnonyl).

In some embodiments, R" is selected from the group consisting of C3.14 alkyl and C3-14 alkenyl. In some ments, R" is C3 alkyl, C4 alkyl, C5 alkyl, C; alkyl, C7 alkyl, or Cg alkyl. In some embodiments, R" is C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, or C14 alkyl.

In some embodiments, M’ is -C(O)O-. In some embodiments, M’ is -OC(O)-.

In other embodiments, M’ is an aryl group or heteroaryl group. For example, M’ can be selected from the group consisting of phenyl, oxazole, and thiazole.

In some embodiments, M is -C(O)O- In some ments, M is -OC(O)-. In some embodiments, M is -C(O)N(R’)—. In some embodiments, M is -P(O)(OR’)O-.

In other embodiments, M is an aryl group or heteroaryl group. For e, M can be selected from the group consisting of phenyl, oxazole, and thiazole.

In some embodiments, M is the same as M’. In other embodiments, M is different from M’.

In some embodiments, each R5 is H. In certain such embodiments, each R6 is also H.

In some ments, R7 is H. In other ments, R7 is C1_3 alkyl (e.g., methyl, ethyl, propyl, or i-propyl).

In some ments, R2 and R3 are independently C544 alkyl or C544 alkenyl.

In some embodiments, R2 and R3 are the same. In some embodiments, R2 and R3 are Cg alkyl. In certain embodiments, R2 and R3 are C2 alkyl. In other embodiments, R2 and R3 are C3 alkyl. In some embodiments, R2 and R3 are C4 alkyl. In certain embodiments, R2 and R3 are C5 alkyl. In other embodiments, R2 and R3 are C6 alkyl. In some embodiments, R2 and R3 are C7 alkyl.

In other embodiments, R2 and R3 are different. In certain embodiments, R2 is Cg alkyl.

In some embodiments, R3 is C1-7 (e.g., C1, C2, C3, C4, C5, C6, or C7 alkyl) or C9 alkyl.

In some embodiments, R7 and R3 are H.

In certain embodiments, R2 is H.

In some embodiments, m is 5, 7, or 9.

In some embodiments, R4 is selected from nQ and -(CH2)nCHQR.

In some embodiments, Q is ed from the group consisting of -OR, -OH, -O(CH2)nN(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, (0)2R, -N(R)C(0)N(R)2, -N(H)C(0)N(R)2, -N(H)C(0)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -C(R)N(R)2C(O)OR, a carbocycle, and a heterocycle.

In certain embodiments, Q is -OH.

In certain embodiments, Q is a substituted or unsubstituted 5- to 10- membered aryl, e.g., Q is an imidazole, a pyrimidine, a , 2-amino-l,9-dihydro-6H—purin oneyl (or guaninyl), adeninyl, cytosin-l-yl, or uracil-l-yl. In certain embodiments, Q is a substituted 5- to l4-membered heterocycloalkyl, e.g., substituted with one or more substituents selected from oxo (=0), OH, amino, and C13 alkyl. For e, Q is 4- methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, or isoindolinyl-l,3-dione.

In certain embodiments, Q is an tituted or substituted C640 aryl (such as phenyl) or C3-6 lkyl.

In some embodiments, n is 1. In other embodiments, n is 2. In further embodiments, n is 3. In certain other embodiments, n is 4. For example, R4 can be -(CH2)2OH. For example, R4 can be -(CH2)3OH. For e, R4 can be -(CH2)4OH. For example, R4 can be benzyl.

For example, R4 can be 4-methoxybenzyl.

In some embodiments, R4 is a C3-6 carbocycle. In some embodiments, R4 is a C3-6 cycloalkyl. For example, R4 can be cyclohexyl ally substituted with e.g., OH, halo, CH, alkyl, etc. For example, R4 can be 2-hydroxycyclohexyl.

In some embodiments, R is H.

In some embodiments, R is unsubstituted C1-3 alkyl or unsubstituted C2-3 alkenyl. For e, R4 can be (OH)CH3 or -CH2CH(OH)CH2CH3.

In some embodiments, R is substituted C1-3 alkyl, e.g., CH2OH. For example, R4 can be -CH2CH(OH)CH2OH.

In some embodiments, R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle. In some embodiments, R2 and R3, together with the atom to which they are attached, form a 5- to l4- membered aromatic or non-aromatic heterocycle having one or more heteroatoms selected from N, O, S, and P. In some embodiments, R2 and R3, together with the atom to which they are attached, form an optionally substituted C3.20 carbocycle (e.g., C348 carbocycle, C345 carbocycle, C3-12 carbocycle, or C340 carbocycle), either aromatic or non-aromatic. In some embodiments, R2 and R3, together with the atom to which they are attached, form a C3-6 carbocycle. In other embodiments, R2 and R3, together with the atom to which they are attached, form a C6 carbocycle, such as a cyclohexyl or phenyl group. In n embodiments, the heterocycle or C3-6 carbocycle is substituted with one or more alkyl groups (e.g., at the same ring atom or at adjacent or non-adjacent ring atoms). For example, R2 and R3, together with the atom to which they are attached, can form a cyclohexyl or phenyl group bearing one or more C5 alkyl substitutions. In certain embodiments, the heterocycle or C3-6 carbocycle formed by R2 and R3, is substituted with a carbocycle groups. For example, R2 and R3, together with the atom to which they are attached, can form a cyclohexyl or phenyl group that is substituted with cyclohexyl. In some embodiments, R2 and R3, together with the atom to which they are attached, form a C745 carbocycle, such as a cycloheptyl, cyclopentadecanyl, or naphthyl group.

In some embodiments, R4 is selected from nQ and -(CH2)nCHQR. In some ments, Q is selected from the group ting of -OR, -OH, -O(CH2)nN(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, (O)2R, -N(H)S(O)2R, -N(R)C(0)N(R)2, -N(H)C(0)N(R)2, -N(H)C(0)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), and a heterocycle. In other embodiments, Q is selected from the group consisting of an imidazole, a pyrimidine, and a purine.

In some embodiments, R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle. In some embodiments, R2 and R3, together with the atom to which they are attached, form a C3-6 carbocycle, such as a phenyl group. In certain embodiments, the heterocycle or C3-6 carbocycle is substituted with one or more alkyl groups (e.g., at the same ring atom or at adjacent or non-adjacent ring atoms). For example, R2 and R3, together with the atom to which they are attached, can form a phenyl group bearing one or more C5 alkyl tutions.

In some embodiments, a subset of compounds of a (1) includes those of Formula (IIa), (IIb), (IIc), or (He): (11a), (Kb), (IIc), or 0 0 (He), or a salt or isomer thereof, wherein R4 is as described herein.

In some embodiments, a subset of compounds of Formula (1) includes those of Formula (11d): 0 R2 (11d), or a salt or isomer thereof, wherein n is 2, 3, or 4, and m, R’, R", and R2 through R6 are as described herein. For example, each of R2 and R3 may be independently selected from the group consisting of C544 alkyl and C544 alkenyl.

In some embodiments, the pharmaceutical itions of the present sure, the compound of formula (I) is selected from the group consisting of: HONNWNgo/m (Compound 1), \/\/\/\/ Hommm (Compound 2), NNKW\/\ (Compound 3), (Compound 4), 0 0 (Compound 5), HO/\/N\/\/\/:\ O 0 (Compound 6), o 0 (Compound 7), N/? W "NWNWVZLm 0 0 (Compound 8), 0 WOW *MWV}m 0 0 (Compound 9), HO/Lval1::2O O (Compound 10), mimom (Compound 11), WEmom (Compound 12), dwim (Compound 13), WVWVO \ "valm 0 0 (Compound 14), \ WNWm o 0 (Compound 15), "NW";m 0 0 (Compound 16), MOM/W /N\/\o/\/N\/\/\/1m 0 O und 17), HO" Manx (Compound 18), 0 0 (Compound 19), HO/Wmom (Compound 20), NC Wm LLWW/\/N O 0 (Compound 21), ::Hgm (Compound 22), W W1m 0 0 (Compound 23), HWNWNZLm 0 0 und 24), HO/\,N\/\/\/1m 0 0 (Compound 25), HO/\/NWV)12% O 0 (Compound 26), HO Wm/\/N O O (Compound 27), WV) LIAWN O 0 (Compound 28), HONNMm 0 0 (Compound 29), HO MOm/\/ N 0 (Compound 30), HO/\/ NWV; 0 0 (Compound 31), Ho/\/Nx/Wl /<:::/\V/ O 0 (Compound 32), HO/\/N\/\/\/1 / O 0 (Compound 33), HO/\/N\/\/\/1 / O O (Compound 34), HO/\/N\/\/\/1 /C/\/\/ O O (Compound 35), HO \/\/\/1 £\/\/\/ 0 O (Compound 36), (Compound 37), und 38), (Compound 39), (Compound 40), /N VVVZLm 0 0 (Compound 41), /N VVVZLm 0 0 (Compound 42), Y? MO 0 0 (Compound 43), H2 m M NWNMQm 0 0 (Compound 44), H2N§NW1W "WNW?m (Compound 45), ONCNWNWVZLLIZ/C (Compound 46), "WNWV}m 0 0 (Compound 47), (Compound 48), und 49), (Compound 50), (Compound 51), (Compound 52), (Compound 53), (Compound 54), "O"Mam und 55), "O"mom (Compound 56), MOM/W HO" mom (Compound 57), MOM/W HO/\/ \/\/\/O:\OJ::\ (Compound 58), MOM/W Ho/\/ \/\/\/0101/ (Compound 59), MOW/V "3" W033 (Compound 60), HO" "72933 (Compound 61), Howmom (Compound 62), HONNvvvl Q/W O 0 (Compound 63), HO/\/N\/\/\/1 O/VV o 0 (Compound 64), wwpmW0 (Compound 65), 0Om und 66), (Compound 67), 033% (Compound 68), HOD/\NWOWO 0Om (Compound 69), (Compound 7O), om (Com ound7p 1)) Om (Compound 72), oOm (Compound 73), om (Compound 74), OOm (Compound 75), Om (Compound 76)7 Om (Compound 77), HO\/\N/\/\/\/\n/O):\/\/\/\0WOW 0 (Compound 78), HOWNWVWOW 0W (Compound 79), HO\/\N/\/\/\/Yo\/\/\/\/\ (Compound 80), HO\/\N/\/\/\/\n/O\/\/W\ (Compound 81), HOWNWOW om (Compound 82), HO\/\N/\/\/\/\n/O\/\/\/\/\ (Compound 83), (Compound 84), HO\/\NWOW 0?x (Compound 85), Ho\/\N/\/\/\/\n/O\6\/\/\/\0WOW 0 (Compound 86), 0Om (Compound 87), und 88), 0 13% (Compound 89), 0 \EV/\:\/\//\\ (Compound 90), 0 (IX (Compound 91), 0 CNN" (Compound 92), (Compound 93), (Compound 94), (Compound 95), (Compound 96), (Compound 97), (Compound 98), und 99), 00m? (Compound 100), UwN/VVWOW und 101), MeO©/\ K/N\/\N/\/\/\/\n/O\/\/\/\/\m 0Om (Compound 102), 0 033$ (Compound 103), HO\/\N/\/\/\n/ (Compound 104), (Compound 105), (Compound 106), om (Compound 107), YHWNMO/E/VWVZ/ O (Compound 108), O " NMm §S’ \/\/ 0 0 (Compound 109), (Compound 1 10), (Compound 111), (Compound 1 12), (Compound 113), (Compound 1 14), O/\/\/\/\/ MO/Q/WV/ I (Compound 115), H2N\S/N\\(\\N O/\/\/\/\/ VNWNWG0% (Compound 116), H NH2 o N\\( O/\/\/\/\/ NVNWNWO/m\ O (Compound "7), O/\/\/\/\/ O 033 HONNWG (Compound 118), 0 D/W O (Compound 119), 0 DVW Ho/VNWO (Compound 120), O/\/\/\/\/ H N NW0O/C::/\/\:/2W (Compound 121), O (Compound 122), \NMVWOW (Compound 123), O \C\/\/:\/:\ (Compound 124), HOWNWO/m 0 (Compound 125), und 126), CW (Compound 127), (Compound 128), 0 (Compound 129), (Compound 13 0), CW (Compound 131), \/\/\/V (Compound 132), (Compound 133), (Compound 134), (Compound 135), 0 und 13 6), HO/\/NWVWV (Compound 137), (Compound 13 8), (Compound 13 9), (Compound 140), (Compound 141), O 0 und 142), HO WWW/0m/\,N 0 (Compound 143), W (Compound 144), (Compound 145), (Compound 146), (Compound 147), (Compound 148), (Compound 149), und 150), (Compound 151), HO\/\N/\/\/\/\n/O\/\/\/\/\ (Compound 152), (Compound 153), und 154), (Compound 155), (Compound 156), (Compound 157), (Compound 158), (Compound 159), W (Compound 160), 0 (X? (Compound 161), (Compound 162), K/VI O Om (Compound 163), Wow0 (Compound 164), WO HO\/\N OJJ\/\/\/\/\ K/\/\L O Om und 165), wwmomO 0 (Compound 166), 0 (Compound 167), N\\\N \NXNMNW/?ro|| H O\/\/\/\/\ 0 (Compound 168), O iMMNMO O\/\/\/\/\ 0 (Compound 169), 0 (Compound 170), (Compound 171), (Compound 172), 0 (Compound 173), 0 (Compound 174), \NAOMN/eroH 0 (Compound 175), {rowmwmO O\/\/\/\/\ 0 (Compound 176), 80fI\I'N\N/\/\N/\/\/\/\g/O(IX 0 und 177), /O\)kN/\/\N/W\/\n/OH 0 (Compound 178), N\ /\/\ 0 Ni N NW \9’ o O\/\/\/\/\ 0 (Compound 179), HO\/\NH WOW0 (Compound 180), \0 HMNMO O\/\/\/\/\ 0 (Compound 181), E "MNMO HN\ O O\/\/\/\/\ 0 (Compound 182), HO\/\ und 183), o (m (Compound 184), HO (Compound 185), (Compound 186), Om (Compound 187), (Compound 188), 0 (Compound 189), HO\/\N/\/\/\/\n/0\/\/\/\n/O\ WAG 0 0 0m (Compound 190), (Compound 191), HOwN/\/\/\/\n/O\/\/\/\n/O\ O o 0Om (Compound 192), «wwwmH o O\/\/\/\/\ 0 (Compound 193), uMmeH o O\/\/\/\/\ 0 und 194), ?NMNAM/YOO O\/\/\/\/\ 0 (Compound 195), )LNMNA/WYO O\/\/\/\/\ 0 (Compound 196), Q/ 0 0%N/\/\N/\/\/\/\n/OH om O\/\/\/\/\ 0 (Compound 197), O (Compound 198), OXNMNMWO \/’ om o (Compound 199), /\/\N/\/\/\/\n/0\<::::\//\\0WOW 0 (Compound 200), iNMNM/V?ro0% o (Compound 201), 0A0 0W O\/\/\/\/\ o (Compound 202), )LNMN/WVWl/Omo\ o O\/\/\/\/\ O (Compound 203), )LNMNWI/OmOH 0 O\/\/\/\/\ o (Compound 204), \OJLNMN/WWO OH O O\/\/\/\/\ O (Compound 205), oé?\l}l/\/\N/\/\/\/\n/O OH 0 0 (Compound 206), mevwom0 0 und 207), O\/\/\/\/\ O (Compound 208), | HN/\/\N/\/\/\/\n/O O\/\/\/\/\ O (Compound 209), \HAMMNAM/Erom| 0 (Compound 210), \NANMN/VWYO| | H (Compound 211), H N/\/\N/\/\/\/\n/OH O\/\/\/\/\ O (Compound 212), \N /\/\/\/\n/O | H O (Compound 213), (Compound 214), (Compound 215), (Compound 216), (Compound 217), (Compound 218), (Compound 219), (Compound 220), (Compound 221), (Compound 222), (Compound 223), und 224), (Compound 225), (Compound 226), HO’N\n/\/\N/\/\/\/\n/O O O O\/\/\/\/\ O (Compound 227), \O,N\n/\/\N/\/\/\/\n/O O O O\/\/\/\/\ O (Compound 228), \O’NWNMO O\/\/\/\/\ O (Compound 229), (Compound 230), und 231), (Compound 232), and salts or stereoisomers thereof.

In some embodiments, a nanoparticle ses the following compound: I 0 szp-ANWW9w (Compound 343), or salts or stereoisomers thereof.

In other embodiments, the compound of a (I) is selected from the group consisting of Compound l-Compound 147, or salt or stereoisomers thereof.

In some embodiments ionizable lipids including a l piperazine moiety are provided. The lipids described herein may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents to mammalian cells or . For example, the lipids described herein have little or no genicity. For example, the lipid compounds disclosed hereinhave a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For e, a formulation comprising a lipid disclosed herein and a therapeutic or lactic agent has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or lactic agent.

In some embodiments, the delivery agent comprises a lipid compound having the formula (III) R3 (111) 7 tis l or 2, A1 and A2 are each independently selected from CH or N, 2O Z is CH2 or absent wherein when Z is CH2, the dashed lines (1) and (2) each represent a single bond, and when Z is absent, the dashed lines (1) and (2) are both absent, R1, R2, R3, R4, and R5 are independently selected from the group consisting of C5-20 alkyl, C5_20 alkenyl, -R"MR’, -R*YR", -YR", and -R*OR", each M is independently selected from the group consisting of -C(O)O-, —, -OC(O)O-, -C(O)N(R’)—, -N(R’)C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)—, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group, X1, X2, and X3 are independently selected from the group consisting of a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, , -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, -CH2-OC(O)—, -CH(OH)-, -C(S)-, and -CH(SH)-, each Y is independently a C3-6 ycle, each R* is independently selected from the group consisting of C142 alkyl and C242 each R is independently selected from the group consisting of C1.3 alkyl and a C3-6 ycle, each R’ is independently selected from the group consisting of C142 alkyl, C242 alkenyl, and H, and each R" is independently selected from the group consisting of C342 alkyl and C342 alkenyl, wherein when ring A is yNd then i) at least one of X1, X2, and X3 is not -CH2-, and/or ii) at least one of R1, R2, R3, R4, and R5 is -R"MR’.

