JP7585217B2 - Gene editing for hemophilia A with improved factor VIII expression - Google Patents

Description

The disclosure provided herein relates to materials and methods for treating hemophilia A, both ex vivo and in vivo. Additionally, materials and methods are provided for gene editing to modulate the expression, function, or activity of blood clotting proteins, such as factor VIII (FVIII).

Hemophilia A (HemA) is caused by a genetic defect in the FVIII gene (F8) that results in low or undetectable levels of FVIII protein in the blood. As a result, effective blood clots do not form at sites of tissue damage, leading to uncontrolled bleeding that can be fatal if not treated. Replacement of defective or non-functional FVIII protein is an effective treatment for HemA subjects and is the current standard of care. However, protein replacement therapy requires frequent intravenous administration of FVIII protein, which is inconvenient in adults, problematic in children, cost-prohibitive (>$200,000/year), and can result in breakthrough bleeding events if the treatment regimen is not followed closely.

A permanent cure for hemophilia A is highly desirable. Viral-based gene therapy using adeno-associated virus (AAV) has shown some promise in preclinical animal models and human subjects, but has many drawbacks. For example, reported AAV-based gene therapy uses a FVIII coding sequence driven by a liver-specific promoter packaged within an AAV viral capsid (generally using serotypes AAV5, AAV8, or AAV9, among others), and AAVrhlO. In general, AAV viruses used in gene therapy deliver packaged coding sequence cassettes into the nucleus of transduced cells, where the cassettes remain mostly episomal, and it is the episomal copy of the therapeutic coding sequence that gives rise to the therapeutic protein. AAV does not have a mechanism for integrating packaged DNA into the genome of the host cell. Because the therapeutic coding sequence is maintained as an episome, it is not replicated coordinately when the host cell divides and can be lost from daughter cells. It has been demonstrated that when liver cells containing AAV episomes are induced to divide, the AAV genome does not replicate but is instead diluted. Therefore, AAV-based gene therapy is unlikely to be effective in children whose livers have not yet reached adult size. Because current therapies are inadequate, there is a critical need for new effective and durable or long-term treatments for HemA in adults and children.

FVIII is initially expressed as a protein with the domain structure A1-A2-B-A3-C1-C2. This protein is activated by proteolytic cleavage of the bulky, highly glycosylated B domain, leaving a heavy (A1-A2) and light (A3-C1-C2) chain heterodimer. The B domain of the FVIII protein is not required for biological activity. Removal of the large B domain from the FVIII coding sequence is essential to allow reliable packaging into AAV vectors used for in vivo delivery. However, removal of the B domain, which contains up to 18 N-linked glycosylation sites, impairs secretion of the FVIII protein. Thus, there is a critical need for improved forms of FVIII that can be expressed efficiently and effectively.

Applicants have discovered compositions and methods of gene editing that can be used to complement a defective F8 gene, resulting in expression of a functional FVIII protein. Accordingly, the inventions provided herein include systems and compositions for modifying host cell DNA sequences, methods for modifying host cell genomes, methods and systems for inserting a synthetic factor VIII coding sequence that results in improved expression, cells having a synthetic factor VIII coding sequence that results in improved expression that can be administered to a subject, methods for treating hemophilia A, and kits embodying any of the foregoing.

In one aspect, provided herein is a system for modifying a host cell DNA sequence comprising a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, a guide RNA (gRNA) or a nucleic acid encoding the gRNA having a spacer sequence complementary to a host cell locus, and a donor template having a nucleic acid sequence encoding a synthetic FVIII protein, the synthetic FVIII protein comprising a B domain substitution, the B domain substitution having 0-9 N-linked glycosylation sites and a length of 3 to about 40 amino acids.

In another aspect, a method of editing a genome in a host cell is provided, the method comprising providing to the cell a gRNA or a nucleic acid encoding the gRNA having a spacer sequence complementary to a host cell locus; a DNA endonuclease or a nucleic acid encoding the DNA endonuclease; and a donor template having a nucleic acid sequence encoding a synthetic FVIII protein, the synthetic FVIII protein having a B domain substitution, the B domain substitution having 0-9 N-linked glycosylation sites and a length of 3 to about 40 amino acids.

In another aspect, a cell is provided, the genome of the cell comprising DNA encoding a synthetic FVIII protein, the synthetic FVIII protein having a B domain substitution, the B domain substitution having 0-9 N-linked glycosylation sites and a length of 3 to about 40 amino acids.

In another aspect, a method is provided for treating hemophilia A in a subject by administering to the subject cells having DNA encoding a synthetic FVIII protein as described above.

In another aspect, a method is provided for treating hemophilia A in a subject by providing to a cell in the subject: a gRNA or a nucleic acid encoding a gRNA having a spacer sequence complementary to a host cell locus; a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; and a donor template having a nucleic acid sequence encoding a synthetic FVIII protein, the synthetic FVIII protein having a B domain substitution, the B domain substitution having 0-9 N-linked glycosylation sites and a length of 3 to about 40 amino acids.

In another aspect, provided herein is a kit comprising one or more elements of the above-described system and further comprising instructions for use.

In another aspect, a nucleic acid is provided having a polynucleotide sequence encoding a synthetic FVIII protein, wherein the synthetic FVIII protein has a B domain substitution, the B domain substitution having 0 to 9 N-linked glycosylation sites and a length of 3 to about 40 amino acids.

In another aspect, provided herein is a method of increasing the amount of FVIII in a subject by providing to cells of the subject having a first serum level of FVIII: a gRNA or a nucleic acid encoding a gRNA having a spacer sequence complementary to a host cell locus; a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; and a donor template having a nucleic acid sequence encoding a synthetic FVIII protein, the synthetic FVIII protein having a B domain substitution, the B domain substitution having 0-9 N-linked glycosylation sites and a length of 3 to about 40 amino acids.

An understanding of the specific features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the present disclosure are utilized, and the accompanying drawings, in which:

1 shows FVIII levels in the blood of mice after hydrodynamic injection of five plasmids encoding FVIII donor templates followed by LNP delivery of Cas9 mRNA and sgRNA. 1 shows FVIII levels in the blood of mice injected with AAV8 virus encapsulating FVIII donor templates pCB099 and pCB102 and administered LNPs encapsulating spCas9 mRNA and gRNA mALbT1 4 weeks later. FVIII levels were measured 10 days after LNP injection. 1 shows FVIII activity in the blood of hemophilia A mice administered four different FVIII donor plasmids by HDI followed by LNPs encapsulating spCas9 and mALbT1 gRNA. 1 shows FVIII activity in blood of hemophilia A mice 11 and 28 days after administration of LNP. Mice received 2×10 ¹² vg/kg of AAV8 virus 4 weeks prior to administration of LNP. Figure 1 shows FVIII activity in the blood of mice hydrodynamically injected with plasmids pCB1007 (n=7 mice), pCB1019 (n=7), and pCB1020 (n=6) and retroorbitally injected with LNPs encapsulating mALbT1 gRNA and Cas9 mRNA. FVIII was measured on days 6 and 9 after LNP administration. Figure 1 shows FVIII activity in the blood of mice hydrodynamically injected with plasmids pCB1007 (n=7 mice), pCB1025 (n=7), and pCB1026 (n=6) and retroorbitally injected with LNPs encapsulating mALbT1 gRNA and Cas9 mRNA. FVIII was measured on days 6 and 9 after LNP administration. FIG. 1 shows the results of cleavage efficiency of guide RNAs T4, T5, T11, and T13 (targeting human albumin intron 1) in primary human hepatocytes from four donors, comparing target sequences of 19 bases versus 20 bases. The intrinsic expression efficiency (FVIII activity divided by targeted integration frequency) for FVIII donor cassettes with 0 to 7 N-linked glycan motifs and different codon optimizations is shown. FVIII constructs in which the B domain substitutions contain either 0, 1, 3, 5, or 6 glycans are shown. 1 shows FVIII activity in the blood of hemophilia A mice 11 days after administration of LNP. 1 shows FVIII activity in the blood of hemophilia A mice 28 days after administration of LNP. 3 shows the intrinsic expression efficiency (FVIII activity divided by targeted integration frequency) for FVIII donor cassettes with 0, 1, 3, 5, or 6 N-linked glycan motifs.

RNA-guided endonuclease editing offers advantages over, for example, lentiviral methods of gene therapy. However, the insertion of large sequences in editing protocols can be problematic, for example, because large sequences can be difficult to package for delivery or can be difficult to manufacture compared to short sequences. Some proteins require the presence of N-linked glycosylation sites in order to be properly secreted from the cells in which they are expressed. The consensus amino acid sequence for N-glycosylation sites is N-X-T/S, where X is any residue except proline. The glycan is added to the N (asparagine) residue (K.F. Medzihradszky, Meth Mol Biol (2008) 446:293-316). Applicants have discovered that the number of N-linked glycosylation sites in such proteins can be significantly reduced or eliminated, thereby reducing the size of the protein coding sequence without adversely affecting transcription, translation or secretion. For example, applicants have discovered that by engineering the B domain of the FVIII coding sequence to reduce or eliminate the number of glycosylation sites, the size of the FVIII sequence used in gene editing can be reduced without significantly affecting the transcription, translation, or secretion of the resulting engineered (synthetic) FVIII, while still producing an engineered FVIII protein with FVIII function. Furthermore, by minimizing the number of N-glycan sites added to the B domain deleted FVIII, the risk of creating new epitopes for antibodies or T cells is minimized, thereby reducing the risk that the new FVIII protein may induce an immune response in the subject. The present disclosure provides, inter alia, compositions and methods for gene editing to modulate the expression, function, or activity of blood coagulation proteins such as FVIII in cells by genome editing. The present disclosure also provides, inter alia, compositions and methods for treating subjects with hemophilia A, both ex vivo and in vivo. In particular, the present invention provides genome editing methods and systems that provide improved integration and improved expression, as well as synthetic FVIII coding sequences and proteins that can ameliorate hemophilia A.

Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the detailed description is exemplary and explanatory only and does not limit the claimed subject matter. In this application, the use of the singular includes the plural unless otherwise specified. As used in the specification, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless otherwise specified. Furthermore, the use of "including" and other forms such as "include" and "included" is not limiting.

Although features of the disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, for clarity, although the disclosure may be described herein in the context of separate embodiments, the disclosure may also be implemented in a single embodiment. Any published patent applications and any other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference for any purpose. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and not limiting.

As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" means "about 5 μL" and also means "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error, such as ±1%, ±2%, ±3%, ±5%, or ±10%.

When a range of numerical values is presented herein, each intervening value between the lower and upper limits of the range, the upper and lower limits of the range, and every recited value within the range are intended to be encompassed by the disclosure. All possible subranges within the lower and upper limits of the range are also contemplated by the disclosure.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a linear series of amino acid residues linked together by peptide bonds, which may include proteins, polypeptides, oligopeptides, peptides, and fragments thereof. Proteins may be composed of naturally occurring and/or synthetic (e.g., modified or non-naturally occurring) amino acids. The terms "amino acid" or "peptide residue" as used herein may refer to both naturally occurring and synthetic amino acids. The terms "polypeptide", "peptide", and "protein" include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, fusion proteins with detectable fusion partners, e.g., fusion proteins that include fluorescent proteins, β-galactosidase, luciferase, etc. as fusion partners. It should further be noted that a dash at the beginning or end of an amino acid sequence indicates either a peptide bond to a further sequence of one or more amino acid residues, or a covalent attachment to the carboxyl or hydroxyl terminal group. However, the absence of dashes should not be construed to mean the absence of such peptide bonds or covalent bonds to the carboxyl or hydroxyl terminal groups, since it is customary to omit them in the representation of amino acid sequences.

The terms "polynucleotide", "oligonucleotide", "oligomer", "oligo", "coding sequence", and "nucleic acid" refer to polymeric forms of nucleotides of different lengths, either ribonucleotides or deoxyribonucleotides. Thus, these terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers with purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

The term "functionally equivalent" or "functional equivalent" refers, without limitation, to any molecule, such as a nucleic acid or protein, having a structure or sequence derived from a compound disclosed herein and whose structure or sequence is sufficiently similar to that disclosed herein such that it has the same or similar activity and utility, or would be expected by one of skill in the art to exhibit the same or similar activity and utility as the referenced compound based on such similarity. Modifications to obtain a functional equivalent, "derivative" or "variant" may include, for example, addition, deletion and/or substitution of one or more nucleic acid or amino acid residues.

A functional equivalent or a functionally equivalent fragment of a protein may have one or more conservative amino acid substitutions. The term "conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid that has similar properties to the original amino acid, i.e., the replacement of an amino acid with another amino acid from the same group. The groups of conservative amino acids are:

Conservative substitutions may be introduced at any position of a given peptide or fragment thereof. However, the introduction of non-conservative substitutions, particularly but not limited to, at any one or more positions, may also be desirable. Non-conservative substitutions that lead to the formation of functionally equivalent fragments of the peptide differ, for example, in polarity, charge, steric bulk, and/or binding to other proteins or nucleic acids, but maintain the anticoagulant functionality of the functional equivalent or variant fragment.

"Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where portions of the polynucleotide or polypeptide sequence in the comparison window may have additions or deletions (i.e., gaps) compared to a reference sequence (which has no additions or deletions) for optimal alignment of the two sequences. In some cases, the percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to give the number of matched positions, dividing this number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to give the percentage of sequence identity. Sequence identity can be determined, for example, using AlignX (included in Vector NTI and based on ClustalW (http://www.clustal.org/clustal2/) using standard parameters (e.g., gap opening penalty = 15; gap extension penalty = 6.6; gap separation penalty range = 8).

The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a particular region (e.g., an entire polypeptide sequence or an individual domain of a polypeptide)) when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence.

The terms "complementary" or "substantially complementary," as used interchangeably herein, mean that a nucleic acid (e.g., DNA or RNA) has a sequence of nucleotides that allows the nucleic acid to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid). As known in the art, standard Watson-Crick base pairing includes adenine (A) and thymidine (T) pairing, adenine (A) and uracil (U) pairing, and guanine (G) and cytosine (C) pairing.

A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide can encode an RNA that is translated into a protein (mRNA), or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or guide RNA; also called "non-coding" RNA or "ncRNA"). A "protein coding sequence or a sequence that encodes a particular protein or polypeptide" is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and, when placed under the control of appropriate regulatory sequences, is translated into a polypeptide in vitro or in vivo (in the case of mRNA).

As used herein, a "codon" refers to a sequence of three nucleotides that together form a unit of the genetic code in a DNA or RNA molecule. As used herein, the term "codon degeneracy" refers to the property of the genetic code that permits variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.

The term "codon-optimized" or "codon optimization" refers to the coding region of a gene or nucleic acid molecule for transformation of a suitable host, and refers to modifying the codons of the coding region of the gene or nucleic acid molecule to reflect the codon usage of the host organism without modifying the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number of codons with one or more codons that are more frequently used in the genes of that organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.or.jp/codon/ (visited January 30, 2019). By utilizing knowledge of the codon usage or codon preference in each organism, one of skill in the art can apply the frequencies to any given polypeptide sequence and produce a nucleic acid fragment of a codon-optimized coding region that encodes the polypeptide but uses the optimal codons for a given species. Codon-optimized coding regions are designed by methods known to those of skill in the art.

The terms "recombinant" or "engineered," when used with respect to, for example, a cell, a nucleic acid, a protein, or a vector, indicate that the cell, nucleic acid, protein, or vector has been modified or is the result of a laboratory method. Thus, for example, a recombinant or engineered protein includes a protein produced by a laboratory method. A recombinant or engineered protein may contain amino acid residues not found in the native (non-recombinant or wild-type) form of the protein and may contain modified (e.g., labeled) amino acid residues. The terms may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications include any chemical modification of a peptide, protein, or nucleic acid sequence; addition, deletion, and/or substitution of one or more amino acids in a peptide or protein; and addition, deletion, and/or substitution of one or more nucleic acids in a nucleic acid sequence.

The term "genomic DNA" or "genomic sequence" refers to the DNA of the genome of an organism, including but not limited to the DNA of the genome of a bacterium, fungus, archaea, plant or animal.

As used herein, "transgene," "exogenous gene," and "exogenous sequence" refer to a nucleic acid sequence or gene that was not present in the genome of a cell but that is artificially introduced into the genome, for example, by genome editing.

As used herein, "endogenous gene" or "endogenous sequence" refers to a nucleic acid sequence or gene that is naturally present in the genome of a cell without being introduced through any artificial means.

The term "vector" or "expression vector" means a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment (e.g., an "insert") can be attached, resulting in the replication of the attached segment in a cell.

The term "expression cassette" refers to a vector having a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition in a relationship that allows the components so described to function in the intended manner. For example, a promoter is operably linked to a coding sequence if it affects its transcription or expression. The terms "recombinant expression vector" and "DNA construct" are used interchangeably herein to refer to a vector and a DNA molecule having at least one insert. Recombinant expression vectors are typically generated for the purpose of expressing and/or propagating an insert or for the construction of other recombinant nucleotide sequences. The nucleic acid may or may not be operably linked to a promoter sequence and may or may not be operably linked to a DNA regulatory sequence.

The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

Cells are "genetically modified" or "transformed" or "transfected" with exogenous DNA, such as a recombinant expression vector (when such DNA is introduced into the cell). The presence of the exogenous DNA results in a permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. Genetically modified (or transformed or transfected) cells may be used that have a therapeutic activity, such as for the treatment of hemophilia A, and may be referred to as "therapeutic cells."

The term "concentration" when used in reference to a molecule, such as a peptide fragment, refers to the amount of the molecule, e.g., moles of the molecule, present in a given volume of solution.

The term "acute phase protein" refers to a protein whose expression or serum concentration changes in response to inflammation. Examples of acute phase proteins include albumin, transferrin, transthyretin, fibrinogen, antithrombin, etc.

The terms "individual," "subject," and "host" refer to any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient. In some embodiments, the subject has or is suspected of having hemophilia A and/or has one or more symptoms of hemophilia A. In some embodiments, the subject is a human who has been diagnosed at or after diagnosis as being at risk for hemophilia A. In some cases, a diagnosis of risk for hemophilia A can be determined based on the presence of one or more mutations in the endogenous FVIII gene or genomic sequences near the FVIII gene in the genome that may affect expression of the FVIII gene.

The term "treatment" as used in reference to a disease or condition means that at least an improvement in symptoms associated with the condition afflicting an individual is achieved, and improvement is used broadly to refer to at least a decrease in a parameter associated with the condition being treated (e.g., hemophilia A), e.g., the magnitude of the symptoms. Treatment also includes conditions in which the pathological condition, or at least the symptoms associated therewith, or at least the symptoms characterizing the condition, are completely inhibited, e.g., prevented from occurring, or completely eliminated such that the host is no longer afflicted with the condition. Thus, treatment includes (i) prevention (i.e., preventing clinical symptoms from developing, e.g., reducing the risk of clinical symptoms developing, including preventing disease progression), and (ii) inhibition (i.e., halting the development or further development of clinical symptoms, e.g., reducing or completely inhibiting active disease).

The terms "effective amount," "pharmacologically effective amount," and "therapeutically effective amount" refer to a sufficient amount of a composition to provide a desired benefit when administered to a subject having a particular condition. In the context of ex vivo treatment of hemophilia A, the term "effective amount" refers to the amount of a population of therapeutic cells or their progeny necessary to prevent or reduce at least one or more signs or symptoms of hemophilia A, and relates to a sufficient amount of a composition having therapeutic cells or their progeny to provide a desired effect, e.g., to treat a symptom of hemophilia A in a subject. Thus, a "therapeutically effective amount" refers to an amount or number of therapeutic cells or a composition having therapeutic cells sufficient to promote a particular effect when administered to a subject in need of treatment, e.g., a subject having or at risk for hemophilia A. An effective amount also includes an amount or number sufficient to prevent or delay the onset of a symptom of a disease, alter the course of a symptom of a disease (e.g., but not limited to, delaying the progression of a symptom of a disease), or reverse a symptom of a disease. In the context of in vivo treatment of hemophilia A in a subject (e.g., a patient) or genome editing performed in cells cultured in vitro, an effective amount refers to the amount of components used in genome editing, such as gRNA, donor template, and/or site-specific polypeptide (e.g., DNA endonuclease), required to edit the genome of cells in the subject or in cells cultured in vitro. It is understood that for any given case, the appropriate "effective amount" can be determined by one of skill in the art.

As used herein, the terms "pharmaceutical composition" and "medicament" refer to a pharma- ceutical acceptable excipient in combination with the cells of the invention (expressing a synthetic FVIII protein) and/or one or more components of the systems of the invention (i.e., gRNA or a nucleic acid encoding a gRNA, DNA endonuclease or a nucleic acid encoding a DNA endonuclease, and/or a donor template encoding a synthetic Factor VIII protein).

As used herein, the term "pharmaceutical acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound of interest to a subject. "Pharmaceutically acceptable excipient" can include substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

The term "synthetic FVIII" refers to a protein that has substantial sequence identity to the A and C domains of wild-type human factor VIII (GenBank: CAD97566.1; G.A. Vehar et al., Nature (1984) 312:337-42), but has a B domain substitution in place of the wild-type B domain. In one embodiment of the invention, the sequences of the A and C domains of the synthetic FVIII protein are 80, 90, 95, 98, or 99% identical to the wild-type sequences of the A and C domains. In some embodiments, the B domain substitution is a polypeptide of any sequence having from about 10 to about 200 amino acids. In some embodiments, the B domain substitution has from about 20 to about 100 amino acids. In some embodiments, the B domain substitution can have fewer than 40 amino acids (e.g., having any number of amino acids from 3 to 40 amino acids) and 1 to 9 N-linked glycosylation sites that provide for glycosylation of the B domain substitution upon expression. The B domain replacement may further comprise a protease cleavage site such that the synthetic FVIII protein may be cleaved into heavy and light chains in the same manner as the wild-type protein. In one embodiment, the B domain replacement protein sequence comprises 1-10 amino acids from the N-terminus and C-terminus of the wild-type B domain in addition to 1-9 N-linked glycosylation ("glycan") sites. In one embodiment, the B domain replacement protein sequence has 1-6 glycan sites. In one embodiment, the B domain replacement protein sequence has 1-5 glycan sites. In one embodiment, the B domain replacement protein sequence has 1-4 glycan sites. In one embodiment, the B domain replacement protein sequence has 2-4 glycan sites. In one embodiment, the B domain replacement protein sequence has any of SEQ ID NOs: 362-369, 371, and 373, or a sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to any of SEQ ID NOs: 362-369, 371, and 373. In one embodiment, the B domain substituted protein sequence has the sequence of any of SEQ ID NOs: 362-366, 371, and 373, or a sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to any of SEQ ID NOs: 362-366, 371, and 373. In one embodiment, the B domain substituted protein sequence has the sequence of any of SEQ ID NOs: 362-364, 371, and 373, or a sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to any of SEQ ID NOs: 362-364, 371, and 373. In one embodiment, the B domain substituted protein sequence has the sequence of any of SEQ ID NOs: 362-363, or a sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to any of SEQ ID NOs: 362-363. In one embodiment, the B domain substituted protein sequence has the sequence of any of SEQ ID NOs: 362-369. In one embodiment, the B domain replacement protein sequence has the sequence of any of SEQ ID NOs: 362-366. In one embodiment, the B domain replacement protein sequence has the sequence of any of SEQ ID NOs: 362-364. In one embodiment, the B domain replacement protein sequence has the sequence of any of SEQ ID NOs: 362-363, 371, and 373. In one embodiment, the B domain replacement protein sequence has the sequence of any of SEQ ID NOs: 371 or 373.

The term "safe harbor locus" refers to a locus in a host cell genome that can be modified (e.g., by cutting or inserting a donor sequence) without disrupting cellular metabolism or regulation (e.g., by causing apoptosis, proliferation, etc.) and/or without causing risk or deleterious effects to other cells (non-edited cells) or the host organism as a whole (e.g., by inadvertently causing overexpression of a growth factor, etc.). In some embodiments, a safe harbor locus is a locus that is expressed in the host cell. In some embodiments, a safe harbor locus is an albumin locus, a fibrinogen locus, an AAVS1 locus, or a transferrin locus.

Nucleic Acid Genome Targeting Nucleic Acid or Guide RNA
The present disclosure provides a genome-targeting nucleic acid that can guide the activity of an associated polypeptide (e.g., a site-directed polypeptide, such as a DNA endonuclease) to a specific target sequence within a target nucleic acid. In some embodiments, the genome-targeting nucleic acid is an RNA. The genome-targeting RNA is referred to herein as a "guide RNA" or "gRNA". The guide RNA has at least a spacer sequence and a CRISPR repeat sequence that can hybridize to a target nucleic acid sequence of interest. In type II systems, the gRNA also has a second RNA called a tracrRNA sequence. In type II gRNA, the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a duplex. In type V gRNA, the crRNA forms a duplex. In both systems, the duplex binds to the site-directed polypeptide such that the gRNA and the site-directed polypeptide form a complex. The genome-targeting nucleic acid provides target specificity to the complex by binding to the site-directed polypeptide. Thus, the genome-targeting nucleic acid induces the activity of the site-directed polypeptide.

In some embodiments, the genome-targeting nucleic acid is a bimolecular gRNA. A bimolecular gRNA has two RNA strands. The first strand has, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence. The second strand has a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. In some embodiments, the genome-targeting nucleic acid is a single-molecule gRNA. A single-molecule gRNA (sgRNA) in a type II system has, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may have elements that contribute additional functionality (e.g., stability) to the gRNA. The single molecule guide linker links the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension has one or more hairpins. The sgRNA in the V-type system has, in the 5' to 3' direction, a minimal CRISPR repeat sequence and a spacer sequence.

By way of example, gRNAs, or other smaller RNAs, used in the CRISPR/Cas/Cpf1 system can be readily synthesized by chemical means, as exemplified below and described in the art. Although chemical synthesis procedures are continually expanding, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as polynucleotide lengths increase significantly beyond 100 nucleotides or so. One approach used to generate longer RNAs is to generate two or more molecules that are linked together. Much longer RNAs, such as those encoding Cas9 or Cpf1 endonucleases, are more easily generated enzymatically. RNA modifications, such as those that enhance stability, reduce the likelihood or extent of an innate immune response, and/or enhance other attributes, can be introduced during or after chemical synthesis and/or enzymatic generation of the RNA, as described in the art.

Spacer extension sequence In some embodiments of the genome-targeting nucleic acid, the spacer extension sequence can modify activity, provide stability, and/or provide a location for modification of the genome-targeting nucleic acid. The spacer extension sequence can modify on-target or off-target activity or specificity. In some embodiments, a spacer extension sequence is provided. The spacer extension sequence can have a length of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or more than 7000 nucleotides. The spacer extension sequence may have a length of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 nucleotides or more. The spacer extension sequence may have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000, or more nucleotides. In some embodiments, the spacer extension sequence is less than 10 nucleotides in length. In some embodiments, the spacer extension sequence is 10-30 nucleotides in length. In some embodiments, the spacer extension sequence is 30-70 nucleotides in length.

In some embodiments, the spacer extension sequence has another portion (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme). In some embodiments, the portion decreases or increases the stability of the nucleic acid targeting nucleic acid. In some embodiments, the portion is a transcription terminator segment (i.e., a transcription termination sequence). In some embodiments, the portion functions in eukaryotic cells. In some embodiments, the portion functions in prokaryotic cells. In some embodiments, the portion functions in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include 5' caps (e.g., 7-methylguanylate caps (m7G)), riboswitch sequences (e.g., to allow for stability and/or regulated accessibility by proteins and protein complexes), sequences that form dsRNA duplexes (i.e., hairpins), sequences that target RNA to subcellular locations (e.g., nuclei, mitochondria, chloroplasts, etc.), modifications or sequences that provide tracking (e.g., direct attachment to a fluorescent molecule, attachment to a moiety that facilitates fluorescent detection, sequences that allow for fluorescent detection, etc.), and/or modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.).

Spacer sequence Spacer sequence can hybridize to a sequence in the target nucleic acid of interest. The spacer of genome targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Thus, the nucleotide sequence of the spacer varies according to the sequence of the target nucleic acid of interest.

In the CRISPR/Cas system herein, the spacer sequence is designed to hybridize to the target nucleic acid located 5' of the PAM of the Cas9 enzyme used in the system. The spacer may be a perfect match to the target sequence or may have a mismatch. Each Cas9 enzyme has a specific PAM sequence that it recognizes in the target DNA. For example, S. pyogenes recognizes a PAM in the target nucleic acid that has the sequence 5'-NRG-3', where R has either A or G and N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

In some embodiments, the target nucleic acid sequence has 20 nucleotides. In some embodiments, the target nucleic acid has fewer than 20 nucleotides. In some embodiments, the target nucleic acid has more than 20 nucleotides. In some embodiments, the target nucleic acid has at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. In some embodiments, the target nucleic acid has up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. In some embodiments, the target nucleic acid sequence has 20 bases 5' to the first nucleotide of the PAM. For example,
In the sequence having the formula: ##STR00011## where the target nucleic acid has a sequence corresponding to N (where N is any nucleotide), the underlined sequence NRG (where R is G or A) is the Streptococcus pyogenes Cas9 PAM. In some embodiments, the PAM sequence used in the compositions and methods of the disclosure as a sequence recognized by Streptococcus pyogenes (S.p) Cas9 is NGG.

In some embodiments, the spacer sequence that hybridizes to the target nucleic acid has a length of at least about 6 nucleotides (nt). The spacer sequence may be at least about 6 nt, about 10 nt, about 15 nt, about 18 nt, about 19 nt, about 20 nt, about 25 nt, about 30 nt, about 35 nt, or about 40 nt, about 6 nt to about 80 nt, about 6 nt to about 50 nt, about 6 nt to about 45 nt, about 6 nt to about 40 nt, about 6 nt to about 35 nt, about 6 nt to about 30 nt, about 6 nt to about 25 nt, about 6 nt to about 20 nt, about 6 nt to about 19 nt, about 10 nt to about 50 nt, about 10 nt to about 45 nt, about 10 nt to about 40 nt, about 10 nt to about 35 nt, about The spacer sequence may be from 10 nt to about 30 nt, from about 10 nt to about 25 nt, from about 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some embodiments, the spacer sequence has 20 nucleotides. In some embodiments, the spacer has 19 nucleotides. In some embodiments, the spacer has 18 nucleotides. In some embodiments, the spacer has 17 nucleotides. In some embodiments, the spacer has 16 nucleotides. In some embodiments, the spacer has 15 nucleotides.

In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, 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 97%, at least about 98%, at least about 99%, or 100%. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six adjacent 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least 60% over about 20 contiguous nucleotides. In some embodiments, the length of the spacer sequence and the target nucleic acid may differ by 1-6 nucleotides, which can be considered a bulge.

In some embodiments, the spacer sequences are designed or selected using a computer program. The computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic frequency (e.g., of sequences that are identical or similar but vary in one or more spots as a result of mismatches, insertions or deletions), methylation status, presence of SNPs, etc.

Minimal CRISPR Repeat Sequence In some embodiments, a minimal CRISPR repeat sequence is a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes; see, e.g., J. J. Ferretti et al., Proc Natl Acad Sci USA (2001) 98(8):4658-63).

In some embodiments, the minimal CRISPR repeat sequence has nucleotides that can hybridize to the minimal tracrRNA sequence in the cell. The minimal CRISPR repeat sequence and the minimal tracrRNA sequence form a duplex. The minimal CRISPR repeat sequence and the minimal tracrRNA sequence together bind to the site-specific polypeptide. At least a portion of the minimal CRISPR repeat sequence hybridizes to the minimal tracrRNA sequence. In some embodiments, at least a portion of the minimal CRISPR repeat sequence is at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimal tracrRNA sequence. In some embodiments, at least a portion of the minimal CRISPR repeat sequence is at most about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimal tracrRNA sequence.

The minimal CRISPR repeat sequence may have a length of about 7 nucleotides to about 100 nucleotides. For example, the length of the minimal CRISPR repeat sequence is about 7 nt to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. In some embodiments, the minimal CRISPR repeat sequence is about 9 nucleotides in length. In some embodiments, the minimal CRISPR repeat sequence is about 12 nucleotides in length.

In some embodiments, the minimal CRISPR repeat sequence is at least about 60% identical to a reference minimal CRISPR repeat sequence (e.g., a wild-type crRNA from S. pyogenes; see, e.g., J. J. Ferretti et al., supra) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimal CRISPR repeat sequence is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a reference minimal CRISPR repeat sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

Minimal tracrRNA Sequence In some embodiments, a minimal tracrRNA sequence is a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a wild-type tracrRNA from S. pyogenes; see, e.g., J. J. Ferretti et al., supra).

In some embodiments, the minimal tracrRNA sequence has nucleotides that hybridize to a minimal CRISPR repeat sequence in a cell. The minimal tracrRNA sequence and the minimal CRISPR repeat sequence form a duplex. The minimal tracrRNA sequence and the minimal CRISPR repeat are combined to bind to a site-specific polypeptide. At least a portion of the minimal tracrRNA sequence can hybridize to the minimal CRISPR repeat sequence. In some embodiments, the minimal tracrRNA sequence is at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimal CRISPR repeat sequence.

The minimal tracrRNA sequence may have a length of about 7 nucleotides to about 100 nucleotides. For example, the minimal tracrRNA sequence may be about 7 nt to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt in length. In some embodiments, the minimal tracrRNA sequence is about 9 nucleotides in length. In some embodiments, the minimal tracrRNA sequence is about 12 nucleotides. In some embodiments, the minimal tracrRNA consists of tracrRNA nt 23-48 as described in M. Jinek et al., Science (2012) 337(6096):816-21.

In some embodiments, the minimal tracrRNA sequence is at least about 60% identical to a reference minimal tracrRNA (e.g., a wild-type, tracrRNA from S. pyogenes; see, e.g., J. J. Ferretti et al., supra) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimal tracrRNA sequence is at least about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to a reference minimal tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

In some embodiments, the duplex between the minimal CRISPR RNA and the minimal tracrRNA has a double helix. In some embodiments, the duplex between the minimal CRISPR RNA and the minimal tracrRNA has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. In some embodiments, the duplex between the minimal CRISPR RNA and the minimal tracrRNA has at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

In some embodiments, the duplex has mismatches (i.e., the two strands of the duplex are not 100% complementary). In some embodiments, the duplex has at least about 1, 2, 3, 4, or 5 or mismatches. In some embodiments, the duplex has up to about 1, 2, 3, 4, or 5 or mismatches. In some embodiments, the duplex has 2 or fewer mismatches.

