AU2019243179B2 - Use of exosomes for targeted delivery of therapeutic agents - Google Patents

Abstract

Provided herein are methods of using exosomes that function like minicells to deliver therapeutic agents to diseased or disordered cells. In particular, the exosomes can be targeted to particular areas of the body using growth factor gradients. These gradients also serve to trigger expression of proteins inside the exosomes, from transfected nucleic acids, at the desired target.

Description

WO wo 2019/191444 PCT/US2019/024603

DESCRIPTION USE OF EXOSOMES FOR TARGETED DELIVERY OF THERAPEUTIC AGENTS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional

application number 62/649,057, filed March 28, 2018, the entire contents of which is

incorporated incorporatedherein by by herein reference. reference.

REFERENCE TO A SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted

in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said

ASCII copy, created on March 21, 2019, is named UTFC.P1363WO_ST25.tx UTFC.P1363WO_ST25.txtand andis is33

kilobytes in size.

BACKGROUND 1. Field

[0003] The present invention relates generally to the fields of biology, medicine, and

oncology. More oncology. Moreparticularly, it concerns particularly, the use it concerns of use the exosomes to targetto of exosomes delivery target ofdelivery therapeutic of therapeutic

agents to diseased or disordered cells.

2. Description of Related Art

[0004] Exosomes are small extracellular vesicles (EVs) with a lipid bilayer that

contain proteins and polynucleotides, including messenger RNAs (mRNAs), non-coding

RNAs and double-stranded genomic DNA (Kalluri, 2016; Raposo and Stoorvogel, 2013).

After their initial discovery as byproducts of reticulocyte differentiation (Harding et al., 1984;

Raposo and Stoorvogel, 2013), it is now generally accepted that exosomes are secreted by

virtually all mammalian cells and found in all body fluids (El-Andaloussi et al., 2013;

Kalluri, 2016).

[0005] Exosomes are part of a larger group of extracellular vesicles, which also

include microvesicles and apoptotic bodies (Colombo et al., 2014). Amongst extracellular

vesicles, exosomes are typically distinguished through their unique biogenesis via the

endocytic pathway. Endocytic vesicles mature into late endosomes, also known as

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

multivesicular bodies, which contain a number of intracellular vesicles (ILVs) generated

through invagination of the endosomal membrane. Through a likely fusion of these

multivesicular bodies with the plasma membrane, exosomes are released into the extracellular

space and enter circulation (Bastos et al., 2017; Colombo et al., 2014). As a result of their

endocytic origin, exosomes membranes have a similar polarity to cellular membranes,

containing membrane proteins anchored with their intracellular domains facing the lumen and

the extracellular domains facing the extracellular space. While the protein content of

exosomes varies depending on their cellular origin, several proteins seem to be generally

enriched. These include members of the tetraspanin family and components of the endocytic

and ILV maturation pathways, such as Rab proteins and members of the ESCRT complex.

Interestingly, different proteomics studies performed with exosomes derived from many

different celltypes different cell types have have identified identified many constituents many constituents associated associated with the with the protein protein translation translation

machinery, such machinery, such as as eukaryotic eukaryotic initiation initiation factors, factors, ADP ribosylation ADP ribosylation factors, proteins factors, ribosomal ribosomal proteins

(Pisitkun et al., 2004; Valadi et al., 2007). Additionally, a subset of transcriptional and

translation regulators identified in exosomes by proteomic analysis has been suggested to be

delivered to recipient cells, altering their pattern of gene and protein expression (Ung et al.,

2014).

[0006] Amongst the proteins commonly identified in exosomes are growth factor

receptors, such as the epithelial growth factor receptor (EGFR). EGFR is a member of the

ErbB family of growth factor receptors, which also includes HER2, HER3 and HER4

(Seshacharyulu et al., 2012). Upon binding one of its ligands, such as the epitheial growth

factor (EGF), the receptor dimerizes, forming either homodimers or heterodimers with other

members of the ErbB family (Seshacharyulu et al., 2012). This dimerization activates the

receptor's intrinsic kinase activity, leading to the autophosphorylation of different key

tyrosine residues on its cytoplasmic domain. This authophosphorylation reaction recruits

different adaptor proteins containing SH2 and PTB (phosphotyrosine binding) domains, such

as Shc and GRB2, which mediate different downstream signaling activities, including the

synthesis of relevant proteins (Normanno et al., 2006; Tomas et al., 2014). Phosphorylated

EGFR is ultimately ubiquitinated and transported to the endosomal pathway, from which it

will either recycle back to the membrane or remain in the late endosomal pathway leading to

integration into multivesicular bodies or lysosomal degradation (Tomas et al., 2014). Since

multivesicular bodies originate exosomes, it is likely that the post-phosphorylation recycling

of EGFR (and other growth factors) contribute to their membrane localization in these extracellular vesicles.

[0007] EGFR signaling has been shown to be important for the progression of different malignancies, such as glioblastoma, lung cancer, and breast cancer (Lim et al., 2019243179

5 2016; Liu et al., 2012; Masuda et al., 2012; Morgillo et al., 2016; Westphal et al., 2017; Zhang et al., 2013). Perhaps for this reason, most studies of EGFR in exosomes have been performed in the context of cancer development. EGFR signaling has particularly been implicated in the patterns of cellular uptake and secretion of exosomes from different origins. In mantle cell carcinoma cells, incubation with gefitinib (an EGFR 10 inhibitor) has been shown to dramatically decrease the rate of exosomes uptake (Hazan- Halevy et al., 2015). Treatment of lung cancer cells with gefitinib leads to an increased secretion of exosomes, which mediate horizontal transfer of cisplatin resistance (Li et al., 2016). The transfer of EGFR via cancer cell-derived exosomes has also been known to cause alterations in components of the microenvironment, such as endothelial cells and 15 T cells (Al-Nedawi et al., 2009; Huang et al., 2013). More recently, exosomes derived from gastric cancer cells containing EGFR were shown to be delivered to stromal cells in the liver, mediating metastasis (Zhang et al., 2017). Finally, exosomes derived from breast cancer cells were shown to contain functional phosphorylated forms of EGFR, which can be transferred to monocytes mediating their survival through activation of the 20 ERK pathway (Song et al., 2016).

[0008] While the delivery of EGFR and members of the protein translation machinery by exosomes seems to have clear biological importance in the context of cell- cell communications, these properties may be harnessed to target the delivery of therapeutic agents to certain tissues and to induce therapeutic protein production at the 25 desired delivery site.

[0008A] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority 30 date of each of the appended claims.

SUMMARY

[0009] Here, protein synthesis was induced in exosomes through growth factor stimulation. Exosomes that contain DNA, RNA, and proteins, can respond to biological stimuli, and initiate properties such as migration, multiplication, initiation of signaling 5 network/cascade, transcription, and protein translation. Thus, in one embodiment, 2019243179

provided herein are exosomes with the ability to function like minicells. As discussed further below, these minicell-like exosomes can be employed in numerous therapeutic means to treat various disease and/or disorders.

[0010] In one embodiment, provided herein are methods of treating a disease or 10 disorder in a patient in need thereof, the method comprising (a) obtaining exosomes having a growth factor receptor on their surface; (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; (c) administering the transfected exosomes to a patient; (d) providing a growth factor gradient at a site of the disease or disorder to attract the exosomes to the site and stimulate production of the therapeutic protein at the 15 site, thereby treating the disease in the patient.

[0010A] In one embodiment, provided herein is a method of treating cancer in a patient in need thereof, the method comprising: (a) obtaining exosomes having an epidermal growth factor receptor on their surface; (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; (c) stimulating the exosomes with 20 recombinant epidermal growth factor to induce protein expression by the exosomes; (d) administering the transfected exosomes to a patient; wherein the cancer provides an epidermal growth factor gradient at a site of the cancer to attract the exosomes to the site and deliver the therapeutic protein to the site, thereby treating the disease in the patient.

[0010B] In one embodiment, provided herein is the use of exosomes having an 25 epidermal growth factor receptor on their surface in the manufacture of a medicament for treating cancer, wherein treating the cancer comprises: (a) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; (b) stimulating the exosomes with recombinant epidermal growth factor to induce protein expression by the exosomes; (c) administering the transfected exosomes to a patient; wherein the cancer provides an

epidermal growth factor gradient at a site of the cancer to attract the exosomes to the site and deliver the therapeutic protein to the site, thereby treating the disease in the patient.

[0011] In some aspects, the method is further defined as a method of 5 administering a therapeutic protein to a diseased cell in a patient. In some aspects, the 2019243179

exosomes obtained in step (a) are obtained from a body fluid sample obtained from the patient. In some aspects, the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum. In some aspects, the nucleic acid is an mRNA, a plasmid, or a cDNA.

10 [0012] In some aspects, the disease or disorder is cancer, an injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a kidney disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a urogenital disorder, or a bone disease or disorder. In certain aspects, the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, 15 esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. In some aspects, the site of the disease or disorder is a tumor. In some aspects, the cancer is metastatic. In certain aspects, the site of the disease or disorder is a metastatic node.

20 [0013] In some aspects, the therapeutic protein is a kinase, a phosphatase, or a transcription factor. In certain aspects, the therapeutic protein corresponds to a wildtype version of a protein that is mutated or inactivated in a cell at the site of the disease or disorder. In certain aspects, the therapeutic protein corresponds to a dominant negative version of a protein that is hyperactive in a cell at the site of the disease or disorder. In 25 certain aspects, the disease or disorder is cancer, wherein the therapeutic protein is a tumor suppressor. In some aspects, the exosomes comprise CD47 on their surface. In some aspects, transfection comprises electroporation.

[0014] In some aspects, the method further comprises administering at least a second therapy to the patient. In some aspects, the second therapy comprises a surgical

therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.

[0015] In one embodiment, methods are provided of treating a disease or disorder in a patient in need thereof, the method comprising (a) obtaining exosomes 5 having a growth factor receptor on their surface; (b) transfecting the exosomes with 2019243179

therapeutic agent; (c) administering the transfected exosomes to a patient; (d) providing a growth factor gradient at a site of the disease or disorder to attract the exosomes to the site and deliver the therapeutic agent to the site, thereby treating the disease in the patient.

[0015A] In one embodiment, provided herein is a method of treating cancer in a 10 patient in need thereof, the method comprising: (a) obtaining exosomes having an epidermal growth factor receptor on their surface; (b) incorporating a therapeutic agent into the exosomes; (c) stimulating the exosomes with recombinant epidermal growth factor to induce protein expression by the exosomes; (d) administering exosomes obtained from step (c) to a 15 patient; wherein the cancer provides an epidermal growth factor gradient at a site of the cancer to attract the exosomes to the site and deliver the therapeutic agent to the site, thereby treating the cancer in the patient.

[0015B] In one embodiment, provided herein is the use of exosomes having an epidermal growth factor receptor on their surface in the manufacture of a medicament 20 for treating cancer, wherein treating the cancer comprises: (a) incorporating a therapeutic agent into the exosomes; (b) stimulating the exosomes with recombinant epidermal growth factor to induce protein expression by the exosomes; (c) administering exosomes obtained from step (b) to a patient, wherein the cancer provides an epidermal growth factor gradient at a site of the cancer to attract the exosomes to the site and deliver the 25 therapeutic agent to the site, thereby treating the cancer in the patient.