In some embodiments, the compound is of any of formulae (IIIal)-(IIIa6): X3 N T1 (\N/ \/ \R5 N x1 R2/ \/ \TAXZ,NQ R3 (IIIal), x3 N T1 \R5 N X1 ,N R2/ \/ \TAXZ R3 (IIIa2), T1 R5 N x1 R2/ \/ \TAXZ R3 (IIIa3), [it x1 N T4 / \/ \ /\ 2/ R X3 N 2 N x I v\R5 R3 (IIIa4), N X1 T4 / \/ \ /\ 2 3 R N x x N I V\R5 R3 (IIIaS), or l R4 Rz/NVX\N/\X2/N1 X3 'L\ I V R3 (IIIa6).

The compounds of Formula (111) or any of (IIIal)—( IIIa6) include one or more of the ing features when applicable. 2/0}: In some embodiments, ring A is or In some embodiments, ring A is "gs/N In some embodiments, ring A is In some embodiments, ring A is or In some ments, ring A iswit/c? wherein ring, in which the N atom is connected with X2.

In some embodiments, Z is CH2~ In some embodiments, Z is absent.

In some embodiments, at least one of A1 and A2 is N.

In some embodiments, each of A1 and A2 is N.

In some embodiments, each of A1 and A2 is CH.

In some embodiments, A1 is N and A2 is CH.

In some embodiments, A1 is CH and A2 is N.

In some ments, at least one of X1, X2, and X3 is not -CH2-. For example, in certain embodiments, X1 is not -CH2-. In some embodiments, at least one of X1, X2, and X3 is -C(O)-.

In some embodiments, X2 is -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, (O)—, -CH2-, -OC(O)-CH2-, -CH2-C(O)O-, or -CH2-OC(O)-.

In some embodiments, X3 is -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, or -CH2-OC(O)-. In other embodiments, X3 is -CH2-.

In some embodiments, X3 is a bond or —(CH2)2-.

In some embodiments, R1 and R2 are the same. In n embodiments, R1, R2, and R3 are the same. In some ments, R4 and R5 are the same. In certain embodiments, R1, R2, R3, R4, and R5 are the same.

In some embodiments, at least one of R1, R2, R3, R4, and R5 is -R"MR’. In some embodiments, at most one of R1, R2, R3, R4, and R5 is -R"MR’. For example, at least one of R1, R2, and R3 may be -R"MR’, and/or at least one of R4 and R5 is -R"MR’. In certain embodiments, at least one M is -C(O)O-. In some embodiments, each M is -C(O)O-. In some embodiments, at least one M is -OC(O)—. In some ments, each M is -OC(O)—. In some embodiments, at least one M is -OC(O)O-. In some ments, each M is -OC(O)O-.

In some embodiments, at least one R" is C3 alkyl. In certain embodiments, each R" is C3 alkyl. In some embodiments, at least one R" is C5 alkyl. In certain embodiments, each R" is C5 alkyl. In some embodiments, at least one R" is C6 alkyl. In certain embodiments, each R" is C6 alkyl. In some embodiments, at least one R" is C7 alkyl. In certain embodiments, each R" is C7 alkyl. In some embodiments, at least one R’ is C5 alkyl. In certain embodiments, each R’ is C5 alkyl. In other embodiments, at least one R’ is C1 alkyl. In certain embodiments, each R’ is C1 alkyl. In some embodiments, at least one R’ is C2 alkyl. In certain embodiments, each R’ is C2 alkyl.

In some embodiments, at least one of R1, R2, R3, R4, and R5 is C12 alkyl. In certain embodiments, each of R1, R2, R3, R4, and R5 are C12 alkyl.

In certain ments, the compound is selected from the group consisting of: WO (\NNN \/\/\/\/\/\/N\)LN/\/N\} W (Compound 233), N\/\N/\erONN W und 234), W (\NJK/N W (Compound 235), W (\NJK/N /\/\/\/\/N\/\N/\n/N W0 (Compound 236), W NJK/N WRAN/er) W0 (Compound 237), W "Ii/W Nv\N/\",N\) W0 (Compound 23 8), W "Ii/(\AAA N\/\N/\?’N\) W0 (Compound 239), /\/\/\/\/N\/\N/\"/N\) W0 (Compound 240), W NJK/N /\/\/\/\/N\/\N/\n/N\) W0 Compound 241), W NJK/N N\/\N/\",N\) WW0 (Compound 242), W NJK/N N\/\N/\n’N\) W0 (Compound 243), WNNOW N/\/N W (Compound 244), (Compound 245), (Compound 247), (Compound 248), \/\/\/\/\N N@JK/NwN/vvm/WV und 274), \/\/\/\/\N N Wow" (Compound 275), (Compound 276), (\NJK/NwN/VVW N/WNd WV MOW 0 (Compound 277), \/\N/\/\/\/\/ "KW/Nd WW0\ Wo 0 (Compound 278), 0 N\/\ /\/\/\/\/ N N (Compound 279), (\NJK/NwN/VVW NWNd WYO\ Wo 0 (Compound 280), \/\/\/\/\N"OkNwN/VVWW (Compound 281), (Compound 282), NWNW/ NW") W (Compound 283), (Compound 284), (Compound 285), (Compound 286), (Compound 287), (Compound 288), (Compound 289), AOJWNMOJK/NwN/VWW (Compound 290), W\/\/\N"OkNwNA/WVW /\/OW (Compound 291), MOW,"MOJK/NwN/VWW (Compound 292), O NJK/NWNW NOMN W (Compound 293), N Wl\/\/\/\/ \/\g/ (Compound 294), (\NJK/NwNWN N/\ITN\) WOW WOW 0 o (Compound 295), \/\/O 0 (\NfK/mv UNWNQ W WOW 0 (Compound 296), (Compound 297), (Compound 298), \/\/\/\/\N/\/©NJK/NW und 300), (Compound 301), (Compound 302), (\NJK/NV\N/\/\/\/\/ und 303), /\/\/O\EO/\ (\NJK/N\/\N/\/\/\/\/ N/\n/N\) W MOW O (Compound 304), W O W /\/\/\/\/N NJK/Nwm/V (Compound 305), OJK/NwN/VVW \/\/\/\/\N/\n/O W (Compound 306), (Compound 307), (Compound 308), WO N/\/N N\)LN/\n/N\) (Compound 310), (Compound 311), WoM O W /\/\/\/\/N\/\©J\/N\/\N/\/\/\/\/W (Compound 312), WOM LW /\/\/\/\/N\/\C/N N\/\N/\/\/\/\/ (Compound 313), mmNJK/NwN/VVW/\/\/O W (Compound 314), NJJ\/N\/\N/\/\/\/\/ \/\/\/\/\N W (Compound 315), O O W WOMVVOJVNNNW /\/\/\/\/N W (Compound 316), WWOJOVW MNNm (Compound 317), O 0 (Compound 318), EL» W (Compound 319), (\NJK/NwNMVW MONWOV WOW (Compound 320), O (\NJK/NV\N/\/\/W Womb/wk} W W0 und 321), (\NJK/NWNW/V N/WNJ WW 0 (Compound 322), (\NJK/Nvm/VVW N/\n/N\} W 0 (Compound 323), o NJK/NWNWW WOMN W W (Compound 324), und 325), (Compound 326), Wm NLW Vb NKAAA/ (Compound 327), WNLNCNMCW (Compound 328), MAAjiOMNAC/NJK/WW (Compound 329), W(Compound 3 3 0), JOJ\/W N/\C/N N\/\N/\/\/\/\/ MOW W (Compound 331), N/\C/N N\/\N/\/\/\/\/ WOW W (Compound 332), muowowwm 0 W (Compound 333), /\/\/\/\/N\)l\oACNJK/NwN/VW/ K/\/\/\/ (Compound 334), W\/\/\ /\n/O Lmv WVJ mO WV (Compound 335), WW0 (Compound 336), (Compound 337), (Compound 338), 0 (Compound 3 3 9), (\NJK/NwNMAA/ N/\n/N\} WV (Compound 340), and (\NJOK/NwN/VWVW o \oioWN/?rk} W WO (Compound 341).

In some embodiments, the delivery agent ses nd 236.

In some embodiments, the delivery agent comprises a compound having the formula R1 ?AZ/v \R5 R2/ \/\N/\/ 1 (1V), or salts or stereoisomer thereof, wherein A1 and A2 are each independently selected from CH or N and at least one of A1 and A2 is N, Z is CH2 or absent wherein when Z is CH2, the dashed lines (1) and (2) each represent a single bond, and when Z is absent, the dashed lines (1) and (2) are both absent; R1, R2, R3, R4, and R5 are independently selected from the group consisting of C6-20 alkyl and C6-20 alkenyl, wherein when ring A is yNd then i) R1, R2, R3, R4, and R5 are the same, wherein R1 is not C12 alkyl, C18 alkyl, or C18 alkenyl, ii) only one of R1, R2, R3, R4, and R5 is ed from C6-20 alkenyl, iii) at least one of R1, R2, R3, R4, and R5 have a different number of carbon atoms than at least one other of R1, R2, R3, R4, and R5, iV) R1, R2, and R3 are selected from C620 alkenyl, and R4 and R5 are selected from C6-20 alkyl, or V) R1, R2, and R3 are selected from C6-20 alkyl, and R4 and R5 are selected from C6-20 alkenyl.

In some embodiments, the nd is of formula (IVa): (IVa).

The compounds of Formula (IV) or (IVa) include one or more of the following features when applicable.

In some embodiments, Z is CH2~ 2O In some embodiments, Z is absent.

In some embodiments, at least one of A1 and A2 is N.

In some ments, each of A1 and A2 is N.

In some embodiments, each of A1 and A2 is CH.

In some embodiments, A1 is N and A2 is CH.

In some embodiments, A1 is CH and A2 is N.

In some embodiments, R1, R2, R3, R4, and R5 are the same, and are not C12 alkyl, C18 alkyl, or C18 alkenyl. In some embodiments, R1, R2, R3, R4, and R5 are the same and are C9 alkyl or C14 alkyl.

In some embodiments, only one of R1, R2, R3, R4, and R5 is selected from C620 alkenyl. In certain such embodiments, R1, R2, R3, R4, and R5 have the same number of carbon atoms. In some embodiments, R4 is selected from C5-20 alkenyl. For example, R4 may be C12 alkenyl or C18 alkenyl.

In some embodiments, at least one of R1, R2, R3, R4, and R5 have a different number of carbon atoms than at least one other of R1, R2, R3, R4, and R5.

In certain embodiments, R1, R2, and R3 are selected from C620 alkenyl, and R4 and R5 are selected from C620 alkyl. In other embodiments, R1, R2, and R3 are selected from C620 alkyl, and R4 and R5 are selected from C620 alkenyl. In some ments, R1, R2, and R3 have the same number of carbon atoms, and/or R4 and R5 have the same number of carbon atoms. For example, R1, R2, and R3, or R4 and R5, may have 6, 8, 9, 12, 14, or 18 carbon atoms. In some embodiments, R1, R2, and R3, or R4 and R5, are C18 l (e.g., linoleyl).

In some ments, R1, R2, and R3, or R4 and R5, are alkyl groups including 6, 8, 9, 12, or 14 carbon atoms.

In some embodiments, R1 has a different number of carbon atoms than R2, R3, R4, and R5. In other embodiments, R3 has a different number of carbon atoms than R1, R2, R4, and 2O R5. In further embodiments, R4 has a different number of carbon atoms than R1, R2, R3, and In some embodiments, the compound is selected from the group consisting of: M (\N/VN V\/\/N\/\N/\/N\) W (Compound 249), W und 250), W (Compound 251), W und 252), W (\NNNM N\/\N/\/N\) W (Compound 253), W (\NNN N\/\N/\/N\) W (Compound 254), W (\NNN \/\/\/\/\/\/N\/\N’\/N\) (Compound 255), W (Compound 256), W (\NNN — N\/\N/\/N\) MW (Compound 257), W (\N/VN \/\/\/\/\/\/N\/\N/\/N\) W (Compound 258), W (\NNN N\/\N’\/N\) (Compound 259), W (\N/VN _ (Compound 260), (Compound 261), W und 262)7 (Compound 263), I/\/\/\/\/\/\/ W(\NNN N \/\N/\/N\J (Compound 264), (Compound 265), and _ — N\/\N/\/N\) (Compound 266).

In other embodiments, the delivery agent comprises a compound having the formula (V) or salts or stereoisomers thereof, in which A3 is CH or N; A4 is CH2 or NH; and at least one of A3 and A4 is N or NH; Z is CH2 or absent wherein when Z is CH2; the dashed lines (1) and (2) each represent a single bond; and when Z is absent; the dashed lines (1) and (2) are both absent; R1; R2; and R3 are independently selected from the group consisting of C5.20 alkyl; C5- alkenyl; -R"MR’; ; -YR"; and -R*OR"; each M is independently selected from -C(O)O-; -; -C(O)N(R’)-; -N(R’)C(O)-; -C(O)-; -C(S)-; -; -SC(S)-; -CH(OH)-; -P(O)(OR’)O-; -S(O)2-; an aryl group; and a heteroaryl group; X1 and X2 are independently selected from the group consisting of -CH2-; -(CH2)2-; -CHR-; -CHY-; ; -C(O)O-; -OC(O)-; -C(O)—CH2-; -CH2-C(O)-; -C(O)O-CH2-; -OC(O)—CH2-; -CH2-C(O)O-; -CH2-OC(O)-; -CH(OH)-; -C(S)-; and -CH(SH)—; each Y is independently a C3-6 carbocycle; each R* is independently selected from the group consisting of C142 alkyl and C242 alkenyl; each R is independently selected from the group consisting of C1.3 alkyl and a C3-6 carbocycle; 2O each R’ is independently selected from the group consisting of C142 alkyl; C242 alkenyl; and H; and each R" is independently selected from the group consisting of C342 alkyl and C342 alkenyl.

In some ments; the compound is of a (Va): T1 N KNAXZ’Nd R3 (Va).

The compounds of Formula (V) or (Va) include one or more of the following features when applicable.

In some embodiments, Z is CH2~ In some ments, Z is absent.

In some embodiments, at least one of A3 and A4 is N or NH.

In some embodiments, A3 is N and A4 is NH.

In some embodiments, A3 is N and A4 is CH2.

In some ments, A3 is CH and A4 is NH.

In some embodiments, at least one of X1 and X2 is not -CH2-. For e, in certain embodiments, X1 is not -CH2-. In some embodiments, at least one of X1 and X2 is -C(O)—.

In some embodiments, X2 is -C(O)—, -C(O)O-, -OC(O)—, -C(O)—CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)—CH2-, (O)O-, or -CH2-OC(O)-.

In some embodiments, R1, R2, and R3 are independently selected from the group consisting of C320 alkyl and C320 alkenyl. In some embodiments, R1, R2, and R3 are the same. In certain embodiments, R1, R2, and R3 are C6, C9, C12, or C14 alkyl. In other embodiments, R1, R2, and R3 are C18 alkenyl. For example, R1, R2, and R3 may be linoleyl.

In some ments, the compound is selected from the group consisting of: i (Compound 267), (\NNNWNMVW "Nd WW (Compound 268), (\NNNwN/VWVW "Nd W (Compound 269), (WA/WV ‘/\N/\,N\/\N HN\) WW (Compound 270), (\N/\/N N "Nd OW (Compound 271), (\NNNwN — "Ndl (Compound 272), 3i23 | | i E| (Compound 273), and H0 3AMNJI\’N N (Compound 309).

In other embodiments, the delivery agent comprises a compound having the formula (VI): R )Vx4\A - R A7 N \X5/\til/V \R2 R3 (VI), or salts or stereoisomers thereof, in which A; and A7 are each independently selected from CH or N, wherein at least one of A6 and A7 is N, Z is CH2 or absent wherein when Z is CH2, the dashed lines (1) and (2) each represent a single bond, and when Z is , the dashed lines (1) and (2) are both absent, X4 and X5 are independently selected from the group consisting of -CH2-, -, -CHR-, -CHY-, -C(O)—, -C(O)O-, -OC(O)—, -C(O)—CH2-, -CH2-C(O)—, -C(O)O-CH2-, -OC(O)— CH2-, (O)O-, C(O)—, -CH(OH)—, -C(S)—, and -CH(SH)—, R1, R2, R3, R4, and R5 each are independently ed from the group consisting of C5_20 alkyl, C5_20 alkenyl, -R"MR’, , -YR", and -R*OR", each M is independently selected from the group consisting of -C(O)O-, -OC(O)—, -C(O)N(R’)—, -N(R’)C(O)—, -C(O)—, -C(S)—, -C(S)S-, -SC(S)—, -CH(OH)—, -P(O)(OR’)O-, -S(O)2- an aryl group, and a heteroaryl group, each Y is independently a C3-6 carbocycle, each R* is independently selected from the group consisting of C142 alkyl and C242 2O alkenyl, each R is independently selected from the group consisting of C1.3 alkyl and a C3-6 carbocycle, each R’ is independently ed from the group consisting of C142 alkyl, C242 alkenyl, and H, and each R" is independently selected from the group consisting of C342 alkyl and C342 alkenyl.

In some embodiments, R1, R2, R3, R4, and R5 each are independently selected from the group consisting of C6-20 alkyl and C6_20 alkenyl.

In some embodiments, R1 and R2 are the same. In certain embodiments, R1, R2, and R3 are the same. In some embodiments, R4 and R5 are the same. In certain embodiments, R1, R2, R3, R4, and R5 are the same.

In some embodiments, at least one of R1, R2, R3, R4, and R5 is C942 alkyl. In certain embodiments, each of R1, R2, R3, R4, and R5 independently is C9, C12 or C14 alkyl. In certain ments, each of R1, R2, R3, R4, and R5 is C9 alkyl.

In some embodiments, A6 is N and A7 is N. In some embodiments, A6 is CH and A7 is N.

In some embodiments, X4 is-CH2- and X5 is -C(O)—. In some embodiments, X4 and X5 are -C(O)—.

In some ments, when A6 is N and A7 is N, at least one of X4 and X5 is not -CH2-, e.g., at least one of X4 and X5 is -C(O)—. In some embodiments, when A6 is N and A7 is N, at least one of R1, R2, R3, R4, and R5 is -R"MR’.

In some embodiments, at least one of R1, R2, R3, R4, and R5 is not .

In some embodiments, the compound is (\NLNWNWW (Compound 299).

In other embodiments, the delivery agent comprises a compound having the formula: AAA/EM (\NNN — _ N\/\N/\/N\) 2O WNW (Compound 342).