Bulges In some embodiments, a "bulge" is present in the duplex between the minimal CRISPR RNA and the minimal tracrRNA. A bulge is an unpaired region of nucleotides within a duplex. In some embodiments, the bulge contributes to binding of the duplex to a site-directed polypeptide. A bulge has an unpaired 5'-XXXY-3' on one side of the duplex, where X is any purine and Y has a nucleotide that can form a wobble pair with a nucleotide on the opposite strand, and an unpaired nucleotide region on the other side of the duplex. The number of unpaired nucleotides on each side of the duplex can be different.

In one example, the bulge has an unpaired purine (e.g., adenine) on the minimal CRISPR repeat strand of the bulge. In some embodiments, the bulge has an unpaired 5'-AAGY-3' on the minimal tracrRNA sequence strand of the bulge, where Y has a nucleotide that can form a wobble pair with a nucleotide on the minimal CRISPR repeat strand.

In some embodiments, the bulge on the minimal CRISPR repeat side of the duplex has at least 1, 2, 3, 4, or 5 or more unpaired nucleotides. In some embodiments, the bulge on the minimal CRISPR repeat side of the duplex has at most 1, 2, 3, 4, or 5 or more unpaired nucleotides. In some embodiments, the bulge on the minimal CRISPR repeat side of the duplex has 1 unpaired nucleotide.

In some embodiments, the bulge on the side of the duplex facing the minimal tracrRNA sequence has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. In some embodiments, the bulge on the side of the duplex facing the minimal tracrRNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. In some embodiments, the bulge on the second side of the duplex (e.g., facing the minimal tracrRNA sequence) has 4 unpaired nucleotides.

In some embodiments, the bulge has at least one wobble pair. In some embodiments, the bulge has at most one wobble pair. In some embodiments, the bulge has at least one purine nucleotide. In some embodiments, the bulge has at least three purine nucleotides. In some embodiments, the bulge sequence has at least five purine nucleotides. In some embodiments, the bulge sequence has at least one guanine nucleotide. In some embodiments, the bulge sequence has at least one adenine nucleotide.

Hairpins In some embodiments, one or more hairpins are located 3' of the minimal tracrRNA in the 3' tracrRNA sequence.

In some embodiments, the hairpin begins at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides 3' from the last paired nucleotide in the duplex of the minimal CRISPR repeat and the minimal tracrRNA sequence. In some embodiments, the hairpin can begin up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides 3' of the last paired nucleotide in the duplex of the minimal CRISPR repeat and the minimal tracrRNA sequence.

In some embodiments, the hairpin has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more contiguous nucleotides. In some embodiments, the hairpin has up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more contiguous nucleotides.

In some embodiments, the hairpin has a CC dinucleotide (i.e., two consecutive cytosine nucleotides).

In some embodiments, the hairpin has duplex nucleotides (i.e., nucleotides in the hairpin that hybridize together). For example, the hairpin has a CC dinucleotide that hybridizes with a GG dinucleotide in the hairpin duplex of the 3' tracrRNA sequence.

One or more of the hairpins may interact with the guide RNA interacting region of the site-specific polypeptide. In some embodiments, there are two or more hairpins, and in some embodiments, there are three or more hairpins.

3' tracrRNA sequence In some embodiments, the 3' tracrRNA sequence has a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

In some embodiments, the 3' tracrRNA sequence has a length of about 6 nucleotides to about 100 nucleotides. For example, the 3' tracrRNA sequence may have a length of about 6 nt to about 50 nt, about 6 nt to about 40 nt, about 6 nt to about 30 nt, about 6 nt to about 25 nt, about 6 nt to about 20 nt, about 6 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. In some embodiments, the 3' tracrRNA sequence has a length of about 14 nucleotides.

In some embodiments, the 3' tracrRNA sequence is at least about 60% identical to a reference 3' tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the 3' tracrRNA sequence is at least about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to a reference 3' tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

In some embodiments, the 3' tracrRNA sequence has two or more double-stranded regions. In some embodiments, the 3' tracrRNA sequence has two double-stranded regions.

In some embodiments, the 3' tracrRNA sequence has a stem-loop structure. In some embodiments, the stem-loop structure in the 3' tracrRNA has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides. In some embodiments, the stem-loop structure in the 3' tracrRNA has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. In some embodiments, the stem-loop structure has a functional portion. For example, the stem-loop structure may have an aptamer, a ribozyme, a protein interaction hairpin, a CRISPR array, an intron, or an exon. In some embodiments, the stem-loop structure has at least about 1, 2, 3, 4, or 5 or more functional portions. In some embodiments, the stem-loop structure has at most about 1, 2, 3, 4, or 5 or more functional portions.

In some embodiments, the hairpin in the 3' tracrRNA sequence has a P domain. In some embodiments, the P domain has a double-stranded region in the hairpin.

tracrRNA Extension Sequence In some embodiments, a tracrRNA extension sequence may be provided whether the tracrRNA is in the context of a single molecule guide or a two molecule guide. In some embodiments, the tracrRNA extension sequence has a length of about 1 nucleotide to about 400 nucleotides. In some embodiments, the tracrRNA extension sequence has a length of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or more than 400 nucleotides. In some embodiments, the tracrRNA extension sequence has a length of about 20 to about 5000 or more nucleotides. In some embodiments, the tracrRNA extension sequence has a length of more than 1000 nucleotides. In some embodiments, the tracrRNA extension sequence has a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or more nucleotides. In some embodiments, the tracrRNA extension sequence may have a length of less than 1000 nucleotides. In some embodiments, the tracrRNA extension sequence has a length of less than 10 nucleotides. In some embodiments, the tracrRNA extension sequence is 10-30 nucleotides in length. In some embodiments, the tracrRNA extension sequence is 30-70 nucleotides in length.

In some embodiments, the tracrRNA extension sequence comprises a functional moiety (e.g., a stability control sequence, a ribozyme, an endoribonuclease binding sequence). In some embodiments, the functional moiety comprises a transcription terminator segment. In some embodiments, the functional portion has a total length of about 10 nt to about 100 nucleotides, about 10 nt to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. In some embodiments, the functional portion functions in a eukaryotic cell. In some embodiments, the functional portion functions in a prokaryotic cell. In some embodiments, the functional portion functions in both eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moieties include 3' polyadenylation tails, riboswitch sequences (e.g., to allow for regulated stability and/or regulated accessibility by proteins and protein complexes), sequences that form dsRNA duplexes, sequences that target the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.), modifications or sequences that provide tracking (e.g., direct binding to a fluorescent molecule, binding to a moiety that facilitates fluorescent detection, sequences that allow fluorescent detection, etc.), and/or modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.). In some embodiments, the tracrRNA extension sequence has a primer binding site or a molecular index (e.g., a barcode sequence). In some embodiments, the tracrRNA extension sequence has one or more affinity tags.

Single Molecule Guide Linker Sequences In some embodiments, the linker sequence of the single molecule guide nucleic acid has a length of about 3 nucleotides to about 100 nucleotides. Exemplary linkers have a length of about 3 nt to about 90 nt, about 3 nt to about 80 nt, about 3 nt to about 70 nt, about 3 nt to about 60 nt, about 3 nt to about 50 nt, about 3 nt to about 40 nt, about 3 nt to about 30 nt, about 3 nt to about 20 nt, about 3 nt to about 10 nt. For example, the linker can have a length of about 3 nt to about 5 nt, about 5 nt to about 10 nt, about 10 nt to about 15 nt, about 15 nt to about 20 nt, about 20 nt to about 25 nt, about 25 nt to about 30 nt, about 30 nt to about 35 nt, about 35 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. In some embodiments, the linker of the single molecule guide nucleic acid is 4-40 nucleotides. In some embodiments, the linker is at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. In some embodiments, the linker is up to about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

The linker can have any of a variety of sequences, but in some embodiments, the linker will not have a sequence with extensive regions of homology to other portions of the gRNA that could cause intramolecular binding that could interfere with other functional regions of the gRNA. In M. Jinek et al., supra, a simple four nucleotide sequence -GAAA was used, but numerous other sequences, including longer sequences, could be used as well.

In some embodiments, the linker sequence has a functional moiety. For example, the linker sequence may have one or more features including an aptamer, a ribozyme, a protein interaction hairpin, a protein binding site, a CRISPR array, an intron, or an exon. In some embodiments, the linker sequence has at least about 1, 2, 3, 4, or 5 or more functional moieties. In some embodiments, the linker sequence has at most about 1, 2, 3, 4, or 5 or more functional moieties.

In some embodiments, the genomic location targeted by a gRNA according to the present disclosure may be at, within, or near a suitable endogenous locus in the genome, e.g., the human genome. Endogenous loci may be selected based on containing highly expressed genes, or alternatively highly selectively expressed genes (e.g., genes expressed only in particular tissues, or genes expressed under particular conditions). Exemplary loci for expression in the liver include, for example, the albumin locus, the transferrin locus, and the fibrinogen locus.

In some embodiments, provided herein is a gRNA comprising a spacer sequence complementary to a genomic sequence within or near an endogenous transferrin locus in a cell. In some embodiments, the gRNA comprises a spacer sequence complementary to a sequence within intron 1 of an endogenous transferrin gene in a cell. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 1-190, or a variant thereof having 3 or fewer mismatches compared to any one of SEQ ID NOs: 1-190. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 96, 5, 6, 9, 8, 11, 15, 16, 12, 7, 10, 17, 18, 29, 76, 50, 54, 81, 64, 51, 1-4, 13, 14, 19-28, 30-49, 52, 53, 55-63, 65-75, 77-80, 82-95, and 97-190; or a variant thereof having 3 or fewer mismatches compared to any one of SEQ ID NOs: 96, 5, 6, 9, 8, 11, 15, 16, 12, 7, 10, 17, 18, 29, 76, 50, 54, 81, 64, 51, 1-4, 13, 14, 19-28, 30-49, 52, 53, 55-63, 65-75, 77-80, 82-95, and 97- 190. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 5, 6, 9, 8, 11, 15, 16, 12, 7, and 10, or a variant thereof having 3 or fewer mismatches compared to any one of SEQ ID NOs: 5, 6, 9, 8, 11, 15, 16, 12, 7, and 10. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 17, 18, 29, 76, 50, 54, 81, 96, 64, and 51, or a variant thereof having no more than three mismatches compared to any one of SEQ ID NOs: 17, 18, 29, 76, 50, 54, 81, 96, 64, and 51. In some embodiments, the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected.

In some embodiments, the genomic location targeted by a gRNA according to the present disclosure can be at, within, or near a genome, e.g., an endogenous fibrinogen-α chain (fibrinogen-α) locus in the human genome. Exemplary guide RNAs targeting such locations include the spacer sequences and associated Cas9 or Cpf1 cleavage sites listed in any of SEQ ID NOs: 192-270. As will be appreciated by those skilled in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. For example, each of the spacer sequences listed in any of SEQ ID NOs: 192-270 can be placed in a single RNA chimera or crRNA (along with the corresponding tracrRNA). See M. Jinek et al., supra, and E. Deltcheva et al., Nature (2011) 471:602-07.

Exemplary guide RNAs targeting albumin loci include a spacer sequence from any one of SEQ ID NOs: 271-298 and an associated Cas9 or Cpf1 cleavage site. For example, a gRNA including a spacer sequence from SEQ ID NO: 271 may include the spacer sequence UAAUUUUCUUUUGCGCACUA (SEQ ID NO: 299). As will be appreciated by those of skill in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. For example, each of the spacer sequences from any one of SEQ ID NOs: 271-298 can be placed into a single RNA chimera or crRNA (along with the corresponding tracrRNA).

Donor Template Site-specific polypeptides, such as DNA endonucleases, can introduce double-stranded or single-stranded breaks in nucleic acids, e.g., genomic DNA. The double-stranded break can stimulate the cell's endogenous DNA repair pathways (e.g., homology-dependent repair (HDR), non-homologous end joining or alternative non-homologous end joining (A-NHEJ), or microhomology-mediated end joining (MMEJ)). NHEJ can repair a cut target nucleic acid without the need for a homologous template. This can result in small deletions or insertions (indels) in the target nucleic acid at the cut site, which can result in disruption or alteration of gene expression. HDR, also known as homologous recombination (HR), can occur when a homologous repair template, or donor, is available.

A homologous donor template has a sequence that is homologous to the sequence adjacent to the target nucleic acid cleavage site. Sister chromatids are commonly used by cells as repair templates. However, for genome editing purposes, repair templates are often supplied as exogenous nucleic acids, such as plasmids, duplex oligonucleotides, single-stranded oligonucleotides, double-stranded oligonucleotides, or viral nucleic acids. Exogenous donor templates are typically used to introduce additional nucleic acid sequences (e.g., transgenes) or modifications (e.g., single or multiple base changes or deletions) between the adjacent regions of homology, so that the additional or modified nucleic acid sequences also become integrated into the target locus. MMEJ produces similar genetic outcomes to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ utilizes a few base pairs of homologous sequences adjacent to the cleavage site to promote favorable end-joining DNA repair outcomes. In some cases, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies in the nuclease target region.

Thus, in some cases, homologous recombination is used to insert an exogenous polynucleotide sequence into a target nucleic acid cleavage site. The exogenous polynucleotide sequence is referred to herein as a donor template (or donor, or donor sequence, or donor DNA template). In some embodiments, the donor template, a portion of the donor template, a copy of the donor template, or a portion of a copy of the donor template is inserted into the target nucleic acid cleavage site. In some embodiments, the donor template is a sequence that does not naturally occur at the target nucleic acid cleavage site.

When an exogenous DNA molecule is provided in sufficient concentration inside the nucleus of a cell in which a double-stranded break has occurred, the exogenous DNA can be inserted into the double-stranded break during the NHEJ repair process, thus becoming a permanent addition to the genome. If the donor template contains the coding sequence of a gene of interest, such as the FVIII gene, optionally together with associated regulatory sequences such as a promoter, enhancer, polyA sequence, and/or splice acceptor sequence (also referred to herein as a "donor cassette"), the coding sequence can be expressed from the integrated copy in the genome, resulting in permanent expression during the life of the cell. Furthermore, the integrated copy of the donor template can be transmitted to daughter cells when the cell divides.

In the presence of a sufficient concentration of donor template that contains adjacent DNA sequences (called homology arms) that are homologous to the DNA sequences on either side of the double-stranded break, the donor template can be integrated via the HDR pathway. The homology arms serve as substrates for homologous recombination between the donor template and the sequences on either side of the double-stranded break. This can result in error-free insertion of the donor template, where the sequences on either side of the double-stranded break are unchanged from those of the unmodified genome.

Donors provided for editing by HDR vary widely but generally contain the intended sequence with small or large flanking homology arms to allow annealing to genomic DNA. The homology regions flanking the introduced genetic change can be as small as 30 bp or less, or as large as multi-kilobase cassettes that may include promoters, cDNAs, etc. Both single-stranded and double-stranded oligonucleotide donors can be used. These oligonucleotides range in size from less than 100 nt to more than many kb, although longer ssDNAs can also be generated and used. Double-stranded donors including PCR amplicons, plasmids, and minicircles are often used. In general, AAV vectors have been found to be a very effective means of delivery of donor templates, although the packaging limit for individual donors is <5 kb. Active transcription of the donor increased HDR by 3-fold, indicating that inclusion of a promoter can increase conversion. Conversely, CpG methylation of the donor can decrease gene expression and HDR.

In some embodiments, donor DNA can be provided together with the nuclease or independently by a variety of different methods (e.g., transfection, nanoparticles, microinjection, or viral transduction). In some embodiments, a range of tethering options can be used to increase the availability of the donor for HDR. Examples include attaching the donor to a nuclease, to a nearby bound DNA binding protein, or to a protein involved in DNA end joining or repair.

In addition to genome editing by NHEJ or HDR, site-specific gene insertion using both the NHEJ pathway and HR can be performed. A combined approach may be applicable under certain circumstances, possibly involving intron/exon boundaries. NHEJ may prove effective for ligation in introns, while error-free HDR may be more appropriate in coding regions.

In embodiments, the exogenous sequence inserted into the genome is a synthetic FVIII coding sequence that encodes a synthetic FVIII protein having a B domain substitution at a position where the wild-type B domain would otherwise be present. The synthetic FVIII coding sequence can include a nucleic acid sequence that encodes a synthetic FVIII protein having substantial activity (e.g., procoagulant activity) of the wild-type FVIII protein. The synthetic FVIII protein can have at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of the activity exhibited by the wild-type FVIII protein. In some embodiments, the synthetic FVIII protein can have at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence identity to a FVIII protein (e.g., a wild-type FVIII protein). In some embodiments, the synthetic FVIII protein can have at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence identity to a FVIII protein that does not include a B domain (e.g., a wild-type FVIII protein after cleavage of the B domain). In some embodiments, one of skill in the art can use a number of methods known in the art to test the functionality or activity of a compound (e.g., a peptide or protein). The synthetic FVIII protein can also include any fragment of a wild-type FVIII protein, or a fragment of a modified FVIII protein having conservative modifications at one or more of the amino acid residues in the full-length wild-type FVIII protein. Thus, in some embodiments, the synthetic FVIII coding sequence can have at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% nucleic acid sequence identity to a FVIII coding sequence (e.g., a wild-type FVIII coding sequence).

In an embodiment of the invention, the synthetic FVIII contains one or more conservative or non-conservative amino acid substitutions that improve aspects of the protein without adversely affecting the anticoagulant properties of the protein. In one embodiment, the phenylalanine at position 309 is (non-conservatively) substituted with serine or alanine to provide the F309S and F309A muteins, respectively. These substitutions are suggested to disrupt a potential binding site for the chaperone immunoglobulin binding protein (BiP) in the A1 domain, thereby improving expression and secretion of the protein (M. Swaroop et al., J Biol Chem (1997) 272:24121-24).

The B domain substitutions of the present invention replace the B domain of wild-type FVIII with a much smaller peptide chain, yet still provide a protease cleavage site and one or more sites for N-linked glycosylation. The B domain substitutions can have from about 10 to about 200 amino acids. In some embodiments, the B domain substitutions have from about 20 to about 100 amino acids. In some embodiments, the B domain substitutions have from about 1 to about 40 amino acids, from about 1 to about 35 amino acids, from about 1 to about 30 amino acids, from about 1 to about 25 amino acids, from about 1 to about 20 amino acids, from about 1 to about 15 amino acids, from about 1 to about 10 amino acids, or from about 1 to about 5 amino acids. In some embodiments, the B domain substitutions have from about 5 to about 40 amino acids, from about 10 to about 40 amino acids, from about 15 to about 40 amino acids, from about 20 to about 40 amino acids, from about 25 to about 40 amino acids, from about 30 to about 40 amino acids, or from about 35 to about 40 amino acids. In some embodiments, the B domain substitution has 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, or 40 amino acids. In some embodiments, the nucleic acid encoding the B domain substitution is codon optimized. In some embodiments, the B domain substitution comprises a protease cleavage site, e.g., RHQR.

In some embodiments of the insertion of the synthetic FVIII coding sequence, the cDNA of the synthetic FVIII coding sequence can be inserted into the genome of a subject having a defective FVIII gene or its regulatory sequence. In such cases, the donor DNA or donor template can be an expression cassette or vector construct having a synthetic FVIII coding sequence. In some embodiments, an expression vector can be used that contains a synthetic FVIII coding sequence as described elsewhere herein.

In some embodiments, according to any of the donor templates described herein that include a donor cassette, the donor cassette is flanked on one or both sides by a gRNA target site. For example, such a donor template may include a donor cassette having a gRNA target site at the 5' of the donor cassette and/or a gRNA target site at the 3' of the donor cassette. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' of the donor cassette. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 3' of the donor cassette. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' of the donor cassette and a gRNA target site at the 3' of the donor cassette. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' of the donor cassette and a gRNA target site at the 3' of the donor cassette, where the two gRNA target sites include the same sequence. In some embodiments, the donor template comprises at least one gRNA target site, and the at least one gRNA target site in the donor template comprises the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is integrated. In some embodiments, the donor template comprises at least one gRNA target site, and the at least one gRNA target site in the donor template comprises a reverse complement of a gRNA target site in a target locus into which the donor cassette of the donor template is integrated. In some embodiments, the donor template comprises a donor cassette having a gRNA target site at the 5' of the donor cassette and a gRNA target site at the 3' of the donor cassette, and the two gRNA target sites in the donor template comprise the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is integrated. In some embodiments, the donor template comprises a donor cassette having a gRNA target site 5' of the donor cassette and a gRNA target site 3' of the donor cassette, and the two gRNA target sites in the donor template comprise the reverse complements of the gRNA target sites in the target locus into which the donor cassette of the donor template is integrated.

Nucleic acids encoding site-specific polypeptides or DNA endonucleases In some embodiments, genome editing methods and compositions can therefore use nucleic acids (or oligonucleotides) encoding site-specific polypeptides, such as DNA endonucleases. The nucleic acid sequence encoding the site-specific polypeptide can be DNA or RNA. If the nucleic acid sequence encoding the site-specific polypeptide is RNA, it can be covalently linked to the gRNA sequence or can be present as a separate sequence. In some embodiments, the site-specific polypeptide (such as a DNA endonuclease) is used directly in place of the nucleic acid sequence encoding it.

Vectors In another aspect, the present disclosure provides a nucleic acid having a nucleotide sequence encoding a genome-targeting nucleic acid of the present disclosure, a site-directed polypeptide of the present disclosure, and/or any nucleic acid or proteinaceous molecule necessary to carry out an embodiment of a method of the present disclosure. In some embodiments, such a nucleic acid is a vector (e.g., a recombinant expression vector).

Contemplated expression vectors include, but are not limited to, vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, viral vectors based on retroviruses (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, vectors pCTx-1, pCTx-2, and pCTx-3. Other vectors may be used so long as they are compatible with the host cell.

In some embodiments, the vector has one or more transcriptional and/or translational control elements. Depending on the host/vector system utilized, any of a number of suitable transcriptional and translational control elements can be used in the expression vector, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, and the like. In some embodiments, the vector is a self-inactivating vector that inactivates either viral sequences or components of the CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, those derived from long terminal repeats (LTRs) from retroviruses, human elongation factor-1 promoter (EF1), a hybrid construct having the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including gRNAs, promoters such as RNA polymerase III promoters, including U6 and H1, may be advantageous. Descriptions and parameters for enhancing the use of such promoters are known in the art, and further information and approaches are regularly described; see, for example, H. Ma et al., Mol Ther Nuc Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also contain appropriate sequences for amplifying expression. The expression vector may also contain a nucleotide sequence encoding a non-native tag (e.g., a histidine tag, a hemagglutinin tag, green fluorescent protein, etc.) that is fused to the site-specific polypeptide, thereby resulting in a fusion protein.

In some embodiments, the promoter is an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). In some embodiments, the promoter is a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some embodiments, the promoter is a spatially restricted promoter and/or a temporally restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.). In some embodiments, the vector does not have a promoter for at least one gene to be expressed in the host cell (if the gene is expressed after insertion into the genome under an endogenous promoter present in the genome).

Modification of targeted DNA by site-specific polypeptides or DNA endonucleases NHEJ and/or HDR can result in, for example, mutations, deletions, modifications, integrations, gene corrections, gene replacements, gene tagging, transgene insertions, nucleotide deletions, gene disruptions, translocations, and/or gene mutations. The process of incorporating non-naturally occurring nucleic acids into genomic DNA is an example of genome editing.

Site-specific polypeptides are nucleases used in genome editing to cleave DNA. Site-specific polypeptides can be administered to a cell or subject either as one or more polypeptides or as one or more mRNAs encoding the polypeptides.

In the context of a CRISPR/Cas or CRISPR/Cpf1 system, the site-specific polypeptide can bind to a gRNA, which then identifies a site in the target DNA to which the polypeptide is directed. In embodiments of the CRISPR/Cas or CRISPR/Cpf1 systems herein, the site-specific polypeptide is an endonuclease (e.g., a DNA endonuclease).

In some embodiments, the site-directed polypeptide has multiple nucleic acid cleavage (i.e., nuclease) domains. Two or more nucleic acid cleavage domains can be linked together via a linker. In some embodiments, the linker is a flexible linker. The linker can have a length of 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, 30, 35, 40 or more amino acids.

Naturally occurring wild-type Cas9 enzymes have two nuclease domains: an HNH nuclease domain and a RuvC domain. As used herein, "Cas9" refers to both naturally occurring and recombinant Cas9. Cas9 enzymes contemplated herein have an HNH or HNH-like nuclease domain, and/or a RuvC or RuvC-like nuclease domain.

HNH and HNH-like domains have a McrA-like fold. HNH and HNH-like domains have two antiparallel β-strands and an α-helix and have a metal binding site (e.g., a divalent cation binding site). HNH and HNH-like domains can cleave one strand of a target nucleic acid (e.g., the complementary strand of a crRNA target strand).

The RuvC and RuvC-like domains have an RNaseH or RNaseH-like fold. The RuvC/RNaseH domain is involved in a diverse set of nucleic acid-based functions and acts on both RNA and DNA. The RNaseH domain has five β-strands surrounded by multiple α-helices. The RuvC/RNaseH and RuvC/RNaseH-like domains have metal binding sites (e.g., divalent cation binding sites) and can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

In some embodiments, the site-directed polypeptide has an amino acid sequence that has at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to a wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes, U.S. Patent Application Publication No. 2014/0068797, SEQ ID NO:8, or R. Sapranauskas et al., Nuc Acids Res (2011) 39(21):9275-82), and other site-directed polypeptides).

In some embodiments, the site-directed polypeptide has an amino acid sequence that has at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to the nuclease domain of a wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes).

In some embodiments, the site-directed polypeptide is a DNA endonuclease having at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes) over 10 consecutive amino acids. In some embodiments, the site-directed polypeptide has up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide over 10 consecutive amino acids. In some embodiments, the site-directed polypeptide has at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide over 10 consecutive amino acids in the HNH nuclease domain of the site-directed polypeptide. In some embodiments, the site-directed polypeptide has at most 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to the wild-type site-directed polypeptide over 10 consecutive amino acids in the HNH nuclease domain of the site-directed polypeptide. In some embodiments, the site-directed polypeptide has at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to the wild-type site-directed polypeptide over 10 consecutive amino acids in the RuvC nuclease domain of the site-directed polypeptide. In some embodiments, the site-directed polypeptide has at most 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to the wild-type site-directed polypeptide over 10 consecutive amino acids in the RuvC nuclease domain of the site-directed polypeptide.

In some embodiments, the site-directed polypeptide has a modified form of a wild-type exemplary site-directed polypeptide. The modified form of the wild-type exemplary site-directed polypeptide has a mutation that reduces the nucleic acid cleavage activity of the site-directed polypeptide. In some embodiments, the modified form of the wild-type exemplary site-directed polypeptide has less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type exemplary site-directed polypeptide. The modified form of the site-directed polypeptide can have no substantial nucleic acid cleavage activity. When the site-directed polypeptide is a modified form that does not have substantial nucleic acid cleavage activity, it is referred to herein as "enzymatically inactive."

In some embodiments, the modified form of the site-directed polypeptide has a mutation such that it is capable of inducing a single-stranded break (SSB) on the target nucleic acid (e.g., by cleaving only one of the sugar-phosphate backbones of a double-stranded target nucleic acid). In some embodiments, the mutation results in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity in one or more of the multiple nucleic acid cleavage domains of a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes). In some embodiments, the mutation results in one or more of the multiple nucleic acid cleavage domains retaining the ability to cleave a complementary strand of the target nucleic acid but reducing the ability to cleave a non-complementary strand of the target nucleic acid. In some embodiments, the mutation results in one or more of the nucleic acid cleavage domains retaining the ability to cleave a non-complementary strand of the target nucleic acid, but reducing the ability to cleave a complementary strand of the target nucleic acid. For example, residues (e.g., Asp10, His840, Asn854, and Asn856) in a wild-type exemplary S. pyogenes Cas9 polypeptide are mutated to inactivate one or more of the nucleic acid cleavage domains (e.g., nuclease domains). In some embodiments, the residues to be mutated correspond to residues Asp10, His840, Asn854, and Asn856 in a wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., as determined by sequence and/or structural alignment). Non-limiting examples of mutations include D10A, H840A, N854A, and N856A. Those skilled in the art will recognize that mutations other than alanine substitutions are suitable.

In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N856A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the H840A mutation is combined with one or more of the D10A, N854A, or N856A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the N854A mutation is combined with one or more of the H840A, D10A, or N856A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the N856A mutation is combined with one or more of the H840A, N854A, or D10A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. Site-directed polypeptides with one substantially inactive nuclease domain are referred to as "nickases."

In some embodiments, RNA-guided endonucleases, such as mutants of Cas9, can be used to increase the specificity of CRISPR-mediated genome editing. Wild-type Cas9 is generally guided by a single guide RNA designed to hybridize to a specific ~20 nucleotide sequence in a target sequence (such as an endogenous genomic locus). However, some mismatches between the guide RNA and the target locus can be tolerated, effectively reducing the length of homology required at the target site, for example to as little as 13 nt of homology, thereby increasing the potential for binding and double-stranded nucleic acid cleavage by the CRISPR/Cas9 complex elsewhere in the target genome - also known as off-target cleavage. Because the nickase mutants of Cas9 each cleave only one strand, to create a double-stranded break, a pair of nickases must bind to adjacent and opposite strands of the target nucleic acid, thereby creating a pair of nicks that correspond to a double-stranded break. To do this, two separate gRNAs (one for each nickase) must bind to adjacent and opposite strands of the target nucleic acid. This requirement essentially doubles the minimum length of homology required for a double-stranded break to occur, thereby reducing the likelihood that a double-stranded break event will occur elsewhere in the genome where the two gRNA sites (if present) are unlikely to be close enough to each other to allow a double-stranded break to occur. Nickases can also be used to promote HDR versus NHEJ, as described in the art. HDR can be used to introduce selected changes at target sites in the genome through the use of specific donor sequences that effectively mediate the desired changes. Descriptions of CRISPR/Cas systems for use in gene editing can be found, for example, in International Patent Application WO 2013/176772, J. D. Sander et al., Nature Biotech (2014) 32:347-55, and references cited therein.

In some embodiments, the site-directed polypeptide (e.g., a mutant, mutated, enzymatically inactive, and/or conditionally enzymatically inactive site-directed polypeptide) targets a nucleic acid. In some embodiments, the site-directed polypeptide targets a DNA. In some embodiments, the site-directed polypeptide targets an RNA.

In some embodiments, the site-directed polypeptide has one or more non-native sequences (e.g., the site-directed polypeptide is a fusion protein).

In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain).

In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a bacterial Cas9 and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain).

In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a bacterial Cas9 and two nucleic acid cleavage domains, where one or both of the nucleic acid cleavage domains have at least 50% amino acid identity to a nuclease domain from a bacterial Cas9.

In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a bacterial Cas9, two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), and a non-native sequence (e.g., a nuclear localization signal) or a linker connecting the site-directed polypeptide to the non-native sequence.

In some embodiments, the site-directed polypeptide has an amino acid sequence that has at least 15% amino acid identity to a bacterial Cas9 and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), and the site-directed polypeptide has a mutation in one or both of the nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%.

In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a bacterial Cas9 and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), one of the nuclease domains having a mutation at aspartic acid 10 and/or one of the nuclease domains having a mutation at histidine 840, the mutation reducing the cleavage activity of the nuclease domain by at least 50%.

In some embodiments, one or more site-specific polypeptides, such as DNA endonucleases, include two nickases that together create one double-stranded break at a specific locus in the genome, or four nickases that together create two double-stranded breaks at a specific locus in the genome. Alternatively, one site-specific polypeptide affects one double-stranded break at a specific locus in the genome.

In some embodiments, a polynucleotide encoding a site-directed polypeptide may be used to edit a genome. In some such embodiments, the polynucleotide encoding the site-directed polypeptide is codon-optimized according to methods known in the art for expression in a cell containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, a human codon-optimized polynucleotide encoding Cas9 may be used to produce the Cas9 polypeptide.

The following provide some examples of site-specific polypeptides that may be used in embodiments of the present disclosure:

CRISPR endonuclease system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic loci can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, CRISPR loci code for products that function as a type of immune system that helps defend prokaryotes against foreign invaders such as viruses and phages. There are three stages of CRISPR locus function: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acids. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

The CRISPR locus contains many short repeating sequences called "repeats". When expressed, the repeats can form secondary hairpin structures (e.g. hairpins) and/or have unstructured single-stranded sequences. The repeats usually occur in clusters and often diverge between species. The repeats are regularly spaced with unique intervening sequences called "spacers", resulting in a repeat-spacer-repeat locus structure. The spacers are identical or highly homologous to known foreign invader sequences. The spacer-repeat units encode the crRNA, which is processed into the mature form of the spacer-repeat units. The crRNA has a "seed" or spacer sequence that is involved in targeting the target nucleic acid (in its naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid). The spacer sequence is located at the 5' or 3' end of the crRNA.

The CRISPR locus also contains polynucleotide sequences encoding CRISPR-associated (Cas) genes. Cas genes encode endonucleases involved in the biosynthesis and interference steps of crRNA function in prokaryotes. Some Cas genes have homologous secondary and/or tertiary structures.

Type II CRISPR Systems In natural Type II CRISPR systems, crRNA biogenesis requires a trans-activated CRISPR RNA (tracrRNA). The tracrRNA is modified by endogenous RNaseIII and then hybridizes to the crRNA repeats in the pre-crRNA array. Endogenous RNaseIII is recruited to cleave the pre-crRNA. The cleaved crRNA is subjected to exoribonuclease trimming to generate the mature crRNA form (e.g., 5' trimming). The tracrRNA remains hybridized to the crRNA, and the tracrRNA and crRNA bind to a site-directed polypeptide (e.g., Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex guides the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of crRNA to the target nucleic acid activates Cas9 for target nucleic acid cleavage. The target nucleic acid in the type II CRISPR system is referred to as a protospacer adjacent motif (PAM). Essentially, the PAM is essential to facilitate the binding of a site-specific polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also referred to as Nmeni or CASS4) are further subdivided into type II-A (CASS4) and type II-B (CASS4a). M. Jinek et al., supra, reported that the CRISPR/Cas9 system is useful for RNA programmable genome editing, and International Patent Application WO 2013/176772 provides numerous examples and applications of the CRISPR/Cas endonuclease system for site-specific gene editing.