[0016] In some aspects, the method is further defined as a method of administering a therapeutic agent to a diseased cell in a patient. In some aspects, the exosomes obtained in step (a) are obtained from a body fluid sample obtained from the

patient. In certain aspects, the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum.

[0017] In some aspects, the therapeutic agent is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug. In certain 2019243179

5 aspects, the antibody binds an intracellular antigen. In certain aspects, the antibody is a full-length antibody, an scFv, a Fab fragment, a (Fab)2, a diabody, a triabody, or a minibody. In certain aspects, the inhibitory RNA is a siRNA, shRNA, miRNA, or pre- miRNA. In certain aspects, the gene editing system is a CRISPR/Cas system. In certain aspects, the therapeutic protein is a kinase, a phosphatase, or a transcription factor. In 10 certain aspects, the therapeutic protein corresponds to a wildtype version of a protein that is mutated or inactivated in a cell at the site of the disease or disorder. In certain aspects, the therapeutic protein corresponds to a dominant negative version of a protein that is hyperactive in a cell at the site of the disease or disorder. In some aspects, the small molecule drug is an imaging agent.

15 [0018] In some aspects, the disease or disorder is cancer, an injury, an autoimmune disorder, a neurological disorder, a gastrointestinal disorder, an infectious disease, a kidney disease, a cardiovascular disorder, an ophthalmic disorder, a skin disease or disorder, a urogenital disorder, or a bone disease or disorder. In certain aspects, the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, 20 esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. In some aspects, the site of the disease or disorder is a tumor. In some aspects, the cancer is metastatic. In some aspects, the site of the disease or disorder is a metastatic node. In some aspects, the disease or 25 disorder is cancer, wherein the therapeutic protein is a tumor suppressor. In some aspects, the disease or disorder is cancer, wherein the therapeutic agent is an inhibitory RNA targeting an oncogene.

[0019] In some aspects, the exosomes comprise CD47 on their surface. In some aspects, transfection comprises electroporation. In some aspects, the method further 30 comprises administering at least a second therapy to the patient. In some aspects, the

-6A-

second therapy comprises a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.

[0020] In further aspects, exosomes for use according to the embodiments are comprised in a tissue scaffold matrix. For example, such a matrix may be a synthetic 2019243179

5 matrix, such a matrix that degradable or can be absorbed in tissues. In further aspects, the matrix may be a living tissue matrix. In some aspects, a exosomes of the embodiments are cultured in a matrix.

[0021] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully 10 formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

15 [0022] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0022A] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated 20 element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0023] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” 25 As used herein “another” may mean at least a second or more.

-6B-

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

[0024] Throughout this application, the term "about" is used to indicate that a value

includes the inherent variation of error for the device, the method being employed to

determine the value, or the variation that exists among the study subjects.

[0025] Other objects, features and advantages of the present invention will become

apparent from the following detailed description. It should be understood, however, that the

detailed description and the specific examples, while indicating preferred embodiments of the

invention, are given by way of illustration only, since various changes and modifications

within the spirit and scope of the invention will become apparent to those skilled in the art

from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included

to further demonstrate certain aspects of the present invention. The invention may be better

understood by reference to one or more of these drawings in combination with the detailed

description of specific embodiments presented herein.

[0027] FIGS. 1A-E. EGFR phosphorylation in exosomes. FIG. 1A ---- Immunoblot of

EGFR expression on exosomes obtained from different human and murine cell lines. The

exosomes marker CD81 is used as a loading control, and to confirm the exosomal origin of

protein extracts. FIG. 1B - Immunoblot showing phosphorylation of EGFR on exosomes

derived from MDA-MB-231 cells, but not MCF10A cells, after incubation with 500 ng/ml

rhEGF for 15 minutes at 37°C. Phosphorylation levels are detected using an antibody specific

for the Tyr1068 residue of EGFR. EGFR levels are shown as a loading control, to confirm

differences in phosphorylation. Band densitometry quantification was performed using

ImageJ software. FIG. 1C --- Immunoblot showing the presence of EGFR adaptor proteins Shc She

and GRB2, as well increased levels of phosphorylated-ERK protein, in exosomes derived

from MDA-MB-231 cells, with and without rhEGF stimulation for 15 minutes at 37°C. The

exosomes marker CD81 is used as a loading control, and to confirm the exosomal origin of

protein extracts. FIG. 1D - GRB immunecomplexes were obtained from protein extracts of

MDA-MB-231 exosomes, with and without incubation with 500 ng/ml rhEGF for 15 minutes

at 37°C, using a GRB2 specific antibody. Immunoblot analysis of the immunocomplexes

shows association of GRB2 with EGFR only upon rhEGF stimulation. Non-specific isotype

control IgG was used as a negative control for the GRB2 pulldown. Equal volumes of the

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

stimulated and unstimulated extracts were probed for B-actin ß-actin as input control. FIG. 1E ----

Similar experiment using an Shc She antibody for the pull down experiment in duplicates, also

showing association with EGFR only after stimulation with 500 ng/ml rhEGF for 15 minutes

at 37°C. Non-specific isotype control IgG was used as a negative control for the Shc

pulldown. Equal volumes of the stimulated and unstimulated extracts were probed for bactin

as an input control.

[0028] FIGS. 2A-G. EGFR phosphorylation alters the content of exosomes. FIG .2A

- Luciferase-based --- ATP Luciferase-based determination ATP assay determination ran assay onon ran protein extracts protein obtained extracts from obtained exosomes from exosomes

either unstimulated or after rhEGF stimulation for 15 minutes at 37°C. Luciferin-derived

luminescence was measured in a plate reader and is presented as arbitrary units. Significance

was determined with a Mann-Whitney test (n === 4). = 4). FIG. FIG. 2B2B ---Immunoblot ---- Immunoblotanalysis analysisof of

exosomes from MDA-MB-231 cells incubated with 500 ng/ml rhEGF at 37°C for 48 h,

showing an increase in both pEGFR and GRB2 levels when compared to unstimulated

exosomes. FIG. 2C ---- Cellular component association analysis of mass spectrometry data

obtained for MDA-MB-231 exosomes unstimulated or after incubation with 500 ng/ml EGF

at 37°C for 48 h. A list of significant proteins identified for stimulated and unstimulated

exosomes was obtained and used as input for the open access FunRich functional enrichment

analysis tool in order to identify the subcellular origin of the identified proteins. FIG. 2D ---

Venn diagram depicting the overlap in proteins identified in control MDA-MB-231 exosomes

and exosomes incubated with 500 ng/ml rhEGF at 37°C for 48 h. FIG. 2E ---- Individual EGFR

and GRB2 protein scores obtained from mass spectrometry analysis of MDA-MB-231

exosomes with or without incubation with 500 ng/ml rhEGF for 48 h. FIG. 2F --- ----BCA BCA

analysis of protein extracts obtained from control MDA-MB-231 exosomes and exosomes

incubated with 500 ng/ml rhEGF at 37°C for 48 h. Significance was determined with a

Mann-Whitney test (n = 3). FIG. 2G --- Immunoblot analysis of B-actin ß-actin expression on protein

extracts obtained from control MDA-MB-231 exosomes and exosomes incubated with 500

ng/ml and 1000 ng/ml rhEGF at 37°C for 48 h. (*p < 0.05, **p < 0.01, ***p < 0.005, ****p

< 0.0001).

[0029] FIGS. 3A-F. Exosomes contain functional components for transcription and

translation. FIG. 3A --- Ultra-performance liquid chromatography-mass spectrometry

(UPLCMS) was used to detect free amino acids in MCF10A-, MDA-MB-231-, HDF, E10-,

and NIH-3T3-derived exosomes. Data are represented as a HeatMap using normalized signal intensities (log10), with hotter colors corresponding to higher intensity levels, as represented in the color legend. FIG. 3B - Immunoblot ---- of eIF4A1, Immunoblot eIF3A, of eIF4A1, and and eIF3A, eIF1A in protein eIF1A extracts in protein extracts of exosomes obtained from E10-, NIH-3T3-, MCF10A-, HDF-, and MDA-MB-231-derived exosomes. CD9 was used as a loading control. FIG. 3C --- In vitro translation assay using protein lysates from MCF10A- and MDA-MB-231-derived exosomes incubated with the pEMT7-GFP cDNA expression plasmid. Protein lysates obtained from cells were used as controls. FIG. 3D - Immunoblot analysis of RNA Polymerase II in exosomes protein extracts, with CD9 shown as a loading control. FIG. 3E ---- Autoradiography of exosomes derived from MDA-MB-231 and E10 cells cultured in the presence of 35-methionine. S-methionine.

Exosomes only, as well as exosomes cultured in the presence of 35S-methionine and ³S-methionine and

cycloheximide, were used as controls. FIG. 3F - BCA --- quantification BCA ofof quantification protein extracts protein extracts

obtained from exosomes immediately after isolation, or after incubation in cell-free

conditions for 24 h and 48 h. Significance was determined with a one-way ANOVA followed

by Tukey's by Tukey'smultiple comparisons multiple test (*ptest comparisons < 0.05, (*p**p< <0.05, 0.01, ***p < 0.005, < 0.01, ****p <0.0001, <0.005, 0.0001,

n == =3). 3).

[0030] FIGS. 4A-J. Exosomes synthesize new proteins through DNA transcription

and cap-dependent mRNA translation. FIG. 4A - qPCR analysis of GFP mRNA levels in

exosomes isolated from MDA-MB-231 cells and either non-electroporated, mock electroporated, or electroporated with a pCMV-GFP plasmid with or without the presence of

a-amanitin. Expression levels -amanitin. Expression levels were were normalized normalized to to GAPDH. GAPDH. FIG. FIG. 4B 4B --- --- Transmission Transmission electron electron

microscopy images of immunogold labeling, using anti-GFP antibody, of exosomes

electroporated with GFP plasmid and incubated in cell-free conditions for 48 h (bottom row).

Secondary antibody only was used as a negative control (top row). Gold particles are

depicted as black dots. Scale bar, 100 nm. FIG. 4C ---- Immunoblot of GFP protein expression

in exosomes electroporated with a pCMV-GFP plasmid and incubated for 12 hours, 2 days,

or 1 week at 37°C. Exosomes only and mock-electroporated exosomes were used as negative

controls. The exosomes marker TSG101 was used as a loading control for the presence of

exosomes. FIG. 4D - Immunoblot of GFP protein expression in exosomes electroporated

with GFP plasmid and incubated for several periods of time up to one month. Non-

electroporated exosomes were used as negative controls. The exosomes marker CD63 was

used as a loading control for the presence of exosomes. FIG. 4E --- Immunoblot of GFP

protein expression in exosomes electroporated with a GFP plasmid immediately after

isolation (0 h) or after incubation in cell-free conditions (24 h) and cultured as previously

WO wo 2019/191444 PCT/US2019/024603

described. Mock electroporated exosomes were used as negative controls. The exosomes

marker TSG101 was used as a loading control for the presence of exosomes exosomes.FIG. FIG.4F 4F--

Immunoblot of GFP protein expression in exosomes electroporated with a pCMV-GFP

plasmid and cultured with the translation inhibitor cycloheximide. Exosomes only and

exosomes mock electroporated were used as negative controls. TSG101 was used as a

loading control for the presence of exosomes. Band densitometry was performed using

ImageJ software. FIG. 4G - Immunoblot of GFP protein expression in exosomes electroporated with a GFP plasmid and cultured with the transcription inhibitor a-amanitin. -amanitin.