Amine moieties of the lipid compounds disclosed herein can be protonated under certain conditions. For example, the l amine moiety of a lipid according to a (I) is typically protonated (1'.e., positively charged) at a pH below the pKa of the amino moiety and is substantially not charged at a pH above the pKa. Such lipids can be referred to ionizable amino lipids.

In one specific embodiment, the ionizable amino lipid is nd 18. In another embodiment, the ionizable amino lipid is Compound 236.

In some embodiments, the amount the ionizable amino lipid, e.g., compound of formula (I) ranges from about 1 mol % to 99 mol % in the lipid ition.

In one embodiment, the amount of the ionizable amino lipid, e.g., compound of a (I)is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 mol % in the lipid composition.

In one embodiment, the amount of the ble amino lipid, e.g., the compound of formula (I) ranges from about 30 mol % to about 70 mol %, from about 35 mol % to about 65 mol %, from about 40 mol % to about 60 mol %, and from about 45 mol % to about 55 mol % in the lipid composition.

In one speci?c embodiment, the amount of the ionizable amino lipid, e.g., compound of formula (I) is about 50 mol % in the lipid composition.

In on to the ionizable amino lipid disclosed herein, e.g., compound of formula (I), the lipid composition of the pharmaceutical compositions disclosed herein can comprise additional components such as phospholipids, ural lipids, PEG-lipids, and any combination thereof.

Phosp_holip_ids The lipid composition of the pharmaceutical composition disclosed herein can se one or more phospholipids, for example, one or more saturated or (poly)unsaturated olipids or a combination thereof. In general, olipids comprise a phospholipid moiety and one or more fatty acid moieties.

A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, oleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more vely charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. tural phospholipid species including natural species with modi?cations and substitutions ing branching, oxidation, ation, and alkynes are also contemplated.

For example, a phospholipid can be onalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper- catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in ating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

Phospholipids include, but are not limited to, glycerophospholipids such as atidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

Examples of phospholipids include, but are not limited to, the following: :3 0 x/\\‘.»" \v/ xv;\ \ .\ 3/ \ .2- \\_, x \o ,\ ’/\\ x" \02’\\‘ 3\ ~»\ f- }- v, " ‘Nv ? ‘\" . \\ \\\I, \ ._,«\\'lr\\ x" \V)l\\ l#\\~/,\,\\)_,. \ 1.1:) 5 ‘1‘ r3 \ /\ V W \v ‘x/ \x ‘w" \x / \x \ . , r ‘x 4* \(x’ \\ r \Q~-‘§3‘~-g\. "N, -+" .‘ V Q ~ {3‘ ¥>\.

\ N .\ A \ ,\ \ /’ \w’ ‘\.»" \a" ___I" ‘\.»’ \’\,-"‘ a. \\r’ \\ : ‘ 7 .A. «'\, ,Mrrrrr \v"' \ /~- /\, v'\ . ,\ / \ z \/ \ _, \.‘, \/ \Ox \?' \Q..gv {k.0" / \ ‘6. ¢ 5 \- \ ~ \ .-\ ~ ‘5.‘ s: a?!" . » O" \/ \\/"’ \xrrmr'/\\:::::.-’ \,-"' \z" \\r‘ \v" ,~ '\ \ .-" \Vxxxxx ,. \ \\\\\ ‘. . v\\\\» K.

W ~,\ """V ,J' \x/ , ~ \x "\V.» | S? 0 /N\/\O/F|)TO/Y\OJkMA/W_ I O \N\/\ IIDI / / \ — o (ITO/TN) \| S? I O \ || /N+\/\O/|I°\O/Y\OO' O\ and In certain embodiments, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC. In certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IX): \® 0 R1-lit/inO\|/O A IF; rm 0 (IX), or a salt f, wherein: each R1 is independently optionally substituted alkyl, or optionally two R1 are joined together with the intervening atoms to form optionally tuted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl, or optionally three R1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl, nis123456789 7 7 7 7 7 7 7 7 70r107 mis0123456789or10,7 7 7 7 7 7 7 7 7 7 L2-R2 "(2)" L2_R2 A 1s of the formula:. \d or , each ce of L2 is independently a bond or optionally substituted CH, alkylene, wherein one methylene unit of the optionally substituted C14, alkylene is optionally replaced with —o—, -N(RN)-, —s—, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, or -NRNC(O)N(RN)-, each ce of R2 is independently optionally substituted C130 alkyl, optionally substituted C130 alkenyl, or optionally substituted C130 alkynyl, ally wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally tuted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, —o—, —s—, -C(O)-, -C(O)N(RN)-, O)-, -NRNC(O)N(RN)-, -, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, -C(O)S-, -, -C(=NRN)-, N)N(RN)-, -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, S)N(RN)-, -S(O)-, -OS(O)—, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)zO-, -OS(O)20-, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, N(RN)-, -N(RN)S(0)O-, -, -N(RN)S(0)2-, -S(0)2N(RN)-, -N(RN)S(0)2N(RN)-, -OS(0)2N(RN)-, or -N(RN)S(O)20-, each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, ally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, and p is l or 2, provided that the compound is not of the formula: 09 \J: O >T/\/O\IIID|/O(a OJLRZ wherein each instance of R2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted l. i) Phospholip_id Head Modi?cations In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phospholipid head (e.g., a modi?ed choline group). In certain embodiments, a phospholipid with a ed head is DSPC, or analog f, with a modified quaternary amine. For example, in embodiments of Formula (IX), at least one of R1 is not methyl. In certain embodiments, at least one of R1 is not hydrogen or methyl. In certain embodiments, the compound of Formula (IX) is of one of the following formulae: \qh )u 0090 W69 09 71»(D 09 WTN noPOMm Q)N O\|,O A (N O\I,O A (Q n '3 Wm Org/)Mn I: Mm V O V 2O O 7 7 3 )u )v POMm/‘)‘Ne000 ( N6 0960 A Mn \P/ Mm ( V ('5 or a salt f, wherein: eachtisindependentlyl 2 3 4 5 6 7 8 9 or 10, 7 7 7 7 7 7 7 7 7 eachuisindependentlyO l 2 3 4 5 6 7 8 9 or 10, and 7 7 7 7 7 7 7 7 7 7 each V is independently l, 2, or 3.

In certain embodiments, the compound of Formula (IX) is of one of the following formulae: E) (B O O\I/O A W 9 me 03,0 A N‘(")’n P ‘(VTm N(9 090 A < " '3 Wm ('3' / @n \E/ Mm K‘ O 9 9 9 6 G 6 lo 0 lo 0 lo 0 I,o A Ou/O A o\n,o A IF; Mm "M. I: Wm "M, I; Mm o o o e lo le 0 e 0 /N(\\)$V)’nN Ou/O A @o o (\N?nOu/O A P Mm N Ou/ A O$ I: Mm " Mn POMm 0 RN 5 or a salt thereof.

In certain embodiments, a compound of a (IX) is one of the following: Le 09 J 0 \/N/\/O\Il'll,/O OJJ\/\/\/\/\/\/\/\ K 0 (Compound 400) L®/\/O\C')/Ov[We O \/N If: 0 K 0 (Compound 401) (6 O G O k/NNO\,FI,"OO \/E /U\/\/\/\/\/\/\/\o K/ 0 (Compound 402) (G) 0 e O k/NNO\.FI.’/OO \/EWo K/ 0 (Compound 403) 930*." (43 0e \/EW l 0 (Compound 404) OY\/\/\/\/\/\/\/\/ (D O9 \/EWO| 0 (Compound 405) G3/\/O\CI)IO\/E /U\/\/\/\/\/\/\/\N e O I: O 0 (Compound 406) OY\/\/\/\/\/\/\/\/ 9 O gee/V05) O\/[ )J\/\/\/\/\/\/\/\/\N IIDI’ o 0 (Compound 407) (\N\®/\/O\Cl)eo Wfl" 0 Cd 0 und 408) ‘/\N\®/\/0 9% /U\/\/\/\/\/\/\/\/\ \B/ 0 0d 0 (Compound 409), or a salt thereof.

In certain ments, a compound of Formula (IX) is of Formula (IX-a): R1 e L2-R2 R1—i\l® 0 Cl) 0% "Mn \?/ m L2_R2 (IX-a), or a salt thereof.

In certain embodiments, phospholipids useful or potentially useful in the present invention comprise a modified core. In certain embodiments, a olipid with a modi?ed core described herein is DSPC, or analog thereof, with a modi?ed core structure. For example, in certain embodiments of Formula , group A is not of the following formula: 0 R2 In certain embodiments, the compound of Formula (IX-a) is of one of the following formulae: r R2 R1 9 raj: R1 6 \® 0 \® 0 Rl-N R2 O\I,O /c\ 2 Rl-N O\I,O [Mn P R m 0 [Mn P m R1 || R1 II o O , , OYRZ OYRZ R1 R1 9 N—SN \® 0 RL-N R-N1 O\I,O ’14 "TV3O‘I’OTVYI:~/OE N m \H/ £fb?1 E m R2 OQT/RZ "Mnc>éilpf£;/N’RN2\IIDI/ m R YR o 0 or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of the following: or salts thereof.

In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a cyclic moiety in place of the glyceride . In certain embodiments, a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety. In certain embodiments, the compound of a (IX) is of Formula (IX-b): R1 9 M’ \® 0 1— (R2) R "MnO\I,O e p / IF; m (IX-b), or a salt thereof.

In certain ments, the compound of Formula (IX-b) is of Formula (IX-b-l): _\® 06 R1 N‘MnoPOkaOERZ( )p O (IX-b-l) or a salt thereof, wherein: wis 0,1, 2, or 3.

In certain embodiments, the compound of Formula (IX-b) is of Formula (IX-b-Z): R16) 09 O ( R2 1— ) R 21%"O\ I }< p .F.’ m o O (IX-b-Z), or a salt f.

In certain embodiments, the compound of Formula (IX-b) is of Formula (IX-b-3): R1 e O \® 0POM/E 7m, Rl-NI1MMn 0' (IX-b-3), or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is of Formula (IX-b-4): R1‘"Mn QED/601%?> 0 (IX-b-4), or a salt thereof.

In certain embodiments, the compound of a (IX-b) is one of the following: 09 O >TAM;Cu OJIFI" o O or salts f. gii) Phospholip_id Tail ations In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified tail. In certain embodiments, a phospholipid useful or potentially useful in the present invention is DSPC, or analog thereof, with a modi?ed tail. As described herein, a "modi?ed tail" may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more enes are replaced by cyclic or heteroatom groups, or any combination thereof. For e, in certain embodiments, the compound of (IX) is of Formula (IX-a), or a salt thereof, wherein at least one instance of R2 is each instance of R2 is optionally substituted C130 alkyl, wherein one or more methylene units of R2 are independently replaced with ally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted e, optionally substituted heteroarylene, -N(RN)-, —o—, —s—, -C(O)—, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)—, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)—, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)zO-, -OS(O)20-, -N(RN)S(O)-, -S(0)N(RN)-, S(0)N(RN)-, -OS(0)N(RN)-, S(0)0-, -S(0)2-, S(0)2-, -S(O)2N(RN)-, -N(RN)S(O)2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)2O-.

In n embodiments, the compound of Formula (IX) is of Formula (IX-c): R1_N 81/62/19 /"moEiWsz/ifl (IX-C), or a salt thereof, wherein: each X is ndently an integer n 0-30, inclusive, and each instance is G is ndently selected from the group consisting of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(0)2-, 0-, 20-, -N(RN)S(0)-, -S(0)N(RN)-, -N(RN)S(0)N(RN)-, -OS(O)N(RN)-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S(O)2-, -S(O)2N(RN)-, -N(RN)S(O)2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)20-. Each possibility represents a separate embodiment of the present invention.

In certain embodiments, the compound of Formula (IX-c) is of a (IX-c—l): V )x Fiia 06 L2 )X V )X R1_N O\ I /O\(\/)/K "Mn I: m L2 )x O (IX-c4), or salt thereof, wherein: each instance of V is independently l, 2, or 3.

In certain embodiments, the compound of Formula (IX-c) is of Formula (IX-c—Z): R1 2 )x )x ‘6 Ce MLK R _N1 O\ I /0 "Mn I: m L2 )x O (IX-c-Z), or a salt thereof.

In certain embodiments, the nd of Formula (IX-c) is of the ing a: R1 0 \e 0e 0 )x R1_N O\ I /O / Mn P m 0 R1 ('3' X or a salt thereof.

IO In certain embodiments, the compound of Formula (IX-c) is the following: 06 \/E O >N/\/0\"3,0 /U\/\/\/\A/\/\/\/ or a salt thereof.

In certain embodiments, the nd of Formula (IX-c) is of Formula (IX-c—3): O ( )x R 1 L2)0)X R1-l\l® 0 o 990 / Mn \P’ m L2 J) O X (IX-o3), or a salt thereof.

In certain embodiments, the compound of a (IX-c) is of the following formulae: R1 e R1-l\l® o 9 OM): / Mn \P/ m 0 O or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is the following: 09 £0 >N~o¢wGD ' OWOW ('5 0 or a salt thereof.

In certain ments, a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine , wherein the alkyl chain linking the quaternary amine to the oryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound ofFormula (IX), n n is l, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, a compound of Formula (IX) is of one of the following formulae: 1 $1 0e O6 (NWOW'VOr/rnf‘ RL® ,N/\/\/O\|/O A P Mm R II R1 \ " 0 R1 0 or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of the following: ('3' H OWVWWV I e 0 /®\/\/ \?/ 0 (Compound 412) e 09 £0 >lll/\/\/O\ IIIDI/O OJJ\/\/\/\/\/\/\/\ (Compound 413) (Compound 414), or salts thereof.

Alternative Lipids In certain embodiments, an alternative lipid is used in place of a olipid of the invention. Non-limiting examples of such alternative lipids include the following: HOWWBES/OJOJW HOWOZVEWNH3 OY\/\/\/\/\/\/\/\/ HOJWO OWf" G NH3 0 CI OW(9 NH3 o HOMNH J\/\/\/\/\/\/\M O O O H\/[W 9 NH3 0 I and ® Ole0W j\/\/\/\/\/\/\/\/\ ural Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural . As used herein, the term tural lipid" refers to sterols and also to lipids containing sterol moieties.

Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As de?ned herein, "sterols" are a subgroup of steroids consisting of steroid alcohols.

In n embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the ural lipid is an analog of cholesterol. In certain ments, the structural lipid is alpha-tocopherol. Examples of structural lipids include, but are not limited to, the following: ..»~N .. «s3% L41! \ \\ J.r‘\'\§’,-é\\ / ’ XI? is = i. §~§ .H RSV ,~R\, w\\~fA\\§./" \( .r'\\v‘ {t} é O and In one ment, the amount of the structural lipid (e.g., an sterol such as cholesterol) in the lipid composition of a pharmaceutical composition disclosed herein ranges from about 20 mol % to about 60 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 50 mol %, or from about 35 mol % to about 45 mol %.

In one embodiment, the amount of the structural lipid (e.g., an sterol such as cholesterol) in the lipid composition disclosed herein ranges from about 25 mol % to about mol %, from about 30 mol % to about 35 mol %, or from about 35 mol % to about 40 mol %.

In one embodiment, the amount of the structural lipid (e.g., a sterol such as cholesterol) in the lipid composition disclosed herein is about 24 mol %, about 29 mol %, about 34 mol %, or about 39 mol %.

In some embodiments, the amount of the structural lipid (e.g., an sterol such as cholesterol) in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol %.

Polyethylene Glycol gPEGz-Lipids 2O The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.

As used herein, the term "PEG-lipid" refers to polyethylene glycol (PEG)-modif1ed lipids. Non-limiting examples of PEG-lipids include di?ed phosphatidylethanolamine and phosphatidic acid, PEG-ceramide ates (e.g., PEG- CerCl4 or PEG-CerC20), PEG-modi?ed dialkylamines and PEG-modi?ed l,2- diacyloxypropanamines. Such lipids are also referred to as PEGylated . For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments, the pid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEGDAG ), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3 -amine (PEG-c-DMA).

In one embodiment, the pid is selected from the group consisting of a PEG- modi?ed phosphatidylethanolamine, a PEG-modi?ed phosphatidic acid, a PEG-modi?ed ceramide, a PEG-modi?ed dialkylamine, a PEG-modi?ed diacylglycerol, a PEG-modi?ed dialkylglycerol, and mixtures thereof.

In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NHZ, has a size of about 1000, 2000, 5000, , 15,000 or 20,000 daltons. In one embodiment, the PEG-lipid is PEGZk-DMG.

In one embodiment, the lipid nanoparticles bed herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.

PEG-lipids are known in the art, such as those described in US. Patent No. 8158601 and International Publ. No. in their entirety.

In general, some of the other lipid components (e.g., PEG lipids) of various formulae, described herein may be synthesized as described International Patent Application No.

PCT/U82016/000129, ?led December 10, 2016, entitled "Compositions and s for Delivery of Therapeutic Agents," which is incorporated by reference in its entirety.

The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene , such as PEG or di?ed lipids. Such s may be ately referred to as PEGylated . A PEG lipid is a lipid modi?ed with polyethylene . A PEG lipid may be selected from the non-limiting group including PEG-modi?ed phosphatidylethanolamines, PEG-modi?ed phosphatidic acids, PEG-modi?ed ceramides, PEG-modi?ed dialkylamines, PEG-modi?ed diacylglycerols, PEG-modi?ed dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PC, or a PEG-DSPE lipid.

In some ments the PEG-modi?ed lipids are a modi?ed form of PEG DMG.