Type V CRISPR system Type V CRISPR system has some important differences from type II system. For example, Cpf1 is a single RNA-guided endonuclease that lacks tracrRNA, in contrast to type II system. In fact, Cpf1-associated CRISPR arrays are processed into mature crRNAs without the need for additional transactivating tracrRNA. Type V CRISPR arrays are processed into short mature crRNAs of 42-44 nucleotides in length, each mature crRNA starting with a 19-nucleotide direct repeat followed by a 23-25 nucleotide spacer sequence. In contrast, mature crRNAs in type II systems start with a 20-24 nucleotide spacer sequence followed by about 22 nucleotide direct repeats. Cpf1 also utilizes a T-rich protospacer adjacent motif such that the Cpf1-crRNA complex efficiently cleaves target DNA preceded by a short T-rich PAM, in contrast to the G-rich PAM that follows the target DNA in type II systems. Thus, type V systems cleave at a point distal to the PAM, whereas type II systems cleave at a point adjacent to the PAM. Furthermore, in contrast to type II systems, Cpf1 cleaves DNA via a staggered DNA double-strand break with a 4- or 5-nucleotide 5' overhang. Type II systems cleave via a blunt double-strand break. Like type II systems, Cpf1 contains a predicted RuvC-like endonuclease domain, but in contrast to type II systems, it lacks a second HNH endonuclease domain.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptide in Figure 1 of I. Fonfara et al., Nucleic Acids Res. (2014) 42:2577-90. The CRISPR/Cas gene nomenclature system has undergone extensive rewriting since the discovery of Cas genes. Figure 5 of Fonfara (supra) provides the PAM sequences of Cas9 polypeptides from different species.

A genome-targeting nucleic acid interacts with a site-specific polypeptide (e.g., a nucleic acid-guided nuclease such as Cas9) to form a complex. The genome-targeting nucleic acid (e.g., a gRNA) guides the site-specific polypeptide to the target nucleic acid.

As previously mentioned, in some embodiments, the site-specific polypeptide and the genome-targeting nucleic acid can each be administered separately to a cell or subject. In some embodiments, the site-specific polypeptide can be pre-complexed with one or more guide RNAs, or one or more crRNAs (along with a tracrRNA). The pre-complexed material can then be administered to a cell or subject. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

Systems for Genome Editing Provided herein are systems for genome editing, in particular for inserting a synthetic FVIII coding sequence into the genome of a cell. The systems can be used in the methods described herein, for example, to edit the genome of a cell and to treat a subject, e.g., a subject with hemophilia A.

In some embodiments, provided herein is a system that includes a donor template that includes (a) a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; (b) a gRNA that targets an albumin locus in the genome of a cell; and (c) a nucleic acid sequence encoding a synthetic FVIII protein. In some embodiments, the gRNA targets intron 1 of the albumin gene. In some embodiments, the gRNA includes a spacer sequence from any one of SEQ ID NOs: 271-298.

In some embodiments, provided herein is a system that includes: (a) a nucleic acid encoding a deoxyribonucleic acid (DNA) endonuclease or a DNA endonuclease; (b) a guide RNA (gRNA) that includes a spacer sequence from any one of SEQ ID NOs: 271-298; and (c) a donor template that includes a nucleic acid sequence encoding a synthetic FVIII protein. In some embodiments, the gRNA includes a spacer sequence from any one of SEQ ID NOs: 274, 275, 281, and 283. In some embodiments, the gRNA includes a spacer sequence from SEQ ID NO: 274. In some embodiments, the gRNA includes a spacer sequence from SEQ ID NO: 275. In some embodiments, the gRNA includes a spacer sequence from SEQ ID NO: 281. In some embodiments, the gRNA includes a spacer sequence from SEQ ID NO: 283.

In some embodiments, according to any of the systems described herein, the DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn In some embodiments, the DNA endonuclease is selected from the group consisting of: 2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease, or a functional equivalent thereof. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the Cas9 is from Streptococcus pyogenes (spCas9). In some embodiments, Cas9 is derived from Staphylococcus lugdunensis (SluCas9).

In some embodiments, according to any of the systems described herein, the nucleic acid sequence encoding the synthetic FVIII protein is codon-optimized for expression in a host cell. In some embodiments, the nucleic acid sequence encoding the synthetic FVIII protein is codon-optimized for expression in a human cell.

In some embodiments, according to any of the systems described herein, the system includes a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding the DNA endonuclease is codon optimized for expression in a host cell. In some embodiments, the nucleic acid encoding the DNA endonuclease is codon optimized for expression in a human cell. In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA, such as a DNA plasmid. In some embodiments, the nucleic acid encoding the DNA endonuclease is RNA, such as an mRNA.

In some embodiments, according to any of the systems described herein, the donor template is encoded by an AAV vector. In some embodiments, the donor template includes a donor cassette that includes a synthetic FVIII coding sequence, and the donor cassette is flanked on one or both sides by gRNA target sites. In some embodiments, the donor cassette is flanked on both sides by gRNA target sites. In some embodiments, the gRNA target site is a target site of a gRNA in the system. In some embodiments, the gRNA target site of the donor template is the reverse complement of a cellular genomic gRNA target site for the gRNA in the system.

In some embodiments, according to any of the systems described herein, the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle. In some embodiments, the liposome or lipid nanoparticle also comprises a gRNA. In some embodiments, the liposome or lipid nanoparticle is a lipid nanoparticle. In some embodiments, the system comprises a lipid nanoparticle comprising a nucleic acid encoding the DNA endonuclease and a gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is an mRNA encoding the DNA endonuclease.

In some embodiments, according to any of the systems described herein, the DNA endonuclease is complexed with the gRNA to form an RNP complex.

Methods of Genome Editing Provided herein are methods of genome editing, in particular, inserting the synthetic FVIII protein into the genome of a cell. This method can be used to treat a subject (e.g., a patient with hemophilia A), in which case the cells can be isolated from the subject or a separate donor. The chromosomal DNA of the cells is then edited using the materials and methods described herein.

Provided herein is a method for knocking in a synthetic FVIII coding sequence into a genome. In one aspect, the disclosure provides for the insertion of a nucleic acid sequence of a synthetic FVIII coding sequence, i.e., a nucleic acid sequence encoding a synthetic FVIII protein, into the genome of a cell. The synthetic FVIII protein can include a peptide having substantial activity of a wild-type FVIII protein, e.g., at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of the activity exhibited by the wild-type FVIII protein. In some embodiments, one of skill in the art can use a number of methods known in the art to test the functionality or activity of a compound (e.g., a peptide or protein). In some embodiments, the synthetic FVIII protein can also include any fragment of a wild-type FVIII protein, or a fragment of a modified FVIII protein having conservative modifications to one or more amino acid residues in the full-length wild-type FVIII protein. In some embodiments, the synthetic FVIII protein can also include any modification, e.g., deletion, insertion, and/or mutation, of one or more amino acids that does not substantially adversely affect the functionality of the wild-type FVIII protein. Thus, in some embodiments, the nucleic acid sequence of the synthetic FVIII coding sequence can have at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% nucleic acid sequence identity to the FVIII coding sequence.

In some embodiments, the synthetic FVIII coding sequence is inserted into a genomic sequence in the cell. In some embodiments, the insertion site is at or within the albumin locus, transferrin locus, or fibrinogen alpha locus in the genome of the cell. In some embodiments, the insertion site is the albumin locus. The insertion method uses one or more gRNAs that target the first intron (or intron 1) of the albumin gene. In some embodiments, the donor DNA is single-stranded or double-stranded DNA having the synthetic FVIII coding sequence.

In some embodiments, the genome editing method utilizes a DNA endonuclease, such as the CRISPR/Cas system, to genetically introduce (knock in) a synthetic FVIII coding sequence. In some embodiments, the DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease, homologs thereof, recombinant naturally occurring molecules, codon optimized, or modified versions thereof, and combinations of any of the above. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the Cas9 is from Streptococcus pyogenes (spCas9). In some embodiments, Cas9 is derived from Staphylococcus lugdunensis (SluCas9).

In some embodiments, the cellular subject of genome editing has one or more mutations in the genome that result in a reduction in expression of the endogenous FVIII gene compared to expression in a normal without such mutation. The normal cell can be a healthy cell or a control cell derived from (or isolated from) a different subject that does not have a FVIII gene defect. In some embodiments, the cellular subject of genome editing can be derived from (or isolated from) a subject in need of treatment for a FVIII gene-associated condition or disorder, such as hemophilia A. Thus, in some embodiments, the expression of the endogenous FVIII gene in such cells is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the endogenous FVIII gene expression in a normal cell.

In some embodiments, the genome editing method provides for targeted integration (in a non-coding region of the genome) of a functional FVIII coding sequence, e.g., a FVIII coding sequence operably linked to a promoter provided to stably produce a FVIII protein in vivo. In some embodiments, the targeted integration of the FVIII coding sequence occurs in an intron of an albumin gene that is highly expressed in a cell type of interest (e.g., hepatocytes or sinusoidal endothelial cells).

In one aspect, the nucleic acid sequence of the synthetic FVIII coding sequence is inserted into the genome of the cell. In an embodiment, the synthetic FVIII coding sequence inserted is a modified FVIII coding sequence. In some embodiments, in the modified FVIII coding sequence, the B domain of the wild-type FVIII coding sequence is deleted and replaced with a B domain replacement. In some embodiments, the synthetic FVIII is superior to the full-length wild-type FVIII due to its smaller size (4371 bp vs. 7053 bp). Thus, in some embodiments, a synthetic FVIII coding sequence lacking the FVIII signal peptide and including a splice acceptor sequence at its 5' end (the N-terminus of the FVIII coding sequence) is specifically integrated into intron 1 of a locus in hepatocytes of mammals, including humans. In one embodiment, the locus is the albumin locus. In another embodiment, the locus is the transferrin locus. In another embodiment, the locus is the fibrinogen alpha locus.

Transcription of the synthetic FVIII coding sequence from the transferrin promoter can result in a pre-mRNA that contains transferrin exon 1, a portion of intron 1, and the integrated synthetic FVIII coding sequence. Once this pre-mRNA has undergone the natural splicing process to remove the intron, the splicing machinery can link the splice donor 3' of transferrin exon 1 to the next available splice acceptor that becomes the splice acceptor at the 5' end of the inserted DNA donor synthetic FVIII coding sequence. This can result in a mature mRNA that contains transferrin exon 1 fused to the mature coding sequence of synthetic FVIII.

Transcription of this synthetic FVIII coding sequence from the albumin promoter can result in a pre-mRNA that contains albumin exon 1, a portion of intron 1, and the integrated synthetic FVIII coding sequence. Once this pre-mRNA has undergone the natural splicing process of removing the intron, the splicing machinery can link the splice donor 3' of albumin exon 1 to the next available splice acceptor that becomes the splice acceptor at the 5' end of the inserted DNA donor synthetic FVIII coding sequence. This can result in a mature mRNA that contains albumin exon 1 fused to the mature synthetic FVIII coding sequence. Albumin exon 1 codes for two additional amino acids in addition to the signal peptide, and one third of the codons that normally code for the N-terminal protein sequence DAH of albumin in humans. Thus, in some embodiments, after expected cleavage of the albumin signal peptide during secretion from a cell, a synthetic FVIII protein can be produced that has three additional amino acid residues added to its N-terminus, and that has the amino acid sequence
Because the third of these three amino acids (underlined) is partially encoded by the end of exon 1 and partially encoded by the synthetic FVIII DNA donor template, it is possible to select the identity of the third additional amino acid residue to be either Leu, Pro, His, Gln, or Arg. Of these options, Leu is used in some embodiments because it is the smallest molecular complex and therefore the least likely to form novel T-cell epitopes, resulting in the amino acid sequence Leu at the N-terminus of the synthetic FVIII protein.
Alternatively, the DNA donor template may be modified to have the amino acid sequence
In some cases, adding additional amino acids to the sequence of a native protein may increase the immunogenicity risk. Thus, an in silico analysis to predict the potential immunogenicity of two potential options for the N-terminus of a synthetic FVIII revealed that a single residue
demonstrate that deletion of has a lower immunogenicity score, which in some embodiments may be by design, at least in some embodiments.

In some embodiments, DNA sequences encoding synthetic FVIII with optimized codon usage can be used to improve expression in mammalian cells (so-called "codon optimization"). Various computer algorithms for performing codon optimization are also available in the art, which generate different DNA sequences (V.P. Mauro et al., Trends Mol Med (2014) 20:604-13). Examples of commercially available codon optimization algorithms are those used by the companies ATUM and GeneArt (part of Thermo Fisher Scientific). Codon optimization of FVIII coding sequences has been demonstrated to significantly improve expression of FVIII after gene-based delivery in mice (A.C. Nathwani et al., Blood (2006) 107(7):2653-61); N. J. Ward et al. , Blood (2011) 117(3):798-807; P. A. Radcliffe et al., Gene Ther. (2008) 15(4):289-97). Codon optimization is an established approach to improve the expression of a coding sequence of interest and is primarily based on replacing less frequently used codons with more frequently used codons without changing the encoded amino acid sequence. From the initial recognition that codon bias can affect protein expression, methods for codon optimization have evolved and algorithms are commercially available, including those offered by DNA synthesis companies such as GeneArt and ATUM. These commercially available algorithms, which are available to users free of charge as part of their DNA synthesis services, are also designed to remove cryptic splicing signals and equalize G/C content throughout the coding sequence. Delivery of exogenous nucleic acids to cells in vivo can induce an innate immune response driven at least in part by recognition of CG dinucleotides (also called CpG sequences) by the Toll receptor system, and reducing the CG dinucleotide content has been proposed as a way to reduce the innate immune response to these nucleic acids, especially when plasmid DNA is the delivery vector. See also P. Collella et al., Mol Ther Methods Clin Dev (2018) 8:87-104. When the naturally occurring (natural) coding sequence of a gene is optimized for expression in a mammalian species, the number of CG dinucleotides is generally increased because more frequently used codons contain a higher frequency of G and C nucleotides in the third (wobble) position of the codon. Thus, an increase in the overall content of G and C nucleotides in the coding sequence will result in a higher content of GC dinucleotides.

In some embodiments, the sequence homology or identity between the synthetic FVIII coding sequence codon-optimized by different algorithms and the native FVIII sequence (present in the human genome) may range from about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the codon-optimized synthetic FVIII coding sequence has about 75% to about 79% sequence homology or identity to the native FVIII sequence. In some embodiments, the codon-optimized synthetic FVIII coding sequence has about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, or about 80% sequence homology or identity to the native FVIII sequence.

In some embodiments, a donor template or donor construct is prepared to contain a DNA sequence encoding a synthetic FVIII. In some embodiments, a DNA donor template is designed to contain a codon-optimized human synthetic FVIII coding sequence. In some embodiments, the codon optimization is performed such that the sequence at the 5' end encoding the signal peptide of FVIII is deleted and replaced with a splice acceptor sequence, and further a polyadenylation signal is added to the 3' end after the FVIII stop codon (MAB8A-SEQ ID NO:301). The splice acceptor sequence can be selected from among known splice acceptor sequences from known genes, or a consensus splice acceptor sequence derived from an alignment of many splice acceptor sequences known in the art can be used. In some embodiments, a splice acceptor sequence from a highly expressed gene is used, as such sequences are believed to provide optimal splicing efficiency. In some embodiments, the consensus splice acceptor sequence is comprised of a branch site with the consensus sequence T/CNC/TT/CA/GAC/T (SEQ ID NO: 302), followed by a 10-12 base polypyrimidine tract (C or T) within 20 bp, followed by AG>G/A (where > is the position of the intron/exon boundary). In one embodiment, the synthetic splice acceptor sequence
In another embodiment, the human albumin gene intron 1/exon 2 boundary is used.
Or mouse
The native splice acceptor sequence is used.

The polyadenylation sequence provides the cell with a signal to add a polyA tail, which is essential for intracellular mRNA stability. In some embodiments, the DNA donor template will be packaged into an AAV particle, but embodiments of the invention keep the size of the packaged DNA within the packaging limits of AAV, which may be less than about 5 Kb, or about 4.7 Kb or less. Thus, in some embodiments, the shortest possible polyA sequence is used (e.g., about 10-mer, about 20-mer, about 30-mer, about 40-mer, about 50-mer, or about 60-mer, or any intervening number of nucleotides as described above). The consensus synthetic polyA signal sequence is described in the literature (N. Levitt et al., Genes Dev (1989) 3(7):1019-25) and is AATAAAAGATCTTTATTTTTCATTAGATCTGTGTGTTGGTTTTTTTTGTGTG (SEQ ID NO: 306), which is commonly used in many expression vectors.

In some embodiments, additional sequence elements can be added to the DNA donor template to improve the frequency of integration. One such element is a homology arm. Sequences from the left of the double-stranded break (LHA) are added to the 5' (N-terminal of the FVIII coding sequence) end of the DNA donor template, and sequences from the right of the double-stranded break (RHA) are added to the 3' (C-terminal of the FVIII coding sequence) end of the DNA donor template, e.g., MAB8B (SEQ ID NO: 308).

An alternative DNA donor template design provided in some embodiments has a sequence complementary to the recognition sequence of the sgRNA used to cleave the genomic site. MAB8C (SEQ ID NO: 309) represents an example of this type of DNA donor template. By including the sgRNA recognition site, the DNA donor template is cleaved by the sgRNA/Cas9 complex in the nucleus of the cell to which the DNA donor template and sgRNA/Cas9 have been delivered. Cleavage of the donor template into linear fragments can increase the frequency of integration at the double-stranded break by non-homologous end joining or HDR mechanisms. This may be particularly beneficial in the case of delivery of AAV-packaged donor templates, since it is known that the AAV genome concatemerizes to form larger circular double-stranded DNA molecules after delivery to the nucleus (H. Nakai et al., J Virol (2001) 75:6969-76). Thus, in some cases, circular concatemers may be less efficient donors for integration at double-strand breaks, especially by the NHEJ mechanism. Previously, it has been reported that the efficiency of targeted integration using circular plasmid DNA donor templates can be increased by including zinc finger nuclease cleavage sites in the plasmid (S. Cristea et al., Biotechnol. Bioeng. (2013) 110:871-80). More recently, this approach has also been applied using CRISPR/Cas9 nucleases (K. Suzuki et al., Nature (2017) 540:144-49). Although the sgRNA recognition sequence is active when present on either strand of the double-stranded DNA donor template, the use of the reverse complement of the sgRNA recognition sequence present in the genome is predicted to favor stable integration, since integration in the reverse orientation can recreate the sgRNA recognition sequence that can be re-cut to release the inserted donor template. Integration of such a donor template in the genome in the forward orientation by NHEJ is predicted not to recreate the sgRNA recognition sequence, such that the integrated donor template cannot be excised from the genome. The benefit of including the sgRNA recognition sequence in the donor with or without homology arms on the integration efficiency of the FVIII donor template can be tested and determined, for example, in mice using AAV for delivery of the donor and LNPs (lipid nanoparticles) for delivery of the CRISPR/CAS9 components.

In some embodiments, the donor template comprises a synthetic FVIII coding sequence in a donor cassette according to any of the embodiments described herein flanked on one or both sides by gRNA target sites. In some embodiments, the donor template comprises a gRNA target site 5' of the donor cassette and/or a gRNA target site 3' of the donor cassette. In some embodiments, the donor template comprises two adjacent gRNA target sites, the two gRNA target sites comprising the same sequence. In some embodiments, the donor template comprises at least one gRNA target site, the at least one gRNA target site in the donor template being a target site of at least one of the one or more gRNAs targeting the first intron of the albumin gene. In some embodiments, the donor template comprises at least one gRNA target site, the at least one gRNA target site in the donor template being a reverse complement of a target site of at least one of the one or more gRNAs in the first intron of the albumin gene. In some embodiments, the donor template comprises a 5' gRNA target site of the donor cassette and a 3' gRNA target site of the donor cassette, where the two gRNA target sites in the donor template are targeted by one or more gRNAs that target the first intron of the albumin gene. In some embodiments, the donor template comprises a 5' gRNA target site of the donor cassette and a 3' gRNA target site of the donor cassette, where the two gRNA target sites in the donor template are reverse complements of at least one target site of one or more gRNAs in the first intron of the albumin gene.

The insertion of the FVIII coding sequence at the target site, i.e., the genomic location where the FVIII coding sequence is to be inserted, can be in the endogenous albumin locus or adjacent sequences thereof. In some embodiments, the FVIII coding sequence is inserted such that expression of the inserted coding sequence is controlled by the endogenous promoter of the albumin gene. In some embodiments, the FVIII coding sequence is inserted into one of the introns of the albumin gene. In some embodiments, the FVIII coding sequence is inserted into one of the exons of the albumin gene. In some embodiments, the FVIII coding sequence is inserted at an intron:exon junction (or vice versa). In some embodiments, the insertion of the FVIII coding sequence is in the first intron (or intron 1) of the albumin locus. In some embodiments, the insertion of the FVIII coding sequence does not significantly affect, e.g., does not upregulate or downregulate, expression of the albumin gene.

In embodiments, the target site for insertion of the FVIII coding sequence is at, within, or near the endogenous albumin gene. In some embodiments, the target site is in an intergenic region upstream of the promoter of the albumin locus in the genome. In some embodiments, the target site is within the albumin locus. In some embodiments, the target site is in one of the introns of the albumin locus. In some embodiments, the target site is in one of the exons of the albumin locus. In some embodiments, the target site is at one of the junctions between an intron and an exon (or vice versa) of the albumin locus. In some embodiments, the target site is in the first intron (or intron 1) of the albumin locus. In certain embodiments, the target site is at least, about, or at most about 0, 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 bp downstream of the first exon (i.e., from the last nucleic acid of the first exon) of the albumin gene. In some embodiments, the target site is at least, about, or at most 0.1 kb, about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 1 kb, about 1.5 kb, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, or about 5 kb upstream of the first intron of the albumin gene. In some embodiments, the target site is between about 0 bp and about 100 bp upstream, between about 101 bp and about 200 bp upstream, between about 201 bp and about 300 bp upstream, between about 301 bp and about 400 bp upstream, between about 401 bp and about 500 bp upstream, between about 501 bp and about 600 bp upstream, between about 601 bp and about 700 bp upstream, between about 701 bp and about 800 bp upstream, between about 801 bp and about 900 bp upstream of the second exon of the albumin gene. The target site is located upstream, about 901 bp to about 1000 bp upstream, about 1001 bp to about 1500 bp upstream, about 1501 bp to about 2000 bp upstream, about 2001 bp to about 2500 bp upstream, about 2501 bp to about 3000 bp upstream, about 3001 bp to about 3500 bp upstream, about 3501 bp to about 4000 bp upstream, about 4001 bp to about 4500 bp upstream, or about 4501 bp to about 5000 bp upstream. In some embodiments, the target site is at least 37 bp downstream of the end (i.e., the 3' end) of the first exon of the human albumin gene in the genome. In some embodiments, the target site is at least 330 bp upstream of the start (i.e., the 5' start) of the second exon of the human albumin gene in the genome.

In some embodiments, provided herein is a method of editing a genome in a cell, the method comprising providing a cell with: (a) a guide RNA (gRNA) that targets an albumin locus in the genome of the cell; (b) a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; and (c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein. In some embodiments, the gRNA targets intron 1 of the albumin gene. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 271-298.

In some embodiments, provided herein is a method of editing a genome in a cell, the method comprising providing to the cell: (a) a gRNA comprising a spacer sequence from any one of SEQ ID NOs: 271-298; (b) a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; and (c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 274, 275, 281, and 283. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 274. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 275. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 281. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 283. In some embodiments, the cell is a human cell, e.g., a human hepatocyte.

In some embodiments according to any of the methods of editing a genome in a cell described herein, the DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csx1, Csx2, Csx1 ... sn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease, or a functional equivalent thereof. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the Cas9 is spCas9. In some embodiments, the Cas9 is SluCas9.

In some embodiments according to any of the methods of editing a genome in a cell described herein, the nucleic acid sequence encoding the synthetic FVIII protein is codon-optimized for expression in the cell. In some embodiments, the cell is a human cell.

In some embodiments according to any of the methods of editing a genome in a cell described herein, the method uses a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding the DNA endonuclease is codon optimized for expression in the cell. In some embodiments, the cell is a human cell, e.g., a human hepatocyte. In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA, such as a DNA plasmid. In some embodiments, the nucleic acid encoding the DNA endonuclease is RNA, such as an mRNA.

In some embodiments according to any of the methods of editing a genome in a cell described herein, the donor template is encoded by an AAV vector. In some embodiments, the donor template includes a donor cassette that includes a nucleic acid sequence encoding a synthetic FVIII protein, flanked on one or both sides by gRNA target sites. In some embodiments, the donor cassette is flanked on both sides by gRNA target sites. In some embodiments, the gRNA target site is a target site for the administered gRNA. In some embodiments, the gRNA target site of the donor template is the reverse complement of the cellular genome gRNA target site for the gRNA.

In some embodiments according to any of the methods of editing a genome in a cell described herein, the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle. In some embodiments, the liposome or lipid nanoparticle also comprises a gRNA. In some embodiments, the liposome or lipid nanoparticle is a lipid nanoparticle. In some embodiments, the method uses a lipid nanoparticle comprising a nucleic acid encoding the DNA endonuclease and a gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is an mRNA encoding the DNA endonuclease.

In some embodiments according to any of the methods for editing a genome in a cell described herein, the DNA endonuclease is pre-complexed with the gRNA to form an RNP complex.

In some embodiments according to any of the methods of editing a genome in a cell described herein, the gRNA and the DNA endonuclease or the nucleic acid encoding the DNA endonuclease are provided to the cell after the donor template is provided to the cell. In some embodiments, the gRNA and the DNA endonuclease or the nucleic acid encoding the DNA endonuclease are provided to the cell more than 4 days after the donor template is provided to the cell. In some embodiments, the gRNA and the DNA endonuclease or the nucleic acid encoding the DNA endonuclease are provided to the cell at least 14 days after the donor template is provided to the cell. In some embodiments, the gRNA and the DNA endonuclease or the nucleic acid encoding the DNA endonuclease are provided to the cell at least 17 days after the donor template is provided to the cell. In some embodiments, (a) and (b) are provided to the cell as lipid nanoparticles comprising the nucleic acid encoding the DNA endonuclease and the gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is an mRNA encoding a DNA endonuclease. In some embodiments, (c) is provided to the cell as an AAV vector encoding the donor template.

In some embodiments according to any of the methods of editing a genome in a cell described herein, one or more additional doses of gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease. In some embodiments, one or more additional doses of gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease until a target level of targeted integration of a nucleic acid sequence encoding a synthetic FVIII protein and/or a target level of expression of a nucleic acid sequence encoding a synthetic FVIII protein is achieved.

In some embodiments according to any of the methods of editing a genome in a cell described herein, the nucleic acid sequence encoding the synthetic FVIII protein is expressed under the control of an endogenous albumin promoter.

In some embodiments, provided herein is a method of inserting a synthetic FVIII coding sequence into an albumin locus of a cell genome, the method comprising introducing into the cell a donor template according to any of the embodiments described herein, the donor template comprising (a) a Cas DNA endonuclease (e.g., Cas9) or a nucleic acid encoding a Cas DNA endonuclease, (b) a gRNA or a nucleic acid encoding a gRNA, where the gRNA can direct the Cas DNA endonuclease to cleave a target polynucleotide sequence at the albumin locus, and (c) a synthetic FVIII coding sequence. In some embodiments, the method comprises introducing into the cell an mRNA encoding the Cas DNA endonuclease. In some embodiments, the method comprises introducing into the cell an LNP according to any of the embodiments described herein, the LNP comprising i) an mRNA encoding the Cas DNA endonuclease and ii) a gRNA. In some embodiments, the donor template is an AAV donor template. In some embodiments, the donor template comprises a donor cassette comprising a synthetic FVIII coding sequence, flanked on one or both sides by gRNA target sites. In some embodiments, the gRNA target sites flanking the donor cassette are the reverse complements of the gRNA target sites in the albumin locus. In some embodiments, the Cas DNA endonuclease or a nucleic acid encoding the Cas DNA endonuclease and the gRNA or a nucleic acid encoding the gRNA are introduced into the cell after introduction of the donor template into the cell. In some embodiments, the Cas DNA endonuclease or a nucleic acid encoding the Cas DNA endonuclease and the gRNA or a nucleic acid encoding the gRNA are introduced into the cell for a sufficient time after introduction of the donor template into the cell to allow the donor template to enter the cell nucleus. In some embodiments, the Cas DNA endonuclease or a nucleic acid encoding the Cas DNA endonuclease and the gRNA or a nucleic acid encoding the gRNA are introduced into the cell for a sufficient time after introduction of the donor template into the cell to allow the donor template to be converted from a single-stranded AAV genome into a double-stranded DNA molecule in the cell nucleus. In some embodiments, the Cas DNA endonuclease is Cas9.

In some embodiments according to any of the methods of inserting a synthetic FVIII coding sequence into the albumin locus of a cell genome described herein, the target polynucleotide sequence is in intron 1 of the albumin gene. In some embodiments, the gRNA comprises a spacer sequence of any of SEQ ID NOs: 271-298. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 274, 275, 281, and 283. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 274. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 275. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 281. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 283.

In some embodiments, provided herein is a method of inserting a synthetic FVIII coding sequence into an albumin locus of a cell genome, the method comprising: (a) introducing into a cell an LNP according to any of the embodiments described herein, the LNP comprising i) an mRNA encoding a Cas9 DNA endonuclease and ii) a gRNA capable of inducing the Cas9 DNA endonuclease to cleave a target polynucleotide sequence at the albumin locus; and (b) an AAV donor template according to any of the embodiments described herein, the AAV donor template comprising a synthetic FVIII coding sequence. In some embodiments, the donor template comprises a donor cassette comprising a synthetic FVIII coding sequence, flanked on one or both sides by gRNA target sites. In some embodiments, the gRNA target sites flanking the donor cassette are the reverse complement of the gRNA target sites at the albumin locus. In some embodiments, the LNP is introduced into the cell after introduction of the AAV donor template into the cell. In some embodiments, the LNPs are introduced into the cell for a sufficient time after introduction of the AAV donor template into the cell to allow the donor template to enter the cell nucleus. In some embodiments, the LNPs are introduced into the cell for a sufficient time after introduction of the AAV donor template into the cell to allow the donor template to be converted from a single-stranded AAV genome into a double-stranded DNA molecule in the cell nucleus. In some embodiments, further introduction of one or more (e.g., 2, 3, 4, 5 or more) of the LNPs into the cell follows the initial introduction of the LNPs into the cell. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 271-298. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 274, 275, 281, and 283. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 274. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 275. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 281. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO:283.

Insertion of the FVIII coding sequence at the target site can be in the endogenous fibrinogen-α locus or adjacent sequences thereof. In some embodiments, the FVIII coding sequence is inserted such that expression of the inserted coding sequence is controlled by the endogenous promoter of the fibrinogen-α gene. In some embodiments, the FVIII coding sequence is inserted into one of the introns of the fibrinogen-α gene. In some embodiments, the FVIII coding sequence is inserted into one of the exons of the fibrinogen-α gene. In some embodiments, the FVIII coding sequence is inserted at an intron:exon (or vice versa) junction. In some embodiments, the insertion of the FVIII coding sequence is in the first intron (or intron 1) of the fibrinogen-α locus. In some embodiments, the insertion of the FVIII coding sequence does not significantly affect, e.g., upregulate or downregulate, expression of the fibrinogen-α gene.

In certain embodiments, the target site is at least, about, or up to 0, 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1071 bp, or any intervening length of nucleic acid downstream of the first exon of the fibrinogen-α gene (i.e., from the last base pair or 3' end of the first exon). In some embodiments, the target site is at least, about or up to 0.1 kb, about 0.2 kb, about 0.3 kb, about 0.4 kb, about 0.5 kb, about 1 kb, or any intervening length of nucleic acid upstream of the second exon of the fibrinogen-α gene (i.e., from the first nucleic acid or 5' end of the second exon). In some embodiments, the target site is about 0 bp to about 100 bp, about 101 bp to about 200 bp, about 201 bp to about 300 bp, about 301 bp to about 400 bp, about 401 bp to about 500 bp, about 501 bp to about 600 bp, about 601 bp to about 700 bp, about 701 bp to about 800 bp, about 801 bp to about 900 bp, about 901 bp to about 1000 bp, or about 1001 bp to about 1071 bp upstream of the second exon of the fibrinogen-α gene (i.e., from the first nucleic acid or the 5' end of the second exon).

In some embodiments, the target site for insertion of the FVIII coding sequence is at least 40 bp downstream of the end of the first exon of the human fibrinogen-α gene in the genome and at least 60 bp upstream of the start of the second exon of the human fibrinogen-α gene in the genome.

In some embodiments, the target site for insertion of the FVIII coding sequence is at least 42 bp downstream of the end of the first exon of the human fibrinogen-α gene in the genome and at least 65 bp upstream of the start of the second exon of the human fibrinogen-α gene in the genome.

In some embodiments, the insertion is at least 12 bp downstream of the end of the first exon of the human fibrinogen-α gene in the genome and at least 52 bp upstream of the start of the second exon of the human fibrinogen-α gene in the genome.

In some embodiments, the insertion is at least 94 bp downstream of the end of the first exon of the human fibrinogen-α gene in the genome and at least 86 bp upstream of the start of the second exon of the human fibrinogen-α gene in the genome.