Exosomes only and exosomes mock electroporated were used as negative controls. TSG101

was used as a loading control for the presence of exosomes. Band densitometry was

performed using ImageJ software. FIG. 4H - Schematic representation of the bicistronic

plasmid used as a reporter for cap-dependent or cap-independent translation (pCDNA3-rLuc-

polIRESfLuc). FIG. 4I - Activities of renilla (r-Luc) and firefly luciferase (f-Luc) measured

by bioluminescence after 48 h incubation of exosomes electroporated with the bicistronic

plasmid. Non-electroporated exosomes were used as negative controls. FIG. 4J ----

Luminescence counts measured from exosomes incubated for 48 h after electroporation with

or without a plasmid with firefly luciferase expressed under a CMV promoter.

[0031] FIGS. 5A-E. Protein translation in exosomes generates functional proteins

and can be increased by growth factor stimulation. FIG. 5A - Confocal microscopy showing

the presence of GFP in MCF10A electroporated cells as well as in MCF10A cells, previously

treated with cycloheximide, incubated with MDA-MB-231-derived exosomes electroporated

with a pCMV-GFP plasmid and pre-incubated for 48 h. MCF10A cells treated with non-

electroporated MDA-MB-231-derived exosomes were used as a negative control. FIG. 5B -

Immunoblot analysis of GFP expression on protein lysates from MDA-MB-231-derived

exosomes electroporated with a p53-GFP expression plasmid. Non-electroporated exosomes

were used as negative controls. TSG101 was used as a loading control for the presence of

exosomes. FIG.5C --- p21 mRNA expression in MDA-MB-231 cells treated with mock

electroporated MDA-MB-231-derived exosomes or exosomes electroporated with the p53-

GFP plasmid with and without the presence of cycloheximide. Expression levels were

normalized normalized totothe the housekeeping housekeeping genegene GAPDH GAPDH. FIG. FIG. 5D - 5D - Immunoblot Immunoblot of exosomes of exosomes isolated isolated

from MDA-MB-231 cells, incubated with 100 ug/ml µg/ml of the translation inhibitor

cycloheximide. Exosomes lysates were probed for B-actin ß-actin and GAPDH. Band densitometry

quantification was performed using ImageJ software. FIG. 5E --- ----Immunoblot Immunoblotof ofGFP GFPprotein protein expression in exosomes electroporated with a pCMV-GFP plasmid, and then incubated in the presence of different concentrations of rhEGF at 37°C for 48 h. Exosomes mock electroporated and exosomes without rhEGF incubation are shown as negative controls. The exosomes marker CD81 is used as a loading control. Band densitometry quantification was performed using ImageJ software.

[0032] FIG. 6A-C. Exosomes derived from MDA-MB-231 cells demonstrate chemotaxis towards a gradient of growth factors. FIG. 6A --- Schematic demonstrating the set

up for exosomes retrograde migration assay. In short, 10 X 109 exosomes isolated 10 exosomes isolated from from

MDA-MB-231 cells were placed in the bottom well of a Corning Transwell® system. An

HTS Transwell® insert with 400 nm pores was placed on top of the exosomes suspension

containing either PBS, 20% FBS, or 10,000 ng/ml rhEGF and incubated at 37°C. The number

of exosomes on the top insert was measured by Nanosight NTA after different time points to

assess exosomes motility. FIGS. 6B&6C ---- Quantification of MDA-MB-231 exosomes on the

top insert of the retrograde migration assay, after 4 h (FIG. 6B) and 24 h (FIG. 6C) incubation

at 37°C, by Nanosight NTA. Significance was determined with a one-way ANOVA followed

by Newman-Keuls multiple comparison test. (*p < 0.05, **p < 0.01, < 0.01, < 0.005, < 0.005, ****p ****p < < 0.0001, n ==== 0.0001, 3). n 3).

[0033] FIGS. 7A-D. Tumor-bearing mice show increased protein synthesis in

delivered exosomes. FIG. 7A Schematic depicting ---- Schematic the experimental depicting plan for the experimental planthe forin vivo the in vivo

translation experiment. In short, female Balb/C mice were injected with 4T1 tumor

orthotopically and the tumor was allowed to grow to 500 mm³, after which mice were

injected with 30 billion MDA-MB-231 exosomes electroporated with a pCMV-mCherry

plasmid. The mice were euthanized 12 h after exosomes injection and serum was collected

for exosomes extraction. FIG. 7B - Graphic showing the tumor growth of mice injected with

4T1 tumors and electroporated exosomes, or 4T1 tumors alone, showing comparable growth

kinetics. FIG. 7C ---- Nanosight NTA analysis of exosomes extracted from the serum of healthy

mice injected with electroporated exosomes, as well as 4T1 tumor-bearing mice injected with

electroporated exosomes and 4T1 tumor bearing mice with no exosomes injection. All

exosomes show similar size peaks, around 100 nm. FIG. 7D - Nanosight NTA quantification

of serum exosomes shown in FIG. 7C, showing no significant differences in the exosomes

amount obtained from the serum of different animals, but a trend towards more exosomes in

4T1 tumor-bearing mice injected with MDA-MB-231 electroporated exosomes.

WO wo 2019/191444 PCT/US2019/024603

[0034] FIGS. 8A-G. Exosomes characterization. FIG. 8A --- Nanoparticle tracking

analysis of exosomes collected from MDA-MB-231 cells, obtained using the Nanosight NTA

2.1 Analytical Software. Left graph represents the size distribution of particles in solution

showing a mean size of 104 nm and also showing no peaks at larger sizes. Right graph

represents the distribution by size and concentration of particles in solution. FIG. 8B -

Atomic Force Microscopy image of exosomes (left image). Right graph represents the

distribution of particles in the area analyzed. FIG. 8C - Transmission electron micrograph of

MDA-MB-231 exosomes. Scale bar --- 100 nm. FIG. 8D --- *** Transmission electron micrograph

of immunogold labeled MDA-MB-231 exosomes using anti-CD9 antibody. Gold particles are

depicted as black dots. Scale bar --- 100 nm. FIG. 8E - Immunoblot --- analysis Immunoblot ofof analysis exosomes exosomes

markers CD9, CD63, and TSG101 in protein extracts of exosomes obtained from different

cell lines. FIG. 8F --- Imaging Flow Cytometry analysis of exosomes from MDA-MB-231

cells coupled to 0.4 um µm beads, using antibodies for markers CD9, CD81, CD82, and CD63.

FIG. 8G - Representative images of LB culture plates incubated with E. coli and either

MDA-MB-231 (top) or MCF10A (bottom) exosomes, showing colony formation on the E.

coli inoculated sides (left) and not on the exosomes inoculated sides (right).

[0035] FIGS. 9A-B. EGFR phosphorylation and downstream biological activity in

exosomes from MDA-MB-231 cells. FIG. 9A ---- Immunoblot of protein extracts obtained

from MDA-MB-231 cells and probed for p-EGFR and GRB2. B-actin ß-actin is used as a loading

control. FIG. 9B --- Immunoblot of immunocomplexes obtained with an anti-EGFR antibody

pull down of protein lysates from MDA-MB-231 exosomes with or without incubation with

500 ng/ml at 37°C for 15 minutes. Immuncomplexes were probed for GRB2. Non-specific

isotype control IgG was used as a negative control for the GRB2 pulldown. Equal volumes of

the stimulated and unstimulated extracts were probed for B-actin ß-actin as input control.

[0036] FIG. 10. Proteomics analysis of exosomes derived from various cells.

Heatmap representing the binary identification of all individual proteins included in the

Protein Translation pathway in the Reactome (Croft et al., 2014) database, in mass

spectrometry data obtained from mouse liver cells (Valadi et al., 2007), mouse fibroblasts

(Luga et al., 2012), human colorectal cancer cells (Choi et al., 2012), human plasma (Kalra et

al., 2013), human thymic tissue (Skogberg et al., 2013), and human urine (Gonzales et al.,

2009). Black represents the presence and white represents the absence of each protein in each

dataset. The summary column represents how ubiquitous each protein is in all analyzed datasets, with warmer colors representing a more widespread distribution among different types of exosomes.

[0037] FIGS. 11A-B. Proteomics analysis of exosomes from various origins. FIG.

11A - Heatmap ---- representing Heatmap the the representing number of proteins number identified of proteins in mass identified spectrometry in mass data spectrometry data

obtained from mouse liver cells (Valadi et al., 2007), mouse fibroblasts (Luga et al., 2012),

human colorectal cancer cells (Choi et al., 2012), human plasma (Kalra et al., 2013), human

thymic tissue (Skogberg et al., 2013), and human urine (Gonzales et al., 2009) that associate

with different pathways related to protein translation in the Reactome (Croft et al., 2014)

database. Warmer colors represent higher numbers of proteins identified per pathway. FIG.

11B - Heatmap representing the protein score of proteins associated with protein translation

identified in mass spectrometry obtained from exosomes isolated from HDF, NIH 3T3,

MDA-MB231, MCF10A, and E10 cells. Warmer colors represent a higher protein score.

[0038] FIGS. 12A-C. Exosomes contain nucleic acids and proteins associated with

the protein translation machinery. FIG. 12A - RNA extracted from exosomes of NIH-3T3,

E10, 67NR, 4T1, HDF, MCF10A, MCF7, and MDA-MB-231 cell lines were used to quantify

18S and 28S rRNAs by qPCR. Expression levels of the rNNAs rRNAs were normalized to U6

snRNA expression. The bars in each group represent, from left to right, NIH 3T3, E10,

67NR, 4T1, HDF, MCF10A, MCF7, and MDA-MB-231. FIG. 12B - RNA extracted from 4T1 exosomes and cells were used to identify the presence of tRNAMet, tRNAGly,

tRNALeu, tRNASer, and tRNAVal by digital qPCR. The bars in each group represent, from

left to right, Leu, Met, Val, Ser, and Gly. FIG. 12C --- Immunoprecipitation of eIF4A1

showing presence of eIF3A MCF10A and MDA-MB-231-derived exosomes. MB231 and MCF10A cell lysates were used as positive controls. The exosomes marker CD82 was used

as a loading control.

[0039] FIGS. 13A-E. DNA transcription and mRNA translation in exosomes derived

from MCF10A and MDA-MB-231 cells. FIG. 13A - Immunoblot of GFP protein expression

in exosomes isolated from MCF10A cells, electroporated with a pCMV-GFP plasmid and

incubated at 37°C for different periods of time. Exosomes only and mock electroporated

exosomes were used as negative controls. CD63 was used as a loading control and to confirm

the presence of exosomes. FIG. 13B - Plot depicting the amount of green exosomes detected

by NanoSight in MCF10A-derived exosomes electroporated with a GFP plasmid. MCF10A-

derived exosomes, MCF10A-derived mock electroporated exosomes as well as exosomes

13

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electroporated with a-amanitin and cycloheximide -amanitin and cycloheximide were were used used as as negative negative controls. controls. FIG. FIG. 13C 13C

- Flow --- cytometry Flow analysis cytometry ofof analysis beads attached beads toto attached exosomes after exosomes electroporation after with electroporation a GFP with a GFP

plasmid using increasing voltages, showing the percentages of beads with green fluorescent

signal. FIG. 13D ---- Immunoblot of ovalbumin protein levels in MDA-MB-231 exosomes

electroporated with a pCMV-Ova plasmid and incubated at 37°C for 48 h. B-actin ß-actin was used

as a loading control. FIG. 13E --- ----p21 p21mRNA mRNAexpression expressionin inMDA-MB-231 MDA-MB-231cells cellstreated treatedwith with

mock electroporated MDA-MB-231-derived exosomes or exosomes electroporated with the

p53-GFP plasmid and either added to the cells immediately (0 h) or allowed to incubate in

cell conditions at 37°C for 48 h before treatment (48 h). Exosomes were added to the cells for

either 30 minutes or 48 hours before RNA extraction. Expression levels were normalized to

the housekeeping gene GAPDH.