PEG-DMG has the following structure: .oN-\ \ \ \; \ \ \ ‘73» \V.‘ \V.‘ \-_\_.s \-_\_ \\_.o \\ \\ . E S . w - \; \; .\~\; .ox; xx; _‘\‘\_ \\\_.s- - {y- \ \ \\;_¢~* \\; \\; \\; \\ \\ \ , - In one embodiment, PEG lipids useful in the present invention can be PEGylated lipids bed in International Publication No. W02012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modi?ed to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally de?ned , a "PEG-OH lipid" (also referred to herein as "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (—OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an —OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (VII). Provided herein are compounds of Formula (VII): RVO>TL1—D\(vrmA (V11), or salts thereof, wherein: R3 is —ORO; RO is hydrogen, optionally substituted alkyl, or an oxygen protecting group, r is an integer between 1 and 100, inclusive, L1 is optionally substituted C140 alkylene, wherein at least one ene of the 2O optionally substituted C140 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, ally substituted arylene, optionally substituted heteroarylene, o, N(RN), s, C(O), RN), NRNC(O), C(O)O, — OC(O), , (RN), NRNC(O)O, or NRNC(O)N(RN); D is a moiety ed by click try or a moiety cleavable under physiological conditions, m is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, LZ—R2 \d "2)" . L2_R2 A 1s of the formula: or , each instance of L2 is independently a bond or ally substituted CH, alkylene, wherein one ene unit of the optionally substituted C14, alkylene is optionally replaced with 0, N(RN), s, 0(0), C(O)N(RN), NRNC(O), C(O)O, 00(0), 00(0)0, OC(O)N(RN), — NRNC(O)O, or NRNC(O)N(RN), each ce of R2 is independently optionally substituted C130 alkyl, optionally substituted C130 alkenyl, or optionally substituted C130 alkynyl, optionally wherein one or more methylene units of R2 are independently ed with ally substituted carbocyclylene, optionally substituted heterocyclylene, ally substituted arylene, optionally substituted heteroarylene, N(RN), 0, s, 0(0), C(O)N(RN), NRNC(O), — )N(RN), C(O)O, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, C(O)S, s0(0), — C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O) — , OS(O), S(O)O, OS(O)O, , , OS(O)2O, (O), S(O)N(RN), N N each instance of RN is independently en, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, and p is l or 2.

In certain embodiments, the compound of Fomula (VII) is a PEG-OH lipid (i.e., R3 is —ORO, and Rois hydrogen). In certain embodiments, the compound of Formula (VII) is of Formula (VII-OH): HowokLl-DWmA (VII-OH), or a salt thereof.

In certain embodiments, D is a moiety obtained by click chemistry (e.g., triazole). In certain embodiments, the compound of Formila (VII) is of Formula (VII-a-l) or (VII-a-Z): NcN\ A IN: R\é/\O):L T\3 1 N-(-/)m RwofL3 ’l_N J4"): (VII-a-l) (VII-a-Z), or a salt thereof.

In certain embodiments, the compound of Formula (VII) is of one of the following formulae: 0 L2 0% R2 m VOW? or a salt thereof, wherein sis 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In certain embodiments, the compound of Formula (VII) is of one of the following formulae: or a salt thereof.

In certain embodiments, a nd of Formula (VII) is of one of the following formulae: or a salt thereof.

In certain embodiments, a nd of a (VII) is of one of the following formulae: HOVOM VEOO::NN O)J\/\/\/\/\/\/\ (Compound 415), O\\l/\/\/\/\/ N:N 0 0V0t und 416), N:N 0 0VOMNi (Compound 417), O N:N \/EO O O r / V (Compound 418), or a salt thereof.

In certain embodiments, D is a moiety cleavable under physiological conditions (e.g., ester, amide, carbonate, carbamate, urea). In certain embodiments, a compound of Formula (VII) is of Formula (VII-b-l) or (VII-b-2): Rié?ofog/OWmA1 O Raf/w):L1\OMmA (VII-b-1) (VII-b-2), or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of Formula -l-OH) or (VII-b-Z-OH): HO L1 0 V0): o A :[Df Wm HOVOfLKOJKM/m?x (VII-b-l-OH) (VII-bOH), or a salt thereof.

In n embodiments, the compound of Formula (VII) is of one of the following formulae: L2’R2 ,R2 2 0 L2 R3 1 2 4A0):L 0%9/ \g/ F<\4AO)r/L\O3 1 M ,R m L2 LZIR2 ,RZ \MHZ’R2 0 L2 Ho‘f? i" L1 0 R2 1 M ’ O m HO L\ . it Wk 0 , , or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of the following CYR2 O R2 0 T Rié?Okag/OVEO/uxw Rae/\ofLLOJK/EOJLRZo o 7 7 O R2 7; O R2 HOVoir/L?g/OVEOARZO O o O 1 HOVoir’LkO/ik/EOARZ 7 7 or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of the following formulae: 0 R2 0 R2 0 73/0 O\/[ JL O M0 1/0 RV 2 3 A or s O R R2 s o o o Rxé?or or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of the following formulae: 0 O O O MJW / V0 O r O or salts thereof.

In certain embodiments, a PEG lipid useful in the present ion is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (VIII). Provided herein are compounds of Formula (VIII): R3"0%" (VIII), or a salts f, wherein: R3 is—ORO, Rois hydrogen, optionally substituted alkyl or an oxygen protecting group, r is an r between 1 and 100, inclusive, R5 is optionally substituted C1040 alkyl, optionally substituted C1040 alkenyl, or optionally substituted C1040 alkynyl, and optionally one or more methylene groups of R5 are replaced with optionally tuted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, ally substituted heteroarylene, N(RN), O, S, C(O), -C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, 00(0), , OC(O)N(RN), -NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRN)N(RN), -C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), 8(0), 08(0), S(O)O, OS(O)O, OS(O)2, -S(O)20, OS(O)20, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, -S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O, and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

In certain embodiments, the compound of Formula (VIII) is of Formula (VIII-OH): O)r)l\R5 or a salt thereof. In some embodiments, r is 45. In other embodiments r is l.

In certain embodiments, a compound of Formula (VIII) is of one of the following formulae: / OW\M r (Compound 419), / OWV r (Compound 420), / VO>JK/\/\/\/=\/\/\/\/ ' (Compound 421), / OWV _ _ r (Compound 422), / OW\M r und 423), HO WV r (Compound 424), HO~(/\o)r/\/ N\n/\/\/\/\/\/\/\/\/ 0 (Compound 425), HOVwO (Compound 426), or a salt thereof. In some embodiments, r is 45.

In yet other embodiments the compound of a (VIII) is: HOMOW (Compound 427), or a salt thereof.

In one embodiment, the compound of Formula (VIII) is HOMOWmompwnd 428).

In one embodiment, the amount of PEG-lipid in the lipid composition of a pharmaceutical composition disclosed herein ranges from about 0.1 mol % to about 5 mol %, from about 0.5 mol % to about 5 mol %, from about 1 mol % to about 5 mol %, from about 1.5 mol % to about 5 mol %, from about 2 mol % to about 5 mol % mol %, from about 0.1 mol % to about 4 mol %, from about 0.5 mol % to about 4 mol %, from about 1 mol % to about 4 mol %, from about 1.5 mol % to about 4 mol %, from about 2 mol % to about 4 mol %, from about 0.1 mol % to about 3 mol %, from about 0.5 mol % to about 3 mol %, from about 1 mol % to about 3 mol %, from about 1.5 mol % to about 3 mol %, from about 2 mol % to about 3 mol %, from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 1.5 mol % to about 2 mol %, from about 0.1 mol % to about 1.5 mol %, from about 0.5 mol % to about 1.5 mol %, or from about 1 mol % to about 1.5 mol %.

In one embodiment, the amount of PEG-lipid in the lipid composition disclosed herein is about 2 mol %. In one embodiment, the amount of PEG-lipid in the lipid composition disclosed herein is about 1.5 mol %.

In one embodiment, the amount of PEG-lipid in the lipid composition sed herein is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 2O 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol %.

In some aspects, the lipid composition of the pharmaceutical compositions sed herein does not comprise a pid.

Other Ionizable Amino Lipids The lipid composition of the pharmaceutical composition disclosed herein can comprise one or more ionizable amino lipids in addition to or instead of a lipid according to Formula (I), (II), (III), (IV), (V), or (VI).

Ionizable lipids can be selected from the non-limiting group consisting of 3 -(didodecylamino)-N l ,N l ,4-tridodecyl- l -piperazineethanamine (KL 1 0), N1 idodecylamino)ethyl]-N l ,N4,N4-tridodecyl- l ,4-piperazinediethanamine (KL22), l4,25-ditridecyl-15, l8,2l,24-tetraaza-octatriacontane (KL25), l,2-dilinoleyloxy-N,N—dimethylaminopropane (DLin-DMA), 2,2-dilinoleyldimethylaminomethyl-[ l ,3]—dioxolane (DLin-K-DMA), heptatriaconta-6, 9,28,3 l-tetraen- l 9-yl 4-(dimethyl amino)butanoate (DLin-MC3 -DMA), 2,2-dilinoleyl(2-dimethylaminoethyl)-[ l ,3]—dioxolane (DLin-KCZ-DMA), l,2-dioleyloxy-N,N—dimethylaminopropane (DODMA), (l 32, l6SZ)-N,N-dimethyl-3 - cosa- l 3 - l 6-dien- l -amine (L608), 2-({ 8-[(3 B)—cholesten-3 -yloxy]octyl } ,N-dimethyl-3 -[(9Z,lZZ)—octadeca-9,12-dienl-yloxy ]propan-l -amine (Octyl-CLinDMA), (2R)({ 8-[(3 B)-cholesten-3 ]octyl } oxy)-N,N—dimethyl-3 -[(9Z, 12Z)-octadeca-9, 12- di enyl oxy]propanamine (Octyl-CLinDMA (2R)), and (2 S)—2-({ 8-[(3 B)—cholesten-3 -yloxy]octyl } oxy)-N,N-dimethyl-3 -[(9Z,lZZ)-octadeca-9,12- dien- l -yloxy]propan- l -amine -CLinDMA (2 S)). In addition to these, an ionizable amino lipid can also be a lipid including a cyclic amine group. ble lipids can also be the compounds disclosed in International Publication No. ionizable amino lipids include, but not limited to: HOwNWOm/V HOM"NM/0%N oYCii/V and any combination thereof.

Ionizable lipids can also be the compounds disclosed in International Publication No. ionizable amino lipids include, but not limited to: NV’A‘ rQW%’W/ x" "\x’N’ N \r {\VJAV’N\,JG:££A_WA\VW C} - AD\ltlL;/\W MUM!""\XV\x’j §~ "xx" WMNAOTrix:"x~,- o ; WNW’N"\w"\«Wkax WWWL \Lll/{?vrkvwxwwwmxC3 ?vemwx [NwwaxmxNTNv?xxmv LVAMLIVILVIwwww l ommwx NVMNWw "xxrwrxerTmt/m?vm ,g~Nv\xNN«"VAV»\xNOj"QV’L\xA\fW\/f«xxx/f "WA%{C}\ "\waN" x"WN"\J’\VI’\§T¢OV(\A?‘xx’?v’NV-"wxw’ K/x? {f} ,WWJI' \sz \Ww‘"\\x‘"‘w\/ :3 ; § QT‘x?v?x MNN/‘xNxNxV/f‘xvx‘x WW» waN‘xx’AKrf\/\"Q W‘NA '9,wa <\,NMy;{NW WWW;N"£\:~fw"\xW K ?jw"xm?wf’ "5\M’\ x NAm?wwa"QIKVNWNN‘ "\x’" V«W’\ and any combination thereof. rticle itions The lipid ition of a pharmaceutical composition disclosed herein can e one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components. For example, a permeability enhancer molecule can be a molecule described by US. Patent Application Publication No. 2005/0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

A polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and/or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, ocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and ylates.

The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt).

In some embodiments, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In some embodiments, the wt/wt ratio ofthe lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15: 1.

In one embodiment, the lipid nanoparticles described herein can se polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5: 1, 10: 1, 15: 1, 20: 1, :1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any ofthese ratios such as, but not limited to, 5:1 to about 10: 1, from about 5:1 to about 15: 1, from about 5:1 to about :1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about :1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about :1, from about 10:1 to about 30:1, from about 10:1 to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about 20:1, from about 15:1 to about 25:1,from about 15:1 to about 30:1, from about 15:1 to about :1, from about 15:1 to about 40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

In one ment, the lipid rticles described herein can comprise the polynucleotide in a concentration from imately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

In some embodiments, the pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as a compound of Formula (I) or (III) as described herein, and (ii) a polynucleotide encoding one or more cancer epitope polypeptides. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding one or more cancer epitope polypeptides.

Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle itions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers ted by aqueous tments. Lipid bilayers can be onalized and/or crosslinked to one another. Lipid rs can include one or more s, proteins, or channels.

In some embodiments, the nanoparticle compositions of the t disclosure se at least one compound according to a (I), (III), (IV), (V), or (VI). For 2O example, the nanoparticle composition can include one or more of Compounds l-l47, or one or more of Compounds l-342. Nanoparticle compositions can also include a y of other components. For example, the nanoparticle composition may include one or more other lipids in on to a lipid according to Formula (I), (II), (III), (IV), (V), or (VI), such as (i) at least one phospholipid, (ii) at least one structural lipid, (iii) at least one PEG-lipid, or (iv) any combination thereof. Inclusion of structural lipid can be optional, for example when lipids according to formula III are used in the lipid rticle compositions of the invention.

In some embodiments, the nanoparticle composition comprises a compound of Formula (I), (e.g., Compounds 18, 25, 26 or 48). In some ments, the nanoparticle composition comprises a compound of Formula (I) (e.g., Compounds 18, 25, 26 or 48) and a phospholipid (e.g., DSPC).

In some embodiments, the nanoparticle ition comprises a nd of Formula (III) (e.g., Compound 236). In some embodiments, the nanoparticle composition ses a compound of Formula (III) (e.g., Compound 236) and a phospholipid (e.g., DOPE or DSPC).

In some embodiments, the nanoparticle composition comprises a lipid composition consisting or consisting essentially of compound of a (I) (e.g., Compounds 18, 25, 26 or 48). In some embodiments, the nanoparticle composition comprises a lipid composition consisting or consisting essentially of a compound of Formula (I) (e.g., Compounds 18, 25, 26 or 48) and a phospholipid (e.g., DSPC).

In some embodiments, the nanoparticle composition comprises a lipid composition consisting or consisting essentially of compound of Formula (III) (e.g., Compound 23 6). In some embodiments, the nanoparticle composition comprises a lipid composition consisting or consisting essentially of a compound of Formula (III) (e.g., Compound 236) and a phospholipid (e.g., DOPE or DSPC).

In one ment, a lipid nanoparticle comprises an ble lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modi?ed lipid, a phospholipid and a structural lipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25- 55% structural lipid, and about 05-15% PEG-modi?ed lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modi?ed lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP ses a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino lipid and the phospholipid is a neutral lipid, and the structural lipid is a cholesterol. In some embodiments, the LNP has a molar ratio of 50:38.5: 10:1.5 of ionizable lipid: cholesterol: DSPC: PEG lipid. In some embodiments, the ionizable lipid is Compound 18 or Compound 236, and the PEG lipid is Compound 428.

In some embodiments, the LNP has a molar ratio of 5:10:1.5 of Compound 18 : Cholesterol :Phospholipid : Compound 428. In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 of Compound 18 : Cholesterol : DSPC :Compound 428.

In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 of Compound 236 : Cholesterol :Phospholipid: Compound 428. In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 of Compound 236 : terol : DSPC : Compound 428.

In some embodiments, the LNP has a spersity value of less than 0.4. In some ments, the LNP has a net l charge at a neutral pH. In some embodiments, the LNP has a mean er of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.

As lly de?ned herein, the term "lipid" refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic.

Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids leads them to form mes, vesicles, or membranes in aqueous media.

In some ments, a lipid nanoparticle (LNP) may comprise an ble lipid.

As used herein, the term "ionizable lipid" has its ordinary meaning in the art and may refer to a lipid sing one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. An ionizable lipid may be positively charged, in which case it can be referred to as nic lipid". In n embodiments, an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipids. As used , a "charged moiety" is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -l), divalent (+2, or -2), trivalent (+3, or -3 ), etc.

The charged moiety may be anionic , negatively charged) or cationic (1'.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, ary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine . Examples of negatively- charged groups or precursors f, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the d moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

It should be understood that the terms "charged" or ed moiety" does not refer to a "partial negative charge" or al positive charge" on a molecule. The terms "partial negative charge" and "partial positive " are given its ordinary meaning in the art. A "partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

In some embodiments, the ionizable lipid is an ionizable amino lipid, sometimes referred to in the art as an "ionizable cationic lipid". In one embodiment, the ionizable amino lipid may have a vely charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

In addition to these, an ionizable lipid may also be a lipid including a cyclic amine group.

In one embodiment, the ionizable lipid may be selected from, but not limited to, a ionizable lipid described in International ation Nos. W02013086354 and W02013116126, the contents of each of which are herein incorporated by reference in their entirety.

In yet another embodiment, the ionizable lipid may be selected from, but not limited to, formula CLI—CLXXXXII of US Patent No. 7,404,969, each of which is herein incorporated by reference in their entirety.

In one embodiment, the lipid may be a cleavable lipid such as those bed in International Publication No. W02012/170889, herein incorporated by reference in its entirety. In one embodiment, the lipid may be synthesized by s known in the art and/or as described in International ation Nos. W02013/086354, the contents of each of which are herein incorporated by reference in their entirety.

Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle 2O composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to e zeta potentials. Dynamic light scattering can also be utilized to ine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvem, tershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

In some embodiments, the nanoparticle composition comprises a lipid composition consisting or consisting essentially of nd of Formula (I) (e.g., nds 18, 25, 26 or 48). In some embodiments, the nanoparticle composition comprises a lipid composition ting or consisting essentially of a compound of Formula (I) (e.g., nds 18, 25, 26 or 48) and a phospholipid (e.g., DSPC or MSPC).

Nanoparticle compositions can be terized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or iometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, , tershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

The size of the nanoparticles can help counter biological reactions such as, but not limited to, in?ammation, or can increase the biological effect of the polynucleotide.

As used herein, "size" or "mean size" in the context of nanoparticle itions refers to the mean diameter of a nanoparticle composition.

In one embodiment, the polynucleotide encoding one or more cancer epitope polypeptides are formulated in lipid nanoparticles haVing a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm.

In one embodiment, the nanoparticles have a er from about 10 to 500 nm. In one embodiment, the nanoparticle has a er greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

In some embodiments, the largest ion of a nanoparticle composition is 1 pm or shorter (e.g., l um, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).

A nanoparticle composition can be vely homogenous. A polydispersity indeX can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index lly indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity indeX from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity indeX of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.

The zeta potential of a nanoparticle ition can be used to indicate the electrokinetic ial of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low s, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition disclosed herein can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about 10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about --15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about --5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about --10 mV.

In some embodiments, the zeta potential of the lipid nanoparticles can be from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, from about mV to about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mV to about 30 mV, from about 10 mV to about 20 mV, from about 20 mV to about 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about 80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV, from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, from about 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about 30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mV to about 70 mV, from about 30 mV to about 60 mV, from about 30 mV to about 50 mV, from about 30 mV to about 40 mV, from about 40 mV to about 100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80 mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV, and from about 40 mV to about 50 mV. In some embodiments, the zeta ial of the lipid nanoparticles can be from about 10 mV to about 50 mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and from about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.