In some embodiments according to any of the systems described herein, the donor template comprises a nucleic acid sequence encoding a synthetic FVIII for targeted integration into intron 1 of the transferrin gene, wherein the donor template comprises, from 5' to 3', i) a first gRNA target site; ii) a splice acceptor; iii) a nucleotide sequence encoding the synthetic FVIII; and iv) a polyadenylation signal. In some embodiments, the donor template further comprises iv) a second gRNA target site downstream of the polyadenylation signal. In some embodiments, the first gRNA target site and the second gRNA target site are the same. In some embodiments, the donor template further comprises a sequence encoding a terminal portion of a transferrin signal peptide encoded in exon 2 of the transferrin gene or a variant thereof that retains at least a portion of the activity of the endogenous sequence between ii) the splice acceptor and iii) the nucleotide sequence encoding the synthetic FVIII protein. In some embodiments, the donor template further comprises a polynucleotide spacer between i) the first gRNA target site and ii) the splice acceptor. In some embodiments, the polynucleotide spacer is 18 nucleotides in length. In some embodiments, the donor template is flanked on one side by a first AAV ITR (inverted terminal repeat) and/or on the other side by a second AAV ITR. In some embodiments, the first AAV ITR is an AAV2 ITR and/or the second AAV ITR is an AAV2 ITR. In some embodiments, iii) a nucleotide sequence encoding a synthetic FVIII with a B domain substitution that includes 3, 4, 5, or 6 N-linked glycosylation sites. Exemplary sequences of donor template components can be found in donor template sequences of SEQ ID NOs: 310 and/or 311.

Target Sequence Selection In some embodiments, a shift in the position of the 5' and/or 3' boundaries relative to a particular reference locus is used to facilitate or enhance a particular application of gene editing, which depends in part on the endonuclease system selected for editing, as further described and exemplified herein.

In a first non-limiting aspect of such target sequence selection, many endonuclease systems have rules or criteria that guide the initial selection of a potential target site for cleavage, such as, in the case of CRISPR type II or type V endonucleases, the requirement for a PAM sequence motif at a specific position adjacent to the DNA cleavage site.

In another non-limiting aspect of target sequence selection or optimization, the frequency of "off-target" activity (i.e., the frequency of double-stranded breaks occurring at sites other than the selected target sequence) for a particular combination of target sequence and DNA endonuclease is evaluated relative to the frequency of on-target activity. In some cases, cells that are correctly edited at the desired locus may have a selective advantage compared to other cells. Examples of selective advantages include, but are not limited to, the acquisition of attributes such as enhanced replication rate, persistence, resistance to certain conditions, improved success of engraftment or persistence in vivo after introduction into a subject, and other attributes associated with the maintenance or increased number or viability of such cells. In other cases, cells that are correctly edited at the desired locus may be positively selected by one or more screening methods used to identify, sort, or otherwise select correctly edited cells. Both the selective advantage and the induced selection methods can take advantage of the phenotype associated with the modification. In some embodiments, cells may be edited more than once to create a second modification that creates a new phenotype that is used to select or purify the intended cell population. Such a second modification can be made by adding a second gRNA for a selection or screening marker. In some cases, a DNA fragment containing the cDNA and also the selection marker can be used to precisely edit the cell at the desired locus.

In embodiments, regardless of whether any selective advantage is applicable or any guided selection is applied in a particular case, target sequence selection is also guided by consideration of off-target frequency to enhance the efficacy of the application and/or to reduce the likelihood of undesired modifications at sites other than the desired target. As further described and exemplified herein and in the art, the occurrence of off-target activity is influenced by a number of factors, including the similarities and differences between the target and off-target sites, as well as the particular endonuclease used. Bioinformatics tools are available that aid in the prediction of off-target activity, and often such tools can also be used to identify the most likely sites of off-target activity, which can then be evaluated in an experimental setting to assess the relative frequency of off-target activity to on-target activity, thereby allowing the selection of sequences with higher relative on-target activity. Examples of such techniques are provided herein and others are known in the art.

Another aspect of target sequence selection concerns homologous recombination events. Sequences that share regions of homology can serve as foci for homologous recombination events that result in the deletion of intervening sequences. Such recombination events occur during the normal course of replication of chromosomes and other DNA sequences, and also when DNA sequences are being synthesized, as in the repair of double-strand breaks (DSBs). DSBs occur periodically during normal cell replication cycles, but can also be enhanced by the presence of factors such as UV light and other inducers of DNA breaks, or chemical inducers. Many of these inducers generate DSBs in the genome indiscriminately, and DSBs are regularly induced and repaired in normal cells. During repair, the original sequence can be reconstructed with perfect fidelity, but in some cases small indels are introduced at the DSB site.

DSBs can also be specifically induced at particular locations, as in the case of the endonuclease system described herein, and used to induce directed or preferential genetic modification events at selected chromosomal locations. The propensity of homologous sequences to undergo recombination in the context of DNA repair (and replication) can be exploited in many contexts and is the basis for one application of gene editing systems such as CRISPR, which use homology directed repair to insert a sequence of interest, provided through the use of a donor template, into a desired chromosomal location.

Regions of homology between specific sequences (which may be small regions of "microhomology" that may have as few as 10 base pairs or less) can also be used to effect the desired deletion. For example, a single DSB is introduced at a site that shows microhomology with a nearby sequence. In the normal process of repair of such DSBs, a frequent outcome is the deletion of the intervening sequence as a result of promoted recombination by the DSB and associated cellular repair processes.

However, in some situations, selecting a target sequence within the region of homology may result in much larger deletions, including gene fusions (if the deletion is in a coding region), which may or may not be desirable given the particular situation.

The examples provided herein further illustrate the selection of target regions for the creation of DSBs designed to insert FVIII coding sequences, and the selection of specific target sequences within such regions designed to minimize off-target events relative to on-target events.

Targeted Integration In some embodiments, the methods provided herein are directed to the integration of a synthetic FVIII coding sequence into a specific location in the genome of a hepatocyte, referred to as "targeted integration." In some embodiments, targeted integration is enabled by the generation of a double-stranded break in the genomic DNA using a sequence-specific nuclease.

The CRISPR/CAS system used in some embodiments has the advantage that a large number of genomic targets can be rapidly screened to identify the optimal CRISPR/CAS design. sgRNA molecules targeting any region of the genome can be designed in silico by placing a 20 bp sequence adjacent to every PAM motif. PAM motifs occur on average every 15 bp in eukaryotic genomes. However, sgRNAs designed by in silico methods generate double-stranded breaks in cells with different efficiencies, and it is not currently possible to predict the cleavage efficiency of a set of sgRNA molecules using in silico methods. Since sgRNAs can be rapidly synthesized in vitro, this allows for the rapid screening of all potential sgRNA sequences for a given genomic region to identify the sgRNA that results in the most efficient cleavage. Typically, a range of 0-90% cleavage efficiency is observed when a set of sgRNAs in a given genomic region is tested in cells. In silico algorithms and laboratory experiments can also be used to determine the off-target potential of any given sgRNA. Although perfect matches to the 20 bp recognition sequence of the sgRNA are primarily unique in most eukaryotic genomes, there will be numerous additional sites in the genome with one or more base pair mismatches with the sgRNA. These sites may be cleaved at variable frequencies that are often unpredictable based on the number or location of the mismatches. Cleavage at additional off-target sites not identified by in silico analysis may also occur. Thus, screening multiple sgRNAs in relevant cell types to identify sgRNAs with the most favorable off-target profile is an important component of selecting optimal sgRNAs for therapeutic applications. A favorable off-target profile takes into account not only the number of actual off-target sites and the frequency of cleavage at these sites, but also the location of these sites in the genome. For example, off-target sites near or within functionally important genes, particularly oncogenes or anti-cancer genes, are considered less favorable than sites in intergenic regions with no known function. Therefore, the identification of optimal sgRNAs cannot be predicted simply by in silico analysis of an organism's genomic sequence, but requires experimental testing. In silico analysis helps narrow down the number of guides to be tested, but it cannot predict guides with high on-target cleavage, nor can it predict guides with desirable low off-target cleavage. Experimental data shows that the cleavage efficiency of sgRNAs, each perfectly matching the genome of a region of interest (such as albumin intron 1), varies from no cleavage to more than 90% cleavage and cannot be predicted by known algorithms. The ability of a given sgRNA to promote cleavage by Cas enzymes can be linked to the accessibility of its specific site in genomic DNA, which can be determined by the chromatin structure of that region. The majority of genomic DNA in quiescent differentiated cells, such as hepatocytes, resides in highly condensed heterochromatin, while actively transcribed regions reside in a more open chromatin state that is known to be more accessible to large molecules such as proteins, such as Cas proteins. Even within an actively transcribed gene, some specific regions of DNA are more accessible than others, depending on the presence or absence of bound transcription factors or other regulatory proteins. Sites in the genome or within specific genomic loci or regions of genomic loci, such as introns and albumin intron 1, are not predictable and will therefore need to be determined experimentally in the relevant cell type. Once several sites have been selected as potential sites for insertion, it is possible to add several mutations to such sites, for example, by moving a few nucleotides upstream or downstream from the selected site, with or without experimental testing.

In some embodiments, a gRNA that may be used in the methods disclosed herein is one or more of SEQ ID NOs:271-298, or any functional equivalent thereof having at least about 85% nucleotide sequence identity to those of SEQ ID NOs:271-298.

Nucleic Acid Modifications In some embodiments, a polynucleotide introduced into a cell has one or more modifications that can be used individually or in combination, e.g., to enhance activity, stability or specificity, alter delivery, reduce innate immune responses in host cells, or for other enhancements, as further described herein and known in the art.

In certain embodiments, modified polynucleotides are used in a CRISPR/Cas9/Cpf1 system, where the guide RNA (either a single-molecule guide or a two-molecule guide) and/or DNA or RNA encoding the Cas or Cpf1 endonuclease introduced into the cell may be modified as described and exemplified below. Such modified polynucleotides may be used in a CRISPR/Cas9/Cpf1 system to edit any one or more genomic loci.

For non-limiting illustrative purposes of such uses, the CRISPR/Cas9/Cpf1 system may be used to enhance the formation or stability of a CRISPR/Cas9/Cpf1 genome editing complex with a gRNA, which may be a single molecule guide or a bimolecular, and a Cas or Cpf1 endonuclease, using modifications of the guide RNA. Modifications of the gRNA may also or alternatively be used to enhance the initiation, stability or kinetics of the interaction between the genome editing complex and the target sequence in the genome, which may be used, for example, to enhance on-target activity. Modifications of the guide RNA may also or alternatively be used to enhance specificity, e.g., the relative rate of genome editing at the on-target site compared to the effect at other (off-target) sites.

Modifications can also, or alternatively, be used to increase the stability of the guide RNA, for example, by increasing its resistance to degradation by ribonucleases (RNases) present in the cell, thereby increasing its half-life within the cell. Modifications that increase the half-life of the guide RNA can be particularly useful in embodiments in which a Cas or Cpf1 endonuclease is introduced into the cell to be edited via an RNA that needs to be translated to produce the endonuclease, since increasing the half-life of the guide RNA that is introduced simultaneously with an RNA encoding the endonuclease can be used to increase the time that the guide RNA and the encoded Cas or Cpf1 endonuclease coexist within the cell.

Modifications may also, or alternatively, be used to reduce the likelihood or extent to which RNA introduced into a cell will induce an innate immune response. Such responses, which have been well characterized in the context of RNA interference (RNAi) (including small interfering RNA (siRNA)), tend to be associated with reduced half-life of the RNA and/or induction of cytokines or other factors associated with the immune response, as described below and in the art.

The RNA encoding the endonuclease introduced into a cell can also be modified in one or more types, including, but not limited to, modifications that enhance the stability of the RNA (e.g., by increasing its degradation by RNAses present in the cell), modifications that enhance translation of the resulting product (i.e., the endonuclease), and/or modifications that reduce the likelihood or extent to which the RNA introduced into a cell will elicit an innate immune response.

Combinations of modifications, such as those mentioned above and others, can be used as well. In the case of CRISPR/Cas9/Cpf1, for example, one or more types of modifications can be made to the guide RNA (including those exemplified above) and/or one or more types of modifications can be made to the RNA encoding the Cas endonuclease (including those exemplified above).

As an example, guide RNAs used in the CRISPR/Cas9/Cpf1 system, or other smaller RNAs, can be easily synthesized by chemical means, allowing numerous modifications to be readily incorporated, as exemplified below and described in the art. Although chemical synthesis procedures are continually expanding, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as polynucleotide lengths increase significantly beyond 100 nucleotides or so. One approach used to generate chemically modified RNAs of longer length is to generate two or more molecules that are linked together. Much longer RNAs, such as RNAs encoding the Cas9 endonuclease, are more easily generated enzymatically. Although fewer types of modifications are generally available for use with enzymatically generated RNAs, there are still modifications that can be used, for example, to enhance stability, reduce the likelihood or extent of an innate immune response, and/or enhance other attributes, as described below and further in the art; new types of modifications are regularly developed.

As an example of the types of modifications, particularly those frequently used in smaller chemically synthesized RNAs, the modifications can have one or more nucleotides modified at the 2' position of the sugar, in some embodiments with 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro modified nucleotides. In some embodiments, the RNA modifications include 2'-fluoro, 2'-amino, or 2'-O-methyl modifications on the ribose of pyrimidines, abasic residues, or inverted bases at the 3' end of the RNA. Such modifications have been incorporated into oligonucleotides, and these oligonucleotides have been reported to have a higher _Tm (i.e., higher target binding affinity) for a given target than 2'-deoxyoligonucleotides.

A number of nucleotide and nucleoside modifications have been reported to render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than natural oligonucleotides; these modified oligonucleotides survive intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those with modified backbones, such as phosphorothioates, phosphotriesters, methyl phosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. Some oligonucleotides include oligonucleotides having phosphorothioate backbones, as well as heteroatom backbones, particularly CH ₂ -NH-O-CH ₂ , CH ₂ -N(CH ₃ )-O-CH ₂ (known as the methylene (methylimino) or MMI backbone), CH ₂ -O-N(CH ₃ )-CH ₂ , CH ₂ -N(CH ₃ )-N(CH ₃ )-CH ₂ and O-N(CH ₃ )-CH ₂ -CH2 backbones (where the natural phosphodiester backbone is represented as O-P-O-CH); amide backbones (A. De Mesmaeker et al., Ace Chem. Chem. 1999, 143:131-132, 1999). Res (1995) 28:366-374); oligonucleotides having morpholino backbone structures (see Summerton and Weller, US Pat. No. 5,034,506); and oligonucleotides having peptide nucleic acid (PNA) backbones (described below). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates with 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates with 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reverse polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; U.S. Pat. US Patent Nos. 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233 Nos. 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds have been described in D. A. Braasch et al., Biochem (2002) 41(14):4503-10; S. C. Ekker et al., Genesis (2001) 30(3):89-93 (and other articles in this issue); J. Heasman, Dev Biol (2002) 243:209-14; A. Nasevicius et al., Nat Genet (2000) 26:216-20; G. Lacerra et al. , Proc Natl Acad Sci USA (2000) 97:9591-96; and U.S. Patent No. 5,034,506.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in J. Wang et al., J Am Chem. Soc (2000) 122:8595-602.

Modified oligonucleotide backbones that do not contain a phosphorus atom have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and mixed N, O, S, and CH. Others have backbones with _two moieties; U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; U.S. Pat. See U.S. Patent Nos. 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

One or more substituted sugar moieties, e.g., one of the following at the 2' position: OH, SH, _SCH3 , _F , OCN, _OCH3OCH3 , _OCH3O ( _CH2 ) _{nCH3 ,} O( _CH2 ) _nNH2 , or O( _CH2 ) _nCH3 (where n is 1 to about 10); _C1 - _C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; _Br ; CN; _{CF3 ; OCF3 ;} O-, S- or N-alkyl; O-, S- or N-alkenyl; _SOCH3 ; _SO2CH3 ; _ONO2 ; _NO2 ; _N3 ; _NH2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving groups; reporter groups; intercalators; groups for improving the pharmacokinetic properties of an oligonucleotide; or groups for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties may also be included. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-CH ₂ -CH ₂ -OCH ₃ , also known as 2'-O-(2-methoxyethyl)) (P. Martin et al., Helv Chim Acta (1995) 78:486). Other modifications comprise 2'-methoxy (2'-O-CH ₃ ), 2'-propoxy (2'-OCH ₂ -CH ₂ CH ₃ ) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

In some embodiments, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound that is an oligonucleotide mimetic that has been reported to have excellent hybridization properties is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, e.g., an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings of PNA compounds can be found in P. E. Nielsen et al., Science (1991) 254:1497-500.

In some embodiments, a guide RNA can also additionally or alternatively include a nucleobase (often referred to in the art simply as a "base") modification or substitution. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are found only rarely or occasionally in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2'-deoxycytosine, often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other hetero-substituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine; see G. Gebeyehu et al. , Nucl Acids Res (1997) 15:4513. "Universal" bases known in the art (e.g., inosine) may also be included. 5-Me-C substitutions have been reported to increase nucleic acid duplex stability by 0.6-1.2°C. (Y.S. Sanghvi et al., "Antisense Research and Applications", CRC Press, Boca Raton, 1993, pp. 276-278) are examples of base substitutions.

In some embodiments, modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothiocymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil ), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and other synthetic and natural nucleobases such as 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, "The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, Englisch et al., Ange. Chemie, Int'l Ed, (1991) 30:613, and Y.S. Sanghvi, Chapter 15, "Antisense Research and "Applications", pp289-302, Crooke, S.T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, having 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been reported to increase nucleic acid duplex stability by 0.6-1.2° C. (Y.S. Sanghvi, supra, pp. 276-78) and are an even more preferred embodiment of base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,130,303; and 5,130,304. Nos. 3,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; No. 5,587,469; U.S. Pat. No. 5,596,091; U.S. Pat. No. 5,614,617; U.S. Pat. No. 5,681,941; U.S. Pat. No. 5,750,692; U.S. Pat. No. 5,763,588; U.S. Pat. No. 5,830,653; U.S. Pat. No. 6,005,096 and U.S. Patent Application Publication No. 2003/0158403.

In some embodiments, the guide RNA and/or mRNA (or DNA) encoding the endonuclease is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc Natl Acad Sci USA, (1989) 86:6553-56); choline acid (Manoharan et al., Bioorg Med Chem Let (1994) 4:1053-60); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann NY Acad Sci (1992) 660:306-09) and the thioethers (Manoharan et al., Ann NY Acad Sci (1992) 660:306-09) described in Manoharan et al. , Bioorg Med Chem Let, (1993) 3:2765-70); thiocholesterol (Oberhauser et al., Nucl Acids Res (1992) 20:533-538); aliphatic chains, such as dodecanediol or undecyl residues (Kabanov et al., F. EBS Lett (1990) 259:327-330 and Svinarchuk et al., Biochimie (1993) 75:49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Biochem. Chem. (1993) 25:49-54). al., Tetrahedron Lett (1995) 36:3651-54 and Shea et al., Nucl Acids Res (1990) 18:3777-83); polyamine or polyethylene glycol chains (Mancharan et al., Nucleosides & Nucleotides (1995) 14:969-73); adamantane acetic acid (Manoharan et al., Tetrahedron Lett (1995) 36:3651-54); palmityl moieties (Mishra et al., Biochim Biophys. Acta (1995) 1264:229-37); or octadecylamine or hexylamino-carbonyl-t-oxycholesterol moieties (Crooke et al., J Pharmacol Exp Ther (1996) 277:923-37). U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077 Specifications; U.S. Patent Nos. 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830 Nos. 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416, See also U.S. Pat. No. 203, U.S. Pat. No. 5,451,463; U.S. Pat. No. 5,510,475; U.S. Pat. No. 5,512,667; U.S. Pat. No. 5,514,785; U.S. Pat. No. 5,565,552; U.S. Pat. No. 5,567,810; U.S. Pat. No. 5,574,142; U.S. Pat. No. 5,585,481; U.S. Pat. No. 5,587,371; U.S. Pat. No. 5,595,726; U.S. Pat. No. 5,597,696; U.S. Pat. No. 5,599,923; U.S. Pat. No. 5,599,928, and U.S. Pat. No. 5,688,941.

In some embodiments, sugars and other moieties can be used to target proteins and complexes with nucleotides, such as cationic polysomes and liposomes, to specific sites. For example, hepatocyte-tropic metastasis can be mediated via the asialoglycoprotein receptor (ASGPR); see, e.g., Hu et al., Protein Pept Lett (2014) 21(10):1025-30. Other systems known in the art can be used to target the biomolecules used in the present invention, and/or complexes thereof, to specific target cells of interest.

In some embodiments, these targeting moieties or conjugates can include a conjugate group covalently attached to a functional group such as a primary or secondary hydroxyl group. Conjugate groups of the present disclosure include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of an oligomer, and groups that enhance the pharmacokinetic properties of an oligomer. Exemplary conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folates, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In the context of the present disclosure, groups that enhance pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or enhance sequence-specific hybridization with target nucleic acids. In the context of the present disclosure, groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism, or excretion of the compounds of the present disclosure. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed October 23, 1992, and U.S. Patent No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, choline acid, thioethers, such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol, or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties. See, for example, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,07 No. 7; U.S. Pat. No. 5,486,603; U.S. Pat. No. 5,512,439; U.S. Pat. No. 5,578,718; U.S. Pat. No. 5,608,046; U.S. Pat. No. 4,587,044; U.S. Pat. No. 4,605,735; U.S. Pat. No. 4,667,025; U.S. Pat. No. 4,762,779; U.S. Pat. No. 4,789,737; U.S. Pat. No. 4,824,941; U.S. Pat. No. 4,835,263; U.S. Pat. No. 4,876,335; U.S. Pat. No. 4,904,582; U.S. Pat. No. 4,958,013; U.S. Pat. No. 5,082,8 30; U.S. Pat. No. 5,112,963; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,082,830; U.S. Pat. No. 5,112,963; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,245,022; U.S. Pat. No. 5,254,469; U.S. Pat. No. 5,258,506; U.S. Pat. No. 5,262,536; U.S. Pat. No. 5,272,250; U.S. Pat. No. 5,292,873; U.S. Pat. No. 5,317,098; U.S. Pat. No. 5,371,241, U.S. Pat. No. 5,391,723; U.S. Pat. No. 5,416 ,203, U.S. Pat. Nos. 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941.

Longer polynucleotides that are not amenable to chemical synthesis and are generally produced by enzymatic synthesis can also be modified. Such modifications can include, for example, the introduction of specific nucleotide analogs, the incorporation of specific sequences or other moieties at the 5' or 3' ends of the molecule, and other modifications. By way of example, the mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription. Modifications to the mRNA can be applied, for example, to increase its translation or stability (e.g., by increasing its resistance to degradation within the cell) or to reduce the tendency of the RNA to elicit an innate immune response that is often observed in cells following the introduction of exogenous RNA, particularly longer RNAs such as RNA encoding Cas9.

Many such modifications have been described in the art, such as the use of polyA tails, 5' cap analogs (e.g., Anti Reverse Cap Analog (ARCA) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTRs), modified bases (such as pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP), or treatment with phosphatases to remove the 5' terminal phosphate. These and other modifications are known in the art, and new modifications of RNA are regularly developed.

It has been reported that improved therapeutic efficacy can be achieved using chemically modified mRNA delivered in vivo; see, e.g., Kormann et al., Nature Biotechnol (2011) 29:154-57. Such modifications can be used, for example, to increase the stability of the RNA molecule and/or to reduce its immunogenicity. It was found that using chemical modifications such as pseudo-U, N6-methyl-A, 2-thio-U and 5-methyl-C to replace only one-quarter of the uridine and cytidine residues with 2-thio-U and 5-methyl-C, respectively, significantly reduced toll-like receptor (TLR)-mediated recognition of mRNA in mice. By reducing activation of the innate immune system, these modifications can be used to effectively increase the in vivo stability and longevity of mRNA; see, e.g., Kormann et al., supra.

It has also been reported that repeated administration of synthetic messenger RNA incorporating modifications designed to bypass the natural antiviral response can reprogram differentiated human cells to pluripotency. See, e.g., Warren et al., Cell Stem Cell (2010) 7(5):618-30. Such modified mRNAs acting as primary reprogramming proteins can be an efficient means of reprogramming multiple human cell types. Such cells are termed induced pluripotent stem cells (iPSCs), and it has been found that enzymatically synthesized RNA incorporating 5-methyl-CTP, pseudo-UTP, and anti-inverted cap analog (ARCA) can be used to effectively circumvent the cellular antiviral response; see, e.g., Warren et al., supra.

Other modifications of polynucleotides described in the art include, for example, the use of a polyA tail, the addition of a 5' cap analog (e.g., m7G(5')ppp(5')G (mCAP)), modification of the 5' or 3' untranslated region (UTR), and treatment with a phosphatase to remove the 5' terminal phosphate.

Several compositions and techniques applicable to the generation of modified RNAs as used herein have been developed in the context of RNAi modifications, including siRNA. siRNAs present particular challenges in vivo, as their effects on gene silencing via mRNA interference are generally transient and may require repeated administration. In addition, siRNAs are double-stranded RNA (dsRNA), and mammalian immune responses have evolved to detect and neutralize dsRNA, which is often a by-product of viral infection. Thus, there are mammalian enzymes that can mediate cellular responses to dsRNA (e.g., PKR (dsRNA-responsive kinase), and potentially retinoic acid-inducible gene I (RIG-I)), as well as Toll-like receptors (e.g., TLR3, TLR7, and TLR8), which may trigger the induction of cytokines in response to such molecules; see, e.g., Angart et al. , Pharmaceuticals (Basel) (2013) 6(4): 440-68; Kanasty et al., Mol Ther (2012) 20(3): 513-24; Burnett et al., Biotechnol J (2011) 6(9): 1130-46; Judge and MacLachlan, Hum Gene Ther (2008) 19(2): 111-24; and references cited therein.

A wide variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and/or achieve other benefits that may be useful in connection with the introduction of polynucleotides into human cells, as described herein (see, e.g., K.A. Whitehead et al., Ann Rev Chem Biomol Eng (2011) 2:77-96; Gaglione and Messere, Mini Rev Med Chem (2010) 10(7):578-95; Chernolovskaya et al., Curr Opin Mol Ther (2010) 12(2):158-67; Deleavey et al., Curr Protoc Nuc Acid (2010) 12(2):158-67). See discussion by Chem, Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides (2008) 18(4):305-19; Fucini et al., Nucleic Acid Ther (2012) 22(3):205-210; Bremsen et al., Front Genet (2012) 3:154.

Many commercial suppliers of modified RNAs specialize in modifications designed to improve the efficacy of siRNAs, many of which are based on findings reported in the literature. Various approaches are offered based on findings reported in the literature. For example, Dharmacon notes that substitution of non-bridging oxygens with sulfur (phosphorothioate, PS) has been used to improve the nuclease resistance of siRNAs, as reported by Kole, Nature Rev Drug Disc (2012) 11:125-40. Modifications at the ribose 2' position have been reported to improve the nuclease resistance of the internucleotide phosphate linkages while increasing duplex stability (T _m ), which has also been reported to provide protection from immune activation. Combinations of moderate PS backbone modifications with small, well-tolerated 2'-substitutions (2'-O-methyl, 2'-fluoro, 2'-hydro) have been reported by Soutschek et al. , Nature (2004) 432:173-78, and 2'-O-methyl modifications have been reported to be effective in improving stability as reported by Volkov, Oligonucleotides (2009) 19:191-202. With respect to reducing the induction of innate immune responses, modifying specific sequences with 2'-O-methyl, 2'-fluoro, 2'-hydro has been reported to reduce TLR7/TLR8 interactions while generally preserving silencing activity; see, e.g., Judge et al., Mol Ther (2006) 13:494-505; and Cekaite et al., J Mol Biol (2007) 365:90-108. Additional modifications such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N ⁶ -methyladenosine have also been reported to minimize immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., K. Kariko et al., Immunity (2005) 23:165-75.

As known in the art and commercially available, numerous conjugates that can enhance delivery and/or uptake by cells can be applied to polynucleotides such as RNA for use herein, including, for example, cholesterol, tocopherol and folate, lipids, peptides, polymers, linkers, and aptamers; see, e.g., Winkler, Ther. Deliv. (2013) 4:791-809, and references cited therein.

Delivery In some embodiments, any nucleic acid molecule used in the methods provided herein, such as the genome-targeting nucleic acid of the present disclosure and/or the nucleic acid encoding the site-specific polypeptide, is packaged in or on the surface of a delivery vehicle for delivery to cells. Delivery vehicles include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. As described in the art, various targeting moieties can be used to enhance the preferential interaction of such vehicles with the desired cell type or location.

The complexes, polypeptides, and nucleic acids of the present disclosure can be introduced into cells by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

In embodiments, the guide RNA polynucleotide (RNA or DNA) and/or the endonuclease polynucleotide (RNA or DNA) are delivered by a viral or non-viral delivery vehicle known in the art. Alternatively, the site-specific polypeptide can be delivered by a viral or non-viral delivery vehicle known in the art, such as electroporation or lipid nanoparticles. In some embodiments, the DNA endonuclease is delivered as one or more polypeptides, either alone or pre-complexed with one or more gRNAs, or with one or more crRNAs and tracrRNAs.

In embodiments, the polynucleotides are delivered by non-viral delivery vehicles, including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Ther (2011) 18:1127-33, which focuses on non-viral delivery vehicles for siRNA that are also useful for delivery of other polynucleotides.

In embodiments, polynucleotides such as guide RNAs, sgRNAs, and mRNAs encoding endonucleases are delivered to cells or subjects by lipid nanoparticles (LNPs).

Although several non-viral delivery methods for nucleic acids have been tested in both animal models and humans, the most developed system is the lipid nanoparticle. LNPs are generally composed of an ionizable cationic lipid and three or more additional components, typically cholesterol, DOPE, and a polyethylene glycol (PEG) containing lipid (see, e.g., Example 1). The cationic lipids can bind to the positively charged nucleic acid to form a dense complex that protects the nucleic acid from degradation. During passage through a microfluidic system, the components self-assemble to form particles in the size range of 50-150 nM, in which the nucleic acid is encapsulated in the core, complexed with the cationic lipid, and surrounded by a lipid bilayer-like structure. After injection into the circulation of a subject, these particles can bind apolipoprotein E (apoE), which is a ligand for the LDL receptor and mediates uptake into hepatocytes in the liver via receptor-mediated endocytosis. This type of LNP has been reported to efficiently deliver mRNA and siRNA to hepatocytes in rodent, primate, and human livers. After endocytosis, the LNP resides in endosomes. The encapsulated nucleic acid undergoes a process of endosomal escape mediated by the ionizable nature of cationic lipids. This delivers the nucleic acid to the cytoplasm where the mRNA can be translated into the encoded protein. Thus, in some embodiments, encapsulation of gRNA and mRNA encoding Cas9 in LNPs is used to efficiently deliver both components to hepatocytes after intravenous injection. After endosomal escape, Cas9 mRNA is translated into Cas9 protein and forms a complex with gRNA. In some embodiments, inclusion of a nuclear localization signal in the Cas9 protein sequence promotes the translocation of Cas9 protein/gRNA complex to the nucleus. Alternatively, the small gRNA passes through the nuclear pore complex and forms a complex with Cas9 protein in the nucleus. Once inside the nucleus, the gRNA/Cas9 complex scans the genome for homologous target sites and preferentially generates double-stranded breaks at the desired target site in the genome. The half-life of RNA molecules in vivo is short, on the order of hours to days. Similarly, the half-life of proteins tends to be short, on the order of hours to days. Thus, in some embodiments, delivery of gRNA and Cas9 mRNA using LNPs may result in only transient expression and activity of the gRNA/Cas9 complex. This may provide the advantage of reducing the frequency of off-target cleavage, thus minimizing the risk of genotoxicity in some embodiments. LNPs are generally less immunogenic than viral particles. Many humans have pre-existing immunity to AAV, but not to LNPs. An additional and adaptive immune response to LNPs is unlikely to occur, allowing for repeated administration of LNPs.

When administering gene editing-based gene therapy to a subject, in which a therapeutic coding sequence is integrated into a host genomic locus, such as a safe harbor locus, it would be advantageous to achieve a level of gene expression that provides an optimal therapeutic benefit to the subject. For example, in hemophilia A, the most desirable level of FVIII protein in the blood would be in the range of 20%-100%, 30%-100%, 40%-100%, or 50%-100% of normal levels. Standard AAV-based gene therapy, which uses a strong promoter to drive expression of a therapeutic coding sequence from an episomal copy of the AAV genome, does not allow control of the expression level achieved, since the AAV virus can be administered only once and the expression level achieved varies significantly between subjects (S. Rangarajan et al., N Engl J Med (2017) 377:2519-30). After administering AAV virus to a subject, he or she will develop high titer antibodies against the viral capsid protein that are expected to prevent effective re-administration of the virus based on preclinical models (H. Petry et al., Gene Ther (2008) 15:54-60). Therapeutic genes delivered by AAV viruses are integrated into the genome at safe harbor loci such as albumin intron 1, and this targeted integration occurs via the generation of double-stranded breaks in the genome. One approach provides an opportunity to control the level of targeted integration and, therefore, the level of the therapeutic coding sequence product. After the liver is transduced by AAV encapsulating an AAV genome containing a donor DNA cassette encoding synthetic FVIII, the AAV genome is episomally maintained in the nuclei of the transduced cells. These episomal AAV genomes are relatively stable over time and therefore provide a pool of donor templates for targeted integration at the double-stranded breaks created by CRISPR/Cas9.

Several different ionizable cationic lipids have been developed for use in LNPs. These include C12-200 (K.T. Love et al., Proc Natl Acad Sci USA (2010) 107:1864-69), MC3 (M. Jayaraman et al., Angew Chem Int Ed Engl (2012) 51:8529-33), LN16, and MD1 (Fougerolles et al., U.S. Pat. No. 8,754,062), among others. C12-200 is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol). In one type of LNP, a GalNac moiety is attached to the outside of the LNP and acts as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs for delivery of gRNA and Cas9 mRNA to the liver.

In some embodiments, the LNPs have a diameter of less than about 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, the size of the nanoparticles can range from about 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids can include poor stability and rapid clearance, as well as low efficacy due to the generation of inflammatory or anti-inflammatory responses. LNPs can also have hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids known in the art can be used to generate LNPs. Examples of lipids used to generate LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

In embodiments, lipids can be combined in any number of molar ratios to produce LNPs. Additionally, polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

In embodiments, the site-specific polypeptide and the genome-targeting nucleic acid can each be administered separately to a cell or subject. The site-specific polypeptide can be pre-complexed with one or more guide RNAs, or one or more crRNAs and tracrRNAs. The pre-complexed material can then be administered to a cell or subject. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

RNA can form specific interactions with RNA and DNA. This property is exploited in many biological processes, but it also carries the risk of promiscuous interactions in the nucleic acid-rich cellular environment. One solution to this problem is to form ribonucleoprotein particles (RNPs) in which the RNA is precomplexed with endonucleases. Another advantage of RNPs is that the RNA is protected from degradation.