DETAILED DESCRIPTION

[0040] Extracellular vesicles (EVs), including exosomes, are nano-sized intercellular

communication vehicles having a lipid bilayer that encloses cytosol-like material. Exosomes

participate in several physiological processes and contain DNA, RNA, and proteins. It is

generally assumed that all contents in exosomes are derived from cells and they remain as

such in the exosomes until they enter other cells and deposit their contents. Exosomes are

released by all cells in large numbers and are considered as garbage bags that carry cellular

constituents into the extracellular space as a payload without any biological significance for

exosomes themselves per se.

[0041] Provided here are exosomes that behave like minicells and exhibit the ability

to biologically respond to stimuli and multiply and migrate just as cells do but without a

defined nucleus. These exosomes exhibit chemotaxis towards serum factors and upon

stimulation with growth factors such as EGF, will phosphorylate the EGFR receptor on their

surface and initiate a signaling cascade that leads to transcription and translation of new

proteins. When these exosomes are injected into tumor bearing mice, they preferentially

accumulate in the tumors. Collectively, the capacity for protein translation and growth factor

response by these exosomes provides them a functional role in tissue homeostasis and

modulation of disease.

WO wo 2019/191444 PCT/US2019/024603

I. Aspects of the Present Invention

[0042] Extracellular vesicles, and in particular exosomes, have gained much attention

over the last few years with identification of several constituents such as DNA, RNA, and

proteins. Moreover, exosomes have been implicated in influencing many diverse biological

processes via transfer of its content into recipient cells in different tissues, and facilitating a

unique form of cell-cell communication (Bastos et al., 2017). Alternatively, the potential of

exosomes as delivery vehicles for therapeutics, particularly in the context of cancer or

neurological pathologies was also reported (El-Andaloussi et al., 2012; Kamerkar et al.,

2017). However, the exact patterns of systemic distribution and organ tropism of exosomes

are still not fully understood.

[0043] However, the nuclear and cytoplasmic components of the exosomes are not

just used for passive transfer to recipient cells but can respond to external stimuli to

phosphorylate growth factor receptors, such as EGFR, and generate new proteins via active

transcription and translation. External stimulation of exosomes can initiate de novo biological

activities such as retrograde migration. It is conceivable that exosomes may function like

mini cells, albeit primitive with respect to their fine-tuned operations in response to external

stimuli. In fact, recently it has been suggested that exosomes could potentially constitute

extant representations of protocellular ribosomes, for which they would need to contain

rRNAs, which is confirm in this study (Sinkovics, 2015). Exosomes are biologically

responsive and migrate towards growth factor gradients in an active manner. Actin

remodeling might be involved. The patterns of actin polymerization could therefore constitute

an interesting target in the modulation of exosomes biodistribution.

[0044] Vesicles such as prostasomes obtained from different species have been shown

to contain different components of the glycolytic pathway, which allow them to produce ATP

in cell-free conditions (Ronquist et al., 2013a; Ronquist et al., 2013b). While direct

transcription in exosomes has not been previously reported, a study showed that exosomes

from bovine milk infected with bovine leukemia virus have been shown to exhibit reverse

transcriptase activity (Yamada et al., 2013). It was also recently demonstrated that

independent production of mature miRNAs in exosomes isolated from cancer cells is possible

(Melo et al., 2014). Here, exosomes were demonstrated to possess an intrinsic capacity for de

novo synthesis of functional proteins via DNA transcription coupled with mRNA translation.

Platelets can translate proteins from mRNA molecules remaining within them after

WO wo 2019/191444 PCT/US2019/024603

megakaryocyte differentiation (Weyrich et al., 2004). Nevertheless, DNA transcription

resulting in new mRNA molecules is not reported in platelets. Additionally, foci of mRNA

translation activity, associated with polyribosomes and mRNA binding proteins, is observed

in dendritic spines even when severed from the major body of the cell (Aakalu et al., 2001;

Smith et al., 2001; Steward and Levy, 1982). It is therefore clear that some cellular structures

have preserved the capacity for protein biosynthesis in the absence of a nucleus, perhaps in

order to support their specific biological functions in a rapid manner. Apart from protein

translation, exosomes are capable of DNA transcription via RNA polymerase II. It is well

established that basic transcription of naked DNA in nucleosome-free regions is possible with

minimal components of transcriptional machinery, namely only RNA Pol II and a cocktail of

six general transcription factors (GTFs) (Lorch et al., 2014; Nagai et al., 2017). It has also

been shown that exosomes contain a plethora of transcription factors that can be delivered to

cells in order to alter their patterns of protein expression (Ung et al., 2014). It is therefore

conceivable that exosomes contain naked DNA residues unbound by chromatin, which could

undergo transcription in the presence of these minimal transcriptional components.

[0045] This study also suggests that the required components for transcription/translation are likely exhausted within 24 h, resulting in a limited rate of

transcription and translation. Since it has been suggested that different subpopulations of

exosomes may possess distinct molecular characteristics (Willms et al., 2016), it is possible

that only a small subset of exosomes possess the capacity for de novo protein synthesis. The

newly synthetized proteins in exosomes are functionally active, suggestive of an appropriate

protein conformation. It has been shown that exosomes contain not only the components of

ribosomes, but also several molecular chaperones, such as Hsp60 and Hsp70. The ribosome

itself has an important role in co-translational protein folding, for instance, it can promote the

formation of secondary structures in newly formed proteins. The ribosome also acts as a

platform for the association of chaperones that can assist with the appropriate folding of

nascent proteins (Kramer et al., 2009). It is conceivable that these exosomes components

could contribute to the stabilization of newly formed proteins. Physical confinement, as

would be the case in the lumen of exosomes, can also have a stabilizing effect on the folding

of proteins (Rao and Cruz, 2013). The possibility, however, cannot be ruled out that many

proteins might exhibit inappropriate conformation or mis-folding. These could still have

important biological implications, as demonstrated with the recent unraveling of unexpected

features of the "dark proteome" (Perdigao et al., 2015), which suggested that proteins with unknown structure or intrinsically disordered regions may have important physiological functions.

[0046] A meticulous and quantitative identification of the proteins naturally

synthetized in exosomes is necessary to fully appreciate the biological significance of this

process. It is clear, however, that this could have significant impact in re-evaluating the

understanding of eukaryotic biology. Recent studies suggest that cells can selectively

incorporate mRNAs into exosomes (Raposo and Stoorvogel, 2013). This raises the possibility

that mRNAs selectively packaged into exosomes could be translated into proteins whose

expression is repressed in their cell of origin, as shown in this study as a proof of concept.

The identification of newly synthetized proteins in exosomes up to one month after

translation, suggests that exosomes-mediated production of proteins could lead to aa

significantly increased protein half-life, possibly due to lower levels of protein degradation

enzymes.

[0047] Taken together, these results collectively demonstrate exosomes possess

previously unappreciated biological activity with a potentially profound impact on body

homeostasis and tissue pathogenesis. One could speculate that growth factor gradients could

play a role on the systemic tropism of exosomes in the body. A disruption of the naturally

occurring pattern of growth factor production could have immediate consequences on both

the redistribution and delivery patterns of exosomes. These patterns of response could have

potential implications in determining cell-cell communication, particularly between distant

body sites. The fact that they can change their patterns of protein expression in response to

these extracellular cues would suggest that exosomes could act as the primary responders in

tissue injury. In conclusion, these findings provide a novel insight into the basic biology of

exosomes and inform on their biological functions in organism homeostasis and their

potential impact in disease states.

I. Lipid-based Nanoparticles

[0048] In some embodiments, a lipid-based nanoparticle is a liposomes, an exosomes,

lipid preparations, or another lipid-based nanoparticle, such as a lipid-based vesicle (e.g., a

DOTAP:cholesterol vesicle). Lipid-based nanoparticles may be positively charged,

negatively charged or neutral. Lipid-based nanoparticles may comprise the necessary

17

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

components to allow for transcription and translation, signal transduction, chemotaxis, or

other cellular functions.

A. Liposomes Liposomes

[0049] A "liposome" is a generic term encompassing a variety of single and

multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates.

Liposomes may be characterized as having vesicular structures with a bilayer membrane,

generally comprising a phospholipid, and an inner medium that generally comprises an

composition. Liposomes aqueous composition. aqueous Liposomes provided provided herein herein include include unilamellar unilamellar liposomes, liposomes,

multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein may be

positively charged, negatively charged, or neutrally charged. In certain embodiments, the

liposomes are neutral in charge.

[0050] A multilamellar liposome has multiple lipid layers separated by aqueous

medium. Such liposomes form spontaneously when lipids comprising phospholipids are

suspended in an excess of aqueous solution. The lipid components undergo self-

rearrangement before the formation of closed structures and entrap water and dissolved

solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions

may also dissolve in or associate with the lipid bilayer.

[0051] In specific aspects, a polypeptide, a nucleic acid, or a small molecule drug

may be, for example, encapsulated in the aqueous interior of a liposome, interspersed within

the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is

associated with both the liposome and the polypeptide/nucleic acid, entrapped in a liposome,

complexed with a liposome, or the like.

[0052] A liposome used according to the present embodiments can be made by

different methods, as would be known to one of ordinary skill in the art. For example, a

phospholipid, such as for example the neutral phospholipid dioleoylphosphatidylcholine

(DOPC), is dissolved in tert-butanol. The lipid(s) is then mixed with a polypeptide, nucleic

acid, and/or other component(s). Tween 20 is added to the lipid mixture such that Tween 20

is about 5% of the composition's weight. Excess tert-butanol is added to this mixture such

that the volume of tert-butanol is at least 95% 95%.The Themixture mixtureis isvortexed, vortexed,frozen frozenin ina adry dry

ice/acetone bath and lyophilized overnight. The lyophilized preparation is stored at -20°C

WO wo 2019/191444 PCT/US2019/024603

and can be used up to three months. When required the lyophilized liposomes are

reconstituted in 0.9% saline.

[0053] Alternatively, a liposome can be prepared by mixing lipids in a solvent in a

container, e.g., a glass, pear-shaped flask. The container should have a volume ten-times

greater than the volume of the expected suspension of liposomes. Using a rotary evaporator,

the solvent is removed at approximately 40°C under negative pressure pressure.The Thesolvent solventnormally normally

is removed within about 5 min to 2 h, depending on the desired volume of the liposomes.

The composition can be dried further in a desiccator under vacuum. The dried lipids

generally are discarded after about 1 week because of a tendency to deteriorate with time.

[0054] Dried lipids can be hydrated at approximately 25-50 mM phospholipid in

sterile, pyrogen-free water by shaking until all the lipid film is resuspended. The aqueous

liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed

under vacuum.