The term sulation ef?ciency" of a polynucleotide describes the amount of the cleotide that is encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. As used herein, "encapsulation" can refer to te, substantial, or partial enclosure, con?nement, nding, or encasement. ulation ef?ciency is desirably high (e.g., close to 100%). The encapsulation ef?ciency can be measured, for example, by comparing the amount of the polynucleotide in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents.

Fluorescence can be used to measure the amount of free polynucleotide in a solution.

For the nanoparticle itions described herein, the encapsulation ef?ciency of a polynucleotide can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation ef?ciency can be at least 80%. In certain embodiments, the encapsulation ef?ciency can be at least 90%.

The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the cleotide.

For e, the amount of an mRNA useful in a nanoparticle ition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary.

The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the t disclosure can be optimized according to considerations of ef?cacy and tolerability. For compositions including an mRNA as a polynucleotide, the NP ratio can serve as a useful metric.

As the NP ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable. N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.

In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2:1 to about 30: 1, such as 2: 1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1,12:1,14:1,16:1,18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1.

In certain embodiments, the NP ratio can be from about 2:1 to about 8: 1. In other ments, the NP ratio is from about 5:1 to about 8:1. In certain embodiments, the NP ratio is between 5:1 and 6:1. In one speci?c aspect, the NP ratio is about is about 5.67:1.

In on to providing rticle compositions, the present disclosure also es s of producing lipid nanoparticles comprising encapsulating a polynucleotide. Such method comprises using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles in accordance with s of production of lipid nanoparticles known in the art. See, e.g., Wang et al (2015) "Delivery of oligonucleotides with lipid rticles" Adv. Drug Deliv. Rev. 87:68-80, Silva et al (2015) "Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles" Curr. Pharm. Technol. 16: 940-954, Naseri et al (2015) "Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application" Adv. Pharm. Bull. 5:305-13, Silva et al. (2015) "Lipid nanoparticles for the delivery of biopharmaceuticals" Curr. Pharm. Biotechnol. 16291-3 02, and references cited therein.

Kit Formulations Kits for accomplishing these methods are also provided in other aspects of the invention. The kit includes a container housing a lipid nanoparticle ation, a container g a vaccine formulation, and instructions for adding a personalized mRNA cancer vaccine to the vaccine formulation to produce a personalized mRNA cancer e formulation, mixing the personalized mRNA cancer vaccine formulation with the lipid nanoparticle formulation within 24 hours of administration to a subject. In some embodiments the kit includes a mRNA having an open reading frame encoding 2-100 cancer antigens.

The articles include pharmaceutical or diagnostic grade nds of the invention in one or more containers. The article may include instructions or labels promoting or bing the use of the compounds of the invention.

As used herein, "promoted" includes all methods of doing business including methods of education, hospital and other clinical ction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional ty including written, oral and onic communication of any form, ated with compositions of the invention in connection with ent of cancer.

"Instructions" can de?ne a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.

Thus the agents described herein may, in some embodiments, be assembled into pharmaceutical or diagnostic or ch kits to facilitate their use in therapeutic, stic or research applications. A kit may include one or more containers housing the components of the invention and instructions for use. Speci?cally, such kits may include one or more agents described , along with instructions describing the intended therapeutic application and the proper administration of these . In certain embodiments agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.

The kit may be designed to facilitate use of the methods described herein by physicians and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise sable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be ed with the kit.

As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written ctions on or associated with packaging of the invention.

Instructions also can include any oral or electronic ctions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e. g., videotape, DVD, etc.), Internet, and/or sed communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also re?ects approval by the agency of manufacture, use or sale for human administration.

The kit may contain any one or more of the ents described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a t. The kit may include a container housing agents described herein. The agents may be prepared ely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.

The kit may have a variety of forms, such as a r pouch, a shrink wrapped pouch, a vacuum le pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kit may be sterilized after the accessories are added, thereby allowing the dual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit may also include other components, depending on the c application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for ng or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.

The compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders. When the composition ed is a dry powder, the powder may be reconstituted by the addition of a suitable solvent, which may also be ed. In embodiments where liquid forms of the composition are sued, the liquid form may be concentrated or ready to use. The solvent will depend on the nd and the mode of use or administration. Suitable solvents for drug compositions are well known and are available in the literature. The solvent will depend on the compound and the mode of use or administration.

The kits, in one set of embodiments, may comprise a carrier means being compartmentalized to receive in close con?nement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the te elements to be used in the method. For e, one of the containers may se a positive control for an assay. Additionally, the kit may include containers for other components, for example, buffers useful in the assay.

The present invention also encompasses a ?nished packaged and labeled pharmaceutical product. This article of manufacture es the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is ically sealed. In the case of dosage forms suitable for parenteral administration the active ingredient is sterile and suitable for administration as a particulate free solution. In other words, the invention encompasses both parenteral solutions and lyophilized powders, each being sterile, and the latter being le for reconstitution prior to injection.

Alternatively, the unit dosage form may be a solid suitable for oral, transdermal, topical or mucosal delivery.

In a preferred embodiment, the unit dosage form is suitable for intravenous, intramuscular or subcutaneous ry. Thus, the invention encompasses solutions, preferably sterile, suitable for each delivery route.

In another preferred embodiment, compositions of the invention are stored in containers with biocompatible detergents, ing but not limited to, lecithin, taurocholic acid, and cholesterol, or with other ns, ing but not limited to, gamma globulins and serum albumins. More preferably, compositions of the ion are stored with human serum albumins for human uses, and stored with bovine serum ns for veterinary uses.

As with any pharmaceutical t, the packaging material and container are designed to protect the ity of the product during storage and shipment. Further, the products of the invention include instructions for use or other informational material that advise the physician, technician or patient on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures (such as methods for monitoring mean ab solute lymphocyte counts, tumor cell counts, and tumor size) and other monitoring information.

More speci?cally, the invention provides an e of manufacture comprising packaging al, such as a box, bottle, tube, vial, container, sprayer, insuf?ator, intravenous (iv) bag, envelope and the like, and at least one unit dosage form of a pharmaceutical agent contained within said packaging material. The invention also provides an article of manufacture comprising ing material, such as a box, bottle, tube, vial, container, sprayer, insuf?ator, intravenous (iv) bag, envelope and the like, and at least one unit dosage form of each pharmaceutical agent contained within said packaging al. The invention further provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insuf?ator, intravenous (iv) bag, envelope and the like, and at least one unit dosage form of each pharmaceutical agent contained within said packaging material. The ion further provides an article of manufacture comprising a needle or syringe, preferably packaged in sterile form, for injection of the formulation, and/or a packaged alcohol pad.

Relative amounts of the active ingredient, the pharmaceutically able excipient, and/or any additional ingredients in a vaccine composition may vary, depending upon the identity, size, and/or condition of the subject being treated and r depending upon the route by which the composition is to be administered. For e, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1- %, between 5-80%, at least 80% (w/w) active ingredient.

In some ments, the package containing the pharmaceutical product contains 0.1 mg to 1 mg of mRNA. In some embodiments, the package containing the pharmaceutical product contains 0.35 mg of mRNA. In some embodiments, the concentration of the mRNA is 1 mg/mL.

In some embodiments, the package containing the pharmaceutical t contains contains 5-15 mg of total lipid. In some embodiments, the package containing the pharmaceutical product contains contains 7 mg of total lipid. In some embodiment, the concentration of total lipid is 20 mg/mL.

In some embodiments, the RNA (e.g., mRNA) vaccine compositions may be administered at dosage levels suf?cient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses bed in International Publication No. W02013/078199, herein incorporated by reference in its entirety). In some embodiments, the RNA (e.g., mRNA) vaccine is stered at a dosage level ent to deliver 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. In some ments, the RNA (e.g., mRNA) vaccine is administered at a dosage level suf?cient to deliver between 10 ug and 400 ug of the mRNA vaccine to the subject. In some embodiments, the RNA (e.g., mRNA) vaccine is is administered at a dosage level suf?cient to deliver 0.033mg, 0.1 mg, 0.2 mg, or 0.4 mg to the subject.

The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three , every 6 months, etc. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, ?ve, siX, seven, eight, nine, ten, eleven, twelve, en, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. In some embodiments, the RNA vaccine itions may be administered at dosage levels suf?cient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg. In some embodiments, the RNA vaccine compositions may be administered once or twice (or more) at dosage levels ent to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, the RNA vaccine compositions may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels suf?cient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower s and frequency of administration are encompassed by the present disclosure.

For example, a the RNA vaccine composition may be administered three or four times, or more. In some embodiments, the mRNA vaccine composition is administered once a day every three weeks In some embodiments, the RNA vaccine compositions may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.

In some embodiments the RNA vaccine for use in a method of vaccinating a subject is administered the subject a single dosage of between 10 ug/kg and 400 ug/kg of the nucleic acid vaccine in an effective amount to vaccinate the subject. In some embodiments the RNA e for use in a method of vaccinating a subject is administered the subject a single dosage of between 10 ug and 400 ug of the nucleic acid vaccine in an effective amount to vaccinate the subject.

In some embodiments, the RNA vaccine composition may comprise the polynucleotide described herein, formulated in a lipid nanoparticle comprising MC3, Cholesterol, DSPC and PEG2000-DMG, the buffer trisodium citrate, sucrose and water for injection. As a non-limiting example, the composition comprises: 2.0 mg/mL of drug substance (e.g., polynucleotides ng cancer antigens), 21.8 mg/mL of MC3, 10.1 mg/mL of cholesterol, 5.4 mg/mL ofDSPC, 2.7 mg/mL ofPEG2000-DMG, 5.16 mg/mL of trisodium citrate, 71 mg/mL of sucrose and 1.0 mL of water for injection.

In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm or 80- 200 nm.

In some ments, the RNA vaccine comprises 5-15 mg of total lipid, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg oftotal lipid. In some ments, the RNA vaccine comprises 7 mg of total lipid. In some embodiment, the concentration of total lipid in the vaccine formulation is 10-30 mg/mL, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg/mL. lin is an approximately 500 amino acid monomeric protein that polymerizes to form the ?agella associated with bacterial motion. Flagellin is expressed by a variety of ?agellated bacteria (Salmonella typhimurium for example) as well as non-?agellated bacteria (such as Escherichia coli). Sensing of in by cells of the innate immune system itic cells, macrophages, etc.) is mediated by the Toll-like receptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf and Naip5. TLRs and NLRs have been identified as playing a role in the tion of innate immune response and adaptive immune response.

As such, ?agellin provides an adjuvant effect in a vaccine.

The nucleotide and amino acid sequences encoding known ?agellin polypeptides are publicly available in the NCBI k database. The in sequences from S.

Typhimurium, H. Pylori, V. Cholera, S. marcesens, S. ?exneri, T. pallidum, L. pneumophila, B. burgdorferei, C. difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P.

Mirabilis, B. us, L. monocytogenes, P. aeruginosa, and E. coli, among others are known.

A ?agellin polypeptide, as used herein, refers to a full length ?agellin protein, immunogenic fragments f, and peptides having at least 50% sequence identity to a ?agellin protein or immunogenic fragments thereof. Exemplary ?agellin proteins include ?agellin from Salmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium (AOAOC9DG09), Salmonella enteritidis (AOAOC9BAB7), and Salmonella choleraesuis (Q6V2X8. In some embodiments, the ?agellin polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% ce identity to a in n or immunogenic fragments thereof.

In some embodiments, the ?agellin polypeptide is an genic fragment. An immunogenic fragment is a portion of a in protein that provokes an immune response.

In some embodiments, the immune response is a TLR5 immune response. An example of an immunogenic fragment is a ?agellin n in which all or a portion of a hinge region has been deleted or replaced with other amino acids. For example, an antigenic polypeptide may be inserted in the hinge region. Hinge regions are the hypervariable regions of a ?agellin.

Hinge regions of a ?agellin are also ed to as "D3 domain or region,77 (Lpropeller domain or region," "hypervariable domain or region" and "variable domain or region." "At least a n of a hinge region," as used herein, refers to any part of the hinge region of the ?agellin, or the ty of the hinge region. In other ments an genic fragment of ?agellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment of ?agellin.

The ?agellin monomer is formed by domains D0 through D3. D0 and D1, which form the stem, are composed of tandem long alpha helices and are highly conserved among different bacteria. The Dl domain includes several stretches of amino acids that are useful for TLR5 activation. The entire Dl domain or one or more of the active regions within the domain are immunogenic fragments of ?agellin. Examples of immunogenic s within the D1 domain include residues 88-114 and residues 41 1-431 in Salmonella typhimurium FliC ?agellin. Within the 13 amino acids in the 88-100 region, at least 6 substitutions are permitted between Salmonella ?agellin and other ?agellins that still preserve TLR5 activation. Thus, genic fragments of ?agellin include ?agellin like sequences that activate TLR5 and contain a 13 amino acid motif that is 53% or more identical to the Salmonella sequence in 88-100 of FliC (LQRVRELAVQSAN, SEQ ID NO: 356).

In some embodiments, the RNA (e.g., mRNA) vaccine includes an RNA that encodes a fusion protein of ?agellin and one or more antigenic ptides. A "fusion protein" as used herein, refers to a linking of two components of the construct. In some ments, a carboxy-terminus of the antigenic polypeptide is fused or linked to an amino terminus of the in polypeptide. In other embodiments, an amino-terminus of the antigenic ptide is fused or linked to a carboxy-terminus of the ?agellin ptide. The fusion protein may include, for example, one, two, three, four, ?ve, siX or more ?agellin ptides linked to one, two, three, four, ?ve, siX or more antigenic polypeptides. When two or more ?agellin polypeptides and/or two or more antigenic polypeptides are linked such a uct may be referred to as a "multimer." Each of the components of a fusion protein may be directly linked to one another or they may be ted through a linker. For instance, the linker may be an amino acid linker. The amino acid linker encoded for by the RNA (e.g., mRNA) vaccine to link the components of the fusion protein may include, for instance, at least one member selected from the group consisting of a lysine residue, a glutamic acid residue, a serine residue and an ne residue. In some embodiments the linker is 1-30, 1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids in length.

Modes of Vaccine Administration Cancer RNA vaccines may be administered by any route which s in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, and/or subcutaneous administration. The present disclosure provides methods comprising administering RNA vaccines to a t in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Cancer RNA vaccines itions are typically ated in dosage unit form for ease of administration and uniformity of .

It will be understood, however, that the total daily usage of cancer RNA vaccines compositions may be decided by the attending physician within the scope of sound medical judgment. The speci?c therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder, the activity of the speci?c compound employed, the speci?c composition employed, the age, body weight, general health, seX and diet of the patient, the time of administration, route of administration, and rate of excretion of the speci?c compound employed; the duration of the treatment; drugs used in combination or coincidental with the speci?c compound employed; and like factors well known in the medical arts.

In some embodiments; cancer RNA es compositions may be administered at dosage levels suf?cient to deliver 0.0001 mg/kg to 100 mg/kg; 0.001 mg/kg to 0.05 mg/kg; 0.005 mg/kg to 0.05 mg/kg; 0.001 mg/kg to 0.005 mg/kg; 0.05 mg/kg to 0.5 mg/kg; 0.01 mg/kg to 50 mg/kg; 01 mg/kg to 40 mg/kg; 0.5 mg/kg to 30 mg/kg; 0.01 mg/kg to 10 mg/kg; 0.1 mg/kg to 10 mg/kg; or 1 mg/kg to 25 mg/kg; of subject body weight per day; one or more times a day; per week; per month; etc. to obtain the desired therapeutic; diagnostic; prophylactic; or imaging effect (see e.g.; the range of unit doses described in International Publication No W02013/078199; herein incorporated by nce in its entirety). The desired dosage may be delivered three times a day; two times a day; once a day; every other day; every third day; every week; every two weeks; every three weeks; every four weeks; every 2 months; every three months; every 6 months; etc. In certain embodiments; the d dosage may be delivered using le administrations (e.g.; two; three; four; ?ve; siX; seven; eight; nine; ten; eleven; twelve; thirteen; fourteen; or more administrations). When multiple administrations are employed; split dosing regimens such as those described herein may be used. In exemplary embodiments; cancer RNA vaccines itions may be administered at dosage levels suf?cient to r 0.0005 mg/kg to 0.01 mg/kg; e.g.; about 0.0005 mg/kg to about 0.0075 mg/kg; e.g.; about 0.0005 mg/kg; about 0.001 mg/kg; about 0.002 mg/kg; about 0.003 mg/kg; about 0.004 mg/kg or about 0.005 mg/kg.

A RNA e pharmaceutical composition described herein can be ated into a dosage form described herein; such as an intranasal; racheal; or injectable (e.g.; intravenous; intraocular; intravitreal; intramuscular; intradermal; intracardiac; intraperitoneal; and subcutaneous).

This invention is not d in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also; the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," ising," or "having; 77 (L containing; 77 (4'1nvolving;" and variations thereof herein; is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

EXAMPLES e 1. Manufacture of Polynucleotides ing to the present disclosure, the manufacture of polynucleotides and or parts or regions thereof may be accomplished utilizing the methods taught in International Application W02014/152027 entitled "Manufacturing Methods for Production ofRNA Transcripts", the contents of which is incorporated herein by reference in its entirety.

Puri?cation methods may include those taught in ational Application W02014/152030 and W02014/152031, each of which is incorporated herein by reference in its entirety.

Detection and characterization s of the polynucleotides may be performed as taught in /14403 9, which is incorporated herein by reference in its entirety.

Characterization of the polynucleotides of the disclosure may be accomplished using a procedure ed from the group consisting of polynucleotide mapping, reverse transcriptase cing, charge distribution analysis, and detection ofRNA ties, wherein characterizing comprises determining the RNA transcript ce, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript.

Such methods are taught in, for example, W02014/144711 and W02014/144767, the contents of each of which is incorporated herein by reference in its entirety.

Example 2 Chimeric polynucleotide synthesis Introduction According to the present disclosure, two regions or parts of a chimeric polynucleotide may be joined or ligated using triphosphate chemistry.

According to this method, a ?rst region or part of 100 nucleotides or less is chemically synthesized with a 5’ monophosphate and terminal 3’desOH or blocked OH. If the region is longer than 80 nucleotides, it may be synthesized as two strands for on.