In some embodiments, the endonuclease in the RNP may be modified or unmodified. Similarly, the gRNA, crRNA, tracrRNA, or sgRNA may be modified or unmodified. Numerous modifications are known in the art and may be used.

The endonuclease and sgRNA are generally combined in a molar ratio of about 1:1. Alternatively, the endonuclease, crRNA, and tracrRNA can be combined in a molar ratio of about 1:1:1. However, a wide range of molar ratios can be used to generate RNPs.

In some embodiments, recombinant AAV vectors are used for delivery. Techniques for generating rAAV particles are standard in the art, in which a cell is provided with the AAV genome to be packaged, including the polynucleotide to be delivered, the rep and cap genes, and helper virus functions. The production of rAAV requires that the following components be present in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separate from the rAAV genome (i.e., not in the rAAV genome), and helper virus functions. The AAV rep and cap genes can be found in any of the AAV serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh. The rAAV genomic ITRs may be derived from any AAV serotype from which a recombinant virus can be derived, including AAV serotype 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, 99, 100, 101, 112, 123, 134, 140, 141, 152, 163, 170, 175, 186, 197, 198, 199, 102, 103,

In some embodiments, the method of producing a packaging cell includes generating a cell line that stably expresses components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) having a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of the cell. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (R. J. Samulski et al., Proc Natl Acad Sci USA (1982) 79:2077-81), addition of synthetic linkers containing restriction endonuclease cleavage sites (C. A. Laughlin et al., Gene (1983) 23:65-73), and direct blunt-end ligation (P. Senapathy et al., J Biol Chem (1984) 259:4661-66). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and it is amenable to large-scale production of rAAV. Other suitable methods use adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and/or rep and cap genes into packaging cells.

The general principles of rAAV production are reviewed, for example, in B. J. Carter, Cur Op Biotechnol (1992) 3(5):533-39; and N. Muzyczka, Curr Topics Microbiol Immunol (1992) 158:97-129. Several approaches are reviewed in J. D. Tratschin et al., Mol Cell Biol (1984) 4:2072-81; P. L. Hermonat et al., Proc Natl Acad Sci USA (1984) 81:6466-70; J. D. Tratschin et al. , Mol Cell Biol (1985) 5:3251-60; K. McLaughlin et al. , J Virol (1988) 62:1963-73; S. Lebkowski et al. , Mol Cell Biol (1988) 8:3988-96; J. Samulski et al. ,J Virol (1989) 63:3822-28); U.S. Pat. No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441. (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; P. Perrin et al., Vaccine (1995) 13:1244-50; R. W. Paul et al., Human Gene Ther (1993) 4:609-15; Clark et al. , Gene Ther (1996) 3:1124-32; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595.

The AAV vector serotype can be matched to the target cell type. For example, the following exemplary cell types may be transduced, among others, by the AAV serotypes indicated. For example, suitable AAV vector serotypes for liver tissue/cell types include, but are not limited to, AAV3, AAV5, AAV8, and AAV9.

In addition to adeno-associated viral vectors, other viral vectors can be used. Such viral vectors include, but are not limited to, lentiviruses, alphaviruses, enteroviruses, pestiviruses, baculoviruses, herpes viruses, Epstein-Barr viruses, papovaviruses, pox viruses, vaccinia viruses, and herpes simplex viruses.

In some embodiments, the Cas9 mRNA, the sgRNA targeting one or two loci of the albumin gene, and the donor DNA are each formulated separately into a lipid nanoparticle, or are all co-formulated into one lipid nanoparticle, or are co-formulated into two or more lipid nanoparticles.

In some embodiments, the Cas9 mRNA is formulated in a lipid nanoparticle, while the sgRNA and donor DNA are delivered in an AAV vector. In some embodiments, the Cas9 mRNA and sgRNA are co-formulated in a lipid nanoparticle, while the donor DNA is delivered in an AAV vector.

Options are available to deliver the Cas9 nuclease as a DNA plasmid, as an mRNA, or as a protein. Guide RNA can be expressed from the same DNA or can be delivered as RNA. The RNA can be chemically modified to alter or improve its half-life or to reduce the likelihood or extent of an immune response. The endonuclease protein can be complexed with the gRNA prior to delivery. Viral vectors allow efficient delivery: split versions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV, as can the donor for HDR. There is also a range of non-viral delivery methods that can deliver each of these components, or non-viral and viral methods can be used in tandem. For example, nanoparticles can be used to deliver protein and guide RNA, while AAV can be used to deliver donor DNA.

In some embodiments related to delivery of genome editing components for therapeutic treatment, at least two components, a sequence-specific nuclease and a DNA donor template, are delivered to the nucleus of a cell to be transformed (e.g., a hepatocyte). In some embodiments, the donor template is packaged in an AAV with tropism for the liver. In some embodiments, the AAV is selected from serotypes AAV8, AAV9, AAVrhlO, AAV5, AAV6, or AAV-DJ. In some embodiments, the AAV-packaged DNA donor template is administered to the subject, for example, first by peripheral vein injection, followed by the sequence-specific nuclease. The advantage of delivering the AAV-packaged donor template first is that the delivered donor template is stably maintained in the nucleus of the transduced hepatocyte, allowing for subsequent administration of the sequence-specific nuclease. This creates a double-strand break in the genome, followed by integration of the donor template by HDR or NHEJ. In some embodiments, it is desirable that the sequence-specific nuclease remains active in the target cell for the time required to promote the targeted integration of the transgene at a level sufficient for the desired therapeutic effect. If the sequence-specific nuclease remains active in the cell for a long period of time, this will result in an increased frequency of double-stranded breaks at off-target sites. Specifically, the frequency of off-target cleavage is a function of the off-target cleavage efficiency multiplied by the time the nuclease is active. By delivering the sequence-specific nuclease in the form of mRNA, the duration of nuclease activity is shortened, ranging from a few hours to a few days, since the mRNA and translated protein are short-lived in the cell. Therefore, it is expected that the delivery of sequence-specific nuclease to cells that already contain donor templates will result in a better ratio of targeted integration compared to off-target integration. Furthermore, AAV-mediated delivery of donor templates to the nuclei of hepatocytes following peripheral intravenous injection generally takes on the order of 1-14 days due to the time it takes for the virus to infect the cells, escape endosomes and translocate to the nucleus, and convert the single-stranded AAV genome into a double-stranded DNA molecule by host components. Thus, in some embodiments, delivery of the donor template to the nucleus is completed before providing the CRISPR/Cas9 components, since these nuclease components are active for approximately 1-3 days.

In some embodiments, the DNA endonuclease is CRISPR/Cas9, which is composed of an sgRNA directed to a DNA sequence within intron 1 of the albumin gene and a Cas9 nuclease. In some embodiments, the Cas9 endonuclease is delivered as an mRNA encoding the Cas9 protein operably fused to one or more nuclear localization signals (NLS). In some embodiments, the sgRNA and Cas9 mRNA are delivered to hepatocytes packaged in lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise the lipid C12-200 (K.T. Love et al., Proc Natl Acad Sci USA (2010) 107:1864-69). In some embodiments, the ratio of sgRNA to Cas9 mRNA packaged in the LNPs is 1:1 (by mass), thereby resulting in maximal DNA cleavage in vivo in mice. In alternative embodiments, different mass ratios of sgRNA to Cas9 mRNA packaged in the LNPs can be used, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1, or the inverse ratio. In some embodiments, the Cas9 mRNA and sgRNA are packaged in separate LNP formulations, and the LNPs containing the Cas9 mRNA are delivered to the subject about 1 to about 8 hours before the LNPs containing the sgRNA, allowing optimal time for the Cas9 mRNA to be translated prior to delivery of the sgRNA.

In some embodiments, an LNP formulation encapsulating gRNA and Cas9 mRNA ("LNP-nuclease formulation") is administered to a subject, e.g., a subject that has previously been administered an AAV-packaged DNA donor template. In some embodiments, the LNP-nuclease formulation is administered to the subject within 1 to 28 days, or within 7 to 28 days, or within 7 to 14 days after administration of the AAV donor template. The optimal timing of delivery of the LNP-nuclease formulation relative to the AAV donor template can be determined using techniques known in the art (e.g., studies conducted in animal models including mice and monkeys).

In some embodiments, the DNA donor template is delivered to the hepatocytes of a subject (e.g., a subject) using a non-viral delivery method. Although some subjects (typically 30%) have pre-existing neutralizing antibodies directed against the most commonly used AAV serotypes that prevent effective gene delivery by AAV, all subjects are treatable with non-viral delivery methods. Several non-viral delivery methodologies are known in the art. In particular, LNPs are known to efficiently deliver their encapsulated cargo into the cytoplasm of hepatocytes after intravenous injection in animals and humans. These LNPs are actively taken up into the liver via a process of receptor-mediated endocytosis, resulting in preferential uptake in the liver.

In some embodiments, to facilitate nuclear localization of the donor template, DNA sequences that can facilitate nuclear localization of the plasmid can be added to the donor template, such as the 366 bp region of the simian virus 40 (SV40) origin of replication and early promoter. Other DNA sequences that bind cellular proteins can also be used to improve nuclear entry of DNA.

In some embodiments, the level of expression or activity of the introduced FVIII is measured in the blood of the subject (e.g., the subject) after the first administration of an LNP-nuclease formulation containing, for example, gRNA and Cas9 nuclease or mRNA encoding Cas9 nuclease after the AAV donor template. If the FVIII level is not sufficient to treat the disease, for example, at a level that is 5% of normal levels, a second or third administration of the LNP-nuclease formulation can be administered to promote further targeted integration into the genomic safe harbor locus. The feasibility of using multiple doses of the LNP-nuclease formulation to obtain the desired therapeutic level of FVIII can be tested and optimized using techniques known in the art (e.g., tests using animal models including mice and monkeys).

In some embodiments according to any of the methods described herein, comprising i) administering an AAV donor template comprising a donor cassette, and ii) administering an LNP-nuclease formulation to a subject, an initial dose of the LNP-nuclease formulation is administered to the subject within about 1 day to about 28 days after administration of the AAV donor template to the subject. In some embodiments, the initial dose of the LNP-nuclease formulation is administered to the subject after a sufficient time to allow delivery of the donor template to the nucleus of a target cell. In some embodiments, the initial dose of the LNP-nuclease formulation is administered to the subject after a sufficient time to allow conversion of the single-stranded AAV genome in the nucleus of the target cell to a double-stranded DNA molecule. In some embodiments, one or more (e.g., 2, 3, 4, 5 or more) additional doses of the LNP-nuclease formulation are administered to the subject after administration of the initial dose. In some embodiments, one or more doses of the LNP-nuclease formulation are administered to the subject until a target level of targeted integration of the donor cassette and/or a target expression level of the donor cassette is achieved. In some embodiments, the method further comprises measuring a level of targeted integration of the donor cassette and/or a level of expression of the donor cassette after each administration of the LNP-nuclease formulation, and administering an additional dose of the LNP-nuclease formulation if the target level of targeted integration of the donor cassette and/or the target expression level of the donor cassette is not achieved. In some embodiments, the amount of at least one of the additional doses of the LNP-nuclease formulation is the same as the first dose. In some embodiments, the amount of at least one of the additional doses of the LNP-nuclease formulation is less than the first dose. In some embodiments, the amount of at least one of the additional doses of the LNP-nuclease formulation is greater than the first dose.

Genetically modified cells and cell populations In one aspect, the present disclosure provides a method for editing the genome in a cell, thereby creating a genetically modified cell. In some aspects, a population of genetically modified cells is provided. Thus, a "genetically modified cell" refers to a cell that has at least one genetic modification introduced by genome editing (e.g., using the CRISPR/Cas9/Cpf1 system). In some embodiments, the genetically modified cell is a genetically modified hepatocyte. Genetically modified cells that have an exogenous genome-targeting nucleic acid and/or an exogenous nucleic acid encoding a genome-targeting nucleic acid are contemplated herein.

In some embodiments, the genome of a cell may be edited by inserting a nucleic acid sequence of a synthetic FVIII coding sequence into the genome sequence of the cell. In some embodiments, the cell subjected to genome editing has one or more mutations in the genome that result in reduced expression of the endogenous FVIII gene compared to expression in a normal without such mutation. The normal cell may be a healthy or control cell originating (or isolated) from a different subject that does not have a FVIII gene defect. In some embodiments, the cell subjected to genome editing may originate (or be isolated) from a subject in need of treatment for a FVIII gene-associated condition or disorder. Thus, in some embodiments, the expression of the endogenous FVIII gene in such a cell is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the endogenous FVIII gene expression in a normal cell.

Upon successful insertion of a transgene (e.g., a nucleic acid encoding a synthetic FVIII coding sequence), expression of the introduced synthetic FVIII coding sequence in a cell can be at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1,000%, about 2,000%, about 3,000%, about 5,000%, about 10,000% or more compared to expression of the cell's endogenous FVIII gene. In some embodiments, the activity of the introduced FVIII coding sequence product, including a synthetic FVIII coding sequence, in a genome edited cell can be at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1,000%, about 2,000%, about 3,000%, about 5,000%, about 10,000% or more compared to the expression of the endogenous FVIII gene in the cell. In some embodiments, expression of the introduced synthetic FVIII coding sequence in the cell is at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold, about 1000-fold or more than the expression of the endogenous FVIII gene in the cell. Also, in some embodiments, the activity of the introduced synthetic FVIII coding sequence in the genome edited cell can be comparable to or greater than the activity of the FVIII gene product in normal healthy cells.

In embodiments for treating or ameliorating hemophilia A, the primary target of gene editing is human cells. In some embodiments of ex vivo and in vivo methods, the human cells are hepatocytes. In some embodiments, by performing gene editing in autologous cells derived from a subject in need (and thus already fully matched to the subject), it is possible to generate cells that can be safely reintroduced into the subject and effectively give rise to a cell population that would be effective in ameliorating one or more clinical conditions associated with the subject's disease. In some embodiments for such treatment, hepatocytes are isolated according to any method known in the art and used to generate genetically modified, therapeutically effective cells. In one embodiment, liver stem cells are genetically modified ex vivo and then reintroduced into the subject where they give rise to genetically modified hepatocytes or sinusoidal endothelial cells that express the inserted FVIII coding sequence.

Treatment Approaches Hemophilia is classified as "mild" (FVIII protein serum concentration 0.40-0.05 IU/mL), "moderate" (0.05-0.01 IU/mL), or "severe"(<0.01 IU/mL, <1% of normal) (G.C. White et al., Thromb Haemost (2001) 85(3):560-75). An analysis of hemophilia A patients receiving FVIII replacement protein therapy reported bleeding-free frequencies of 71%, 79%, 91%, 97%, and 100% for predicted FVIII trough levels of 3%, 5%, 10%, 15%, and 20% of normal, respectively (G. Spotts et al., Blood (2014) 124:689). This suggests that if FVIII levels are maintained above 15-20% of nadir, the rate of bleeding events will drop to near zero. Although the exact VIII level required to cure hemophilia A has not been determined and will likely vary between subjects, levels of about 5% to about 30% are expected to result in a significant reduction in bleeding events.

In one aspect, provided herein is a gene therapy approach to treat hemophilia A in a subject by editing the subject's genome. In some embodiments, the gene therapy approach integrates a functional synthetic FVIII coding sequence into the genome of relevant cell types in the subject, providing a permanent cure for hemophilia A. In some embodiments, the synthetic FVIII coding sequence is integrated into hepatocytes for efficient expression and secretion of many proteins into the blood. Furthermore, this integration approach with hepatocytes can be considered for pediatric subjects whose livers are not fully developed, since the integrated coding sequence is transmitted to daughter cells as the hepatocytes divide.

In another aspect, provided herein are cellular ex vivo and in vivo methods for using genome engineering tools to create permanent changes in the genome by knocking in a synthetic FVIII coding sequence into a locus and restoring FVIII protein activity. Such methods use endonucleases, such as CRISPR-associated (CRISPR/Cas9, Cpf1, etc.) nucleases, to permanently delete, insert, edit, correct, or replace any sequence from the genome or to insert an exogenous sequence (e.g., a synthetic FVIII coding sequence) into a genomic locus. In this manner, the examples described in this disclosure restore the activity of the FVIII gene with a single treatment (rather than delivering a potential treatment for the subject's lifetime).

In some embodiments, ex vivo cell-based therapy uses liver cells isolated from a subject. The chromosomal DNA of these cells is edited using the materials and methods described herein. Finally, the edited cells and/or their progeny are administered or implanted into the subject.

One advantage of the ex vivo cell therapy approach is the ability to perform a comprehensive analysis of the therapy prior to administration. Nuclease-based therapeutics have some level of off-target effects. By performing gene correction ex vivo, the corrected cell population can be characterized prior to administration. Aspects of the present disclosure include determining the genomic sequence of the corrected cells to ensure that any off-target cleavage is at a genomic location associated with minimal risk to the subject. Additionally, specific populations of cells, including clonal populations, may be screened or isolated prior to administration or implantation.

Another embodiment is an in vivo based treatment. In this method, the chromosomal DNA of the subject's cells is modified using the materials and methods described herein. In some embodiments, the cells are hepatocytes.

The advantage of in vivo gene therapy is the ease of therapeutic production and administration. The same therapeutic approach and treatment can be used to treat more than one subject, for example multiple subjects who share the same or similar genotypes or alleles. In contrast, ex vivo cell therapy generally uses a subject's own cells that are isolated, manipulated, and then returned to the same subject.

In some embodiments, the subject has symptoms of hemophilia A. In some embodiments, the subject is a human suspected of having hemophilia A. Alternatively, the subject is a human diagnosed as being at risk for hemophilia A. In some embodiments, the subject in need of treatment has one or more genetic defects (e.g., deletions, insertions, and/or mutations) in the endogenous FVIII gene or its regulatory sequences such that the activity (including expression levels or functionality) of the FVIII protein is substantially reduced compared to normal healthy subjects.

In some embodiments, provided herein is a method of treating hemophilia A in a subject, the method comprising providing to a cell in the subject: (a) a gRNA that targets an albumin locus in the cellular genome; (b) a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; and (c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein. In some embodiments, the gRNA targets intron 1 of the albumin gene. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 271-298.

In some embodiments, provided herein is a method of treating hemophilia A in a subject, the method comprising providing a cell in the subject with: (a) a gRNA comprising a spacer sequence from any one of SEQ ID NOs: 271-298; (b) a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; and (c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 274, 275, 281, and 283. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 274. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 275. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 281. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 283. In some embodiments, the cell is a human cell, e.g., a human hepatocyte. In some embodiments, the subject has or is suspected of having hemophilia A. In some embodiments, the subject is diagnosed as being at risk for hemophilia A.

In some embodiments according to any of the methods of treating hemophilia A described herein, the DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csx1, Csx2, Csx1 ... sn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpf1 endonucleases, and functional equivalents thereof. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the Cas9 is spCas9. In some embodiments, the Cas9 is SluCas9.

In some embodiments according to any of the methods of treating hemophilia A described herein, the nucleic acid sequence encoding the synthetic FVIII coding sequence is codon-optimized for expression in a cell. In some embodiments, the cell is a human cell.

In some embodiments according to any of the methods of treating hemophilia A described herein, the method employs a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in the cell. In some embodiments, the cell is a human cell, e.g., a human hepatocyte. In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA, such as a DNA plasmid. In some embodiments, the nucleic acid encoding the DNA endonuclease is RNA, such as an mRNA.

In some embodiments according to any of the methods of treating hemophilia A described herein, the donor template is encoded by an AAV vector. In some embodiments, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding a synthetic FVIII protein, flanked on one or both sides by gRNA target sites. In some embodiments, the donor cassette is flanked on both sides by gRNA target sites. In some embodiments, the gRNA target site is a target site of the administered gRNA. In some embodiments, the gRNA target site of the donor template is the reverse complement of a cellular genomic gRNA target site of the administered gRNA. In some embodiments, providing the donor template to the cell comprises administering the donor template to the subject. In some embodiments, the administration is intravenous.

In some embodiments according to any of the methods of treating hemophilia A described herein, the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is formulated in a liposome or LNP. In some embodiments, the liposome or LNP also comprises a gRNA. In some embodiments, providing the cell with the gRNA and the DNA endonuclease or a nucleic acid encoding the DNA endonuclease comprises administering the liposome or LNP to the subject. In some embodiments, the administration is intravenous. In some embodiments, the liposome or LNP is an LNP. In some embodiments, the method uses an LNP comprising a nucleic acid encoding the DNA endonuclease and a gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is an mRNA encoding the DNA endonuclease.

In some embodiments according to any of the methods of treating hemophilia A described herein, the DNA endonuclease is pre-complexed with the gRNA to form an RNP complex.

The process by which AAV infects cells, including those of the liver, involves escape from endosomes, viral uncoating, and transport of the AAV genome to the nucleus. In the case of the AAV used in these studies, where a single-stranded genome is packaged into the virus, the single-stranded genome undergoes a process of second-strand DNA synthesis to form a double-stranded DNA genome. The time required for complete conversion of the single-stranded genome to a double-stranded genome is not well established, but is thought to be the rate-limiting step (Ferrari et al., J Virol (1996) 70:3227-34). The double-stranded linear genome is then concatemerized into a multimeric circular form composed of monomers linked head-to-tail and tail-to-head (Sun et al., Human Gene Ther. (2010) 21:750-62).

In some embodiments according to any of the methods of treating hemophilia A described herein, the administered gRNA and the administered DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell after the donor template is provided to the cell. In some embodiments, the administered gRNA and the administered DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell more than 4 days after the donor template is provided to the cell. In some embodiments, the gRNA and the DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell at least 14 days after the donor template is provided to the cell. In some embodiments, the gRNA and the DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell at least 17 days after the donor template is provided to the cell. In some embodiments, providing the gRNA and the DNA endonuclease to the cell comprises administering (e.g., by an intravenous route) an LNP comprising a nucleic acid encoding a DNA endonuclease and a gRNA to the subject. In some embodiments, the nucleic acid encoding the DNA endonuclease is an mRNA encoding a DNA endonuclease. In some embodiments, providing the donor template to the cell includes administering to the subject (e.g., by an intravenous route) a donor template encoded by an AAV vector.

In some embodiments according to any of the methods of treating hemophilia A described herein, one or more additional doses of gRNA and DNA endonuclease or nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA and DNA endonuclease or nucleic acid encoding a DNA endonuclease. In some embodiments, one or more additional doses of gRNA and DNA endonuclease or nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA and DNA endonuclease or nucleic acid encoding a DNA endonuclease until a target level of targeted integration of a nucleic acid sequence encoding a synthetic FVIII protein and/or a target expression level of a nucleic acid sequence encoding a synthetic FVIII protein is achieved. In some embodiments, providing the cell with gRNA and DNA endonuclease comprises administering (e.g., by an intravenous route) lipid nanoparticles comprising a nucleic acid encoding a DNA endonuclease and a gRNA to the subject. In some embodiments, the nucleic acid encoding a DNA endonuclease is an mRNA encoding a DNA endonuclease.

In some embodiments according to any of the methods of treating hemophilia A described herein, the nucleic acid sequence encoding the synthetic FVIII protein is expressed under the control of an endogenous albumin promoter. In some embodiments, the nucleic acid sequence encoding the synthetic FVIII protein is expressed under the control of an endogenous transferrin promoter. In some embodiments, the nucleic acid sequence encoding the synthetic FVIII protein is expressed under the control of an endogenous fibrinogen-α chain promoter.

In some embodiments according to any of the methods of treating hemophilia A described herein, the nucleic acid sequence encoding the synthetic FVIII protein is expressed in the liver of the subject.

Delivery of cells to a subject In some embodiments, the ex vivo method of the present disclosure includes administering genome-edited cells to a subject in need thereof. This can be accomplished using any method of parenteral administration known in the art. For example, the genetically modified cells can be directly injected into the subject's blood, directly injected (implanted) into or near the liver, or otherwise administered to the subject.

In some embodiments, the methods disclosed herein involve implanting or "transplanting" genetically modified therapeutic cells into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect is produced. The therapeutic cells or their differentiated progeny can be introduced by any suitable route that results in delivery to a desired location in a subject, where at least a portion of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., 24 hours to days, as long as several years, or for the life of the subject, i.e., long-term engraftment.

When provided prophylactically, the therapeutic cells described herein are administered to a subject prior to any symptoms of hemophilia A. Thus, in some embodiments, prophylactic administration of a population of genetically modified hepatic cells serves to prevent the onset of hemophilia A symptoms.

In some embodiments, when provided therapeutically, the genetically modified hepatocytes are provided at (or after) the onset of symptoms or signs of hemophilia A, e.g., at the onset of the disease.

In some embodiments, the therapeutic hepatocyte population administered according to the methods described herein comprises allogeneic hepatocytes obtained from one or more donors. "Allogeneic" refers to hepatocytes or biological samples having hepatocytes obtained from one or more different donors of the same species, where the genes at one or more genetic loci are not identical. For example, the hepatocyte population administered to the subject may be derived from one or more unrelated donor subjects, or from one or more non-identical siblings. In some embodiments, isogenic hepatocyte populations can be used, such as those obtained from genetically identical animals or from identical twins. In other embodiments, the hepatocytes are autologous; i.e., the hepatocytes are obtained or isolated from a subject and administered to the same subject, i.e., the donor and recipient are the same.

In one embodiment, an effective amount refers to the amount of a therapeutic cell population necessary to prevent or reduce at least one or more signs or symptoms of hemophilia A, and relates to a sufficient amount of a composition to have a desired effect, e.g., to treat a subject with hemophilia A. Thus, in an embodiment, a therapeutically effective amount refers to an amount of a therapeutic cell or a composition having therapeutic cells sufficient to promote a particular effect when administered to a subject, such as a subject with or at risk for hemophilia A. An effective amount also includes an amount sufficient to prevent or delay the onset of a disease symptom, alter the course of a disease symptom (e.g., but not limited to, delay the progression of a disease symptom), or reverse a disease symptom. It is understood that for any given case, the appropriate effective amount can be determined by one of skill in the art.

For use in the embodiments described herein, an effective amount of therapeutic cells, e.g., genome edited hepatocytes, is at least about ¹⁰² cells, at least about 5x102 cells, at least about ¹⁰³ cells, at least about ^5x103 cells, at least about ¹⁰⁴ cells, at least about ^5x104 cells, at least about ¹⁰⁵ cells, at least about ^2x105 cells, at least about ^3x105 cells, at least about ^4x105 cells, at least about ^5x105 cells, at least about ^6x105 cells, at least about 7x105 cells, at least about ^8x105 cells, at least about ^9x105 cells, at least about ^1x106 cells, at least about ^2x106 cells, at least about ^3x106 cells, at least about ^4x106 cells, at least about ^5x106 cells ^, at least about ^6x106 cells, at least about ^7x106 cells, at least ^{about 8x106} cells, The ^therapeutic cells may be at least about 9×10 ⁶ cells, at least about 9×10 6 cells, or multiples thereof. The therapeutic cells may be derived from one or more donors or may be obtained from an autologous source. In some embodiments described herein, the therapeutic cells are expanded in culture prior to administration to a subject in need thereof.

In some embodiments, moderate and incremental increases in the level of functional FVIII expressed in cells of a subject with hemophilia A are beneficial for improving one or more symptoms of the disease, increasing long-term survival, and/or reducing side effects associated with other treatments. When such cells are administered to a human subject, the presence of therapeutic cells that produce increased levels of functional FVIII is beneficial. In some embodiments, effective treatment of a subject results in at least about 1%, 3%, 5%, or 7% functional FVIII relative to the total FVIII in the treated subject. In some embodiments, the functional FVIII is at least about 10% of the total FVIII. In some embodiments, the functional FVIII is at least, about, or at most 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the total FVIII. Similarly, even introducing a relatively limited subpopulation of cells with significantly elevated levels of functional FVIII is beneficial in a subject, as in some circumstances normalized cells have a selective advantage over diseased cells. However, even moderate levels of therapeutic cells with elevated levels of functional FVIII are beneficial for improving one or more aspects of hemophilia A in a subject. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the therapeutic agent in the subject to which the cells are administered produce increased levels of functional FVIII.

In embodiments, delivery of a therapeutic cell composition to a subject by a method or route results in at least partial localization of the cell composition at a desired site. The cell composition can be administered by any suitable route that results in an effective treatment in the subject, i.e., administration results in delivery to a desired location in the subject, where at least a portion of the composition delivered, i.e., at least about 1×10 ⁴ cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, infusion, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For delivery of cells, administration can be by injection or infusion.

In one embodiment, the cells are administered systemically, in other words, a population of therapeutic cells is administered not directly to a target site, tissue, or organ, so that it instead enters the subject's circulatory system and is therefore subject to metabolic and other similar processes.

The efficacy of a treatment with a composition for the treatment of hemophilia A can be determined by a skilled clinician. However, a treatment is considered effective if any one or more of the signs or symptoms of functional FVIII, such as the level of functional FVIII, is altered in a beneficial manner (e.g., increased by at least 10%) or other clinically acceptable symptoms or markers of the disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to deteriorate, as assessed by the need for hospitalization or medical intervention (e.g., progression of the disease is stopped or at least slowed). Methods for measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or mammals), including: (1) inhibiting the disease (e.g., stopping or slowing the progression of a symptom); or (2) relieving the disease (e.g., causing regression of a symptom); and (3) preventing or reducing the likelihood of onset of a symptom.

Compositions In one aspect, the present disclosure provides compositions for carrying out the methods disclosed herein. The compositions may include one or more of the following: a genome-targeting nucleic acid (e.g., gRNA); a site-directed polypeptide (e.g., DNA endonuclease) or a nucleotide sequence encoding a site-directed polypeptide; and a polynucleotide (e.g., a donor template) that is inserted to effect the desired genetic modification of the methods disclosed herein.

In some embodiments, the composition has a nucleotide sequence encoding a genome-targeting nucleic acid (e.g., a gRNA).

In some embodiments, the composition comprises a site-specific polypeptide (e.g., a DNA endonuclease). In some embodiments, the composition comprises a nucleotide sequence encoding the site-specific polypeptide.

In some embodiments, the composition comprises a polynucleotide (e.g., a donor template) to be inserted into a genome.

In some embodiments, the composition has (i) a nucleotide sequence encoding a genome-targeting nucleic acid (e.g., a gRNA), and (ii) a site-directed polypeptide (e.g., a DNA endonuclease) or a nucleotide sequence encoding a site-directed polypeptide.

In some embodiments, the composition comprises (i) a nucleotide sequence encoding a genome-targeting nucleic acid (e.g., a gRNA) and (ii) a polynucleotide to be inserted into the genome (e.g., a donor template).

In some embodiments, the composition comprises (i) a site-directed polypeptide (e.g., a DNA endonuclease) or a nucleotide sequence encoding the site-directed polypeptide, and (ii) a polynucleotide to be inserted into a genome (e.g., a donor template).

In some embodiments, the composition has (i) a nucleotide sequence encoding a genome-targeting nucleic acid, (ii) a site-directed polypeptide or a nucleotide sequence encoding a site-directed polypeptide, and (iii) a polynucleotide to be inserted into the genome (e.g., a donor template).

In some embodiments of any of the above compositions, the composition comprises a single molecule guide genome-targeting nucleic acid. In some embodiments of any of the above compositions, the composition comprises a two molecule genome-targeting nucleic acid. In some embodiments of any of the above compositions, the composition comprises two or more two molecule guides or a single molecule guide. In some embodiments, the composition comprises a vector encoding a nucleic acid-targeting nucleic acid. In some embodiments, the genome-targeting nucleic acid is a DNA endonuclease, particularly Cas9.

In some embodiments, the composition comprises one or more gRNAs suitable for genome editing, in particular for the insertion of a synthetic FVIII coding sequence into the genome of a cell. The gRNA for the composition can target a genomic site at, in, or near an endogenous albumin gene. In some embodiments, the gRNA has a spacer sequence that is complementary to a genomic sequence at, in, or near the albumin gene.

In some embodiments, the gRNA of the composition is a sequence selected from any of SEQ ID NOs:271-298 and variants thereof, the variant having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% identity or homology to any of SEQ ID NOs:271-298. In some embodiments, the gRNA variant has at least about 85% homology to any of SEQ ID NOs:271-298.

In some embodiments, the gRNA of the composition has a spacer sequence that is complementary to a target site in the genome. In some embodiments, the spacer sequence is 15-20 bases in length. In some embodiments, the complementarity between the spacer sequence and the genome sequence is at least about 80%, at least about 85%, 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%.

In some embodiments, the composition comprises a donor template having a DNA endonuclease or a nucleic acid encoding a DNA endonuclease, and/or a nucleic acid sequence of a synthetic FVIII coding sequence. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA or RNA.

In some embodiments, one or more of any of the oligonucleotide or nucleic acid sequences are encoded by an AAV vector. Thus, in some embodiments, the gRNA is encoded by an AAV vector. In some embodiments, the nucleic acid encoding the DNA endonuclease is encoded by an AAV vector. In some embodiments, the donor template is encoded by an AAV vector. In some embodiments, two or more oligonucleotide or nucleic acid sequences are encoded by a single AAV vector. Thus, in some embodiments, the gRNA sequence and the nucleic acid encoding the DNA endonuclease are encoded by a single AAV vector.

In some embodiments, the composition comprises liposomes or lipid nanoparticles. Thus, in some embodiments, any of the compounds of the composition (e.g., the DNA endonuclease or the nucleic acid encoding it, the gRNA, and the donor template) may be formulated in liposomes or LNPs. In some embodiments, one or more such compounds are associated with the liposome or LNP via covalent or non-covalent bonds. In some embodiments, any of the compounds are included separately or together in the liposome or LNP. Thus, in some embodiments, each of the DNA endonuclease or the nucleic acid encoding it, the gRNA, and the donor template are separately formulated in the liposome or LNP. In some embodiments, the DNA endonuclease is formulated in the liposome or LNP together with the gRNA. In some embodiments, the DNA endonuclease or the nucleic acid encoding it, the gRNA, and the donor template are formulated together in the liposome or LNP.