[0055] The dried lipids or lyophilized liposomes prepared as described above may be

dehydrated and reconstituted in a solution of a protein or peptide and diluted to an

appropriate concentration with a suitable solvent, e.g., DPBS. The mixture is then vigorously

shaken in a vortex mixer. Unencapsulated additional materials, such as agents including but

not limited to hormones, drugs, nucleic acid constructs and the like, are removed by

centrifugation at 29,000 X g and the liposomal pellets washed. The washed liposomes are

resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM. The

amount of additional material or active agent encapsulated can be determined in accordance

with standard methods. After determination of the amount of additional material or active

agent encapsulated in the liposome preparation, the liposomes may be diluted to appropriate

concentrations and stored at 4°C until use. A pharmaceutical composition comprising the

liposomes will liposomes willusually include usually a sterile, include pharmaceutically a sterile, acceptable pharmaceutically carrier orcarrier acceptable diluent,or such diluent, such

as water or saline solution.

[0056] Additional liposomes which may be useful with the present embodiments

include cationic liposomes, for example, as described in WO02/100435A1, U.S Patent

5,962,016, U.S. Application 2004/0208921, WO03/015757A1, WO04029213A2, U.S. Patent

5,030,453, and U.S. Patent 6,680,068, all of which are hereby incorporated by reference in

their entirety without disclaimer.

WO wo 2019/191444 PCT/US2019/024603

[0057] In preparing such liposomes, any protocol described herein, or as would be

known to one of ordinary skill in the art may be used. Additional non-limiting examples of

preparing liposomes are described in U.S. Patents 4,728,578, 4,728,575, 4,737,323,

4,533,254, 4,162,282, 4,310,505, and 4,921,706; International Applications PCT/US85/01161 and PCT/US89/05040, each incorporated herein by reference.

[0058] In certain embodiments, the lipid based nanoparticle is a neutral liposome

(e.g., a DOPC liposome). "Neutral liposomes" or "non-charged liposomes", as used herein,

are defined as liposomes having one or more lipid components that yield an essentially-

neutral, net charge (substantially non-charged). By "essentially neutral" or "essentially non-

charged", it is meant that few, if any, lipid components within a given population (e.g., a

population of liposomes) include a charge that is not canceled by an opposite charge of

another component (i.e., fewer than 10% of components include a non-canceled charge, more

preferably fewer than 5%, and most preferably fewer than 1%). In certain embodiments,

neutral liposomes may include mostly lipids and/or phospholipids that are themselves neutral

under physiological conditions (i.e., at about pH 7).

[0059] Liposomes and/or lipid-based nanoparticles of the present embodiments may

comprise a phospholipid. In certain embodiments, a single kind of phospholipid may be used

in the creation of liposomes (e.g., a neutral phospholipid, such as DOPC, may be used to

generate neutral liposomes). In other embodiments, more than one kind of phospholipid may

be used to create liposomes. Phospholipids may be from natural or synthetic sources.

Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols, and

phosphatidylethanolamines; because phosphatidylethanolamines and phosphatidyl cholines

are non-charged under physiological conditions (i.e., at about pH 7), these compounds may

be particularly useful for generating neutral liposomes. In certain embodiments, the

phospholipid DOPC is used to produce non-charged liposomes. In certain embodiments, a

lipid that is not a phospholipid (e.g., a cholesterol) may be used

[0060] Phospholipids include glycerophospholipids and certain sphingolipids.

Phospholipids include, but are not limited to, dioleoylphosphatidylycholine ("DOPC"), egg

phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"),

dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"),

distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoy! 1-myristoyl-2-palmitoyl phosphatidylcholine

("MPPC"), 1-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), 1-palmitoy1-2-stearoyl 1-palmitoyl-2-stearoyl phosphatidylcholine phosphatidylcholine ("PSPC"), ("PSPC"), l-stearoyl-2-palmitoyl 1-stearoyl-2-palmitoyl phosphatidylcholine phosphatidylcholine ("SPPC"), ("SPPC"), dilauryloylphosphatidylglycerol dilauryloylphosphatidylglycerol ("DLPG"), ("DLPG"), dimyristoylphosphatidylglycerol dimyristoylphosphatidylglycerol ("DMPG"), ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), dipalmitoylphosphatidylglycerol distearoylphosphatidylglycerol ("DPPG"), ("DSPG"), distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoylphophatidylethanolamine ("DSPE"), ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2- diarachidoyl-sn-glycero-3-phosphocholine diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), ("DBPC"), 1,2-dieicosenoyl-sn-glycero-3- 1,2-dieicosenoyl-sn-glycero-3- phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoy! palmitoyloeoyl phosphatidylcholine ("POPC"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

B. Exosomes

[0061] The terms "microvesicle" and "exosomes," as used herein, refer to a

membranous particle having a diameter (or largest dimension where the particles is not

spheroid) of between about 10 nm to about 5000 nm, more typically between 30 nm and 1000

nm, and most typically between about 50 nm and 750 nm, wherein at least part of the

membrane of the exosomes is directly obtained from a cell. Most commonly, exosomes will

have a size (average diameter) that is up to 5% of the size of the donor cell. Therefore,

especially contemplated exosomes include those that are shed from a cell.

[0062] Exosomes may be detected in or isolated from any suitable sample type, such

as, for example, body fluids. As used herein, the term "isolated" refers to separation out of its

natural environment and is meant to include at least partial purification and may include

substantial purification. As used herein, the term "sample" refers to any sample suitable for

the methods provided by the present invention. The sample may be any sample that includes

exosomes suitable for detection or isolation. Sources of samples include blood, bone marrow,

pleural fluid,peritoneal pleural fluid, peritoneal fluid, fluid, cerebrospinal cerebrospinal fluid, fluid, urine, amniotic urine, saliva, saliva, fluid, amniotic fluid, malignant malignant

ascites, broncho-alveolar lavage fluid, synovial fluid, breast milk, sweat, tears, joint fluid, and

bronchial washes. In one aspect, the sample is a blood sample, including, for example, whole

blood or any fraction or component thereof. A blood sample suitable for use with the present

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

invention may be extracted from any source known that includes blood cells or components

thereof, such as venous, arterial, peripheral, tissue, cord, and the like. For example, a sample

may be obtained and processed using well-known and routine clinical methods (e.g.,

procedures for drawing and processing whole blood). In one aspect, an exemplary sample

may be peripheral blood drawn from a subject with cancer.

[0063] Exosomes may also be isolated from tissue samples, such as surgical samples,

biopsy samples, tissues, feces, and cultured cells. When isolating exosomes from tissue

sources it may be necessary to homogenize the tissue in order to obtain a single cell

suspension followed by lysis of the cells to release the exosomes. When isolating exosomes

from tissue samples it is important to select homogenization and lysis procedures that do not

result in disruption of the exosomes. Exosomes contemplated herein are preferably isolated

from body fluid in a physiologically acceptable solution, for example, buffered saline, growth

medium, various aqueous medium, etc.

[0064] Exosomes may be isolated from freshly collected samples or from samples

that have been stored frozen or refrigerated. In some embodiments, exosomes may be

isolated from cell culture medium. Although not necessary, higher purity exosomes may be

obtained if fluid samples are clarified before precipitation with a volume-excluding polymer,

to remove any debris from the sample. Methods of clarification include centrifugation,

ultracentrifugation, filtration, or ultrafiltration. Most typically, exosomes can be isolated by

numerous methods well-known in the art. One preferred method is differential centrifugation

from body fluids or cell culture supernatants. Exemplary methods for isolation of exosomes

are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004).

Alternatively, exosomes may also be isolated via flow cytometry as described in (Combes et

al., 1997).

[0065] One accepted protocol for isolation of exosomes includes ultracentrifugation,

often in combination with sucrose density gradients or sucrose cushions to float the relatively

low-density exosomes. Isolation of exosomes by sequential differential centrifugations is

complicated by the possibility of overlapping size distributions with other microvesicles or

macromolecular complexes. Furthermore, centrifugation may provide insufficient means to

separate vesicles based on their sizes. However, sequential centrifugations, when combined

with sucrose gradient ultracentrifugation, can provide high enrichment of exosomes.

WO wo 2019/191444 PCT/US2019/024603

[0066] Isolation of exosomes based on size, using alternatives to the ultracentrifugation routes, is another option. Successful purification of exosomes using

ultrafiltration procedures that are less time consuming than ultracentrifugation, and do not

require use of special equipment have been reported. Similarly, a commercial kit is available

(EXOMIRTM, (EXOMIR, Bioo Bioo Scientific) Scientific) which which allows allows removal removal of of cells, cells, platelets, platelets, and and cellular cellular debris debris on on

one microfilter and capturing of vesicles bigger than 30 nm on a second microfilter using

positive pressure to drive the fluid. However, for this process, the exosomes are not

recovered, their RNA content is directly extracted from the material caught on the second

microfilter, which can then be used for PCR analysis. HPLC-based protocols could

potentially allow one to obtain highly pure exosomes, though these processes require

dedicated equipment and are difficult to scale up. A significant problem is that both blood

and cell culture media contain large numbers of nanoparticles (some non-vesicular) in the

same size range as exosomes. For example, some miRNAs may be contained within

extracellular protein complexes rather than exosomes; however, treatment with protease (e.g.,

proteinase K) can be performed to eliminate any possible contamination with "extraexosomal" protein.

[0067] In another embodiment, cancer cell-derived exosomes may be captured by

techniques commonly used to enrich a sample for exosomes, such as those involving

immunospecific interactions (e.g., immunomagnetic capture). Immunomagnetic capture, also

known as immunomagnetic cell separation, typically involves attaching antibodies directed to

proteins found on a particular cell type to small paramagnetic beads. When the antibody-

coated beads are mixed with a sample, such as blood, they attach to and surround the

particular cell. The sample is then placed in a strong magnetic field, causing the beads to

pellet to one side. After removing the blood, captured cells are retained with the beads. Many

variations of this general method are well-known in the art and suitable for use to isolate

exosomes. In one example, the exosomes may be attached to magnetic beads (e.g.,

aldehyde/sulphate beads) and then an antibody is added to the mixture to recognize an

epitope on the surface of the exosomes that are attached to the beads. Exemplary proteins

that are known to be found on cancer cell-derived exosomes include ATP-binding cassette

sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4

(SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33

(CD33), and glypican-1 (GPC1). Cancer cell-derived exosomes may be isolated using, for

example, antibodies or aptamers to one or more of these proteins.