If the ?rst region or part is synthesized as a sitionally modi?ed region or part using in vitro transcription (IVT), conversion the 5’monophosphate with subsequent capping of the 3’ terminus may follow.

Monophosphate protecting groups may be selected from any of those known in the The second region or part of the chimeric polynucleotide may be synthesized using either al synthesis or IVT s. IVT methods may include an RNA polymerase that can utilize a primer with a modi?ed cap. Alternatively, a cap of up to 130 nucleotides may be chemically synthesized and coupled to the IVT region or part.

The entire chimeric polynucleotide need not be manufactured with a phosphate-sugar backbone. If one of the regions or parts encodes a polypeptide, then it is able that such region or part comprise a phosphate-sugar backbone. on is then performed using any known click chemistry, orthoclick chemistry, solulink, or other bioconjugate chemistries known to those in the art.

Synthetic route The chimeric polynucleotide is made using a series of starting segments. Such segments include: (a) Capped and protected 5’ segment comprising a normal 3’OH (SEG. l) (b) 5’ triphosphate segment which may include the coding region of a polypeptide and comprising a normal 3’OH (SEG. 2) (c) 5’ monophosphate segment for the 3’ end of the ic polynucleotide (e.g., the tail) comprising cordycepin or no 3’OH (SEG. 3) After synthesis (chemical or IVT), segment 3 (SEG. 3) is treated with cordycepin and then with pyrophosphatase to create the 5’monophosphate.

Segment 2 (SEG. 2) is then ligated to SEG. 3 using RNA ligase. The ligated polynucleotide is then puri?ed and treated with pyrophosphatase to cleave the diphosphate. 2O The d SEG.2-SEG. 3 uct is then purified and SEG. l is ligated to the 5’ terminus.

A further purification step of the chimeric polynucleotide may be performed.

The yields of each step may be as much as 90-95%. e 3: PCR for cDNA Production PCR procedures for the preparation of cDNA are performed using 2X KAPA HIFITM HotStart ReadyMiX by Kapa Biosystems (Woburn, MA). This system includes 2X KAPA ReadyMin2.5 ul, Forward Primer (lO uM) 0.75 ul, Reverse Primer (lO uM) 0.75 ul, Template cDNA -100 ng, and deO diluted to 25.0 ul. The reaction ions are at 95° C for 5 min. and 25 cycles of 98° C for 20 sec, then 58° C for 15 sec, then 72° C for 45 sec, then 72° C for 5 min. then 4° C to termination.

The reaction is cleaned up using Invitrogen’s PURELINKTM PCR Micro Kit bad, CA) per manufacturer’s instructions (up to 5 ug). Larger reactions will require a p using a product with a larger capacity. Following the cleanup, the cDNA is fied using the NANODROPTM and analyzed by agarose gel electrophoresis to con?rm the cDNA is the expected size. The cDNA is then submitted for sequencing analysis before proceeding to the in vitro transcription reaction.

Example 4. In vitro Transcription gIVTg The in vitro ription reaction generates polynucleotides containing uniformly modi?ed cleotides. Such uniformly modi?ed polynucleotides may comprise a region or part of the polynucleotides of the disclosure. The input nucleotide triphosphate (NTP) mix is made in-house using natural and un-natural NTPs.

A typical in vitro ription reaction es the following: 1 Template cDNA 1.0 pg 2 10x transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgC12, 50 mM DTT, 10 mM Spermidine) 2.0 ul 3 Custom NTPs (25mM each) 7.2 ul 4 RNase Inhibitor 20 U 5 T7 RNA polymerase 3000 U 6 dH20 Up to 20.0 ul. and 7 Incubation at 37° C for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C overnight for cleanup the next day. l U of RNase-free DNase is then used to digest the original template. After 15 minutes of incubation at 37° C, the mRNA is d using Ambion’s EARTM Kit (Austin, TX) following the manufacturer’s ctions. This kit can purify up to 500 ug of RNA.

Following the cleanup, the RNA is quanti?ed using the NanoDrop and analyzed by agarose gel electrophoresis to con?rm the RNA is the proper size and that no degradation of the RNA has occurred.

Example 5. STING Studies In this example, whether an immune potentiator, such as constitutively active STING, can boost T-cell responses to a concatameric vaccine was investigated. An mRNA construct encoding the RNA 31 concatemer, which encodes Class I and Class II epitopes, was used as the vaccine and the effect of STING on T-cell responses to Class I and Class II epitopes was investigated. The RNA 31 and STING mRNAs were either coformulated and delivered simultaneously, or were not coformulated, with delayed deviery of STING mRNA. Animals were given a priming dose on Day 1 and a boost on Day 15. cytes were harvested on Day 22.

Different materials were tested in order to determine the immunogeni city when adding STING at various ratios to a concatemeric vaccine, to compare STING to top-ranked commercially ble nts, to determine whether the immunogenicity is dependent upon the timing of STING dosing, and to examine the immunogenicity of unforrnulated mRNA when dosed with STING. The following materials/conditions were tested: RNA 31 (311g), RNA 31 (311g) with Poly I:C (lOug), RNA 31 (311g) with MPLA (511g), STING (1 ug)/RNA 31 (311g), STING (O.6ug)/RNA 31 (3 ug), STING (O.6ug)/RNA 54 (311g), STING (O.6ug)/RNA 31 (3 pg) (24 hours later), STING (O.6ug)/RNA 31 (3 pg) (48 hours later), STING (0.6ug)/RNA 31 (311g) (unformulated), and STING (6ug)/RNA 31 (30ug) (unformulated). CA-54 is a emer of 5 Class II epitopes (all of which are ned within RNA 31).

Results are shown in FIGs. 12-13. When the antigen-specif1c lFNv responses were examined with Class II epitopes STING was found to boost the immune response to the MHC class II epitopes encoded by mRNA. STING behaved comparably to commercially available adjuvants (5-10 fold difference in dose). Although both ratios tested worked, the 1:5 STING:antigen ratio performed better than 1:3 combination (). Similar s were obtained using Class I epitopes as described above and shown in . Likewise, the 1:5 STING:antigen ratio was found to perform better than the 1:3 combination for class I epitopes. 2O Further, it was found that dosing STING at a later time point (24 hours) had similar iimmunogenicity to codelivery ().

In a further experiment, the effect of different STING-to-antigen ratios was examined using 52 murine epitopes (adding eptioes_4a_DX_RX_perm). Mice received a prime dose on Day 1, a boost dose on Day 8, and splenocytes were harvested on Day 15. T cell responses to re-stimulation were evaluated using ELISpot and FACS. Restimulation was performed with peptide sequences corresponding to epitopes eocnding the amer. T cell response to two Class II epitopes (RNA 2, RNA 3) and four Class I epitopes (RNA 7, RNA , RNA 13, RNA 22) were examined.

Quite surprisingly, it was found that the addition of STING across the majority of ratios tested ed T cell responses ed to antigen alone and never performed worse than antigen alone. The breadth of responsiveness was unexpected. For four of the six antigens (epitopes) , the on of STING to n at the 10-3 Oug total dose tently produced higher T cell responses than that of the 50ug dose of antigen alone.

Thus, there is a wide bell curve in the ratio of STING:antigen for improved immunogenicity.

The study groups were as shown in the following table: swam""""""""""""""""""""=‘ """"""""""""""""""" "1 0:1 20:1 5:1 1:1 155 125 Hummm‘ ‘ ‘ ~ ~ gzzm 1.5 3 27 3 2.35 0.15 3,4 {1.6 1.5 1.5 0.6 2.4 0.15 235$ 20 10 9.5 0,5 3,3 1.4 5.0 5.0 1.4 8.3 0.5 9.5 0 so 28,5 1.4 25.0 4.3 15.0 meg 4.2 25.0 1.4 28.6 ............................................................................................................................................................................................................................................

Among the Class II epitopes, RNA 2 (results shown in and RNA 3 (results shown in showed that adding STING increased T cell responses at ratios less than l:l (STINGzantigen) relative to the antigen only group, including at doses up to 50 ug n alone. The left panel of shows that adding STING increased T cell response at all ratios relative to the antigen only group.

Similar s were seen with the Class I epitopes. RNA 7 (results shown in , RNA 13 (results shown in , RNA 22 (results shown in ), and RNA 10 (results shown in ) all showed that ratios of STINGzantigen produced higher T cell responses relative to the antigen only group when compared to the total mRNA dose. e 6. Concatamer Studies Studies were conducted to examine whether full read through of longer constructs was possible and to e immunogenicity to epitopes contained in 20 and 52 epitope constructs. For the experiments, five groups of different formulations were tested in LNPs ning Compound 257: Group Test/Control al Final Class 11 (number Class I (number Concentration of constructs — of constructs — number of amino number of acids) amino acids) 1 RNA31 3 5—3laa 15—31321 2 20 epitopes_21 ?anks 3 5 — 21 aa 15 — 213a 3 20 epitopes_21 ?ank Class II_15 ?ank 3 5 — 21 aa 15 — 1533 Class I 4 52 epitopes_21 ?anks 7.5 13 — 21 aa 39 — 213a 52 eptiopes_21 ?ank Class II_15 ?ank 7.5 13 — 21 aa 39 — 1533 Class I Dosing was equi-picomolar, meaning that all groups received the same concentration of each individual epitope despite construct length. Animals were given one dose on day 0 (priming dose), a second dose on day 6 (boost), and then splenocytes were harvested on day 12 and IFNy ELISpot was med on samples.

The immunogenicity of the 52 e-containing vaccine was examined. RNA 1/SIINFEKL (SEQ ID NO: 231) was the ?nal epitope for each of the four constructs tested.

SIINFEKL (SEQ ID NO: 231) T-cell responses in 52 epitope constructs con?rm the full read through of the concatamer, as INFy responses were observed from all test groups when re- ation with RNA FEKL (SEQ ID NO: 231) was performed (. Note that, as expected, there was no RNA 1 found in the RNA 31 concatamer because the concatamer did not have the RNA 1/SIINFEKL (SEQ ID NO: 231) epitope.

The immunogenicity n the 52mer and 20mer ucts was similar. For example, both behave similarly when re-stimulated with Class I epitopes ( Trimming the length of the Class II epitopes may improve genicity, while trimming Class I epitopes from 21 to 15 amino acids did not affect genicity. Further, immunogenicity to additional epitopes was detected in the 52 epitope constructs (. Both 52mer and 20mer constructs behaved comparably when re-stimulated with Class II epitopes (.

Table 3. Selected Sequences SEQ SLQLNCE MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAEISAVCEKGNFN MAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGD HAG|KDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQN NC RLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (huSTING(V155M); no epitope tag) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAEISAVCEKGNFN VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDH AG|KDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDtLEQAKLFCRTLEDILADAPESQNNCRL IAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(R284T); no epitope tag) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAEISAVCEKGNFN VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDH AG|KDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDmLEQAKLFCRTLEDILADAPESQNNCR LIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (hu STING (R284M); no epitope tag) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAEISAVCEKGNFN VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDH AGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDkLEQAKLFCRTLEDILADAPESQNNCR LIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING (R284K); no epitope tag) SEQ SLQLNCE MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWG LGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAE|SAVCEKGNFS VAHGLAWSYYIGYLRLILPELQARIRTYNQHYN N LLRGAVSQRLYI GVPDN LSMADPN | RFLDKLPQQTG DH AGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAG FSREDRLEQAKLFCRTLEDILADAPESQNNCR LIAYQE PADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(N154S); no epitope tag) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWG HTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS WRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAE|SA|CEKGNFN VAHGLAWSYYIGYLRLILPELQARIRTYNQHYN N LLRGAVSQRLYI LLPLDCGVPDN LSMADPN | RFLDKLPQQTG DH AGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAG FSREDRLEQAKLFCRTLEDILADAPESQNNCR LIAYQE PADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(V147L); no epitope tag) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWG LGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAE|SAVCEKGNFN VAHGLAWSYYIGYLRLILPELQARIRTYNQHYN N LLRGAVSQRLYI LLPLDCGVPDN LSMADPN | RFLDKLPQQTG DH AGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAG FSREDRLEQAKLFCRTLEDILADAPESQNNCR LIAYQq PADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING (53150); no epitope tag) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWG LGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS WRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAE|SAVCEKGNFN VAHGLAWSYYIGYLRLILPELQARIRTYNQHYN N LLRGAVSQRLYI LLPLDCGVPDN LSMADPN | RFLDKLPQQTG DH AGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAG FSREDRLEQAKLFCRTLEDILADAPESQNNCR LIAYQE PADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLaTDFS (Hu STING (R375A); no epitope tag) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWG LGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAE|SALCEKGNFS WSYYIGYLRLILPELQARI RTYNQHYN N LLRGAVSQRLYI LLPLDCGVPDN LSMADPNIRFLDKLPQQTGD HAGIKDRVYSNSIYE LLENGQRAGTCVLEYATPLQTLFAMSQYSQAG FSRE DRLEQAKLFCRTLEDILADAPESQN NC RLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQE PE LLISGMEKPLPLRTDFS (Hu V147L/N154S/V155M); no epitope tag) O MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWG LGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS WRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAE|SALCEKGNFS MAHGLAWSYYIGYLRLILPELQARI RTYNQHYN N LLRGAVSQRLYI LLPLDCGVPDN LSMADPNIRFLDKLPQQTGD HAG|KDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAG FSREDMLEQAKLFCRTLEDILADAPESQNN CRLIAYQE PADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(R284M/Vl47L/N154S/V155M); no epitope tag) 199 ATGCCCCACAGTAGCCTCCACCCCAGCATCCCCTGCCCCAGAGGCCACGGCGCACAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGTCTGGTGACCCTGTGGGGTCTGGGCGAGCCCCCCGAGCACACCCTGCGGTACCTCGT GCTGCATCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAAGAGCTGAGACA CATCCACAGCAGATACAGAGGCTCCTACTGGAGAACCGTCAGAGCCTGCCTCGGCTGTCCCCTGAGAAGAGGC GCCCTGCTGCTCCTGAGCATCTACTTCTACTACAGCCTGCCCAACGCCGTGGGCCCCCCCTTCACCTGGATGCTG GCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCTTGGCCCCCGCCGAGATCTCCG CCGTGTGCGAGAAGGGCAACTTCAACATGGCCCATGGCCTTGCCTGGTCCTACTACATCGGCTACCTGAGACTG ATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGA GCCAAAGACTGTACATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTTAGCATGGCCGACCCCAACATC AGATTCCTGGACAAGCTGCCCCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGC ATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCCCTGCAGACCC TGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAAGCCAAGCTGTTCTG CAGAACCCTGGAGGACATCCTGGCGGACGCCCCCGAGAGCCAAAACAACTGCAGACTGATCGCCTACCAGGA GCCCGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAAGTGCTGAGACACCTGAGACAGGAAGAGAAGGAGG CCGTGGGAAGCCTGAAGACCAGCGCCGTGCCCAGCACCAGCACCATGAGCCAGGAGCCCGAGCTGC TGATCAGCGGCATGGAGAAGCCCCTGCCCCTGAGAACCGACTTCAGC (huSTING(V155M); no epitope tag; tide sequence) SLQLNCE ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGT CCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGG CGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCT GGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGC GCCGTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGAC TGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGT GAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAAC ATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACA GCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGAC CCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACACCCTGGAGCAGGCCAAGCTGTTC TGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGG AGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAG GAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTG CTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(R284T); no epitope tag; nucleotide ce) 201 ATGCCCCACAGCAGCCTGCACCCCTCCATCCCCTGTCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGAGCGCCTGCCTGGTGACCTTATGGGGCCTGGGCGAGCCCCCCGAGCACACCCTGAGATACCTGGT CCTGGCCAGCCTCCAGCTGGGCCTGCTGCTCAACGGCGTGTGTAGCCTGGCCGAGGAGCTGAGACAC ATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGTTGCCCACTGAGAAGAGGA GCTCTGCTGCTGCTGAGCATCTACTTCTACTACTCGCTGCCCAACGCTGTGGGCCCCCCCTTCACCTGGATGCTG GCCCTGCTGGGTCTGAGCCAGGCCCTGAACATCCTCCTGGGCCTGAAGGGCCTGGCCCCCGCCGAGATAAGCG CCGTTTGCGAGAAGGGCAACTTCAACGTGGCCCATGGCCTGGCCTGGAGCTACTACATCGGCTACTTACGCCTG ATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCATTACAACAACCTGCTGAGAGGCGCCGTGA GCCAGAGACTGTATATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTGAGCATGGCCGACCCCAACATC AGATTCCTGGACAAGCTCCCCCAGCAGACCGGCGACCACGCCGGAATCAAAGACAGAGTGTATAGCAACAGCA TCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTACTGGAGTACGCCACCCCCTTGCAGACCCT GTTTGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACATGCTGGAGCAGGCCAAGCTGTTCTGC AGAACCCTGGAGGACATCCTGGCCGACGCCCCCGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAAGAGC CCGCCGACGACAGCAGCTTCAGCTTAAGCCAGGAGGTGCTGAGACATCTGAGACAGGAGGAGAAGGAGGAG GTGACCGTGGGCAGCCTCAAGACCAGCGCTGTGCCCTCTACCAGCACCATGAGCCAGGAGCCCGAGCTGCTGA TCAGCGGCATGGAGAAGCCCCTGCCCCTGAGAACAGACTI'CAGC (hu STING (R284M); no epitope tag; nucleotide sequence) 202 ATGCCCCATAGCAGCCTGCACCCCAGCATCCCCTGCCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TCCTGCTGAGCGCATGCCTGGTCACCCTGTGGGGCCTGGGCGAGCCCCCCGAGCACACCCTGAGATACCTGGT GCTGCACCTCGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATATAGAGGCAGCTACTGGAGAACCGTGAGAGCTTGCCTCGGCTGCCCCCTGAGAAGAGGC GCCCTGCTGCTGCTGAGCATCTACTTTTACTACAGCCTGCCCAACGCTGTGGGCCCCCCTTTCACGTGGATGCTC GCCCTGCTGGGACTGAGCCAGGCCCTGAACATCCTGCTGGGCCTTAAGGGCCTAGCCCCCGCCGAGATCAGCG CCGTGTGCGAGAAGGGCAACTTCAATGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACT GATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAATCAGCACTACAACAACCTGCTGAGAGGCGCCGTG AGACTGTACATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTCAGCATGGCCGACCCCAACAT CAGATTCCTGGACAAGCTGCCCCAGCAGACCGGCGACCACGCCGGCATCAAGGATCGCGTGTACAGCAACAGC ATCTACGAGCTGCTGGAAAACGGCCAGAGAGCCGGAACCTGCGTGCTGGAGTACGCCACACCCCTGCAGACCC TGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAAGCTGGAGCAGGCCAAGCTGTTCT GCAGAACCCTGGAGGATATCCTCGCCGACGCCCCCGAGAGCCAGAACAACTGCAGGCTGATCGCGTACCAGG AGCCCGCTGACGACAGCAGCTTTAGCCTGAGCCAGGAGGTGCTGAGACATCTGCGTCAAGAGGAAAAGGAGG AGGTGACCGTGGGCTCCCTGAAGACCAGCGCCGTGCCCAGCACCAGCACCATGAGCCAGGAGCCCGAGCTGC TGATCAGCGGCATGGAGAAGCCACTGCCCCTCAGAACCGACTTCAGC (Hu STING (R284K); no epitope tag; nucleotide sequence) SLQLNCE ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGT GCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGG GCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCT GGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGC GCCGTGTGCGAGAAGGGCAACTTCAGCGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGAC TGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGT GAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAAC ATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACA GCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGAC CCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTC TGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGG AGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAG GAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTG CTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(N154S); no epitope tag; nucleotide sequence) 204 ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGT GCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGG CGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCT GGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGC GCCCTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGAC TGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGT GAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAAC ATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACA GCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGAC CCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTC ACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGG AGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAG GAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTG CTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(V147L); no epitope tag; nucleotide sequence) 205 CACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGT GCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGG CGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCT GGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGC GCCGTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGAC TGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGT GAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAAC ATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACA GCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGAC CCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTC TGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGC AGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAG GAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTG CTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING (E3150); no epitope tag,- nucleotide sequence) ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGT GCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGG CGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCT GGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGC GCCGTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGAC TGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGT GAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAAC TTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACA GCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGAC CCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTC TGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGG AGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAG GAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTG CTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGGCCACCGACTTCAGC (Hu STING (R375A); no epitope tag; nucleotide sequence) 207 ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGT GCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGG CGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCT GCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGC GCCCTGTGCGAGAAGGGCAACTTCAGCATGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGAC TGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGT GAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAAC ATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACA GCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGAC CCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTC TGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGG AGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAG GAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTG CTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(V147L/N154S/V155M); no epitope tag; nucleotide sequence) 208 ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGT GCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGG CGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCT GGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGC GCCCTGTGCGAGAAGGGCAACTTCAGCATGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGAC TGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGT GAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAAC ATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACA GCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGAC CCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACATGCTGGAGCAGGCCAAGCTGTTC TGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGG AGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAG GAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTG CTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(R284M/V147L/N154S/V155M); no epitope tag; nucleotide sequence) 209 TGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTC CTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCWGAATAAAGTCTGAGTGGGCGGC (3" UTR used in STING V15Sl‘v’i uct, containing miRlZZ binding site) wo 2018/144082 ZOl7/058595 SLQ SE UENCE MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWG LGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHS RYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALN|LLGLKGLAPAE|SAVCEKGNFN VAHGLAWSYYIGYLRLILPELQARIRTYNQHYN N LLRGAVSQRLYI GVPDN LSMADPN | RFLDKLPQQTG DH AGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAG FSREDkLEQAKLFCRTLEDILADAPESQNNCR LIAYQE PADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFST (Hu STING (R284K) var; no epitope tag) 225 ATGCCCCATAGCAGCCTGCACCCCAGCATCCCCTGCCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCTGG TCCTGCTGAGCGCATGCCTGGTCACCCTGTGGGGCCTGGGCGAGCCCCCCGAGCACACCCTGAGATACCTGGT GCTGCACCTCGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACA CATCCACAGCAGATATAGAGGCAGCTACTGGAGAACCGTGAGAGCTTGCCTCGGCTGCCCCCTGAGAAGAGGC GCCCTGCTGCTGCTGAGCATCTACTTTTACTACAGCCTGCCCAACGCTGTGGGCCCCCCTTTCACGTGGATGCTC GCCCTGCTGGGACTGAGCCAGGCCCTGAACATCCTGCTGGGCCTTAAGGGCCTAGCCCCCGCCGAGATCAGCG GCGAGAAGGGCAACTTCAATGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACT GATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAATCAGCACTACAACAACCTGCTGAGAGGCGCCGTG AGCCAGAGACTGTACATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTCAGCATGGCCGACCCCAACAT CAGATTCCTGGACAAGCTGCCCCAGCAGACCGGCGACCACGCCGGCATCAAGGATCGCGTGTACAGCAACAGC ATCTACGAGCTGCTGGAAAACGGCCAGAGAGCCGGAACCTGCGTGCTGGAGTACGCCACACCCCTGCAGACCC TGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAAGCTGGAGCAGGCCAAGCTGTTCT GCAGAACCCTGGAGGATATCCTCGCCGACGCCCCCGAGAGCCAGAACAACTGCAGGCTGATCGCGTACCAGG AGCCCGCTGACGACAGCAGCTTTAGCCTGAGCCAGGAGGTGCTGAGACATCTGCGTCAAGAGGAAAAGGAGG AGGTGACCGTGGGCTCCCTGAAGACCAGCGCCGTGCCCAGCACCAGCACCATGAGCCAGGAGCCCGAGCTGC TGATCAGCGGCATGGAGAAGCCACTGCCCCTCAGAACCGACTTCAGCACC (Hu STING (R284K) var; no epitope tag) Example 7. Activating Oncogene KRAS Mutations KRAS is the most frequently d ne in human cancer (~15%). KRAS mutations are mostly conserved in a single "hotspot", and activate the oncogene. Prior research has shown limited ability to raise T cells speci?c to the oncogenic on.