In some embodiments, the composition further comprises one or more additional reagents, such additional reagents being selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, wash buffers, control reagents, control vectors, control RNA polynucleotides, reagents for in vitro production of polypeptides from DNA, adapters for sequencing, and the like. The buffers can be stabilization buffers, reconstitution buffers, dilution buffers, and the like. In some embodiments, the composition also comprises one or more components that can be used to facilitate or enhance on-target binding or cleavage of DNA by the endonuclease or to improve the specificity of targeting.

In some embodiments, any component of the composition is formulated with a pharma- ceutically acceptable excipient, such as a carrier, solvent, stabilizer, adjuvant, diluent, etc., depending on the particular mode of administration and dosage form. In embodiments, the guide RNA composition is generally formulated to achieve a physiologically compatible pH, and a pH of about 3 to about 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH is adjusted to a range of about pH 5.0 to about pH 8.0. In some embodiments, the composition has a therapeutically effective amount of at least one compound described herein, together with one or more pharma- ceutically acceptable excipients. Optionally, the composition can have a combination of compounds described herein, can include a second active ingredient useful for treating or preventing bacterial growth (e.g., but not limited to, an antibacterial or antimicrobial agent), and can include a combination of reagents of the present disclosure. In some embodiments, the gRNA is formulated with one or more other oligonucleotides, such as a nucleic acid encoding a DNA endonuclease, and/or a donor template. Alternatively, the nucleic acid encoding the DNA endonuclease and the donor template are formulated separately or in combination with other oligonucleotides as described above for gRNA formulations.

Suitable excipients include carrier molecules, including large, slowly metabolized macromolecules, such as, for example, proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants (e.g., but not limited to, ascorbic acid), chelating agents (e.g., but not limited to, EDTA), carbohydrates (e.g., but not limited to, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (e.g., but not limited to, oils, water, saline, glycerol, and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.

In some embodiments, any of the components of the composition (e.g., the DNA endonuclease or a nucleic acid encoding it, the gRNA, and the donor template) are delivered via transfection, such as electroporation. In some exemplary embodiments, the DNA endonuclease is pre-complexed with the gRNA to form an RNP complex prior to delivery to the cell, and the RNP complex is electroporated. In such embodiments, the donor template may be delivered via electroporation.

In some embodiments, "composition" refers to a therapeutic composition having therapeutic cells used in an ex vivo treatment method.

In embodiments, the therapeutic composition contains a physiologically tolerable carrier together with the cell composition, and optionally at least one additional bioactive agent, as described herein, dissolved or dispersed therein as an active ingredient. In some embodiments, the therapeutic composition is substantially non-immunogenic when administered to a mammalian or human subject for therapeutic purposes.

Generally, the genetically modified therapeutic cells described herein are administered as a suspension with a pharma- ceutically acceptable carrier. One of skill in the art will recognize that the pharma- ceutically acceptable carrier used in the cell composition does not include buffers, compounds, cryopreservatives, preservatives, or other agents in amounts that substantially interfere with the viability of the cells delivered to the subject. The formulation with the cells can include, for example, an osmotic buffer that allows the integrity of the cell membrane to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and/or may be adapted for use with the cells as described herein.

In some embodiments, the cell composition may also be emulsified or presented as a liposomal composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients may be mixed with excipients that are pharma- ceutically acceptable and compatible with the active ingredients, in amounts suitable for use in the therapeutic methods described herein.

Additional agents included in the cell composition can include pharma- ceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredient and water, or that contain a buffer such as sodium phosphate at a physiological pH value, physiological saline, or both, e.g., phosphate buffered saline. Additionally, aqueous carriers can contain two or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in the cell composition that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

Kits Some embodiments provide kits comprising any of the compositions described above, e.g., a composition for genome editing or a therapeutic cell composition, and one or more additional components.

In some embodiments, the kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the composition for a desired purpose, e.g., genome editing or cell therapy.

In some embodiments, the kit may further include instructions for using the components of the kit to practice the method. The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit as a package insert, on the labeling of the kit's container or a component thereof (i.e., associated with the packaging or subpackaging), etc. The instructions may be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., a CD-ROM, a diskette, a flash drive, etc. In some cases, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) is provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, methods for obtaining the instructions may be recorded on a suitable substrate.

Further Therapeutic Approaches Gene editing can be performed using site-specific polypeptides engineered to target specific sequences. To date, there are four main types of such nucleases: meganucleases and their functional equivalents, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/CAS nuclease systems. Nuclease platforms vary in design difficulty, targeting density, and mode of action, especially since the specificity of ZFNs and TALENs is due to protein-DNA interactions, while RNA-DNA interactions mainly guide the Cas protein. Cas9 cleavage also requires a flanking motif, PAM, which differs between different CRISPR systems. Cas9 from Streptococcus pyogenes cleaves using the NRG PAM, while CRISPR from Neisseria meningitidis can cleave at sites with PAMs including NNNNGATT (SEQ ID NO: 312), NNNNNGTTTT (SEQ ID NO: 313), and NNNGCTT (SEQ ID NO: 314). Many other Cas9 orthologs target protospacers flanked by alternative PAMs.

CRISPR endonucleases such as Cas9 can be used in embodiments of the disclosed methods. Additionally, the teachings described herein, such as therapeutic targeting sites, can be applied to other forms of endonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or combinations of nucleases can be used. However, to apply the teachings of the disclosure to such endonucleases, it would be necessary, among other things, to engineer proteins directed to specific target sites.

To increase specificity, additional binding domains can be fused to the Cas9 protein. The target sites of these constructs map to the identified gRNA specific sites, but require additional binding motifs such as zinc finger domains. In the case of Mega-TALs, a meganuclease can be fused to a TALE DNA binding domain. The meganuclease domain can increase specificity and provide cleavage. Similarly, an inactivated or "dead" Cas9 (dCas9) can be fused to a cleavage domain, requiring the sgRNA/Cas9 target site and adjacent binding sites for the fused DNA binding domain. This would require some protein engineering of dCas9 in addition to catalytic inactivation to reduce binding without additional binding sites.

In some embodiments, compositions and methods for editing a genome (e.g., inserting a FVIII coding sequence into the albumin locus) according to the present disclosure use any of the following approaches:

Zinc finger nucleases Zinc finger nucleases (ZFNs) are modular proteins with engineered zinc finger DNA binding domains linked to the catalytic domain of the type II endonuclease FokI. Because FokI only functions as a dimer, a pair of ZFNs must be engineered to bind to cognate target "half-site" sequences on opposite DNA strands with precise spacing between them to allow the formation of catalytically active FokI dimers. When the FokI domain dimerizes (itself having no sequence specificity), a DNA double-strand break is generated between the half-sites of the ZFNs as the initiating step of genome editing.

The DNA-binding domain of each ZFN generally has three to six zinc fingers of abundant Cys2-His2 structure, each finger recognizing primarily a triplet of nucleotides on one strand of the target DNA sequence, although interstrand interactions with the fourth nucleotide may also be important. Altering the amino acids of the fingers at positions that make important contacts with the DNA alters the sequence specificity of a given finger. Thus, a four-finger zinc finger protein selectively recognizes a 12-bp target sequence, where the target sequence is a complex of triplet preferences contributed by each finger, but triplet preferences can be influenced to various degrees by adjacent fingers. An important aspect of ZFNs is that they can be easily retargeted to almost any genomic address simply by modifying individual fingers, although doing this successfully requires considerable expertise. In most applications of ZFNs, proteins with four to six fingers are used, each recognizing 12 to 18 bp. Thus, a pair of ZFNs generally recognizes a combined target sequence of 24-36 bp, not including the 5-7 bp spacer between the half-sites. The binding sites can be further separated by a larger spacer, including 15-17 bp. A target sequence of this length is likely to be unique in the human genome, assuming that repetitive sequences or gene homologs are excluded during the design process. Nevertheless, since the ZFN protein-DNA interaction is not absolute in its specificity, off-target binding and cleavage events occur either as heterodimers between the two ZFNs or as homodimers of one or the other of the ZFNs. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to create "plus" and "minus" mutants (also known as obligate heterodimer mutants), which can only dimerize with each other, but not with themselves. Forcing obligate heterodimers prevents the formation of homodimers. This has greatly enhanced specificity for ZFNs, and any other nucleases that employ these FokI variants.

Various ZFN-based systems have been described in the art, modifications of which are reported regularly, and numerous references describe the rules and parameters used to guide the design of ZFNs; see, e.g., Segal et al., Proc Natl Acad Sci USA (1999) 96(6):2758-63; B. Dreier et al., J Mol Biol. (2000) 303(4):489-502; Q. Liu et al., J Biol Chem. (2002) 277(6):3850-6; Dreier et al., J Biol Chem (2005) 280(42):35588-97; and Dreier et al. , J Biol Chem. (2001) 276(31):29466-78.

Transcription Activator-Like Effector Nucleases (TALENs)
TALENs represent another type of modular nuclease in which, like ZFNs, an engineered DNA-binding domain is linked to a FokI nuclease domain, and a pair of TALENs act in tandem to achieve targeted DNA cleavage. The main difference with ZFNs is the nature of the DNA-binding domain and the associated target DNA sequence recognition properties. TALEN DNA-binding domains are derived from TALE proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALEs have a tandem array of 33-35 amino acid repeats, with each repeat generally recognizing a single base pair in the target DNA sequence up to 20 bp in length, giving a total target sequence length of up to 40 bp. The nucleotide specificity of each repeat is determined by a repeat variable dinucleotide (RVD) that contains only two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine, and thymine are primarily recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. This constitutes a much simpler recognition code than zinc fingers and is therefore more advantageous than the latter for nuclease design. Nevertheless, similar to ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs also benefit from the use of obligate heterodimer mutants of the FokI domain to reduce off-target activity.

Further mutants of the FokI domain have been created that are inactivated in their catalytic function. If either half of a TALEN or ZFN pair contains an inactive FokI domain, only single-stranded DNA breaks (nicking) will occur at the target site, not DSBs. The result is equivalent to using a CRISPR/Cas9/Cpf1 "nickase" mutant in which one of the Cas9 cleavage domains is inactivated. DNA nicks can be used to drive genome editing by HDR, but less efficiently than DSBs. The main advantage is that off-target nicks are repaired quickly and accurately, unlike DSBs, which are prone to misrepair via NHEJ.

Various TALEN-based systems have been described in the art, and modifications are reported periodically: see, for example, Boch, Science (2009) 326(5959): 1509-12; Mak et al., Science (2012) 335(6069): 716-9; and Moscou et al., Science (2009) 326(5959): 1501. The use of TALENs based on the "Golden Gate" platform or cloning scheme has been described by several groups: see, for example, T. Cermak et al., Nucleic Acids Res. (2011) 39(12): e82; Li et al., Nucleic Acids Res. (2011) 39(14):6315-25; Weber et al., PLoS One (2011) 6(2):e16765; Wang et al., J Genet Genomics (2014) 41(6):339-47, Epub 2014 Can 17; and T. Cermak et al., Methods Mol Biol. (2015) 1239:133-59.

Homing endonucleases Homing endonucleases (HEs) are site-specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity - often at sites unique to the genome. There are at least six known families of HEs classified by their structure, including LAGLIDADG (SEQ ID NO: 6), GIY-YIG, His-Cis box, H-N-H, PD-(D/E)xK, and Vsr-like, derived from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria, and phages. Similar to ZFNs and TALENs, HEs can be used to create DSBs at target loci as an initial step in genome editing. Furthermore, some natural and engineered HEs cleave only a single strand of DNA, thereby functioning as site-specific nickases. The large target sequences of HEs and the specificity they offer make them attractive candidates for creating site-specific DSBs.

Various HE-based systems have been described in the art, and modifications thereof are reported regularly: see, for example, Steentoft et al., Glycobiology (2014) 24(8):663-80; Belfort and Bonocora, Methods Mol Biol. (2014) 1123:1-26; Hafez and Hausner, Genome (2012) 55(8):553-69; and references cited therein.

MegaTAL/Tev-mTALEN/MegaTev
As further examples of hybrid nucleases, the MegaTAL platform and the Tev-mTALEN platform use a fusion of a TALE DNA binding domain with a catalytically active HE to provide tunable DNA binding and specificity of the TALE and cleavage sequence specificity of the HE. and Boissel and Scharenberg, Methods Mol. Biol. (2015) 1239:171-96.

In a further variation, the MegaTev construct is a fusion of Meganuclease (Mega) with a nuclease domain derived from GIY-YIG homing endonuclease I-TevI (Tev). The two active sites are located approximately 30 bp apart on the DNA substrate and generate two DSBs with incompatible sticky ends; see, e.g., Wolfs et al., Nuc Acids Res. (2014) 42:8816-29. It is anticipated that other combinations of existing nuclease-based approaches will evolve and be useful in achieving the targeted genome modifications described herein.

dCas9-FokI or dCpf1-Fok1 and other nucleases Combining the structural and functional properties of the above nuclease platforms provides an additional approach to genome editing that could potentially overcome some of the inherent shortcomings. As an example, the CRISPR genome editing system generally uses a single Cas9 endonuclease to create DSBs. The specificity of targeting is driven by a 20 or 22 nucleotide sequence in the guide RNA that forms Watson-Crick base pairs with the target DNA (in the case of Cas9 from S. pyogenes, it adds two additional bases in the adjacent NAG or NGG PAM sequence). Although such sequences are long enough to be unique in the human genome, the specificity of the RNA/DNA interaction is not absolute and sometimes significant promiscuity is tolerated, especially in the 5' half of the target sequence, effectively reducing the number of bases that drive specificity. One solution to this has been to completely inactivate Cas9 or Cpf1 catalytic function (retaining only RNA-guided DNA binding function) and instead fuse the FokI domain to the inactivated Cas9 (see, e.g., Tsai et al., Nature Biotech (2014) 32:569-76; and Guilinger et al., Nature Biotech (2014) 32:577-82). Because FokI must dimerize to become catalytically active, two guide RNAs are needed to tightly tether the two FokI fusions to form dimers and cleave DNA. This essentially doubles the number of bases in the combined target site, thereby increasing the stringency of targeting by CRISPR-based systems.

As a further example, fusion of a TALE DNA-binding domain to a catalytically active HE such as I-TevI is expected to exploit both the tunable DNA binding and specificity of the TALE and the cleavage sequence specificity of I-TevI, further reducing off-target cleavage.

The details of one or more embodiments of the present disclosure are set forth in the accompanying description below. Other features, objects, and advantages of the present disclosure will become apparent from the following description. As used herein, the singular forms include the plural forms unless the context clearly dictates otherwise. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification will control.

It is understood that the examples and embodiments described herein are for illustrative purposes only, and that modifications or variations in light thereof will be suggested to one of ordinary skill in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Some embodiments of the disclosure provided herein are further illustrated by the following non-limiting examples.

Exemplary embodiments embodiment 1. A system comprising a donor template comprising a deoxyribonucleic acid (DNA) endonuclease or a nucleic acid encoding a DNA endonuclease; a guide RNA (gRNA) or a nucleic acid encoding the gRNA comprising a spacer sequence complementary to a host cell locus; and a nucleic acid sequence encoding a synthetic FVIII protein, wherein the synthetic FVIII protein comprises a B domain substitution, the B domain substitution comprising 0-9 N-linked glycosylation sites and 3 to about 40 amino acids in length.

Embodiment 2. The system of embodiment 1, wherein the B domain substitution comprises 0 to 6 N-linked glycosylation sites.

Embodiment 3. The system of embodiment 2, wherein the B domain substitution comprises 0 to 3 N-linked glycosylation sites.

Embodiment 4. The system of embodiment 1, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-369, 371, and 373.

Embodiment 5. The system of embodiment 4, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-366, 371, and 373, or a variant thereof having at least 80% identity to any one of SEQ ID NOs: 362-366, 371, and 373.

Embodiment 6. The system of embodiment 5, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-364, 371, and 373.

Embodiment 7. The system of any one of embodiments 1 to 6, wherein the host cell locus is a locus of a gene expressed in the liver.

Embodiment 8. The system of any one of embodiments 1 to 7, wherein the host cell locus is a locus of a gene encoding an acute phase protein.

Embodiment 9. The system of embodiment 8, wherein the acute phase protein is albumin, transferrin, or fibrinogen.

Embodiment 10. The system of any one of embodiments 1 to 7, wherein the host cell locus is a safe harbor locus.

Embodiment 11. The DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cm The system of any one of embodiments 1 to 10, wherein the endonuclease is selected from the group consisting of r1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease, and functional derivatives thereof.

Embodiment 12. The system of embodiment 11, wherein the DNA endonuclease is Cas9.

Embodiment 13. The system of any one of embodiments 1 to 11, wherein the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in a host cell.

Embodiment 14. The system of any one of embodiments 1 to 13, wherein the nucleic acid encoding the DNA endonuclease is deoxyribonucleic acid (DNA).

Embodiment 15. The system of any one of embodiments 1 to 13, wherein the nucleic acid encoding the DNA endonuclease is ribonucleic acid (RNA).

Embodiment 16. The system of embodiment 15, wherein the RNA encoding the DNA endonuclease is mRNA.

Embodiment 17. The system of any one of embodiments 1 to 16, wherein the donor template nucleic acid sequence is codon-optimized for expression in a host cell.

Embodiment 18. The system of any one of embodiments 1 to 17, wherein the donor template nucleic acid sequence comprises a reduced content of CpG dinucleotides compared to a wild-type nucleic acid sequence encoding a FVIII protein.

Embodiment 19. The system of embodiment 18, wherein the donor template nucleic acid sequence does not contain CpG dinucleotides.

Embodiment 20. The system of any one of embodiments 1 to 19, wherein the donor template is encoded by an AAV vector.

Embodiment 21. The system of any one of embodiments 1 to 20, wherein the donor template comprises a donor cassette comprising a nucleic acid sequence encoding a synthetic FVIII protein, the donor cassette being flanked on one or both sides by gRNA target sites.

Embodiment 22. The system of embodiment 21, wherein the donor cassette is flanked on both sides by gRNA target sites.

Embodiment 23. The system of embodiment 21, wherein the donor cassette is flanked on the 5' side by gRNA target sites.

Embodiment 24. The system of any one of embodiments 21 to 23, wherein the gRNA target site is a target site of a gRNA in the system.

Embodiment 25. The system of embodiment 24, wherein the gRNA target site of the donor template is the reverse complement of a genomic gRNA target site for the gRNA in the system.

Embodiment 26. The system of any one of embodiments 1 to 25, wherein the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is contained in a liposome or lipid nanoparticle.

Embodiment 27. The system of embodiment 26, wherein the liposome or lipid nanoparticle also comprises a gRNA.

Embodiment 28. The system of any one of embodiments 1 to 27, wherein the DNA endonuclease is complexed with the gRNA, thereby providing a ribonucleoprotein (RNP) complex.

Embodiment 29. A method of editing a genome in a host cell, comprising providing a cell with a donor template comprising: (A) a gRNA or a nucleic acid encoding the gRNA, the gRNA comprising a spacer sequence complementary to a host cell locus; (b) a DNA endonuclease or a nucleic acid encoding the DNA endonuclease; and (c) a nucleic acid sequence encoding a synthetic FVIII protein, the synthetic FVIII protein comprising a B domain substitution, the B domain substitution comprising 0-9 N-linked glycosylation sites and 3 to about 40 amino acids in length.

Embodiment 30. The method of embodiment 29, wherein the B domain substitution comprises 0 to 6 N-linked glycosylation sites.

Embodiment 31. The method of embodiment 30, wherein the B domain substitution comprises 0 to 3 N-linked glycosylation sites.

Embodiment 32. The method of embodiment 29, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-369, 371, and 373.

Embodiment 33. The method of embodiment 32, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-366, 371, and 373, or a variant thereof having at least 80% identity to any one of SEQ ID NOs: 362-366, 371, and 373.

Embodiment 34. The method of embodiment 33, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-364, 371, and 373.

Embodiment 35. The method of any one of embodiments 29 to 34, wherein the host cell endogenous locus is a locus of a gene expressed in the liver.

Embodiment 36. The method of any one of embodiments 29 to 35, wherein the host cell endogenous locus is a locus of a gene encoding an acute phase protein.

Embodiment 37. The method of embodiment 36, wherein the acute phase protein is albumin, transferrin, or fibrinogen.

Embodiment 38. The method of any one of embodiments 29 to 34, wherein the host cell endogenous locus is a safe harbor locus.

Embodiment 39. The DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr 1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; or a functional derivative thereof.

Embodiment 40. The method of embodiment 39, wherein the DNA endonuclease is Cas9.

Embodiment 41. The method of any one of embodiments 29 to 40, wherein the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in a host cell.

Embodiment 42. The method of any one of embodiments 29 to 41, wherein the nucleic acid encoding the DNA endonuclease is deoxyribonucleic acid (DNA).

Embodiment 43. The method of any one of embodiments 29 to 41, wherein the nucleic acid encoding the DNA endonuclease is ribonucleic acid (RNA).

Embodiment 44. The method of embodiment 43, wherein the RNA encoding the DNA endonuclease is mRNA.

Embodiment 45. The method of embodiment 29, wherein the donor template is encoded by an AAV vector.

Embodiment 46. The method of any one of embodiments 29 to 45, wherein the donor template nucleic acid sequence is codon-optimized for expression in a host cell.

Embodiment 47. The method of any one of embodiments 29 to 46, wherein the donor template nucleic acid sequence comprises a reduced content of CpG dinucleotides compared to a wild-type nucleic acid sequence encoding FVIII.

Embodiment 48. The method of embodiment 47, wherein the donor template nucleic acid sequence does not contain CpG dinucleotides.

Embodiment 49. The method of any one of embodiments 29 to 48, wherein the donor template comprises a donor cassette comprising a nucleic acid sequence encoding a synthetic FVIII protein, the donor cassette being flanked on one or both sides by gRNA target sites.

Embodiment 50. The method of embodiment 49, wherein the donor cassette is flanked on both sides by gRNA target sites.

Embodiment 51. The method of embodiment 49, wherein the donor cassette is flanked 5' by gRNA target sites.

Embodiment 52. The method of any one of embodiments 49 to 51, wherein the gRNA target site is a target site of the administered gRNA.

Embodiment 53. The method of embodiment 52, wherein the gRNA target site of the donor template is the reverse complement of the gRNA target site in the cellular genome of the administered gRNA.

Embodiment 54. The method of any one of embodiments 29 to 53, wherein the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.

Embodiment 55. The method of embodiment 54, wherein the liposome or lipid nanoparticle also comprises a gRNA.

Embodiment 56. The method of any one of embodiments 29 to 55, wherein the DNA endonuclease and gRNA are provided to the host cell as a ribonucleoprotein (RNP) complex that includes the DNA endonuclease precomplexed with the gRNA.

Embodiment 57. The method of any one of embodiments 29 to 56, wherein the gRNA or a nucleic acid encoding the gRNA and the DNA endonuclease or a nucleic acid encoding the DNA endonuclease are provided to the cell more than 4 days after the donor template is provided to the cell.

Embodiment 58. The method of any one of embodiments 29 to 57, wherein the gRNA or a nucleic acid encoding the gRNA and the DNA endonuclease or a nucleic acid encoding the DNA endonuclease are provided to the cell at least 14 days after the donor template is provided to the cell.

Embodiment 59. The method of embodiment 57 or 58, wherein one or more further doses of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease.

Embodiment 60. The method of embodiment 59, wherein one or more further doses of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease until a target level of targeted integration of the nucleic acid sequence encoding the synthetic FVIII protein is achieved or a target expression level of the nucleic acid sequence encoding the synthetic FVIII protein is achieved.

Embodiment 61. The method of any one of embodiments 29 to 60, wherein the cell is a liver cell.

Embodiment 62. The method of embodiment 61, wherein the cells are human hepatocytes or human sinusoidal epithelial cells.

Embodiment 63. A cell, wherein the genome of the cell comprises DNA encoding a synthetic FVIII protein, the synthetic FVIII protein comprising a B domain substitution, the B domain substitution comprising 0 to 9 N-linked glycosylation sites and 3 to about 40 amino acids in length.

Embodiment 64. The cell of embodiment 63, wherein the synthetic FVIII protein is operably linked to an endogenous albumin promoter, an endogenous transferrin promoter, or an endogenous fibrinogen alpha promoter.

Embodiment 65. The cell of embodiment 63, wherein the nucleic acid sequence encoding the synthetic FVIII protein is codon-optimized for expression in the cell.

Embodiment 66. The cell of embodiment 63, wherein the cell is a human liver cell.

Embodiment 67. The cell of embodiment 66, wherein the cell is a human hepatocyte or a human sinusoidal epithelial cell.

Embodiment 68. The cell of embodiment 67, prepared by the method of any one of embodiments 29 to 62.

Embodiment 69. A method of treating hemophilia A in a subject, comprising providing to a cell of the subject a donor template comprising: (a) a gRNA or a nucleic acid encoding the gRNA, the gRNA comprising a spacer sequence complementary to a host cell locus; (b) a DNA endonuclease or a nucleic acid encoding the DNA endonuclease; and (c) a nucleic acid sequence encoding a synthetic FVIII protein, the synthetic FVIII protein comprising a B domain substitution, the B domain substitution comprising 0-9 N-linked glycosylation sites and 3 to about 40 amino acids in length.

Embodiment 70. The method of embodiment 69, wherein the B domain substitution comprises 0 to 6 N-linked glycosylation sites.

Embodiment 71. The method of embodiment 70, wherein the B domain substitution comprises 0 to 3 N-linked glycosylation sites.

Embodiment 72. The method of embodiment 29, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-369, 371, and 373.

Embodiment 73. The method of embodiment 72, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-366, 371, and 373, or a variant thereof having at least 80% identity to any one of SEQ ID NOs: 362-366, 371, and 373.

Embodiment 74. The method of embodiment 73, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-364, 371, and 373.

Embodiment 75. The method of any one of embodiments 69 to 74, wherein the host cell locus is a locus of a gene expressed in the liver.

Embodiment 76. The method of any one of embodiments 69 to 75, wherein the host cell locus is a locus of a gene encoding an acute phase protein.

Embodiment 77. The method of embodiment 76, wherein the acute phase protein is albumin, transferrin, or fibrinogen.

Embodiment 78. The method of any one of embodiments 69 to 74, wherein the host cell locus is a safe harbor locus.

Embodiment 79. The DNA endonuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cm r1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpf1 endonuclease; or a functional derivative thereof.

Embodiment 80. The method of embodiment 79, wherein the DNA endonuclease is Cas9.

Embodiment 81. The method of embodiment 80, wherein the Cas9 is spCas9 or SluCas9.

Embodiment 82. The method of any one of embodiments 69 to 81, wherein the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in the cell.

Embodiment 83. The method of any one of embodiments 69 to 82, wherein the nucleic acid encoding the DNA endonuclease is deoxyribonucleic acid (DNA).

Embodiment 84. The method of any one of embodiments 69 to 82, wherein the nucleic acid encoding the DNA endonuclease is ribonucleic acid (RNA).

Embodiment 85. The method of embodiment 84, wherein the RNA encoding the DNA endonuclease is mRNA.

Embodiment 86. The method of any one of embodiments 69 to 85, wherein one or more of the gRNA or a nucleic acid encoding the gRNA, the DNA endonuclease or a nucleic acid encoding the DNA endonuclease, and the donor template are formulated in a liposome or lipid nanoparticle.

Embodiment 87. The method of any one of embodiments 69 to 86, wherein the donor template is encoded by an AAV vector.

Embodiment 88. The method of any one of embodiments 69 to 87, wherein the donor template nucleic acid sequence is codon-optimized for expression in a host cell.

Embodiment 89. The method of any one of embodiments 69 to 88, wherein the donor template nucleic acid sequence comprises a reduced content of CpG dinucleotides compared to a wild-type nucleic acid sequence encoding FVIII.

Embodiment 90. The method of embodiment 89, wherein the donor template nucleic acid sequence does not contain CpG dinucleotides.

Embodiment 91. The method of any one of embodiments 69 to 90, wherein the donor template comprises a donor cassette comprising a nucleic acid sequence encoding a synthetic FVIII protein, the donor cassette being flanked on one or both sides by gRNA target sites.

Embodiment 92. The method of embodiment 91, wherein the donor cassette is flanked on both sides by gRNA target sites.

Embodiment 93. The method of embodiment 91, wherein the donor cassette is flanked 5' by gRNA target sites.

Embodiment 94. The method of any one of embodiments 91 to 93, wherein the gRNA target site is a target site of a gRNA.

Embodiment 95. The method of embodiment 94, wherein the gRNA target site of the donor template is a reverse complement of the gRNA target site in the cell genome for the gRNA.

Embodiment 96. The method of any one of embodiments 69 to 95, wherein providing the donor template to the cells comprises administering the donor template intravenously to the subject.

Embodiment 97. The method of any one of embodiments 69 to 96, wherein the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.

Embodiment 98. The method of embodiment 97, wherein the liposome or lipid nanoparticle also comprises a gRNA.

Embodiment 99. The method of embodiment 98, wherein providing the cell with the gRNA or a nucleic acid encoding the gRNA, and the DNA endonuclease or a nucleic acid encoding the DNA endonuclease comprises administering a liposome or lipid nanoparticle intravenously to the subject.

Embodiment 100. The method of any one of embodiments 69 to 99, wherein the DNA endonuclease and gRNA are provided to the host cell as a ribonucleoprotein (RNP) complex, the RNP complex comprising the DNA endonuclease complexed with the gRNA.

Embodiment 101. The method of any one of embodiments 69 to 100, wherein the gRNA or a nucleic acid encoding the gRNA and the DNA endonuclease or a nucleic acid encoding the DNA endonuclease are provided to the cell more than 4 days after the donor template is provided to the cell.

Embodiment 102. The method of any one of embodiments 69 to 101, wherein the gRNA or a nucleic acid encoding the gRNA and the DNA endonuclease or a nucleic acid encoding the DNA endonuclease are provided to the cell at least 14 days after the donor template is provided to the cell.

Embodiment 103. The method of embodiment 101 or 102, wherein one or more further doses of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease.

Embodiment 104. The method of embodiment 103, wherein one or more further doses of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease are provided to the cell following the first dose of gRNA or a nucleic acid encoding a gRNA and a DNA endonuclease or a nucleic acid encoding a DNA endonuclease until a target level of targeted integration of the nucleic acid sequence encoding the synthetic FVIII protein and/or a target expression level of the nucleic acid sequence encoding the synthetic FVIII protein is achieved.

Embodiment 105. The method of any one of embodiments 101 to 104, wherein providing the cell with the gRNA and the DNA endonuclease or a nucleic acid encoding the DNA endonuclease comprises administering to the subject lipid nanoparticles comprising the nucleic acid encoding the DNA endonuclease and the gRNA.

Embodiment 106. The method of any one of embodiments 101 to 105, wherein providing the cell with a donor template comprises administering to the subject a donor template encoded by an AAV vector.

Embodiment 107. The method of any one of embodiments 69 to 106, wherein the cells are hepatocytes.

Embodiment 108. The method of any one of embodiments 69 to 107, wherein the nucleic acid sequence encoding the synthetic FVIII protein is expressed in the liver of the subject.

Embodiment 109. A method for treating hemophilia A in a subject, comprising administering to the subject a cell according to any one of embodiments 63 to 68.

Embodiment 110. The method of embodiment 109, wherein the cells are autologous to the subject.

Embodiment 111. The method of embodiment 110, further comprising obtaining a biological sample from the subject, the biological sample comprising liver cells, and the cells being prepared from the liver cells.

Embodiment 112. A kit comprising one or more elements of the system described in any one of embodiments 1 to 28, further comprising instructions for use.

Embodiment 113. A nucleic acid comprising a polynucleotide sequence encoding a synthetic FVIII protein, the synthetic FVIII protein comprising a B domain substitution, the B domain substitution comprising 0 to 9 N-linked glycosylation sites and 3 to about 40 amino acids in length.

Embodiment 114. The nucleic acid of embodiment 113, wherein the B domain substitution comprises 0 to 6 N-linked glycosylation sites.

Embodiment 115. The nucleic acid of embodiment 113, wherein the B domain substitution comprises 0 to 3 N-linked glycosylation sites.

Embodiment 116. The nucleic acid of embodiment 113, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-369, 371, and 373.

Embodiment 117. The nucleic acid of embodiment 116, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-364, 371, and 373, or a variant thereof having at least 80% identity to any one of SEQ ID NOs: 362-364, 371, and 373.

Embodiment 118. The nucleic acid of embodiment 116, wherein the B domain substitution comprises an amino acid sequence of any one of SEQ ID NOs: 362-363, 371, and 373.

Embodiment 119. The nucleic acid of any one of embodiments 113 to 118, wherein the polynucleotide sequence encoding the synthetic FVIII protein is codon-optimized for expression in a host cell.

Embodiment 120. The nucleic acid of any one of embodiments 113 to 119, wherein the polynucleotide sequence encoding the synthetic FVIII protein comprises a reduced content of CpG dinucleotides compared to a wild-type nucleic acid sequence encoding FVIII.

Embodiment 121. The nucleic acid of embodiment 120, wherein the polynucleotide sequence encoding the synthetic FVIII protein does not contain any CpG dinucleotides.

Embodiment 122. The nucleic acid of any one of embodiments 113 to 121, wherein the nucleic acid is a viral vector.

Embodiment 123. The nucleic acid of embodiment 122, wherein the viral vector is an AAV vector.

Embodiment 124. A method of increasing an amount of FVIII in a subject, comprising providing to a cell of a subject having a first serum level of FVIII: (a) a gRNA or a nucleic acid encoding a gRNA comprising a spacer sequence complementary to a host cell locus; (b) a DNA endonuclease or a nucleic acid encoding a DNA endonuclease; and (c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein, the synthetic FVIII protein comprising a B domain substitution, the B domain substitution comprising 0 to 9 N-linked glycosylation sites and 3 to about 40 amino acids in length.

Embodiment 125. The method of embodiment 124, wherein the first serum level of VIII is less than about 0.40 IU/mL.

Embodiment 126. The method of embodiment 125, wherein the first serum level of VIII is less than about 0.05 IU/mL.

Embodiment 127. The method of embodiment 125, wherein the first serum level of VIII is less than about 0.01 IU/mL.