[0068] As used herein, analysis includes any method that allows direct or indirect

visualization of exosomes and may be in vivo or ex vivo. For example, analysis may include,

but not limited to, ex vivo microscopic or cytometric detection and visualization of exosomes

bound to a solid substrate, flow cytometry, fluorescent imaging, and the like. In an exemplary

aspect, cancer cell-derived exosomes are detected using antibodies directed to one or more of

ATP-binding cassette sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and

NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell

surface antigen CD33 (CD33), glypican-1 (GPC1), Histone H2A type 2-A (HIST1H2AA),

Histone H2A type 1-A (HISTIH1AA), (HISTIHIAA), Histone H3.3 (H3F3A), Histone H3.1 (HIST1H3A),

Zinc finger protein 37 homolog (ZFP37), Laminin subunit beta-1 (LAMB1), Tubulointerstitial nephritis antigen-like (TINAGL1), Peroxiredeoxin-4 (PRDX4), Collagen

alpha-2(IV) chain (COL4A2), Putative protein C3P1 (C3P1), Hemicentin-1 (HMCN1),

Putative rhophilin-2-like protein (RHPN2P1), Ankyrin repeat domain-containing protein 62

(ANKRD62), Tripartite motif-containing protein 42 (TRIM42), Junction plakoglobin (JUP),

Tubulin beta-2B chain (TUBB2B), Endoribonuclease Dicer (DICER1), E3 ubiquitin-protein

ligase TRIM71 (TRIM71), Katanin p60 ATPase-containing subunit A-like 2 (KATNAL2),

5'-nucleotidasedomain-containing Protein S100-A6 (S100A6), "-nucleotidase domain-containingprotein protein33(NT5DC3), (NT5DC3),Valine- Valine-

tRNA ligase (VARS), Kazrin (KAZN), ELAV-like protein 4 (ELAVL4), RING finger

protein 166 (RNF166), FERM and PDZ domain-containing protein 1 (FRMPD1), 78 kDa

glucose-regulated protein (HSPA5), Trafficking protein particle complex subunit 6A

(TRAPPC6A), Squalene monooxygenase (SQLE), Tumor susceptibility gene 101 protein

(TSG101), Vacuolar protein sorting 28 homolog (VPS28), Prostaglandin F2 receptor negative

regulator (PTGFRN), Isobutyryl-CoA dehydrogenase, mitochondrial (ACAD8), 26S protease

regulatory subunit 6B (PSMC4), Elongation factor 1-gamma (EEF1G), Titin (TTN),

Tyrosine-protein phosphatase type 13 (PTPN13), Triosephosphate isomerase (TPI1), or

Carboxypeptidase E (CPE) and subsequently bound to a solid substrate and/or visualized

using microscopic or cytometric detection.

[0069] It should be noted that not all proteins expressing in a cell are found in

exosomes secreted by that cell (see FIG. 11). For example, calnexin, GM130, and LAMP-2

are all proteins expressed in MCF-7 cells but not found in exosomes secreted by MCF-7 cells

(Baietti et al., 2012). As another example, one study found that 190/190 pancreatic ductal

adenocarcinoma patients had higher levels of GPC1+ exosomes than healthy controls (Melo

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

et al., 2015, which is incorporated herein by reference in its entirety). Notably, only 2,3% 2.3% of

healthy controls, on average, had GPC1+ exosomes.

1. Exemplary Protocol for Collecting Exosomes from Cell Culture

[0070] On Day 1, seed enough cells (e.g., about five million cells) in T225 flasks in

media containing 10% FBS SO so that the next day the cells will be about 70% confluent. On

Day 2, aspirate the media on the cells, wash the cells twice with PBS, and then add 25-30 mL

base media (i.e., no PenStrep or FBS) to the cells. Incubate the cells for 24-48 hours. A 4848

hour incubation is preferred, but some cells lines are more sensitive to serum-free media and

SO the incubation time should be reduced to 24 hours. Note that FBS contains exosomes that

will heavily skew NanoSight results.

[0071] On Day 3/4, collect the media and centrifuge at room temperature for five

minutes at 800 X g 8 to pellet dead cells and large debris. Transfer the supernatant to new

conical tubes and centrifuge the media again for 10 minutes at 2000 X g to remove other large

debris and large vesicles. Pass the media through a 0.2 um µm filter and then aliquot into

ultracentrifuge tubes (e.g., 25 X 89 mm Beckman Ultra-Clear) using 35 mL per tube. If the

volume of media per tube is less than 35 mL, fill the remainder of the tube with PBS to reach

35 mL. Ultracentrifuge the media for 2-4 hours at 28,000 rpm at 4°C using a SW 32 Ti rotor

(k-factor 266.7, RCF max 133,907). Carefully aspirate the supernatant until there is roughly

1-inch of liquid remaining remaining.Tilt Tiltthe thetube tubeand andallow allowremaining remainingmedia mediato toslowly slowlyenter enteraspirator aspirator

pipette. If desired, the exosomes pellet can be resuspended in PBS and the ultracentrifugation

at 28,000 rpm repeated for 1-2 hours to further purify the population of exosomes.

[0072] Finally, resuspend the exosomes pellet in 210 uL µL PBS. If there are multiple

ultracentrifuge tubes for each sample, use the same 210 uL µL PBS to serially resuspend each

exosomes pellet. For each sample, take 10 uL µL and add to 990 uL µL H2O to use for nanoparticle

tracking analysis. Use the remaining 200 uL µL exosomes-containing suspension for

downstream processes or immediately store at -80°C.

2. Exemplary Protocol for Extracting Exosomes from Serum Samples Samples

[0073] First, allow serum samples to thaw on ice. Then, dilute 250 uL µL of cell-free

serum samples in 11 mL PBS; filter through a 0.2 um µm pore filter. Ultracentrifuge the diluted

sample at 150,000 X g overnight at 4°C. The following day, carefully discard the supernatant

WO wo 2019/191444 PCT/US2019/024603

and wash the exosomes pellet in 11 mL PBS. Perform a second round of ultracentrifugation

at 150,000 X g at 4°C for 2 hours. Finally, carefully discard the supernatant and resuspend the

exosomes pellet in 100 uL µL PBS for analysis.

C. Exemplary Protocol for Electroporation of Exosomes and Liposomes

[0074] Mix 1 X 10 exosomes (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula Nano Sciences) and 1 ug µg of siRNA (Qiagen) or

shRNA in 400 uL µL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM

potassium chloride, 21% Optiprep). Electroporate the exosomes or liposomes using a 4 mm

cuvette (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After

electroporation, treat the exosomes or liposomes with protease-free RNAse followed by

addition of 10x concentrated RNase inhibitor. Finally, wash the exosomes or liposomes with

PBS under ultracentrifugation methods, as described above.

II. Diagnosis, Prognosis, and Treatment of Diseases

[0075] Certain aspects of the present invention provide for treating a patient with

exosomes that express or comprise a therapeutic agent or a diagnostic agent. A "therapeutic

agent" as used herein is an atom, molecule, or compound that is useful in the treatment of

cancer or other conditions. Examples of therapeutic agents include, but are not limited to,

drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins,

radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense

oligonucleotides, chelators, boron compounds, photoactive agents, and dyes. A "diagnostic

agent" as used herein is an atom, molecule, or compound that is useful in diagnosing,

detecting or visualizing a disease. According to the embodiments described herein, diagnostic

agents may include, but are not limited to, radioactive substances (e.g., radioisotopes,

radionuclides, radiolabels or radiotracers), dyes, contrast agents, fluorescent compounds or

molecules, bioluminescent compounds or molecules, enzymes and enhancing agents (e.g.,

paramagnetic ions).

[0076] In some aspects, a therapeutic recombinant protein may be a protein having an

activity that has been lost in a cell of the patient, a protein having a desired enzymatic

activity, a protein having a desired inhibitory activity, etc. For example, the protein may be a a

transcription factor, an enzyme, a proteinaceous toxin, an antibody, a monoclonal antibody,

etc. The monoclonal antibody may specifically or selectively bind to an intracellular antigen.

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

The monoclonal antibody may inhibit the function of the intracellular antigen and/or disrupt a

protein-protein interaction. Other aspects of the present invention provide for diagnosing a

disease based on the presence of cancer cell-derived exosomes in a patient sample.

[0077] As exosomes are known to comprise the machinery necessary to complete

mRNA transcription and protein translation (see PCT/US2014/068630, which is incorporated

herein by reference in its entirety), mRNA or DNA nucleic acids encoding a therapeutic

protein proteinmay maybebe transfected intointo transfected exosomes. Alternatively, exosomes. the therapeutic Alternatively, protein itself the therapeutic may be protein itself may be

electroporated into the exosomes or incorporated directly into a liposome. Exemplary

therapeutic proteins include, but are not limited to, a tumor suppressor protein, peptides, a

wild type protein counterparts of a mutant protein, a DNA repair protein, a proteolytic

enzyme, proteinaceous toxin, a protein that can inhibit the activity of an intracellular protein,

a protein that can activate the activity of an intracellular protein, or any protein whose loss of

function needs to be reconstituted. Specific examples of exemplary therapeutic proteins

include 123F2, Abcb4, Abcc1, Abcg2, Actb, Ada, Ahr, Akt, Akt1, Akt2, Akt3, Amhr2,

Anxa7, Apc, Ar, Atm, Axin2, B2m, Bard1, Bardl, Bcl211, Becnl, Bhlhal5, Binl, Blm, Braf, Brcal,

Brca2, Brca3, Braf, Brcata, Brinp3, Brip1, Bripl, Bublb, Bub1b, Bwscrla, Cadm3, Cascl, Casp3, Casp7,

Casp8, Cavl, Cav1, Ccam, Ccnd1, Ccr4, Ccsl, Cd28, Cdc25a, Cd95, Cdhl, Cdh1, Cdkn1a, Cdknla, Cdkn1b,

Cdkn2a, Cdkn2b, Cdkn2c, Cftr, Chek1, Chek2, Crcs1, Crcs10, Crcs11, Crcs2, Crcs3, Crcs4,

Crcs5, Crcs6, Crcs7, Crcs8, Crcs9, Ctnnb1, Ctsl, Cts1, Cyplal, Cyp2a6, Cyp2b2, Cyld, Dcc,

Dkcl, Dicer1, Dicerl, Dmtf1, Dmtfl, Dnmt1, Dpc4, E2f1, E2fl, Eaf2, Eeflal, Egfr, Egfr4, Erbb2, Erbb4, Ercc2,

Ercc6, Ercc8, Errfil, Esrl, Etv4, Faslg, Fbxo10, Fcc, Fgfr3, Fntb, Foxm1, Foxn1, Fusl, Fzd6,

Fzd7, Fzrl, Fzr1, Gadd45a, Gast, Gnai2, Gpcl, Gpc1, Gpr124, Gpr87, Gprc5a, Gprc5d, Grb2, Gstml, Gstm1,

Gstm5, Gstpl, Gstp1, Gsttl, Gstt1, H19, H2afx, Hck, Limsl, Lims1, Hdac, Hexa, Hicl, Hinl, Hin1, Hmmr, Hnpcc8, Hprt, Hras, Htatip2, Il1b, Illb, II10, II2, I12, I16, I18rb Il8rb Inha, Itgav, Jun, Jak3, Kit, Klf4, Kras, Kras2,

Kras2b, Ligl, Lig4, Lkb1, Lmo7, Lncr1, Lncrl, Lncr2, Lncr3, Lncr4, Ltbp4, Lucal, Luca2, Lyz2,

Lztsl, Lzts1, Mad111, Mad211, Madr2/Jv18, Mapk14, Mcc, Mcm4, Men1, Men2, Met, Mgat5, Mif,

Mlh1, Mlh3, Mmacl, Mmac1, Mmp8, Mnt, Mpo, Msh2, Msh3, Msh6, Msmb, Mthfr, Mtsl, Mts1, Mutyh,

Myh11, Nat2, Nbn, Ncoa3, Neil1, Nfl, Nf2, Nfe211, Nhej1, Nkx2-1, Nkx2-9, Nkx3-1, Npr12, Nprl2,

Nqo1, Nras, Nudt1, Oggl, Ogg1, Oxgrl, p16, p19, p21, p27, p27mt, p57, p14ARF, Palb2, Park2,

Pggtlb, Pgr, Pi3k, Pik3ca, Piwil2, P16, Pl6, Pla2g2a, Plg, Plk3, Pmsl, Pms1, Pms2, Pold1, Pole, Ppard,

Pparg, Ppfia2, Ppmld, Prdm2, Prdx1, Prkarla, Ptch, Pten, Prom 1, Psca, Prom1, Psca, Ptch1, Ptch1, Ptfla, Ptfla, Ptger2, Ptger2,

Ptpn13, Ptprj, Rara, Rad51, Rassfl, Rb, Rb1, Rb1ccl, Rblccl, Rb12, Recgl4, Ret, Rgs5, Rhoc, Rintl, Rint1,

Robol, Rp138, Rpl38, S100a4, SCGB1A1, Skp2, Smad2, Smad3, Smad4, Smarcb1, Smo, Snx25,

PCT/US2019/024603

Spata13, Srpx, Ssicl, Sstr2, Sstr5, Stat3, St5, St7, St14, Stk11, Suds3, Tapl, Tbx21, Terc,

Tnf, Tp53, Tp73, Trpm5, Tsc2, Tscl, Tsc1, Vhl, Wrn, Wtl, Wt1, Wt2, Xrccl, Xrcc1, Xrcc5, Xrcc6, and Zacl.