However, much of this was done in the context of the most common HLA allele (A2, which occurs in ~50% of Caucasians). More recently, it has been demonstrated that (a) speci?c T cells can be generated against point mutations in the context of less common HLA alleles (All, C8), and (b) growing these cells eX-vivo and transferring them back to the patient has mediated a dramatic tumor response in a patient with lung cancer. (N Engl J Med 2016, 375:2255-2262December 8, 2016DOI: 6/NEJMoal609279).

As shown in Table 4 below, in CRC (colorectal cancer), only 3 mutations (G12V, G12D, and G13D) t for 96% of cases. Furthermore, all CRC patients get typed for KRAS mutations as standard of care.

Table 4.

CQSM¥C* case counts A5§§ cancers ‘5?) CRC 93 (31.25 2849 1% {312? {3213 41% SSS-:15 38% Eizc 4535 2333 {3133 2383-13 3’%. 8083 «ism 612.5% 22.29 3.2% {3123 223.61 31% 6'13!) $0813, 2%. £228?" 23% 3.8% 96% Tested ZQSSZQ 183?}. fcancemauger.aauix‘fnosmicfgenefanaéysis?§n=rK.RAS In another COSMIC data set, 73.68% ofKRAS mutations in colorectal cancer are ted for by these 3 mutations (G12V, G12D, and G13D) (Figure 15 and Table 5).

Table 5 12D 635 35.04 178 33.46 813 34.68 13D 338 18.65 88 16.54 426 FIGs. 16, 17, and 18 depict isoform-secif1c point mutation city for HRAS, KRAS, and NRAS, respecively. Data representing total number of tumors with each point mutation were collated from COSMIC V52 release. Single base mutations generating each amino acid substitution are indicated. The most frequent ons for each m for each cancer type are ghted with grey shading. H/L: hematopoietic/lymphoid tissues. (Prior et al. Cancer Res. 2012 May 15, 72(10): 2457—2467).

In addition, secondary KRAS mutations have been identi?ed in EGFR blockade resistant patients. RAS is downstream of EGFR and it has been found to constitute a mechanism of resistance to EGFR blockade therapies. EGFR blockade resistant KRAS mutant tumors can be targeted using compositions and methods disclosed herein. In a few cases, more than one KRAS mutation was identified in the same patient (up to four different mutations co-occur). This mutational spectrum appears to be at least somewhat different than primary tumor missense mutants in colorectal cancer. (Diaz et al The molecular evolution of acquired ance to targeted EGFR blockade in colorectal cancers, Nature 486:537 , Misale et al Emergence ofKRAS muations and acquired resistance to anti-EGFR therapy in colorectal cancer, Nature 486:532 (2012)). depicts secondary KRAS mutations after acquisition of EGFR blockade resistance. (Diaz et al The molecular evolution of ed resistance to targeted EGFR blockade in ctal cancers, Nature 486:537 (2012)). depicts secondary KRAS mutations after EGFR blockade. (Misale et al Emergence of KRAS muations and acquired resistance to anti-EGFR therapy in colorectal cancer, Nature 486:532 ).

As shown in , NRAS is also mutated in ctal cancer, but at a lower frequency than KRAS.

In this e, animals are administered an RNA cancer vaccine that includes an mRNA encoding at least one activating oncogene mutation peptide, e.g., at least one activating KRAS mutation. HLA*A*11:01 Tg mice (Taconic, strain 966OF, n=4) or HLA- A*2:01 Tg mice (Taconic, strain 9659F, n=4) are administered mRNA encoding mutated KRAS as follows: mRNA encoding mutated KRAS administered on day 1, bleed taken on day 8, mRNA encoding mutated KRAS administered on day 15, animal sacri?ced on day 22.

The test groups are shown in Table 6 as follows: Table 6 Test/Control Genetic Dosing TESTgroup Group Vehicle. Route al adjuvant Regimen 1 KRAS G12D None(NTFIX) Com§SOund IM Day 1, 15 C d KRAS-MUT 2 KRAS G12v None(NTFIX) 0mg?" IM Day 1, 15 Compound 3 KRAS 613D None(NTFIX) IM Day 1, 15 Compound No Ag 4 NTFIX NTFIX IM Day 1, 15 mRNA is administered to animals at a dose of 0.5 mg/kg (10ug per 20-g animal). Ex vivo restimulation (lug/ml per peptide) is tested for 4 hours at 37 degrees Celsius in the 2O presence of GolgiPlug (Brefeldin A). Intracellular cytokine staining (ICS) is tested for KRAS G12D, KRAS G12V, KRAS G13D, KRAS G12WT, KRAS G13WT, and no peptide. mRNA encoding KRAS ons is tested for the ability to generate T cells.

Ef?cacy of mRNA encoding KRAS mutations is compared, for e, to peptide vaccination.

Exemplary KRAS t peptide sequences and mRNA constructs are shown in Tables 7-9.

Table 7. KRAS mutant peptide sequences mer (SEQ ID KLVVVGADGVGKSAL VITEYKLVVVGADGVGKSALTIQLIQ G12D \Oz316) (SEQ ID \Oz317) (SEQ ID \Oz318) VVGAVGVGK (SEQ ID KLVVVGAVGVGKSAL VITEYKLVVVGAVGVGKSALTIQLIQ G12V \Oz319) (SEQ ID \Oz320) (SEQ ID \Oz321) VGAGDVGKS (SEQ ID GDVGKSALT LVVVGAGDVGKSALTIQLIQ G13D \Oz322) (SEQ ID \Oz323) (SEQ ID \Oz324) VVGACGVGK (SEQ ID KLVVVGACGVGKSA VITEYKLVVVGACGVGKSALTIQLIQ G12C ) (SEQ ID \Oz326) (SEQ ID \Oz327) WT —VITEYKLVVVGAGGVGKSALTIQLIQ(SEQ ID \Oz328) Table 8. KRAS mutant amino acid sequences KRAS MUTANT AMINO ACID SEQUENCE KRAS(G12D) 15mer VIKLVVVGADGVGKSAL (SEQ ID NO:329) KRAS(G12V) 15mer VIKLVVVGAVGVGKSAL (SEQ ID NO:330) KRAS(G13D) 15mer VILVVVGAGDVGKSALT (SEQ ID NO:331) KRAS(G12D) 25mer VITEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO:332) KRAS(G12V) 25mer VITEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO:333) KRAS(G13D) 25mer VITEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO:334) KRAS(G12D) VIKLVVVGADGVGKSALKLVVVGADGVGKSALKLVVVGADGVGKSAL 15mer/‘3 (SEQ ID NO:335) KRAS(G12V) VIKLVVVGAVGVGKSALKLVVVGAVGVGKSALKLVVVGAVGVGKSAL 15mer/‘3 (SEQ ID NO:336) KRAS(G13D) VILVVVGAGDVGKSALTLVVVGAGDVGKSALTLVVVGAGDVGKSALT 15mer/‘3 (SEQ ID NO:337) KRAS(G12D) VITEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQL 25mer"3 IQMTEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO:33 8) KRAS(G12V) VITEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQL 3 IQMTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO:339) KRAS(G13D) VITEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQL 25mer"3 IQMTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO:340) KRAS(G12C) 25mer VITEYKLVVVGACGVGKSALTIQLIQ (SEQ ID ) KRAS(G12C) VITEYKLVVVGACGVGKSALTIQLIQMTEYKLVVVGACGVGKSALTIQL 25mer"3 IQMTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO:342) KRAS(WT) 25mer VITEYKLVVVGAGGVGKSALTIQLIQ (SEQ ID NO:343) Table 9. KRAS mutant antigen mRNA sequences mRNA S_eq_(__qu_(_enceAminoOrf Orf ce (Nucleotidel NameS Acid MTEYKLVWGADGV ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGAC (G12D) IQLIQ (SEQ GGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATC 25mer ID NO357) CAG (SEQ ID NO344) (G12\S/) MTEYKLVWGAVGV ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGTG GKSALTIQLIQ (SEQ GGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATC 25mer ID NO:35 8) CAG (SEQ ID NO:345) MTEYKLVWGAGDV ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGC (G13D) GKSALTIQLIQ GACGTGGGCAAGAGCGCCCTGACCATCCAGCTGATC 25mer (SEQ ID NO:359) CAG (SEQ ID NO:346) MTEYKLVVVGADGV ATGACCGAGTACAAGTTAGTGGTTGTGGGCGCCGAC (G12D) GKSALTIQLIQMTEY GGCGTGGGCAAGAGCGCCCTCACCATCCAGCTTATC 25mer"3 KLVVVGADGVGKSA CAGATGACGGAATATAAGTTAGTAGTAGTGGGAGCC LTIQLIQMTEYKLVV GACGGTGTCGGCAAGTCCGCTTTGACCATTCAACTT VGADGVGKSALTIQL ATTCAGATGACAGAGTATAAGCTGGTCGTTGTAGGC 1Q (SEQ ID NO:360) GCAGACGGCGTTGGAAAGTCGGCACTGACGATCCAG TTGATCCAG (SEQ ID NO:347) KRAS MTEYKLVVVGAVGV ATGACCGAGTACAAGCTCGTCGTGGTGGGCGCCGTG (G12V) GKSALTIQLIQMTEY GGCGTGGGCAAGAGCGCCCTAACCATCCAGTTGATC 3 KLVVVGAVGVGKSA CAGATGACCGAATATAAGCTCGTGGTAGTCGGAGCG LTIQLIQMTEYKLVV GTGGGCGTTGGCAAGTCAGCGCTAACAATACAACTA GKSALTIQL ATCCAAATGACCGAATACAAGCTAGTTGTAGTCGGT 1Q (SEQ ID NO:361) GCCGTCGGCGTTGGAAAGTCAGCCCTTACAATTCAG CTCATTCAG (SEQ ID NO:348) KRAS VVVGAGDV GAGTACAAGCTCGTAGTGGTTGGCGCCGGC (G13D) GKSALTIQLIQMTEY GACGTGGGCAAGAGCGCCCTAACCATCCAGCTCATC 25mer"3 KLVVVGAGDVGKSA CAGATGACAGAATATAAGCTTGTGGTTGTGGGAGCA LTIQLIQMTEYKLVV GGAGACGTGGGAAAGAGTGCGTTGACGATTCAACTC VGAGDVGKSALTIQL ATGACCGAATACAAGTTGGTGGTGGTCGGC 1Q (SEQ ID NO:362) GCAGGTGACGTTGGTAAGTCTGCACTAACTATACAA CTGATCCAG (SEQ ID NO:349) KRAS MTEYKLVVVGACGV ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCTGC (G12C) GKSALTIQLIQ (SEQ GGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATC 25mer ID NO:363) CAG (SEQ ID NO:350) KRAS VVVGACGV ATGACCGAGTACAAGCTCGTGGTTGTTGGCGCCTGC (G12C) GKSALTIQLIQMTEY GGCGTGGGCAAGAGCGCCCTCACCATCCAGCTCATC 25mer"3 KLVVVGACGVGKSA CAGATGACAGAGTATAAGTTAGTCGTTGTCGGAGCT QMTEYKLVV TGCGGAGTTGGAAAGTCGGCGCTCACCATTCAACTC VGACGVGKSALTIQL ATACAAATGACAGAATATAAGTTAGTGGTGGTGGGT 1Q (SEQ ID NO:364) GGCGTTGGCAAGAGTGCGCTTACTATCCAG CTCATTCAG (SEQ ID NO:351) KRAS MTEYKLVVVGAGGV ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGC (WT) GKSALTIQLIQ (SEQ GGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATC 25mer ID NO:365) CAG (SEQ ID NO:352) Chemistry: uridines modi?ed N1 -methyl pseudouridine (m 1‘I’) Cap: C1 Tail: T100 ’ UTR Se uence standard 5' Flank includes Production FP + T7 site + 5'UTR : TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG AGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (SEQ ID NO:353) ’ UTR ce {No Promoter): TAAGAGAGAAAAGAAGAGTAAGAA GAAATATAAGAGCCACC (SEQ ID NO:354) 3’ UTR Sequence (Human 3' UTR no XbaI): TGATAATAGGCTGGAGCCTCGGTGGCCA TGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTAC CCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO:355) Example 8. ent splice site and silent mutation "hotspots" in 953 The p53 gene (of?cial symbol TP53) is mutated more frequently than any other gene in human cancers. Large cohort studies have shown that, for most p53 mutations, the c position is unique to one or only a few patients and the mutation cannot be used as ent neoantigens for therapeutic vaccines designed for a speci?c population of patients.