Embodiment 128. Use of the system described in any one of embodiments 1 to 28 for the treatment of hemophilia A.

Embodiment 129. Use of the system described in any one of embodiments 1 to 28 for the manufacture of a medicament for the treatment of hemophilia A.

Embodiment 130. Use of any one of the cells of embodiments 63 to 68 for the treatment of hemophilia A.

Embodiment 131. Use of a cell according to any one of embodiments 63 to 68 for the manufacture of a medicament for the treatment of hemophilia A.

Embodiment 132. Use of the kit described in embodiment 112 for the treatment of hemophilia A.

Embodiment 133. Use of the kit described in embodiment 112 for the manufacture of a medicament for the treatment of hemophilia A.

Embodiment 134. Use of a nucleic acid according to any one of embodiments 113 to 123 for the treatment of hemophilia A.

Embodiment 135. Use of a nucleic acid according to any one of embodiments 113 to 123 for the manufacture of a medicament for the treatment of hemophilia A.

Embodiment 136. A synthetic FVIII protein comprising a B domain substitution, the B domain substitution comprising 0-9 N-linked glycosylation sites and having a length of about 40 amino acids or less.

Example 1: Construct design in which an amino acid sequence containing an N-glycosylation motif improves FVIII expression after targeted integration into mouse albumin intron 1 mediated by CRISPR/Cas9 cleavage The challenge of inserting a FVIII-encoding nucleic acid into the genome is that the native FVIII coding sequence is 7053 bp, making it difficult to package, especially in adeno-associated viruses (AAV has a packaging limit in the range of 4800-5000 bp for in vivo delivery, as a template for integration at the double-stranded break created by a sequence-specific nuclease such as Cas9). To solve this problem, applicants designed a set of FVIII coding sequences with modified B domains. The B domain of FVIII is not required for function, but improves secretion of FVIII. These FVIII coding sequences were designed to express synthetic FVIII with short B domains (substituted B domains). To evaluate the synthetic FVIII coding sequence with a substituted B domain for its ability to produce and secrete FVIII protein after integration into the genome, constructs were designed to target the integration of the FVIII coding sequence into intron 1 of the mouse albumin gene. The albumin locus provides a strong promoter that is active in liver cells, such that an appropriate FVIII coding sequence inserted into this locus can be expressed when operably linked to the albumin promoter.

A series of plasmids, referred to herein as pCB076 (SEQ ID NO:316), pCB100 (SEQ ID NO:320), pCB1003 (SEQ ID NO:324), pCB085 (SEQ ID NO:3319), or pCB080 (SEQ ID NO:318), were constructed using known molecular biology techniques. The same pUC19-based bacterial plasmid backbone (containing a bacterial origin of replication and a kanamycin resistance gene) was used for all five plasmids. The plasmids were constructed with the following elements (in order): gRNA target site (for gRNA mAlbT1, SEQ ID NO:338, targeting exon 1 of the mouse albumin gene) | 18 bp spacer | splice acceptor site (SA) | FVIII coding sequence | polyadenylation signal ("sPA"). The plasmids differed only in the codon optimization of the human FVIII coding sequence and the presence (pCB076) or absence (pCB100, pCB1003, pCB085, and pCB080) of sequences encoding a B domain replacement. The B domain replacement used in this example consisted of the first six N-glycosylation motifs derived from the N-terminus of the human FVIII B domain.

Plasmids pCB100, pCB1003, pCB085 and pCB080 all contain the coding sequence for a B domain deleted human FVIII, with the B domain replaced with an "SQ linker" (encoding amino acids SFSQNPPVLKRHQR, SEQ ID NO:337). The SQ linker contains a protease cleavage site (RHQR) but lacks an N-linked glycosylation site. Plasmid pCB076 (SEQ ID NO:316) contains the same codon-optimized B domain deleted human FVIII coding sequence ("co1", see Example 4 below) as pCB100, with an additional DNA sequence encoding 17 amino acids corresponding to the first six N-glycosylation motifs from the N-terminus of the human FVIII B domain inserted into the SQ linker in place of the B domain (thus forming a B domain replacement). Other plasmids have codon optimization: pCB100-co1 (SEQ ID NO:320), pCB1003-co2 (SEQ ID NO:324), pCB085-co3 (SEQ ID NO:319), and pCB080-co4 (SEQ ID NO:318) (see Example 4 below). The plasmids were designed to be donors for targeted integration into a double stranded break generated in intron 1 of the mouse albumin gene using the CRISPR/Cas9 system utilizing gRNA mALbT1 (tgccagttcccgatcgttac, SEQ ID NO:338). The liver, and specifically hepatocytes, are the target organ for this targeted integration. In vivo, hepatocytes are mostly quiescent, and the predominant cellular mechanism for repairing double-stranded breaks in the DNA of non-dividing cells is known to be non-homologous end joining (NHEJ) (Z. Mao et al., Cell Cycle (2008) 7:2902-06). In the presence of a linear double-stranded DNA molecule (donor) and a double-stranded break in the genome, the donor DNA can be inserted into the double-stranded break by the NHEJ mechanism.

Alternatively, the ends of double-stranded breaks in the genome can be rejoined by the same NHEJ mechanism, an event that generally occurs more frequently than the insertion of the donor template. Repair by NHEJ is an error-prone process that leads to the introduction of insertions or deletions at the double-stranded break site. Targeted integration of donor templates delivered as plasmids at double-stranded breaks in the genome of cells can be enhanced by including a nuclease cleavage site in the donor plasmid. Plasmids are circular molecules and therefore do not serve as templates for integration at double-stranded breaks. Including a single guide RNA cleavage site in the plasmid linearizes the plasmid in the presence of the Cas9/gRNA complex. Therefore, a single guide RNA cleavage site for the mALbT1 guide was inserted at the 5' end of the FVIII cassette with the reverse complement of the sequence present in the mouse genome.

The use of the reverse complement of a guide sequence in the genome theoretically favors integration in the forward orientation if two guide sites flanking the cassette are used. However, this advantage is unlikely to be maintained if only one guide cleavage site is used. Inclusion of guide cleavage sites flanking the coding sequence would generate two linear fragments consisting of the coding sequence cassette and the bacterial plasmid backbone (encoding antibiotic resistance genes and an origin of replication), in which case the bacterial backbone fragment would compete for integration at the double-stranded break in the genome. For this reason, applicants have designed the plasmid so that a single guide cleavage site is used. The synthetic FVIII coding sequence cassette was composed of the following elements (in order) starting at the 5' end: mAlbT1 gRNA target site, an 18 bp spacer sequence, a splice acceptor sequence (ACTAAAGAATTATTCTTTTACATTTCAG, SEQ ID NO: 307), a B domain deleted human FVIII coding sequence in which the signal peptide has been replaced with the dinucleotide TG, and a polyadenylation signal (aataaaagatcttttattttcattagatctgtgtgttggttttttttgtgtg, SEQ ID NO: 306).

This construct was designed to generate a hybrid pre-mRNA containing albumin exon 1, a portion of albumin intron 1, and the FVIII coding sequence cassette after integration into albumin intron 1. After integration into albumin intron 1, it is expected that at some frequency the cellular splicing machinery will splice out intron 1, thereby generating a mature mRNA in which albumin exon 1 is fused in frame to the mature FVIII coding sequence. A TG dinucleotide is included in the construct to maintain the translation reading frame. Translation of this mRNA was predicted to produce a protein in which the signal peptide and albumin propeptide are fused to the mature FVIII coding sequence. Upon passage through the cellular secretion machinery, the signal peptide and propeptide were predicted to be cleaved, leaving three amino acids (Glu-Ala-Leu) added to the native N-terminus of mature FVIII. FVIII protein produced using this method was active in mice despite the presence of these three additional amino acids.

gRNA
The gRNAs used in these experiments were chemically synthesized and incorporated chemically modified nucleotides to improve resistance to nucleases. In one example, the gRNA is composed of the following structure:
(where "A, G, U, C" are natural RNA nucleotides, "a, g, u, c" are 2'-O-methyl nucleotides, and "s" represents a phosphorothioate backbone.) The mouse albumin-targeting sequence of the gRNA is underlined, and the remainder of the gRNA sequence is the common scaffold sequence.

mRNA
The mRNA can be produced by methods known in the art. One such method used herein is in vitro transcription using T7 polymerase, where the sequence of the mRNA is encoded in a plasmid containing a T7 polymerase promoter. Briefly, the plasmid was incubated in an appropriate buffer containing T7 polymerase and ribonucleotides to produce an RNA molecule encoding the amino acid sequence of the desired protein. Either natural ribonucleotides or chemically modified ribonucleotides can be used in the reaction mixture to generate mRNA molecules having either the natural chemical structure of natural mRNA or modified chemical structures. In the studies described herein, natural (unmodified) ribonucleotides were used. Additionally, a capping component was included in the transcription reaction so that the 5' end of the mRNA was capped.

The spCas9 mRNA was designed to encode the spCas9 protein fused to a nuclear localization domain (NLS), which is necessary for transporting the spCas9 protein to the nuclear compartment where cleavage of genomic DNA can occur. Further components of the Cas9 mRNA are a KOZAK sequence at the 5' end before the first codon to facilitate ribosome binding, and a polyA tail at the 3' end consisting of a series of A residues. An example of an spCas9 mRNA with an NLS sequence is set forth in SEQ ID NO: 340. Furthermore, the sequence of the spCas9 coding sequence was optimized for codon usage by utilizing the most frequently used codon for each amino acid. Furthermore, the coding sequence was optimized to remove potential ribosome binding sites and upstream open reading frames to facilitate efficient translation of the mRNA into the spCas9 protein.

LNP
The main component of the LNPs used in these studies is the lipid C12-200 (Love et al., 2010, supra). C12-200 forms a complex with negatively charged RNA molecules. Typically, C12-200 was combined with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DMPE-mPEG2000, and cholesterol. When mixed with nucleic acids such as gRNA and mRNA under controlled conditions, for example in a NanoAssemblr® device (Precision NanoSystems, Vancouver, BC), self-assembly of LNPs occurred with the nucleic acid encapsulated within the LNP. To assemble the gRNA and Cas9 mRNA in the LNPs, ethanol and lipid stocks were pipetted into glass vials as needed. An exemplary ratio consisted of C12-200, DOPE, cholesterol, and mPEG2000-DMG in a molar ratio of 50:10:38.5:1.5. gRNA and mRNA were diluted in 100 mM sodium citrate (pH 3.0) and 300 mM NaCl in RNase-free tubes. NanoAssemblr® cartridges (Precision NanoSystems) were washed with ethanol on the lipid side and water on the RNA side. A working stock of lipid was drawn into the syringe, air was removed from the syringe, and the syringe was inserted into the cartridge. The same procedure was used to load the gRNA and Cas9 mRNA mixture into the syringe. A NanoAssemblr® run was then performed under the manufacturer's recommended conditions. The LNP suspension was dialyzed in 4 liters of PBS using a 10K molecular weight cut-off (MWCO) dialysis cartridge for 4 hours and then concentrated by centrifugation through a 100K MWCO spin cartridge (Amicon) (including 3 washes in PBS during centrifugation). Finally, the LNP suspension was sterile filtered through a 0.2 μm syringe filter. Endotoxin levels were determined using a commercially available endotoxin kit (Limulus amebocyte lysate (LAL) assay) and particle size distribution was determined by dynamic light scattering.

The concentration of encapsulated RNA was determined using the RiboGreen® assay (Thermo Fisher). Alternatively, gRNA and Cas9 mRNA were formulated separately into LNPs and then mixed together prior to treatment of cells in culture or injection into animals. Using separately formulated gRNA and Cas9 mRNA, specific ratios of gRNA to Cas9 mRNA could be tested.

Alternative LNP formulations utilizing alternative cationic lipid molecules are also used for in vivo delivery of gRNA and Cas9 mRNA.

In Vivo Testing of Constructs A mouse model was used to test the ability of the designed constructs to produce FVIII. Mouse models of hemophilia A are known in the art (e.g., L. Bi et al., Nat Genet (1995) 10:119-21, doi:10.1038/ng0595-119). Plasmids pCB076, pCB100, pCB1003, pCB085, and pCB080 were purified using Qiagen EndoFree® Plasmid maxiPrep Kit (cat #12362) and then diluted to a final concentration of 15 μg/mL in 0.9% saline. Hemophilia A mice (B6 strain; 129S-F8 ^tm1Kaz /J strain), a mouse strain lacking the mouse FVIII protein, were obtained from The Jackson Laboratory (Bar Harbor, ME). A cohort of hemophilia A mice was injected with 2 mL of diluted plasmid DNA per mouse via the tail vein for 5-6 seconds by hydrodynamic injection ("HDI"). The HDI process has been reported to result in the delivery of plasmid DNA to the nuclei of liver cells, including hepatocytes (see, e.g., F. Niola et al., Meth Mol Biol (2019) 1961:329-41). The day after injection, the mice received a retro-orbital ("RO") injection of LNP formulations encapsulating spCas9 mRNA and guide RNA mAlbT1. The dose of LNP administered to mice was 1 mg/kg body weight of spCas9 mRNA + 1 mg/kg body weight of gRNA.

Groups of mice administered LNP alone were sacrificed 3 days later, and DNA was extracted from whole livers and assayed for indels at the predicted cleavage site of mAlbT1 gRNA using TIDE analysis (E.K. Brinkman et al., Nuc Acid Res (2014) 42:e168). In the TIDE analysis, the genomic region of the predicted CRISPR/Cas9 cleavage site is amplified from genomic DNA of treated cells by PCR and then subjected to Sanger sequencing. Sequencing chromatograms were analyzed using the TIDE software program, which determines the frequency of insertions and deletions in the region surrounding the predicted cleavage site.

In these experiments, the frequency of indels at the on-target site was determined to be 25.4%. Six days after administration of LNP to plasmid-injected mice, blood samples were collected by RO blood collection into sodium citrate (sodium citrate to blood ratio 1:9) and plasma was collected by centrifugation. FVIII activity in plasma was measured using a FVIII activity assay (Diapharma, Chromogenix Coatest® SP Factor FVIII, cat# K824086). Recombinant human FVIII, Kogenate® (Bayer), was used as the standard, and units per mL of FVIII activity in blood were converted to percent of normal activity (1 U/mL = 100%). The results are summarized in Figure 1. Mice injected with plasmid pCB076, which contains six N-glycan B domain replacement sequences in place of the B domain, had average synthetic FVIII levels equivalent to 20% of normal human FVIII levels. In contrast, mice injected with pCB100 plasmid, which is identical to pCB076 except for the absence of the six N-glycan B domain replacement sequences, had no detectable FVIII levels in their blood. Mice injected with plasmids pCB1003, pCB085, or pCB080, which contain different codon-optimized B domain deleted FVIII coding sequences lacking the six N-glycan B domain replacement sequences, had low or no measurable FVIII activity in their blood when compared to non-gene edited (naive) hemophilia A mice. Some of the mice injected with pCB1003 and pCB080 had detectable FVIII in their blood at 1-3% of normal, indicating that codon-optimized co2 (pCB1003) and co4 (pCB080) may be more active than codon-optimized co1 (pCB100) and co3 (pCB085).

The levels of FVIII produced in the blood of mice in this study depended on both the frequency of targeted integration into albumin intron 1 in the forward orientation (the orientation that allows FVIII protein to be produced) and the intrinsic expression efficiency of the FVIII coding sequence, which is a function of the transcription efficiency, translation efficiency (which varies depending on the type of codon optimization used), and the efficiency of the secretion process. In the case of FVIII protein, it has been reported that protein secretion may be a rate-limiting step and is associated with the unfolded protein response that may be induced when FVIII is expressed at high levels in cells. (M. Swaroop et al., J Biol Chem (1997) 272:24121-24; R. J. Kaufman, Blood (2009) 114:SCI-19).

To distinguish between targeted integration frequency, which may vary between mice due to variability in donor delivery efficiency due to HDI or other factors, and the intrinsic expression efficiency of the synthetic FVIII coding sequence, targeted integration frequency was quantified using droplet digital PCR (DD-PCR). DD-PCR is a method for quantifying the absolute copy number of a nucleic acid sequence in a sample. To quantify only the forward orientation of the synthetic FVIII coding sequence cassette inserted into albumin intron 1, a pair of PCR primers was designed with a forward primer located in albumin intron 1 at a site 5' of the gRNA mALBTI cleavage site and a reverse primer located at the 5' end of the FVIII coding sequence. A fluorescent probe was designed that was complementary to the sequence between the two primers. A reference primer/probe set was designed against the native mouse albumin gene sequence at a site distant from the mALBTI gRNA site. The reference primer probe was used to normalize the amount of input mouse genomic DNA in each assay.

To perform this analysis, mice from the above experiment were sacrificed 8 days after the mice were administered LNP. Whole livers were homogenized and total genomic DNA was purified using the Qiagen DNeasy® Tissue kit. Equal masses of genomic DNA were then assayed for targeted integration frequency using the DD-PCR assay described above. The results for each mouse are summarized in Table 2. Targeted integration frequencies in the forward orientation ranged from 0.09% to 0.95% (0.09-0.95 copies per 100 monoploid genomes). Maximum FVIII levels in blood positively correlated with integration frequency, indicating that FVIII levels are dependent on the copy number of the FVIII cassette integrated into albumin intron 1. The average targeted integration frequency in mice injected with pCB076 was 0.47 ± 0.26 compared with 0.28 ± 0.15 in mice injected with pCB100, indicating a trend toward higher integration frequencies in mice injected with pCB076 containing the B domain substitution instead of the SQ linker, although this difference was not statistically significant.

Normalizing the FVIII levels in the blood of each mouse to the integration frequency provides a measure of the intrinsic expression efficiency of the FVIII coding sequence. The average ratio of FVIII levels divided by the targeted integration frequency was 42 for pCB076 and 5.3 for pCB100, a difference that was statistically significant (p=0.0004) as determined using a two-tailed Student's T-test. These results demonstrate that the intrinsic expression efficiency of the synthetic FVIII coding sequence in pCB076 is approximately 8-fold greater than that of the coding sequence in pCB100. This demonstrates that the inclusion of the sequence encoding the B domain substitution in place of the SQ linker improved the intrinsic expression efficiency of this codon-optimized FVIII coding sequence by approximately 8-fold. The magnitude of this improvement is significantly greater than the two-fold improvement reported for the same six glycan motif sequence when the FVIII coding sequence is delivered in a non-integrating AAV virus driven by a strong liver-specific promoter (J. McIntosh et al., Blood (2013) 121:3335-44).

Example 2: Replacement of the SQ linker with a B domain replacement increases FVIII expression from a FVIII donor cassette delivered by AAV and integrated into intron 1 of albumin To determine whether the same beneficial effects of the B domain replacement peptide occurred when a synthetic FVIII coding sequence was delivered to mouse liver using AAV, plasmids pCB099 (SEQ ID NO:311) and pCB102 (SEQ ID NO:341) were constructed and packaged into AAV8 (Vector Biolabs, Malvern, PA, or SabTech, Philadelphia, PA). The plasmids were constructed with the following elements (in order): ITR|gRNA target site (for mAlbT1)|18 bp spacer|splice acceptor site (SA)|FVIII coding sequence|polyadenylation signal ("sPA")|gRNA target site|ITR. The FVIII coding sequences of pCB099 and pCB102 were identical to those of pCB076 (with B domain substitution) and pCB100 (with only the SQ linker), respectively. These FVIII cassettes lack a promoter and therefore cannot express FVIII as a non-integrated AAV episomal genome. Expression of FVIII delivered by these AAV viruses requires integration adjacent to an appropriate promoter.

In these experiments, hemophilia A mice were intravenously injected with 2×10 ¹² vector genomes ("vg") of AAV8-pCB099 or AAV8-pCB102 per kg body weight. Four weeks later, mice were intravenously injected with a 1:1 mixture of two LNPs, one encapsulating mAlbT1 gRNA and the other encapsulating spCas9 mRNA. LNPs were prepared as described in Example 1, with a total dose of 2 mg RNA per kg body weight. FVIII activity was measured in the blood of mice 10 days after LNP administration using the method described in Example 1. FVIII levels in the blood of mice 10 days after LNP administration (Figure 2) averaged 20% of normal human FVIII levels in mice that received AAV9-pCB099, but were at background levels in mice that received AAV8-pCB102 (lacking the B domain substitution).

On day 24 after LNP administration, mice were sacrificed, whole livers were homogenized, and total genomic DNA was extracted from a portion of the liver lysate. The frequency of targeted integration into albumin intron 1 in the forward orientation was quantified using the DD-PCR assay described in Example 1. The results for each mouse are summarized in Table 3.

The results show that the mean targeted integration frequency (%/monoploid genome) in mice injected with AAV8-pCB099 was 1.86 (±0.25), whereas in mice injected with AAV8-pCB102, the mean targeted integration frequency was 0.46 (±0.2). This difference was statistically significant using a two-tailed Student's T-test (p<0.01). These results demonstrate that inclusion of the B domain substitution results in a four-fold higher frequency of targeted integration, an unexpected result given that the inclusion of a glycan in place of the B domain of FVIII has previously only been shown to improve FVIII expression levels. The mean FVIII levels in the blood of mice injected with AAV8-pCB099 were 18.6 (±2.2)% of normal, whereas in mice injected with AAV8-pCB102, the mean FVIII levels were 1.7 (±1.1)% of normal. This 11-fold difference was statistically significant (p<0.01) using a two-tailed Student's T-test. FVIII levels were normalized to the targeted integration frequency by dividing the FVIII levels in individual mice by the targeted integration frequency (Table 3). The mean ratio of FVIII activity divided by targeted integration frequency was 10.2 (±1.7) in AAV8-pCB099-injected mice and 3.1 (±1.7) in AAV8-pCB102-injected mice. This difference was statistically significant (p<0.01) using a two-tailed Student's T-test.

These data demonstrate that the intrinsic expression efficiency of the FVIII coding sequence in AAV8-pCB099 is 3-fold higher than that of AAV8-pCB102. Because AAV8-pCB099 differs from AAV8-pCB102 only by the presence of an N-glycan motif-containing sequence, these data demonstrate that the N-glycan motif in AAV8-pCB099 confers a 3-fold improvement in intrinsic expression efficiency. Thus, the overall 11-fold improvement in FVIII levels in the blood of mice is due to a combination of 4-fold higher targeted integration of the integrated FVIII coding sequence and 3-fold improved expression efficiency.

Example 3: Optimization of the number of N-glycans in the B domain replacement The data from Examples 1 and 2 demonstrate that inserting a B domain replacement containing six N-linked glycan motifs improved the frequency of FVIII expression and targeted integration. However, the dependence of this improvement on the number of N-glycan sequences in the B domain replacement was unclear. Therefore, the inventors designed experiments to probe this aspect of FVIII expression. In particular, it was desired to determine the minimum number of N-linked glycan motifs required for improved FVIII expression.

Plasmid constructs To explore the effect of different numbers of N-glycan motifs on expression, a series of donor plasmids containing one to nine N-glycan motifs were constructed. These are summarized in Table 4. All plasmids consisted of the following sequence elements, in 5' to 3' order: mAlbT1 gRNA target sequence | 18 bp spacer | splice acceptor | B-domain deleted FVIII coding sequence with the signal peptide replaced with a TG dinucleotide | polyadenylation signal sequence. In each of these plasmids, the FVIII coding sequence was based on the codon-optimized sequence used in pCB076 (see Example 1), with the signal peptide replaced with a TG dinucleotide, but with one to nine N-linked glycosylation sites in the B-domain replacement. All plasmids contained the same pUC19-based bacterial plasmid backbone, including a bacterial origin of replication and a kanamycin resistance gene.

In vivo testing of constructs: 5, 6 and 7 glycans Hemophilia A mice were administered plasmid pCB077, pCB1006, pCB1007, or pCB1008 at 30 μg per mouse by hydrodynamic injection using the method of Example 1. The next day, the same mice were injected retro-orbitally with a 1:1 mixture of LNPs encapsulating spCas9 mRNA and mALbT1 gRNA at a total RNA dose of 2 mg/kg body weight. LNPs were prepared as described in Example 1. FVIII activity was measured in the blood of mice 6 days later using the method described in Example 1. The results are summarized in Figure 3 and demonstrate that the levels of FVIII produced by the four plasmid donors were similar.

The level of FVIII produced in the blood of mice in this study depends on both the frequency of targeted integration into albumin intron 1 in the forward orientation (the orientation that allows FVIII protein to be produced) and the intrinsic expression efficiency of the FVIII coding sequence. The intrinsic expression efficiency of the FVIII coding sequence is a function of the transcription rate, translation efficiency (affected by the type of codon optimization used), and the efficiency of the secretion process. In the case of FVIII protein, protein secretion may be the rate-limiting step (M. Swaroop et al., supra) and has been suggested to be associated with the unfolded protein response (R. J. Kaufman, supra), which occurs when FVIII is expressed at high levels in cells. To distinguish between the targeted integration frequency, which is expected to vary between individual mice, and the intrinsic expression efficiency of the integrated synthetic FVIII coding sequence, the targeted integration frequency was quantified using droplet digital PCR (DD-PCR) as described in Example 1.

Eight days after administering LNP to the mice, the mice were sacrificed, whole livers were homogenized, and total genomic DNA was purified using the Qiagen DNeasy® Tissue kit. Equal masses of genomic DNA were then assayed for targeted integration frequency using DD-quantity. The results for each mouse are summarized in Table 5. Forward targeted integration frequencies ranged from 0.17% to 0.70% in individual mice, but the averages within each group of mice for the four plasmids were similar at 0.49%, 0.47%, 0.52%, and 0.38% for pCB077, pCB1006, pCB1007, and pCB1008, respectively. The average ratios of FVIII activity to TI for mice injected with pCB077, pCB1006, pCB1007, and pCB1008 were 51.33, 48.54, 48.9, and 38.9, respectively, and the differences between the plasmids were not statistically significant. These results demonstrate that synthetic FVIII coding sequences containing five N-glycan sites (pCB1007) or seven glycan sites (pCB1008), or in which one of the glycan tripeptide motifs was changed from NDS to NDT (pCB1006), have similar intrinsic expression efficiencies compared to a synthetic FVIII coding sequence encoding six N-glycan sites (pCB077).

The same mouse study was performed using plasmids pCB1015 (SEQ ID NO:328) and pCB1016 (SEQ ID NO:329), where the number of N-glycan motifs was varied to 8 and 9, respectively. Additionally, plasmids identical to pCB077, except with only one or two N-glycan motifs, were constructed and tested for their ability to express FVIII after targeted integration into mouse albumin intron 1 using the same gRNA and spCas9 mRNA delivered into LNPs.

In vivo testing of constructs: 3, 4, and 5 glycans Plasmids pCB1007, pCB1017, and pCB1018 were purified and administered to hemophilia A mice as described above. The next day, mice received a retro-orbital (RO) injection of C12-200-based LNPs encapsulating spCas9 mRNA (1 mg/kg) and guide RNA (gRNA) mAlbT1 (1 mg/kg). Blood samples were collected 5 and 7 days after LNP administration by RO bleeding into sodium citrate (sodium citrate to blood ratio 1:9) and plasma was collected by centrifugation. FVIII activity in plasma was measured using the method described in Example 1.

On day 5, FVIII activity in blood averaged 8.1%, 5.0%, and 23.5% in mice injected with pCB1007, pCB1018, and pCB1018, respectively. On day 7, FVIII activity averaged 7.9%, 3.0%, and 13.5% in mice injected with pCB1007, pCB1018, and pCB1018, respectively. Thus, FVIII expression in mice injected with plasmids containing four N-glycan motifs (pCB1017) or three N-glycan motifs (pCB1018) was similar to that in mice receiving a plasmid containing five N-glycan motifs in the B domain substitution (pCB1007).

After blood samples were taken on day 7 after LNP administration, mice were sacrificed and whole livers were removed and stored in RNAlater™ buffer (Qiagen). Whole livers were homogenized using a bead-based homogenizer, and DNA was purified from aliquots of the homogenate using the Qiagen DNA/RNA Mini Kit (cat #80204). Liver genomic DNA was analyzed by DD-PCR for the frequency of forward integration of the FVIII donor cassette as described in Example 1. The average targeted integration frequency was 0.27%, 0.27%, and 0.55% in mice injected with pCB1007, pCB1017, and pCB1018, respectively, and these values were not statistically different (two-tailed Student's T-test).

Normalizing the FVIII levels in the blood of each mouse to the integration frequency provides a measure of the intrinsic expression efficiency of the FVIII coding sequence. The mean ratio of FVIII levels divided by targeted integration frequency was 23.6 in mice injected with pCB1007 (five N-glycans), 11.6 in mice injected with pCB1017 (four N-glycans), and 23.3 in mice injected with pCB1018 (three N-glycans). The FVIII divided by targeted integration ratios of pCB1017 and pCB1018 injected mice were not statistically different from those of pCB1007 injected mice.

These data demonstrate that the use of synthetic FVIII coding sequences containing B domain substitutions with either four N-glycan motifs or three N-glycan motifs results in similar expression when incorporated into albumin intron 1 as FVIII coding sequences containing five N-glycan motifs. Thus, synthetic FVIII constructs with B domain substitutions with three N-glycan motifs provide improved FVIII expression equivalent to that provided by B domain substitutions with five N-glycan motifs. By inference, because the five N-glycan motifs containing B domain substitution was equivalent to the six N-glycan motifs containing B domain substitution, the inventors infer that the three N-glycan motifs are equally potent as the six N-glycan motifs.

In vivo testing of constructs: one and two glycans Plasmids pCB1018 (containing a FVIII donor with a B domain substitution with three N-glycan motifs), pCB1029 (containing a FVIII donor with a B domain substitution with two N-glycan motifs), and pCB1030 (containing a FVIII donor with a B domain substitution with one N-glycan motif) were purified and administered to hemophilia A mice by hydrodynamic injection as described above. The next day, mice received retro-orbital (RO) injections of C12-200-based LNPs encapsulating spCas9 mRNA (1 mg/kg) and gRNA mAlbT1 (1 mg/kg). Five and eight days after LNP administration, blood samples were collected into sodium citrate (1:9 sodium citrate to blood ratio) by RO bleeding and plasma was collected by centrifugation. FVIII activity in plasma was measured as described above and expressed as a percentage of normal activity (1 U/mL=100%).

On day 5, FVIII activity in blood averaged 12.8%, 15.8%, and 13.4% in mice injected with pCB1018, pCB1029, and pCB1030, respectively. On day 8, FVIII activity averaged 13.8%, 14.5%, and 16.0% in mice injected with pCB1018, pCB1029, and pCB1030, respectively. Thus, FVIII expression in mice injected with plasmids containing B domain substitutions with three N-glycan motifs (pCB1018), two N-glycan motifs (pCB1029), or one N-glycan motif (pCB1030) was similar to each other.

After blood samples were taken on the 7th day after LNP administration, mice were sacrificed and whole livers were removed and stored in RNAlater™ buffer (Qiagen). Whole livers were homogenized and liver genomic DNA was analyzed by DD-PCR for the frequency of forward integration of the FVIII donor cassette as described in Example 1. The mean targeted integration frequency was 0.29%, 0.47%, and 0.36% in mice injected with pCB1018, pCB1029, and pCB1030, respectively; these values were not statistically different (two-tailed Student's T-test).

Normalizing the FVIII levels in the blood of each mouse to the integration frequency provides a measure of the intrinsic expression efficiency of the FVIII coding sequence. The mean ratio of FVIII levels divided by the targeted integration frequency was 41.9 for mice injected with pCB1018 (three N-glycans), 31.4 for mice injected with pCB1029 (two N-glycans), and 40.2 for mice injected with pCB1030 (one N-glycan). The intrinsic expression efficiency for mice injected with pCB1029 (three N-glycans) and pCB1030 (two N-glycans) was not statistically different from that of mice injected with pCB1018 (three N-glycans). These data demonstrate that FVIII donor cassettes containing B domain substitutions with either two N-glycan motifs (amino acid sequence NATNVS) or one N-glycan motif (amino acid sequence NAT) are expressed with equal efficiency as FVIII donor cassettes containing B domain substitutions with three N-glycan motifs.

Comparison of in vivo FVIII expression results for FVIII donor cassettes containing B domain substitutions with 0, 1, 2, 3, 4, 5, 6, or 7 N-linked glycan motifs The intrinsic expression efficiency of the different FVIII cassettes tested above was compared. The data set described throughout Example 3 was generated in a total of five studies using the same strain of mice (hemophilia A mice) and the same experimental protocol. FVIII activity was measured on either day 5 or 6, and again on day 8 or 9. Targeted integration frequency was measured in DNA extracted from whole liver of mice sacrificed on the day of the final FVIII activity measurement (day 8 or 9). A collection of intrinsic expression efficiencies is shown in FIG. 8. This comparison includes FVIII cassettes with different codon optimizations. A comparison of the impact of different numbers of glycans on normalized FVIII expression can be made for the donor with the codon optimization called "co1", which is the first nine bars in FIG. 8. These donors contain FVIII cassettes that differ only in the number of N-glycan motifs in the B domain substitution. Intrinsic expression efficiency was not significantly different for glycan variants containing 1 to 7 N-glycan motifs. The donor with two N-glycan motifs ("co1-2") showed a trend toward lower normalized FVIII activity (a value of 30 compared to values of approximately 45 for variants containing 5, 6, or 7 N-glycans), but this difference was not statistically significant. The donor without an N-glycan motif in place of the B domain ("co1-0") showed significantly lower normalized FVIII activity (a value of 7.4 compared to values of 40 to 50 for variants with the same glycan and codon optimization). The FVIII donor with five glycans and codon-optimized co2 was equivalent to col with five N-glycan motifs, while co3 with five N-glycans was expressed at approximately 50% of the efficiency of col with five N-glycans. These data demonstrate that FVIII coding sequences containing B domain substitutions with a single N-glycan motif were sufficient to confer FVIII expression levels equivalent to those achieved with B domain substitutions containing two to seven N-glycan motifs. The FVIII coding sequence containing a B domain substitution with a single N-glycan motif ("co1-1"/pCB1030 in FIG. 8) was expressed approximately 5.4-fold more efficiently (40.1/7.4) than the same FVIII coding sequence lacking the B domain substitution ("co1-0"/pCB100). Thus, FVIII coding sequences that include B domain replacements having fewer than six N-glycans, e.g., five N-glycans, four N-glycans, three N-glycans, two N-glycans, or one N-glycan, have advantages for use in gene editing approaches due to the reduced number of unnatural amino acids added to the FVIII protein and the reduced size of the DNA donor.