[0078] One specific type of protein that it may be desirable to introduce into the

intracellular space of a diseased cell is an antibody (e.g., a monoclonal antibody). Such an

antibody may disrupt the function of an intracellular protein and/or disrupt an intracellular

protein-protein interaction. protein-protein interaction. Exemplary Exemplary targets targets of of such such monoclonal monoclonal antibodies antibodies include, include, but but are are

not limited to, proteins involved in the RNAi pathway, telomerase, transcription factors that

control disease processes, kinases, phosphatases, proteins required for DNA synthesis,

protein required for protein translation. Specific examples of exemplary therapeutic antibody

targets include proteins encoded by the following genes: Dicer, Agol, Ago2, Trbp, Ras, raf,

wnt, btk, Bcl-2, Akt, Sis, src, Notch, Stathmin, mdm2, abl, hTERT, c-fos, c-jun, c-myc, erbB,

HER2/Neu, HER3, VEGFR, PDGFR, c-kit, c-met, c-ret, flt3, API, AMLI, axl, alk, fins, fps,

gip, lck, 1ck, Stat, Hox, MLM, PRAD-I, and trk. In addition to monoclonal antibodies, any

antigen binding fragment there of, such as a scFv, a Fab fragment, a Fab', a F(ab')2, a Fv, a

peptibody, a diabody, a triabody, or a minibody, is also contemplated. Any such antibodies or

antibody fragment may be either glycosylated or aglycosylated.

[0079] As exosomes are known to comprise DICER and active RNA processing

RISC complex (see PCT Publn. WO 2014/152622, which is incorporated herein by reference

in its entirety), shRNA transfected into exosomes can mature into RISC-complex bound

siRNA with the exosomes themselves. Alternatively, mature siRNA can itself be transfected

into exosomes or liposomes. Thus, by way of example, the following are classes of possible

target genesthat target genes that maymay be used be used in methods in the the methods of the of the present present inventioninvention toormodulate to modulate attenuateor attenuate

target gene expression: wild-type or mutant versions of developmental genes (e.g., adhesion

molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix

family members, Hox family members, cytokines/lymphokines and their receptors, growth or

differentiation factors and their receptors, neurotransmitters and their receptors), tumor

suppressor genes (e.g., APC, CYLD, HIN-1, KRAS2b, p16, p19, p21, p27, p27mt, p53, p57,

p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN2A, DCC, DPC4,

MADR2/JV18, MEN1, MEN2, MTSI, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS- 1, zacl, ras, MMAC1, FCC, MCC, FUS1, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-

1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, Gene 21 (NPRL2), or a gene

encoding a SEM A3 polypeptide), pro-apoptotic genes (e.g., CD95, caspase-3, Bax, Bag-1,

CRADD, TSSC3, bax, hid, Bak, MKP-7, PARP, bad, bcl-2, MST1, bbc3, Sax, BIK, and

BID), cytokines (e.g., GM-CSF, G-CSF, IL-1a, IL-1B,IL-2, IL-1, IL-1ß, IL-2,IL-3, IL-3,IL-4, IL-4,IL-5, IL-5,IL-6, IL-6,IL-7, IL-7,IL- IL-

8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,

IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32 IFN-a, IFN-B, IFN-, IFN-B,

IFN-y, MIP-1a, IFN-, MIP-1, MIP-1ß, MIP-1B, TGF-6, TGF-, TNF-a, TNF-, TNF-B, TNF-B, PDGF, PDGF, and and mda7), mda7), oncogenes oncogenes (e.g., (e.g., ABLI, ABLI,

BLC1, BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR,

FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIMI, PIM1, PML, RET, SRC, TAL1, TCL3 and YES), and enzymes (e.g., ACP

desaturases and hycroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehycrogenases, amylases, amyloglucosidases, catalases, cellulases, cyclooxygenases,

decarboxylases, dextrinases, esterases, DNA and RNA polymerases, galactosidases,

glucanases, glucose oxidases, GTPases, helicases, hemicellulases, integrases, invertases,

isomersases, kinases, lactases, lipases, lipoxygenases, lysozymes, nucleases, pectinesterases,

peroxidases, phosphatases, phospholipases, phosphorylases, polygalacturonases, proteinases

and peptideases, pullanases, recombinases, reverse transcriptases, topoisomerases,

xylanases). In some cases, sh/siRNA may be designed to specifically target a mutant version

of a gene expressed in a cancer cell while not affecting the expression of the corresponding

wild-type version. In fact, any inhibitory nucleic acid that can be applied in the compositions

and methods of the present invention if such inhibitory nucleic acid has been found by any

source to be a validated downregulator of a protein of interest.

[0080] In designing RNAi there are several factors that need to be considered, such as

the nature of the siRNA, the durability of the silencing effect, and the choice of delivery

system. To produce an RNAi effect, the siRNA that is introduced into the organism will

typically contain exonic sequences. Furthermore, the RNAi process is homology dependent,

SO so the sequences must be carefully selected SO so as to maximize gene specificity, while

minimizing the possibility of cross-interference between homologous, but not gene-specific

sequences. Preferably the siRNA exhibits greater than 80%, 85%, 90%, 95%, 98%, or even

100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences

less than about 80% identical to the target gene are substantially less effective. Thus, the

greater homology between the siRNA and the gene to be inhibited, the less likely expression

of unrelated genes will be affected.

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

[0081] Exosomes may also be engineered to comprise a gene editing system, such as

a CRISPR/Cas system. In general, "CRISPR system" refers collectively to transcripts and

other elements involved in the expression of or directing the activity of CRISPR-associated

("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR)

sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing

a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an

endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context

of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR

locus. In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for

the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at

the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using

complementary base pairing. The target site may be selected based on its location

immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or

NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20,

19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the

target DNA sequence sequence.In Ingeneral, general,a aCRISPR CRISPRsystem systemis ischaracterized characterizedby byelements elementsthat that

promote the formation of a CRISPR complex at the site of a target sequence. Typically,

"target sequence" generally refers to a sequence to which a guide sequence is designed to

have complementarity, where hybridization between the target sequence and a guide

sequence promotes the formation of a CRISPR complex. Full complementarity is not

necessarily required, provided there is sufficient complementarity to cause hybridization and

promote formation of a CRISPR complex. The CRISPR system in exosomes engineered to

comprise such a system may function to edit the genomic DNA inside a target cell, or the

system may edit the DNA inside the exosomes itself.

[0082] In addition to protein- and nucleic acid-based therapeutics, exosomes may be

used to deliver small molecule drugs, either alone or in combination with any protein- or

nucleic acid-based therapeutic. Exemplary small molecule drugs that are contemplated for

use in the present embodiments include, but are not limited to, toxins, chemotherapeutic

agents, agents,agents agentsthat inhibit that the activity inhibit of an of the activity intracellular protein, protein, an intracellular agents that activate agents theactivate the that

activity of intracellular proteins, agents for the prevention of restenosis, agents for treating

renal disease, agents used for intermittent claudication, agents used in the treatment of

hypotension and shock, angiotensin converting enzyme inhibitors, antianginal agents, anti-

arrhythmics, anti-hypertensive agents, antiotensin ii receptor antagonists, antiplatelet drugs, wo 2019/191444 WO PCT/US2019/024603 b-blockers b1 selective, beta blocking agents, botanical product for cardiovascular indication, calcium channel blockers, cardiovascular/diagnostics, central alpha-2 agonists, coronary vasodilators, diuretics and renal tubule inhibitors, neutral endopeptidase/angiotensin converting enzyme inhibitors, peripheral vasodilators, potassium channel openers, potassium salts, anticonvulsants, antiemetics, antinauseants, anti-parkinson agents, antispasticity agents, cerebral stimulants, agents that can be applied in the treatment of trauma, agents that can be applied in the treatment of Alzheimer disease or dementia, agents that can be applied in the treatment of migraine, agents that can be applied in the treatment of neurodegenerative diseases, agents that can be applied in the treatment of kaposi's sarcoma, agents that can be applied in the treatment of AIDS, cancer chemotherapeutic agents, agents that can be applied in the treatment of immune disorders, agents that can be applied in the treatment of psychiatric disorders, analgesics, epidural and intrathecal anesthetic agents, general, local, regional neuromuscular blocking agents sedatives, preanesthetic adrenal/acth, anabolic steroids, agents that can be applied in the treatment of diabetes, dopamine agonists, growth hormone and analogs, hyperglycemic agents, hypoglycemic agents, oral insulins, large volume parenterals (lvps), lipid-altering agents, metabolic studies and inborn errors of metabolism, nutrients/amino acids, nutritional lvps, obesity drugs (anorectics), somatostatin, thyroid agents, vasopressin, vitamins, corticosteroids, mucolytic agents, pulmonary anti- inflammatory agents, pulmonary surfactants, antacids, anticholinergics, antidiarrheals, antiemetics, cholelitholytic agents, inflammatory bowel disease agents, irritable bowel syndrome agents, liver agents, metal chelators, miscellaneous gastric secretory agents, pancreatitis agents, pancreatic enzymes, prostaglandins, prostaglandins, proton pump inhibitors, sclerosing agents, sucralfate, anti-progestins, contraceptives, oral contraceptives, not oral dopamine agonists, estrogens, gonadotropins, GNRH agonists, GHRH antagonists, oxytocies, oxytocics, progestins, uterine-acting agents, anti-anemia drugs, anticoagulants, antifibrinolytics, antiplatelet agents, antithrombin drugs, coagulants, fibrinolytics, hematology, heparin inhibitors, metal chelators, prostaglandins, vitamin K, anti-androgens, aminoglycosides, antibacterial agents, sulfonamides, cephalosporins, clindamycins, dermatologics, detergents, erythromycins, anthelmintic agents, antifungal agents, antimalarials, antimycobacterial 30 antimalarials, antimycobacterial agents, agents,antiparasitic agents, antiparasitic antiprotozoal agents, agents,agents, antiprotozoal antitrichomonads, antituberculosis agents, immunomodulators, immunostimulatory agents, macrolides, antiparasitic agents, corticosteroids, cyclooxygenase inhibitors, enzyme blockers, immunomodulators for rheumatic diseases, metalloproteinase inhibitors, nonsteroidal anti- inflammatory agents, analgesics, antipyretics, alpha adrenergic agonists/blockers, antibiotics,

WO wo 2019/191444 PCT/US2019/024603

antivirals, beta adrenergic blockers, carbonic anhydrase inhibitors, corticosteroids, immune

system regulators, mast cell inhibitors, nonsteroidal anti-inflammatory agents, and

prostaglandins.