A small subset of p53 loci do, however, exhibit a "hotspot" pattern, in which several positions in the gene are mutated with relatively high frequency. Strikingly, a large portion of these recurrently mutated regions occur near exon-intron boundaries, disrupting the canonical nucleotide sequence motifs recognized by the mRNA splicing machinery. Mutation of a splicing motif can alter the ?nal mRNA sequence even if no change to the local amino acid sequence is predicted (i.e. for synonymous or intronic mutations). Therefore, these ons are often annotated as "noncoding" by common tion tools and neglected for further 2O analysis, even though they may alter mRNA splicing in unpredictable ways and exert severe functional impact on the translated protein. If an alternatively spliced isoform produces an in- frame sequence change (1'.e., no PTC is produced), it can escape depletion by NMD and be readily expressed, processed, and presented on the cell surface by the HLA system. Further, mutation-derived alternative splicing is usually "cryptic", 1'. e., not expressed in normal tissues, and ore may be recognized by T-cells as non-self neoantigens.

Several mutation sites were ed by RNA-seq to produce retained introns or cryptic ng. Two representative mutation-derived peptides had multiple HLA-A2 binding es with no matches elsewhere in the coding genome.

Recurrent mutations in p53 that were identi?ed included: 3O (1) mutations at the canonical 5’ splice site neighboring codon p.Tl25, inducing a retained intron having peptide sequence TAKSVTCTVSCPEGLASMRLQCLAVSPCISFVWNFGIPLHPLASCQCFFIVYPLNV (SEQ ID NO: 232) that contains epitopes AVSPCISFVW (SEQ ID NO: 233) *57:Ol, HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) (HLA-A*O2:Ol, HLA-A*O2:O6, HLA-B*35:Ol), (2) mutations at the canonical 5’ splice site neighboring codon p.331, inducing a ed intron having peptide sequence EYFTLQVLSLGTSYQVESFQSNTQNAVFFLTVLPAIGAFAIRGQ (SEQ ID NO: 236) that contains epitopes LQVLSLGTSY (SEQ ID NO: 237) (HLA-B*15:01), FQSNTQNAVF (SEQ IDNO: 238) (HLA-B*15:01), (3) mutations at the cal 3’ splice site neighboring codon p. 126, inducing a cryptic alternative exonic 3’ splice site producing the novel spanning peptide sequence AKSVTCTMFCQLAK (SEQ ID NO: 239) that ns epitopes CTMFCQLAK (SEQ ID NO: 240) (HLA-A*11:01), MF (SEQ ID NO: 241) (HLA-B*58:01), and (4) mutations at the canonical 5’ splice site neighboring codon p.224, inducing a cryptic alternative intronic 5’ splice site producing the novel spanning peptide sequence VPYEPPEVWLALTVPPSTAWAA (SEQ ID NO: 242) that contains epitopes VPYEPPEVW (SEQ ID NO: 243 (HLA-B*53:Ol, HLA-B*51:Ol), LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:01, HLA-B*57 :01), n the transcript codon positions refer to the canonical full-length p53 transcript ENST00000269305 (SEQ ID NO: 245) from the Ensembl v83 human genome annotation.

EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than e mentation, many equivalents to the speci?c embodiments of the disclosure described herein. Such equivalents are ed to be encompassed by the following claims.

The term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated nce value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

All references, including patent documents, sed herein are incorporated by reference in their entirety.

Claims

1. An mRNA cancer vaccine, comprising: a lipid nanoparticle comprising an mRNA having an open reading frame encoding 10-50 cancer antigen peptide epitopes, wherein at least 10 of the cancer antigen peptide epitopes are encoded in a concatemeric structure, wherein the cancer n peptide epitopes comprise ns identified as being expressed in a tumor of a subject, wherein the cancer antigen peptide epitopes se an activating oncogene on e having an amino acid sequence selected from the group consisting of a recurrent KRAS mutant, a recurrent HRAS mutant, and a ent NRAS mutant identified as being expressed in the tumor of the subject, and wherein the lipid rticle comprises an ionizable cationic lipid, a neutral lipid, a sterol, and a PEG-modified lipid.

2. The mRNA cancer vaccine of claim 1, wherein at least three of the cancer antigen peptide epitopes are complex variants and/or at least two of the cancer antigen peptide es comprise point mutations.

3. The mRNA cancer vaccine of claim 1 or claim 2, wherein the mRNA comprises a 5’ UTR comprising the nucleotide sequence set forth in SEQ ID NO: 176.

4. The mRNA cancer vaccine of any one of claims 1 to 3, wherein the mRNA comprises a poly A tail.

5. The mRNA cancer vaccine of claim 4, wherein the poly A tail comprises about 100 nucleotides.

6. The mRNA cancer vaccine of any one of claims 1 to 5, wherein the mRNA comprises a 5’ Cap 1 structure.

7. The mRNA cancer vaccine of any one of claims 1 to 6, wherein the mRNA comprises at least one chemical modification.

8. The mRNA cancer vaccine of claim 7, wherein the chemical cation is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4’- thiouridine, 5-methylcytosine, 2-thiomethyldeaza-pseudouridine, 2-thiomethylpseudouridine , 2-thioaza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thiopseudouridine , 4-methoxythio-pseudouridine, 4-methoxy-pseudouridine, 4-thiomethylpseudouridine , 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methyluridine, 5-methoxyuridine, and 2’-O-methyl uridine.

9. The mRNA cancer vaccine of claim 7 or claim 8, wherein the chemical modification is N1-methylpseudouridine.

10. The mRNA cancer e of claim 9, wherein the mRNA is fully modified with N1- pseudouridine.

11. The mRNA cancer e of any one of claims 1 to 10, wherein two or more of the cancer antigen peptide epitopes of the concatemeric structure are interspersed by ge ive sites.

12. The mRNA cancer vaccine of any one of claims 1 to 11, wherein the mRNA encodes 20- 40 cancer antigen peptide epitopes.

13. The mRNA cancer vaccine of any one of claims 1 to 12, wherein the mRNA encodes 30- 40 cancer antigen peptide epitopes.

14. The mRNA cancer e of any one of claims 1 to 13, wherein at least 20 of the cancer antigen peptide epitopes are encoded in the concatemeric structure.

15. The mRNA cancer vaccine of any one of claims 1 to 14, wherein all of the cancer n peptide epitopes are encoded in the concatemeric structure.

16. The mRNA cancer vaccine of any one of claims 1 to 15, wherein at least two of the cancer antigen peptide epitopes comprise a centrally located SNP mutation with 7-15 flanking amino acids on each side of the SNP mutation.

17. The mRNA cancer vaccine of any one of claims 1 to 16, wherein at least one of the cancer n peptide epitopes is a T cell epitope.

18. The mRNA cancer vaccine of any one of claims 1 to 16, wherein at least one of the cancer antigen peptide epitopes is a B cell epitope.

19. The mRNA cancer vaccine of any one of claims 1 to 16, wherein the cancer antigen peptide es comprise a ation of T cell epitopes and B cell epitopes.

20. The mRNA cancer e of any one of claims 1 to 19, further comprising a recall antigen.

21. The mRNA cancer vaccine of claim 20, wherein the recall antigen is an infectious disease antigen.

22. The mRNA cancer vaccine of any one of claims 1 to 21, wherein the activating oncogene on peptide comprises an amino acid sequence from a recurrent KRAS mutant.

23. The mRNA cancer vaccine of claim 22, wherein the recurrent KRAS mutant is a G12 mutant.

24. The mRNA cancer vaccine of claim 23, wherein the G12 KRAS mutant is selected from the group consisting of a G12D, G12V, G12S, G12C, G12A, and a G12R KRAS mutant.

25. The mRNA cancer vaccine of claim 22, wherein the recurrent KRAS mutant is a G13 mutant.

26. The mRNA cancer vaccine of claim 25, wherein the G13 KRAS mutant is a G13D KRAS mutant.

27. The mRNA cancer vaccine of any one of claims 1 to 21, wherein the ting oncogene mutation peptide comprises an amino acid sequence from a recurrent HRAS mutant or a recurrent NRAS mutant.

28. The mRNA cancer vaccine of any one of claims 1 to 27, wherein at least two of the cancer antigen peptide epitopes are separated from one another by an amino acid linker.

29. The mRNA cancer vaccine of claim 28, wherein at least two of the cancer antigen peptide epitopes are separated from one another by a single Glycine.

30. The mRNA cancer vaccine of any one of claims 1 to 10 and 12 to 29, wherein all of the cancer antigen e epitopes are separated from one another by a single Glycine.

31. The mRNA cancer vaccine of any one of claims 1 to 29, wherein at least two of the cancer n peptide epitopes are linked ly to one another without a linker.

32. The mRNA cancer vaccine of any one of claims 1 to 31, wherein the cancer antigen peptide epitopes further comprise a second activating oncogene on peptide comprising an amino acid ce from a recurrent p53 mutant.

33. The mRNA cancer vaccine of claim 32, wherein the second activating ne mutation peptide is selected from the group ting of: AVSPCISFVW (SEQ ID NO: 233) (HLAB *57:01, HLA-B*58:01), HPLASCQCFF (SEQ ID NO: 234) (HLA-B*35:01, HLA-B*53:01), FVWNFGIPL (SEQ ID NO: 235) (HLA-A*02:01, HLA-A*02:06, HLA-B*35:01), LQVLSLGTSY (SEQ ID NO: 237) (HLA-B*15:01), FQSNTQNAVF (SEQ ID NO: 238) (HLAB *15:01), CTMFCQLAK (SEQ ID NO: 240) (HLA-A*11:01), KSVTCTMF (SEQ ID NO: 241) (HLA-B*58:01), VPYEPPEVW (SEQ ID NO: 243) (HLA-B*53:01, HLA-B*51:01), and LTVPPSTAW (SEQ ID NO: 244) (HLA-B*58:01, HLA-B*57:01).

34. The mRNA cancer vaccine of any one of claims 1 to 33, wherein the lipid nanoparticle comprises 20-60% ble cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

35. The mRNA cancer vaccine of claim 34, n the ionizable amino lipid is selected from the group consisting of 2,2-dilinoleyldimethylaminoethyl-[1,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyldimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-nonen yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

36. The mRNA cancer vaccine of any one of claims 1 to 35, wherein the lipid nanoparticle comprises a compound of Formula (I): wherein: R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 l, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 ycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, (O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, 9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently ed from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, (R’)-, -N(R’)C(O)-, , -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a aryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and each R’ is independently selected from the group ting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or stereoisomers thereof.

37. The mRNA cancer vaccine of claim 36, wherein the compound of Formula (I) is

38. The mRNA cancer vaccine of any one of claims 1 to 34, wherein the lipid nanoparticle ses about 50% , about 10% DSPC, about 38.5% cholesterol, and about 1.5% PEG-DMG.

39. The mRNA cancer vaccine of any one of claims 1 to 38, wherein the lipid nanoparticle has a polydispersity index of less than 0.4.

40. The mRNA cancer e of any one of claims 1 to 39, n the lipid nanoparticle has a net neutral charge at a neutral pH value.

41. The mRNA cancer vaccine of any one of claims 1 to 40, wherein the mRNA further comprises an open reading frame encoding an immune checkpoint modulator.

42. The mRNA cancer vaccine of any one of claims 1 to 41, further comprising an additional cancer therapeutic agent.

43. The mRNA cancer vaccine of claim 42, wherein the additional cancer therapeutic agent is an immune checkpoint modulator.

44. The mRNA cancer vaccine of claim 41 or 43, n the immune checkpoint modulator is an inhibitory checkpoint polypeptide.

45. The mRNA cancer e of claim 44, wherein the inhibitory checkpoint polypeptide inhibits PD1, PD-L1, CTLA4, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, or a combination thereof.

46. The mRNA cancer vaccine of claim 45, wherein the inhibitory oint ptide is an antibody.

47. The mRNA cancer vaccine of claim 46, wherein the inhibitory oint polypeptide is an antibody selected from an anti-CTLA4 antibody or n-binding fragment thereof that specifically binds CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that ically binds PD-L1, and a combination thereof.

48. The mRNA cancer vaccine of any one of claims 44 to 47, wherein the inhibitory checkpoint polypeptide is an antibody selected from atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, nivolumab, and pembrolizumab.

49. The mRNA cancer vaccine of any one of claims 44 to 47, wherein the inhibitory checkpoint polypeptide comprises pembrolizumab.

50. The mRNA cancer vaccine of any one of claims 1 to 49, wherein the recurrent HRAS mutant has an amino acid mutation in codon 12, codon 13, and/or codon 61.

51. The mRNA cancer e of claim 50, wherein the recurrent HRAS mutant is a 12V, 61L, or 61R mutation.

52. The mRNA cancer vaccine of any one of claims 1 to 51, wherein the recurrent NRAS mutant has an amino acid mutation in codon 12, codon 13, and/or codon 61.

53. The mRNA cancer e of claim 52, wherein the recurrent NRAS mutant is a 12D, 13D, 61K, or 61R mutant.

54. The mRNA cancer vaccine of any one of claims 1 to 53, wherein the cancer antigen peptide epitopes are arranged in order to minimize -epitopes.

55. The mRNA cancer vaccine of any one of claims 1 to 54, wherein the cancer antigen peptide es comprise at least one MHC class I e and at least one MHC class II epitope.

56. The mRNA cancer vaccine of any one of claims 1 to 55, wherein at least 40% of the cancer antigen peptide es are MHC class I epitopes and at least 10% of the cancer antigen peptide epitopes are MHC class II epitopes.

57. Use of the mRNA cancer vaccine of any one of claims 1 to 56 in the manufacture of a medicament for prophylactic and/or therapeutic treatment of cancer in a subject.

58. The use of claim 57, wherein the medicament is formulated for administration at a dosage level sufficient to deliver between 10 µg and 400 µg of the mRNA cancer vaccine to the subject.

59. The use of claim 58, wherein the medicament is formulated for administration at a dosage level sufficient to deliver 0.033mg, 0.1mg, 0.2 mg, or 0.4 mg to the subject.

60. The use of claim 57 or 58, n the medicament is formulated for administration to the subject twice, three times, four times or more.

61. The use of claim 60, wherein the medicament is formulated for administration once a day every three weeks.

62. The use of any one of claims 57 to 61, wherein the medicament is formulated for administration by intradermal, intramuscular, and/or subcutaneous administration.

63. The use of claim 62, wherein the medicament is formulated for stration by intramuscular administration.

64. Use of the mRNA cancer e of any one of claims 42 to 49 in the manufacture of a medicament for prophylactic and/or therapeutic treatment of cancer in a subject, wherein the medicament is formulated for administration at a dosage level sufficient to deliver 100-300 mg of the additional cancer therapeutic agent to the subject.

65. The use of claim 64, wherein the medicament is ated for administration at a dosage level sufficient to deliver 200 mg of the additional cancer therapeutic agent to the subject.

66. The use of any one of claims 57 to 65, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial carcinoma, gative head and neck squamous cell oma (HNSCC), and a solid ancy that is microsatellite high (MSI H) / mismatch repair (MMR) deficient.

67. The use of any one of claims 57 to 65, wherein the cancer is selected from cancer of the pancreas, cancer of the peritoneum, cancer of the large intestine, cancer of the small intestine, cancer of the biliary tract, lung cancer, cancer of the endometrium, cancer of the ovary, cancer of the genital tract, cancer of the gastrointestinal tract, cancer of the cervix, cancer of the h, cancer of the urinary tract, colon , cancer of the rectum, and cancer of the hematopoietic and lymphoid tissues.

68. The use of claim 66, wherein the cancer is NSCLC, and the NSCLC lacks an EGFR sensitizing mutation and/or an ALK translocation.

69. The use of claim 66, wherein the cancer is the solid malignancy that is microsatellite high (MSI H) / mismatch repair (MMR) deficient, and is selected from the group consisting of ctal cancer, stomach adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer.

70. A method of producing an mRNA encoding a concatemeric cancer antigen comprising between 1000 and 3000 nucleotides, the method comprising: (a) binding a first cleotide comprising an open reading frame encoding 10-50 cancer antigen peptide epitopes encoded in a concatemeric ure and a second polynucleotide comprising a 5'-UTR to a polynucleotide conjugated to a solid support, wherein the cancer n peptide epitopes comprise an activating oncogene mutation peptide, wherein the activating oncogene mutation peptide ses an amino acid sequence from a recurrent KRAS mutant, a recurrent HRAS , or a recurrent NRAS mutant identified as being expressed in a tumor of a subject; (b) ligating the 3'-terminus of the second polynucleotide to the 5'-terminus of the first polynucleotide under suitable ions, wherein the suitable conditions comprise a DNA Ligase, thereby producing a first ligation product; (c) ligating the 5’ us of a third polynucleotide comprising a 3'-UTR to the 3’- terminus of the first ligation product under suitable conditions, n the suitable conditions comprise an RNA Ligase, thereby producing a second ligation product; and (d) releasing the second ligation product from the solid support, thereby ing an mRNA encoding the concatemeric cancer antigen comprising between 1000 and 3000 nucleotides. m m mwmwu mwmmu ?g ?g $33 m; $me $me 3ng WEE Egg Mama Pm wmammna?m wwsmmagam «Nammmawam wmammggam {Km mm mm mm mm 43mmzmm2142m ?mumcmmegnEEm mmvw? mam anew mam (W3 93?) S??Sm SUBSTITUTE SHEET (RULE 26) Ae%%%%@@g?@@¢zaz«z@%5% mama 5% 3&0 %v ?ag, Mama @3232on am? gmmm wmammgaam mm mm 5 &% N Eng. m &% $me OE magnum .Ww mama &% mmmmm .Ww mvm?mm mama ,m?mw %% wNimememw wmammm?am ON mm .. % § a Qmmw amen. C3 (Mia 93?) mag Am: SUBSTITUTE SHEET (RULE 26) m?sbmgam mmmmu amxymwawxm mwmmu Emma xcmm wmma?um eeeaaaz?wgeazax??zzaganwvv%%%%%%%%%&%%%%%%%%%&wwvwwvwwvwwwv de wmimmmogqm FNEwmaEam ::@waw mm mm H .. W%WW@@ chm ammw 03mm, mag mam gay {M13 <33?) Ems N?i SUBSTITUTE SHEET (RULE 26) m?sbmcau m; EEwm ?ag mama. mmmzwnmuxm “Ngwmmgam wmammmgam magnum mm mm 3&5 OE wwumbmcau m; mmmmu Eon Emma mama Eng ? wwammmgam 33me mm mm r é.) é; mam C} Q C) C? m “<1" N {MIG gm?) 3mg N?? SUBSTITUTE SHEET (RULE 26) VQLATELE STQRAGE PRQCESSGR E “2.3.; SUBSTITUTE SHEET (RULE 26) §\\\\\\\\\