Example 4: Identification of optimal codon optimization of FVIII coding sequence for expression after targeted integration into safe harbor loci (e.g., albumin locus) in mice Plasmid constructs Experiments were performed to determine the effect of different forms of codon optimization on the expression of synthetic FVIII. A mature (lacking signal peptide) B-domain deleted human FVIII coding sequence (total coding sequence of 1438 amino acids) containing a 14 amino acid SQ linker in place of the B-domain was codon optimized (co3) by applying a commercially available algorithm available at GeneArt, which increased the number of CG dinucleotides from 54 present in the native sequence to 198. The co3 form of variant of the B-domain deleted FVIII ("co4") was codon optimized according to the published H. The B-domain deleted FVIII ("FVIII-BDD") coding sequence was created by manually removing 198 CG dinucleotides by selecting alternative codons that were either the most frequent or more frequently used codons according to the C. sapiens codon usage table (H.C. Brown et al., Mol Ther Meth & Clin Dev (2018) 9:57-69 (doi:10.1016/j.omtm.2018.01.004). The B-domain deleted FVIII ("FVIII-BDD") coding sequence was codon optimized using an algorithm based on the codon bias of genes highly expressed in the liver (H.C. Brown et al., supra) to generate FVIII-BDD codon 2, which contains 176 CG dinucleotides. A further modified version of this construct to remove all CG dinucleotides, designated co5, was also synthesized. A codon-optimized FVIII-BDD coding sequence of J. McIntosh et al., Blood (2013) 121(17):3335-44 and US Pat. No. 9,393,323 (SEQ ID NO:1 therein) was also constructed and designated herein as "co1". A further codon-optimized variant of the B-domain deleted FVIII coding sequence published in WO 2011/005968 (SEQ ID NO:5 therein) containing 245 CG dinucleotides was synthesized (herein "FVIII-BDD co6"). The plasmid was constructed as follows: pUC19 plasmid backbone|ITR|gRNA TG dinucleotide|B domain deleted FVIII sequence|polyA (sPA)|gRNA mALBTI target site|ITR (where donor sequence codon optimized was co2 (pCB1002, SEQ ID NO:323), co3 (pCB1001, SEQ ID NO:322), co4 (pCB1000, SEQ ID NO:321), or co5 (pCB103, SEQ ID NO:336)).

The FVIII donor cassette in each plasmid was flanked by AAV2 ITRs and used to package the cassette into AAV8 using a HEK293-based packaging system and purified using cesium chloride density centrifugation. The resulting AAV8 viruses (designated AAV8-pCB103, AAV8-pCB1002, AAV8-pCB1001, and AAV8-pCB1000) were titered using Q-PCR or DD-PCR with primer/probe sets located within the coding sequence of the FVIII gene. These FVIII donor cassettes are designed to express FVIII only after targeted integration into albumin intron 1. Because the donor cassettes lack a promoter, they cannot be transcribed into mRNA from the unintegrated episomal viral genome. Furthermore, all FVIII donor cassettes lack a signal peptide sequence at the N-terminus of the FVIII coding sequence, and therefore any proteins that may be expressed from unintegrated episomal viral copies cannot be secreted into circulation. After integration into albumin intron 1, transcription from the albumin promoter in the genome generates a hybrid pre-mRNA that contains mouse albumin exon 1, a portion of intron 1, and the synthetic FVIII coding sequence, and terminates with a polyadenylation signal contained at the 5' end of the FVIII donor cassette. Splicing of this pre-mRNA between the splice donor of albumin exon 1 and the splice acceptor contained at the 5' end of the FVIII donor cassette generates an mRNA that encodes a pre-pro-peptide in which exon 1 of albumin is fused in frame to the signal peptide and mature FVIII coding sequence. The protein encoded by this hybrid mRNA is processed through the cell's secretory machinery, during which the signal peptide and albumin pre-pro-peptide are cleaved, resulting in the predicted two-chain FVIII molecule that contains three amino acids at the N-terminus of the heavy chain that are not normally present in FVIII.

In vivo testing of constructs To test these formulations, cohorts of 4 or 5 hemophilia A mice were injected via the tail vein with each of the AAV8 viruses (AAV8-pCB103, AAV8-pCB1002, AAV8-pCB1001, and AAV8-pCB1000) at a dose of ^2x1012 vg/kg. Four weeks later, all mice were intravenously injected with a 1:1 mixture of LNPs encapsulating mAlbT1 gRNA and spCas9 mRNA at a total RNA dose of 2 mg/kg. LNPs were formulated according to the methods described in Example 1. FVIII activity in the blood was measured using the methods described in Example 1. The results are summarized in Figure 4.

In these experiments, mice receiving AAV8-pCB103 and AAV8-pCB1002 (containing FVIII-BDD with codon-optimized co5 and co2, respectively) had no detectable FVIII activity in their blood. Mice receiving virus pCB1001 (codon-optimized co3) had an average of 8% FVIII activity at day 11 and 20% FVIII activity at day 28 after LNP administration. Three of five mice receiving virus AAV8-pCB1000 (codon-optimized co4) had FVIII activity levels between 1% and 3% of normal. These data demonstrate that FVIII-BDD DNA sequences codon-optimized using the GeneArt algorithm (AAV8-pCB1001, co3) resulted in higher levels of FVIII expression than FVIII-BDD codons optimized based on the most frequent codons of genes highly expressed in the liver (AAV8-pCB103 and AAV8-pCB1002). Modification of the GeneArt codon-optimized FVIII-BDD sequence to remove CG dinucleotides (AAV8-pCB1000, co4) resulted in reduced FVIII expression compared to the same cassette where FVIII-BDD was codon-optimized using the GeneArt algorithm retaining the CG dinucleotide. FVIII-BDD with co4 optimization was able to generate measurable FVIII activity, unlike the co2 and co5 optimizations. Mice receiving AAV8-pCB102 (co1 codon-optimized FVIII-BDD DNA sequence, see Example 2) delivered in AAV8 at the same dose of 2×10 ¹² vg/kg and using the same dose of LNP did not produce FVIII activity in hemophilia A mice (Example 2, FIG. 2, AAV8-pCB102), demonstrating that co1 was inferior to the co3 and co4 codon-optimized FVIII-BDD sequences for expression of FVIII following targeted integration into albumin intron 1 in mice.

Example 5: Expression of FVIII in mice after targeted integration into albumin intron 1 of a donor template encoding a synthetic FVIII with five N-glycans and alternative codon optimizations co4 and co5 To test the effect of different codon optimizations using a synthetic FVIII with a B domain substitution, a FVIII-BDD coding sequence lacking the signal peptide was constructed using three codon-optimized DNA sequences designated co1, co4 and co5, and further including a B domain substitution in place of the B domain. The B domain substitution contained five N-glycan motifs (sequence: ATNVSNNSNTSNDS, SEQ ID NO:343). The coding sequences were flanked on the 5' side by a target site for the mALbT1 gRNA, an 18 bp spacer, a splice acceptor, and two nucleotides (TG). The TG dinucleotide maintains the correct reading frame after splicing into mouse albumin exon 1. A short polyadenylation signal (sPA) was included at the 3' end of the coding sequence. The synthetic FVIII coding sequences in these three plasmids encode FVIII proteins with identical amino acid sequences, but the coding sequences are encoded by different DNA sequences due to different codon optimization. These plasmids, designated pCB1007 (co1, SEQ ID NO:326), pCB1019 (co4, SEQ ID NO:332), and pCB1020 (co5, SEQ ID NO:333), were tested for their ability to express active FVIII protein in hemophilia A mice after targeted integration into albumin intron 1 mediated by CRISPR/Cas9 cleavage at the target site of mALbT1 gRNA.

The experimental protocol was the same as in Example 1. Plasmid DNA of plasmids pCB1007, pCB1019, and pCB1029 was purified using the Qiagen EndoFree® maxiprep kit (cat #12362) and then diluted to a final concentration of 15 μg/mL in 0.9% saline. Cohorts of hemophilia A mice were injected with 2 mL of diluted plasmid DNA per mouse by HDI. The next day, mice received a retro-orbital injection of C12-200-based LNPs encapsulating spCas9 mRNA (1 mg/kg body weight) and gRNA mAlbT1 (1 mg/kg body weight). A cohort of five hemophilia A mice injected with only spCas9 mRNA and mALbT1 gRNA-encapsulated LNPs were sacrificed 3 days after administration, and genomic DNA extracted from whole livers was analyzed for indels at the on-target site in albumin intron 1. The average indel frequency was 52.9%, indicating efficient cleavage at the on-target site in the liver.

Six and nine days after administration of LNP to plasmid-injected mice, blood samples were collected by RO blood sampling into sodium citrate (sodium citrate to blood ratio 1:9) and plasma was collected by centrifugation. FVIII activity in plasma was measured using the method described in Example 1. The results are summarized in Figure 5.

The mean FVIII activity in mice receiving plasmids pCB1007, pCB1019, or pCB1020 was 22.3%, 17.6%, and 17.8% of normal at day 6 after LNP administration. The mean FVIII activity in mice receiving plasmids pCB1007, pCB1019, or pCB1020 was 19.7, 14.1, and 14.9% of normal at day 9 after LNP administration. FVIII levels in mice receiving the three plasmids were not statistically significantly different on either day 6 or day 9 when assessed using equal variance (two-sample equal variance) two-tailed T-tests (all p values >0.28).

These results demonstrate that in the context of a donor template encoding a synthetic FVIII with a B domain substitution containing five N-glycan motifs in place of the B domain, codon optimizations col, co4, and co5 (all of which lack CG dinucleotides) produced similar levels of FVIII after targeted integration into albumin intron 1. Thus, there is no clear advantage to a particular codon optimization, and the CpG-free codon optimizations (e.g., col, co4, and co5) provide similar levels of synthetic FVIII protein after targeted integration.

Example 6: Combination of B-Domain Substitutions and Mutation of F309 to S or A A point mutation (F309S) within a potential binding site for the chaperone immunoglobulin-binding protein (BiP) in the A1 domain has been reported to improve secretion of B-domain deleted FVIII by approximately three-fold in cells in culture (M. Swaroop et al., J Biol Chem (1997) 272:24121-24). The F309A mutein of FVIII similarly improved secretion. It was reported that the combination of F309S with the 226 amino acid N-terminal portion of the B domain improved in vivo FVIII levels in mice 20-30 fold compared to B domain deleted FVIII, whereas the addition of the 226 amino acid N-terminal portion of the B domain improved FVIII levels only 5 fold (H.Z. Miao et al., Blood (2004) 103(9):3412-19).

To evaluate whether the combination of B domain substitutions, replacing the phenylalanine residue at 309 with serine or alanine, would result in further improvement of FVIII expression after targeted integration, plasmids pCB1025 (SEQ ID NO: 334) and pCB1026 (SEQ ID NO: 335) were constructed. Both plasmids contained a codon-optimized FVIII DNA sequence at codon co4 with a B domain substitution containing five N-linked glycosylation sites. The plasmids had the following elements: pUC19 plasmid backbone | target site for gRNA mALBTI | 18 bp spacer | splice acceptor (SA) | TG dinucleotide | FVIII sequence with 5 B domain substitutions (co4) | polyA (sPA). Plasmid pCB1007 was identical to pCB1025 and pCB1026, except that pCB1025 had Ala instead of Phe at position 309, and pCB1026 had Ser instead of Phe at position 309. Plasmid pCB1007 was used as a control in this study.

The experimental protocol was the same as in Example 1. Plasmids pCB1007, pCB1025, and pCB1026 were purified using the Qiagen EndoFree® maxiprep kit (cat #12362) and then diluted to a final concentration of 15 μg/mL in 0.9% saline. Cohorts of hemophilia A mice were injected with 2 mL of diluted plasmid DNA per mouse by HDI. The next day, mice were given an RO injection of C12-200-based LNPs encapsulating spCas9 mRNA (1 mg/kg) and gRNA mAlbT1 (1 mg/kg). Five days after LNP administration (pCB1025, pCB1026) or six days after LNP administration (pCB1019), blood samples were collected by RO blood sampling into sodium citrate (sodium citrate to blood ratio 1:9) and plasma was collected by centrifugation. FVIII activity in plasma was measured using the method described in Example 1. FVIII activity was similar in the three groups of mice injected with pCB1019, pCB1025, or pCB1026, with mean FVIII activity of 17.6%, 27.2%, and 24.5%, respectively.

FVIII activity in the blood of the same hemophilia A mice was also assayed 9 days after LNP (pCB1019-injected mice) or 7 days after LNP (pCB1025- and pCB1026-injected mice). Mice were then sacrificed and whole livers were prepared and analyzed for integration frequency as described in Example 1 above. Targeted integration frequencies were similar among the three groups, with average frequencies of 0.42 in pCB1019-injected mice, 0.47 in pCB1025-injected mice, and 0.36 in pCB1026-injected mice.

Normalizing the FVIII levels in the blood of each mouse to the integration frequency provides a measure of the intrinsic expression efficiency of the FVIII coding sequence. The mean ratio of FVIII levels divided by the targeted integration frequency was 37.4 in mice injected with pCB1019, 41.5 in mice injected with pCB1025, and 49.9 in mice injected with pCB1026. The difference in FVIII activity normalized to targeted integration in mice injected with pCB1025 and pCB1026 compared to mice injected with pCB1019 was not statistically significant (two-tailed Student's T-test), demonstrating that changing amino acid F309 to serine or alanine (in the context of a FVIII-BDD cassette containing five N-glycan motifs in place of the B domain) does not improve FVIII expression. Therefore, not all amino acid changes made to the VIII protein affect expression of VIII after targeted integration into albumin intron 1.

Example 7: Targeted integration of synthetic FVIII into transferrin intron 1 by CRISPR/Cas nuclease results in therapeutic level expression of human FVIII DNA construct To investigate integration into and expression from the transferrin locus as an alternative to the albumin locus, a human FVIII donor cassette (SEQ ID NO:224) was constructed with sequence elements in 5' to 3' order as follows: AAV2 inverted terminal repeat (ITR)|gRNA mTF-T2 target site|18 bp spacer|splice acceptor|sequence encoding the last four amino acids of mouse transferrin signal peptide (ggctgtgtctggct, SEQ ID NO:225)|synthetic FVIII coding sequence|polyadenylation signal (spA)|gRNA mTF-T2 target site|AAV2 inverted terminal repeat (ITR). The sequence of the target site of gRNA mTF-T2 is the reverse complement of the target sequence in the mouse genome, which may favor integration in the forward orientation. The polyadenylation signal is a short 49 bp sequence that has been reported to efficiently induce polyadenylation (N. Levitt et al., Genes Dev (1989) 3:1019-25). The synthetic FVIII coding sequence contains the amino acid sequence αβ ...
The B domain contained a heterologous 31 amino acid sequence that replaces the B domain, encoding a B domain replacement containing: This sequence contains six tripeptides that correspond to N-linked glycosylation sites (represented in bold) and has been shown to improve expression of FVIII (J. McIntosh et al., Blood (2013) 121:3335-44).

Packaging of pCB1009 FVIII donor DNA into AAV8 was achieved using established virus packaging methods in HEK293 cells transfected with three plasmids; one encoding AAV packaging proteins, a second encoding adenoviral helper proteins, and a third containing FVIII donor DNA sequences flanked by AAV ITR sequences. Transfected cells produced AAV particles of a serotype specified by the composition of the AAV capsid proteins encoded on the first plasmid. These AAV particles were harvested from cell supernatants or from supernatants and lysed cells and purified on a CsCl gradient. Purified virus particles were quantified by measuring the genome copy number of donor DNA by digital droplet PCR (DD-PCR).

In vivo testing of constructs Cohorts of five hemophilia A mice were injected intravenously (i.v.) into the tail vein with AAV8-pCB1009 at a dose of ^2x1012 vg/kg body weight. The AAV8 virus preferentially transduces hepatocytes. Four weeks later, the same mice were injected intravenously with a 1:1 (by mass of RNA) mixture of two LNPs, one encapsulating spCas9 mRNA and one encapsulating the guide RNA mTF-T2, at a total RNA dose of 2 mg/kg body weight. The LNPs are primarily taken up by hepatocytes. Ten days after administration of the LNPs, blood samples were obtained and assayed as described above. FVIII activity averaged 954% (±251%) of normal human FVIII levels (Figure 6), corresponding to 9.54 IU/mL or 9.5-fold the average level in non-hemophilic humans. In naive hemophilia A mice, FVIII activity was undetectable (<0.5% of normal).

These data demonstrate that targeted integration of FVIII coding sequences into intron 1 of transferrin can result in high levels of FVIII expression and activity, and demonstrate the utility of this method for treating conditions with defective FVIII, such as hemophilia A.

Example 8: Further Delivery Modes In another embodiment, the donor template is delivered in vivo using a non-viral LNP delivery system. The DNA molecule is encapsulated in LNP particles similar to those described above and delivered to the liver by intravenous injection. Although the escape of DNA from endosomes to the cytoplasm occurs relatively efficiently, the transfer of large charged DNA molecules into the nucleus is not efficient. In some cases, mimicking the AAV genome by incorporating AAV ITR sequences into the donor template improves the delivery of DNA to the nucleus. In this case, the ITR sequences stabilize the DNA or otherwise improve nuclear transfer. Removing CG dinucleotides (CpG sequences) from the donor template sequence also improves nuclear delivery. DNA containing CG dinucleotides is recognized and eliminated by the innate immune system. Removal of CpG sequences present in artificial DNA sequences improves the persistence of DNA delivered by non-viral and viral vectors. The process of codon optimization generally increases the content of CG dinucleotides, as the most frequent codons often have a C residue at the third position, which increases the chance of generating CG if the next codon begins with a G. The combination of LNP delivery of a donor template, followed by delivery of LNPs containing gRNA and Cas9 mRNA 1 hour to 5 days later, is evaluated in hemophilia A mice.

In vivo delivery of gRNA and Cas9 mRNA can be achieved by known methods. In one method, gRNA and Cas9 protein are expressed from an AAV viral vector. In this case, transcription of gRNA is driven by the U6 promoter, and transcription of Cas9 mRNA is driven by either a ubiquitous promoter, e.g., EF1-α, or a liver-specific promoter/enhancer, such as the transthyretin promoter/enhancer. The size of the spCas9 coding sequence (4.4 Kb) precludes inclusion of spCas9 and gRNA cassettes in a single AAV, thereby requiring separate AAVs to deliver gRNA and spCas9. In the second case, an AAV vector is used that has sequence elements that promote self-inactivation of the viral genome. In this case, cleavage of the vector DNA in vivo occurs by including a cleavage site for the gRNA in the vector DNA. By including a cleavage site at a location that blocks expression of Cas9 when cleaved, Cas9 expression is limited to a shorter period of time. A third alternative approach for delivering gRNA and Cas9 to cells in vivo employs a non-viral delivery method. In one example, LNPs are used as a non-viral delivery method. Several different ionizable cationic lipids are available for use in LNPs. These include C12-200, MC3, LN16, and MD1, among others. In one type of LNP, a GalNac moiety is attached to the outside of the LNP and acts as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs for delivery of gRNA and Cas9 mRNA to the liver.

Example 9: Targeted integration of a therapeutic coding sequence in mouse fibrinogen alpha intron 1 To examine integration into and expression from the fibrinogen alpha locus as an alternative to the albumin or transferrin loci, an AAV8 virus (AAV8-pCB1010, SEQ ID NO:361) was constructed harboring a cassette with the following elements: a target site for gRNA mFGA-T6, an 18 bp spacer, a FIX splice acceptor, a mature human FVIII coding sequence with a modified N-terminus to complete the FGA signal peptide after splicing into endogenous FGA exon 1, in which the B domain has been replaced with six N-glycan motifs, a polyadenylation sequence, and a target site for gRNA mFGA-T6.

Hemophilia A mice were injected with AAV8-pCB1010 and 28 days later received LNPs encapsulating T6 gRNA (targeting mouse fibrinogen alpha intron 1) and Cas9 mRNA. Ten days after administration of LNPs, blood samples were collected by retro-orbital bleed into capillary tubes containing sodium citrate (1:9 ratio of sodium citrate to blood) and plasma was collected by centrifugation. Plasma samples were then assayed for FVIII as described above. Assay results were reported as a percentage of normal human FVIII activity (normal defined as 1 IU/mL). FVIII activity averaged 1124% (±527%) of normal human FVIII levels, corresponding to 11.24 IU/mL or 11-fold the average level in non-hemophilic humans. In naive hemophilia A mice, FVIII activity was undetectable (<0.5% of normal). The AAV8-pCB1010 virus contains a VIII cassette whose coding sequence lacks a signal peptide and lacks a promoter, so the virus alone cannot produce a secreted VIII protein.

These data demonstrate the suitability of fibrinogen as a site for the insertion of coding sequences. Furthermore, they demonstrate that B domain-substituted FVIII sequences can be used to express useful quantities of FVIII. Thus, such constructs and methods can be used to treat disorders associated with defective FVIII.

Example 10: Identification and selection of guide RNAs that efficiently cleave at human albumin intron 1 in primary human hepatocytes in culture To demonstrate the operation of the system of the present invention in human hepatocytes, four gRNAs (T4-SEQ ID NO:357, T5-SEQ ID NO:358, T11-SEQ ID NO:359, and T13-SEQ ID NO:360) were prepared based on their complete identity between humans and non-human primates, as well as screening for cleavage efficiency in HuH7 and HepG2 cells for evaluation of cleavage efficiency in primary human hepatocytes. Primary human hepatocytes (obtained from BioIVT) were thawed, transferred to cryopreserved Hepatocyte Recovery Media (CHRM) (Gibco), pelleted at low speed, and then plated in InVitroGRO™ CP Media (BioIVT) + Torpedo™ Antibiotic Mix (BioIVT) at a density of 0.7 x ¹⁰⁶ cells/mL in 24-well plates pre-coated with Collagen IV (Corning). Plates were incubated at 37°C in 5% CO2. After the cells had attached ( _3-4 hours after plating), dead cells that had not attached to the plate were washed away with fresh warm complete media, additional media was added, and the cells were incubated at 37°C in 5% _CO2 . To transfect cells, Cas9 mRNA (Trilink) and guide RNA were thawed on ice and then added to 30 μL of Opti-Mem™ medium (Gibco) at 0.6 μg of mRNA and 0.2 μg of guide RNA per well. 30 μL of MessengerMax™ (Thermo Fisher) diluted 2:1 volume to total nucleic acid weight in Opti-Mem™ was incubated with the Cas9 mRNA/gRNA Opti-Mem™ solution at room temperature for 20 minutes. This mixture was added dropwise to 500 μL of hepatocyte plating medium per well of cultured hepatocytes in a 24-well plate and the cells were incubated at 37° C. in 5% CO _2. Cells were washed and refed the following morning. Cells were harvested for genomic DNA extraction 48 hours after transfection by adding 200 μL of warm 0.25% trypsin-EDTA (Gibco) to each well and incubating at 37° C. for 5-10 minutes. After removing the cells, 200 μL of FBS (Gibco) was added to inactivate the trypsin. After adding 1 mL of PBS (Gibco), the cells were pelleted at 1200 rpm for 3 minutes and then resuspended in 50 μL of PBS. Genomic DNA was extracted using the MagMAX™ DNA Multi-Sample Ultra 2.0 Kit (Applied Biosystems) according to the kit instructions. The quality and concentration of genomic DNA were analyzed using a spectrophotometer. For TIDE analysis, genomic DNA was PCR amplified using primers flanking the predicted on-target cleavage site (AlbF: CCCTCCGTTTGTCCTAGCTTTTC, SEQ ID NO:353, and AlbR: CCAGATACAGAATATCTTCCTCAACGCAGA, SEQ ID NO:354) and Platinum® PCR SuperMix High Fidelity (Invitrogen™) using 35 cycles of PCR and an annealing temperature of 55° C. The PCR products were analyzed by agarose gel electrophoresis to ensure that the correct size product (1053 bp) was produced, then purified and sequenced using primers (forward: CCTTTGGCACAATGAAGTGG, SEQ ID NO: 355; reverse: GAATCTGAACCCTGATGACAAG, SEQ ID NO: 356). Sequence data was analyzed using a modified version of the TIDES algorithm named Tsunami (E.K. Brinkman et al., Nuc Acids Res (2014) 42(22): e168), which determines the frequency of indels present at the predicted cleavage site of the gRNA/Cas9 complex.

Guide RNAs containing either the 20 nucleotide target sequence or the 19 nucleotide target sequence (1 bp shorter at the 5' end) of the T4 (SEQ ID NO:357), T5 (SEQ ID NO:358), T11 (SEQ ID NO:359), and T13 (SEQ ID NO:360) guides were tested. The 19 nucleotide gRNAs may be more sequence specific, but the shorter guides may have lower potency (efficiency at double strand breaks measured as indels). Control guides targeting the human AAVS1 locus and human complement factors were included for comparison between donors. Indel frequency at the target site in albumin intron 1 was measured 48 hours after transfection using the TIDES method. Figure 7 summarizes the results of transfection of primary hepatocytes from four different human donors.

Results demonstrate cleavage efficiencies ranging from 20% to 80% for the different guides. The 20-nucleotide versions of each albumin gRNA were consistently more potent than the 19-nucleotide variants. The superior potency of the 20-nucleotide gRNAs may offset the possibility that the 19-nucleotide gRNAs may be beneficial in terms of less off-target cleavage. Guide RNA T4 showed the most consistent cleavage across the four cell donors, with an indel frequency of approximately 60%.

Example 11: Evaluation of FVIII expression from AAV8 viruses encapsulating codon-optimized FVIII coding sequences (without CpGs) with B-domain substitutions consisting of different numbers of N-glycans followed by a single LNP dose with gRNA targeting the transferrin locus This study evaluated FVIII expression from AAV8 viruses encoding FVIII with B-domain substitutions containing either 0, 1, 3, 5, or 6 glycans. The FVIII coding sequence was codon-optimized and then the CpGs were manually removed. The constructs used in this study are shown in FIG. 9.

On day 0, hemophilia A mice (8-10 weeks old) were administered each virus via tail vein injection. On day 28, hemophilia A mice were injected retro-orbitally with lipid nanoparticles (LNPs) encapsulating Cas9 mRNA (411 μg/ml) and guide RNA mTF-T2 (379 μg/ml). The test groups and doses are shown in Table 7.

Eleven days after administration of LNPs, blood samples were obtained and assayed as described above. Then, 18 days after administration of LNPs, blood samples were obtained via terminal cardiac bleed and assayed as described above.

The FVIII activity levels measured on day 11 are shown in Figure 10. The FVIII activity levels measured on day 18 are shown in Figure 11. The FVIII activity levels are provided in Tables 8 and 9.

After mice were sacrificed, whole livers were homogenized and total genomic DNA was extracted from a portion of the liver lysate. The frequency of targeted integration into albumin intron 1 in the forward orientation was quantified using the DD-PCR assay described in Example 1. The results are shown in Figure 12 and Table 10.

These data demonstrate that FVIII coding sequences containing either 0, 1, 3, 5 or 6 glycans can result in high levels of FVIII expression and activity, demonstrating the utility of this approach for treating conditions with defective FVIII, such as hemophilia A.

Although the present disclosure has been described at some length and with some specificity with respect to certain illustrated embodiments, the present disclosure should not be limited to any such details or embodiments or to any particular embodiment, but should be construed with reference to the appended claims to provide the broadest possible interpretation of such claims in view of the prior art, thus effectively encompassing the intended scope of the present disclosure.

SEQUENCE LISTING In addition to the sequences disclosed elsewhere in this disclosure, the following sequences are provided because they are referred to or used in the exemplary embodiments of the present disclosure, which are provided for illustrative purposes.

Claims (28)

1. A system for modifying a host cell DNA sequence comprising:
A deoxyribonucleic acid (DNA) endonuclease or a nucleic acid encoding said DNA endonuclease;
a guide RNA (gRNA) or a nucleic acid encoding said gRNA, comprising a spacer sequence complementary to a host cell locus; and a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein.
Including,
the synthetic FVIII protein comprises a B domain substitution;
the B domain substitution comprises 1-5 N - linked glycosylation sites and is 3-40 amino acids in length; and
The B domain substitution comprises the amino acid sequence of any one of SEQ ID NOs: 364, 362-363 , 371 , and 373. The system of claim 1 , wherein the DNA endonuclease is Cas9. The system of claim 1, wherein the donor template is encoded by an adeno-associated virus (AAV) vector. The system according to any one of claims 1 to 3, wherein the host cell locus is a locus of a gene expressed in the liver or a safe harbor locus, or the host cell locus is a locus of a gene encoding an acute phase protein. The system of claim 4, wherein the host cell locus is a locus of a gene encoding an acute phase protein, which is albumin, transferrin, or fibrinogen. The system of any one of claims 1 to 5, wherein the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in the host cell, and/or the nucleic acid encoding the DNA endonuclease is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The system of claim 6, wherein the nucleic acid encoding the DNA endonuclease is RNA, which is mRNA. The system of any one of claims 1 to 7, wherein the donor template nucleic acid sequence is codon-optimized for expression in a host cell, and/or the donor template nucleic acid sequence comprises a reduced content of CpG dinucleotides or is free of CpG dinucleotides compared to a wild-type nucleic acid sequence encoding FVIII. The system of any one of claims 1 to 8, wherein the donor template comprises a donor cassette comprising the nucleic acid sequence encoding a synthetic FVIII protein, the donor cassette being flanked on one or both sides by gRNA target sites. The system of claim 9, wherein the donor cassette is flanked on both sides by gRNA target sites or the donor cassette is flanked on the 5' side by gRNA target sites. The system of claim 10, wherein the gRNA target site is a target site of the gRNA in the system, or the gRNA target site of the donor template is a reverse complement of a gRNA target site in the cell genome for the gRNA in the system. The system according to any one of claims 1 to 11, wherein the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is contained in a liposome or lipid nanoparticle. The system of claim 12, wherein the liposome or lipid nanoparticle also comprises the gRNA and/or the DNA endonuclease is complexed with the gRNA, thereby providing a ribonucleoprotein (RNP) complex. A method for editing a genome in a host cell in vitro, the method comprising providing the cell with a system according to any one of claims 1 to 13. 15. The method of claim 14, wherein (i) the gRNA or nucleic acid encoding the gRNA, and the DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell more than 4 days after the donor template is provided to the cell, and/or (ii) the gRNA or nucleic acid encoding the gRNA, and the DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell at least 14 days after the donor template is provided to the cell. 16. The method of claim 15, wherein one or more further doses of the gRNA or nucleic acid encoding the gRNA, and the DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell following the first dose of the gRNA or nucleic acid encoding the gRNA, and the DNA endonuclease or nucleic acid encoding the DNA endonuclease; or wherein one or more further doses of the gRNA or nucleic acid encoding the gRNA, and the DNA endonuclease or nucleic acid encoding the DNA endonuclease are provided to the cell following the first dose of the gRNA or nucleic acid encoding the gRNA, and the DNA endonuclease or nucleic acid encoding the DNA endonuclease until a target level of targeted integration of the nucleic acid sequence encoding a synthetic FVIII protein is achieved or a target expression level of the nucleic acid sequence encoding a synthetic FVIII protein is achieved. The method of claim 15 or 16, wherein the cells are liver cells, or the cells are human liver cells or human sinusoidal epithelial cells. A cell, the genome of said cell comprising DNA encoding a synthetic FVIII protein, said synthetic FVIII protein comprising a B domain substitution, said B domain substitution comprising 1-5 N - linked glycosylation sites and 3-40 amino acids in length;
A cell, wherein the B domain replacement protein sequence has the sequence of any of SEQ ID NOs: 364, 362-363 , 371 , and 373. The cell of claim 18, wherein the synthetic FVIII protein is operably linked to an endogenous albumin promoter, an endogenous transferrin promoter, or an endogenous fibrinogen alpha promoter, and/or the nucleic acid sequence encoding the synthetic FVIII protein is codon-optimized for expression in the cell. 20. The cell of claim 18 or 19 , wherein the cell is a human liver cell, or the cell is a human hepatocyte or a human sinusoidal epithelial cell. Use of the system according to any one of claims 1 to 13 or the cells according to any one of claims 18 to 20 for the treatment of hemophilia A. A deoxyribonucleic acid (DNA) endonuclease or a nucleic acid encoding said DNA endonuclease;
A guide RNA (gRNA) comprising a spacer sequence complementary to a host cell locus or a nucleic acid encoding said gRNA;
a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein; and
A kit including instructions for use,
the synthetic FVIII protein comprises a B domain substitution;
the B domain substitution comprises 1-5 N-linked glycosylation sites and is 3-40 amino acids in length; and
The B domain substitution comprises the amino acid sequence of any one of SEQ ID NOs: 364, 362-363, 371, and 373 . 23. The kit of claim 22, wherein the DNA endonuclease is Cas9. A nucleic acid comprising a polynucleotide sequence encoding a synthetic FVIII protein, said synthetic FVIII protein comprising a B domain substitution, said B domain substitution comprising 1-5 N - linked glycosylation sites and 3-40 amino acids in length;
A nucleic acid wherein the B domain substitution comprises the amino acid sequence of any one of SEQ ID NOs: 364, 362-363 , 371 , and 373. (i) the polynucleotide sequence encoding a synthetic FVIII protein is codon-optimized for expression in a host cell;
The nucleic acid of claim 24, wherein (ii) the polynucleotide sequence encoding the synthetic FVIII protein comprises a reduced content of CpG dinucleotides compared to a wild-type nucleic acid sequence encoding FVIII , and /or (iii) the nucleic acid is a viral vector. 26. The nucleic acid of claim 25, wherein the polynucleotide sequence encoding a synthetic FVIII protein is free of CpG dinucleotides. The nucleic acid of claim 25 , wherein the viral vector is an AAV vector. A synthetic FVIII protein, comprising a B domain replacement, said B domain replacement comprising 1 to 5 N-linked glycosylation sites and 3 to 40 amino acids in length, said B domain replacement protein sequence having any of SEQ ID NOs: 364, 362-363 , 371 , and 373.

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