[0083] Exosomes may also be used to deliver diagnostic agents. Exemplary

diagnostic agents include, but are not limited to, magnetic resonance image enhancement

agents, positron emission tomography products, radioactive diagnostic agents, radioactive

therapeutic agents, radio-opaque contrast agents, radiopharmaceuticals, ultrasound imaging

agents, and angiographic diagnostic agents.

[0084] The term "subject" as used herein refers to any individual or patient to which

the subject methods are performed. Generally the subject is human, although as will be

appreciated by those in the art, the subject may be an animal. Thus other animals, including

mammals, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs,

rabbits, farm animals (including cows, horses, goats, sheep, pigs, etc.), and primates

(including monkeys, chimpanzees, orangutans, and gorillas) are included within the

definition of subject.

[0085] "Treatment" and "treating" refer to administration or application of a

therapeutic agent to a subject or performance of a procedure or modality on a subject for the

purpose of obtaining a therapeutic benefit of a disease or health-related condition. For

example, a treatment may include administration of chemotherapy, immunotherapy, or

radiotherapy, performance of surgery, or any combination thereof.

[0086] The term "therapeutic benefit" or "therapeutically effective" as used

throughout this throughout this application application refers refers to anything to anything that promotes that promotes or the or enhances enhances the well-being well-being of the of the

subject with respect to the medical treatment of this condition. This includes, but is not

limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For

example, treatment of cancer may involve, for example, a reduction in the invasiveness of a

tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of

cancer may also refer to prolonging survival of a subject with cancer.

[0087] The term "cancer," as used herein, may be used to describe a solid tumor,

metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may

originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus,

WO wo 2019/191444 PCT/US2019/024603

duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver,

lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

[0088] The cancer may specifically be of the following histological type, though it is

not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and

spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma;

lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell

carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;

cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and

cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma

in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid

tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;

chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil

carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;

papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal

cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine

adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid

carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous

cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring

cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;

inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous

carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal

tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma,

malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;

paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma;

glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading

melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue

nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;

liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar

rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor;

nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner

tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;

dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant;

choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0089] The terms "contacted" and "exposed," when applied to a cell, are used herein

to describe the process by which a therapeutic agent are delivered to a target cell or are

placed in direct juxtaposition with the target cell. To achieve cell killing, for example, one or

more agents are delivered to a cell in an amount effective to kill the cell or prevent it from

dividing.

[0090] An effective response of a patient or a patient's "responsiveness" to

treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or

suffering from, a disease or disorder. Such benefit may include cellular or biological

responses, a complete response, a partial response, a stable disease (without progression or

relapse), or a response with a later relapse. For example, an effective response can be reduced

tumor size or progression-free survival in a patient diagnosed with cancer.

[0091] Treatment outcomes can be predicted and monitored and/or patients

benefiting from such treatments can be identified or selected via the methods described

herein.

34

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

[0092] Regarding neoplastic condition treatment, depending on the stage of

the neoplastic condition, neoplastic condition treatment involves one or a combination of the

following therapies: surgery to remove the neoplastic tissue, radiation therapy, and

chemotherapy. Other therapeutic regimens may be combined with the administration of the

anticancer agents, e.g., therapeutic compositions and chemotherapeutic agents. For example,

the patient to be treated with such anti-cancer agents may also receive radiation therapy

and/or may undergo surgery.

[0093] For the treatment of disease, the appropriate dosage of a therapeutic

composition will depend on the type of disease to be treated, as defined above, the severity

and course of the disease, the patient's clinical history and response to the agent, and the

discretion of the attending physician. The agent is suitably administered to the patient at one

time or over a series of treatments.

[0094] Therapeutic and prophylactic methods and compositions can be provided in a

combined amount effective to achieve the desired effect. A tissue, tumor, or cell can be

contacted with one or more compositions or pharmacological formulation(s) comprising one

or more of the agents, or by contacting the tissue, tumor, and/or cell with two or more distinct

compositions or formulations. Also, it is contemplated that such a combination therapy can

be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0095] Administration in combination can include simultaneous Administration in combination include can simultaneous administration of two or more agents in the same dosage form, simultaneous administration

in separate dosage forms, and separate administration. That is, the subject therapeutic

composition and another therapeutic agent can be formulated together in the same dosage

form and administered simultaneously. Alternatively, subject therapeutic composition and

another therapeutic agent can be simultaneously administered, wherein both the agents are

present in separate formulations. In another alternative, the therapeutic agent can be

administered just followed by the other therapeutic agent or vice versa. In the separate

administration protocol, the subject therapeutic composition and another therapeutic agent

may be administered a few minutes apart, or a few hours apart, or a few days apart.

[0096] A first anti-cancer treatment (e.g., exosomes that express a recombinant

protein or with a recombinant protein isolated from exosomes) may be administered before,

during, after, or in various combinations relative to a second anti-cancer treatment. The

WO wo 2019/191444 PCT/US2019/024603

administrations may be in intervals ranging from concurrently to minutes to days to weeks.

In embodiments where the first treatment is provided to a patient separately from the second

treatment, one would generally ensure that a significant period of time did not expire between

the time of each delivery, such that the two compounds would still be able to exert an

advantageously combined effect on the patient. In such instances, it is contemplated that one

may provide a patient with the first therapy and the second therapy within about 12 to 24 or

72 h of each other and, more particularly, within about 6-12 h of each other other.In Insome some

situations it may be desirable to extend the time period for treatment significantly where

several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between

respective administrations.

[0097] In certain embodiments, a course of treatment will last 1-90 days or more (this

such range includes intervening days). It is contemplated that one agent may be given on any

day of day 1 to day 90 (this such range includes intervening days) or any combination

thereof, and another agent is given on any day of day 1 to day 90 (this such range includes

intervening days) or any combination thereof. Within a single day (24-hour period), the

patient may be given one or multiple administrations of the agent(s). Moreover, after a

course of treatment, it is contemplated that there is a period of time at which no anti-cancer

treatment is administered. This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12

months or more (this such range includes intervening days), depending on the condition of

the patient, such as their prognosis, strength, health, etc. It is expected that the treatment

cycles would be repeated as necessary.

[0098] Various combinations may be employed. For the example below a first anti-

cancer therapy is "A" and a second anti-cancer therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[0099] Administration of any compound or therapy of the present invention to a

patient will follow general protocols for the administration of such compounds, taking into

account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of

monitoring toxicity that is attributable to combination therapy.

1. Chemotherapy

[00100] A wide variety of chemotherapeutic agents may be used in accordance

with the present invention. The term "chemotherapy" refers to the use of drugs to treat

cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is

administered in the treatment of cancer. These agents or drugs are categorized by their mode

of activity within a cell, for example, whether and at what stage they affect the cell cycle.

Alternatively, an agent may be characterized based on its ability to directly cross-link DNA,

to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting

nucleic acid synthesis.

[00101] Examples of chemotherapeutic agents include alkylating agents, such

as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and

piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa;

ethylenimines and methylamelamines, including altretamine, triethylenemelamine,

trietylenephosphoramide, triethiylenethiophosphoramide, andtrimethylolomelamine; triethiylenethiophosphoramide and trimethylolomelamine;

acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic

analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin

and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and

cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and

CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such

as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,

mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin,

phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as

carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics,

such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and

calicheamicin omegal1); omegaI1); dynemicin, including dynemicin A; bisphosphonates, such as

clodronate; an esperamicin; as well as neocarzinostatin chromophore and related

chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin,

authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,

chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,

doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-

doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,

mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins,

peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, wo 2019/191444 WO PCT/US2019/024603 tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; 2,2',2"-trichlorotriethylamine trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS

2000; difluorometlhylornithine (DMFO);retinoids, difluoromethylornithine (DMFO); retinoids,such suchas asretinoic retinoicacid; acid;capecitabine; capecitabine;

carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase

inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any

of the above.

2. Radiotherapy

[00102] Other factors that cause DNA damage and have been used extensively

include what are commonly known as y-rays, X-rays, and/or the directed delivery of

radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated,

such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and

UV-irradiation. It is most likely that all of these factors affect a broad range of damage on

DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly

and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to

PCT/US2019/024603

200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000

roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the

isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

3. Immunotherapy

[00103] The skilled artisan will understand that additional immunotherapies

may be used in combination or in conjunction with methods of the invention. In the context

of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells

and molecules to target and destroy cancer cells. Rituximab (Rituxan R) is (Rituxan®) is such such an an example. example.

The immune effector may be, for example, an antibody specific for some marker on the

surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may

recruit other cells to actually affect cell killing. The antibody also may be conjugated to a

drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin,

etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte

carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell

target. Various effector cells include cytotoxic T cells and NK cells.

[00104] In one aspect of immunotherapy, the tumor cell must bear some marker

that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor

markers exist and any of these may be suitable for targeting in the context of the present

invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase

(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,

erb B, and p155. An alternative aspect of immunotherapy is to combine anticancer effects

with immune stimulatory effects. Immune stimulating molecules also exist including:

cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1,

MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[00105] Examples of immunotherapies currently under investigation or in use

are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui

and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g., interferons a, B, , ß,

and Y, IL-1, GM-CSF, , IL-1, GM-CSF, and and TNF TNF (Bukowski (Bukowski et et al., al., 1998; 1998; Davidson Davidson et et al., al., 1998; 1998; Hellstrand Hellstrand et et

al., 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and

Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g.,

WO wo 2019/191444 PCT/US2019/024603

anti-CD20, anti-ganglioside GM2, and anti-p 185 (Hollander, anti-p185 (Hollander, 2012; 2012; Hanibuchi Hanibuchi et et al., al., 1998; 1998;

U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be

employed with the antibody therapies described herein.

[00106] In some embodiments, the immunotherapy may be an immune

checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory

molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by

immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known

as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated

protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell

immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1),

T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of

T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1

axis and/or CTLA-4.

[00107] The immune checkpoint inhibitors may be drugs such as small

molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as

human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev

Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of the

immune checkpoint proteins or analogs thereof may be used, in particular chimerized,

humanized or human forms of antibodies may be used. As the skilled person will know,

alternative and/or equivalent names may be in use for certain antibodies mentioned in the

present disclosure. Such alternative and/or equivalent names are interchangeable in the

context of the present disclosure. For example, it is known that lambrolizumab is also known

under the alternative and equivalent names MK-3475 and pembrolizumab.

[00108] In some embodiments, the PD-1 binding antagonist is a molecule that

inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1

ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding

antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific

aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2

binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a

specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an

antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449,

WO wo 2019/191444 PCT/US2019/024603

all incorporated herein by reference. Other PD-1 axis antagonists for use in the methods

provided herein are known in the art such as described in U.S. Patent Publication Nos.

20140294898, 2014022021, and 20110008369, all incorporated herein by reference.

[00109] In some embodiments, the PD-1 binding antagonist is an anti-PD-1

antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some

embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab,

pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an

immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion

of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin

sequence). In some embodiments, the PD-1 binding antagonist is AMP-224 Nivolumab, AMP- 224. also Nivolumab, also

known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVOR, OPDIVO®, is an anti-

PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475,

Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody

described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1

antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc

fusion soluble receptor described in WO2010/027827 and WO2011/066342.

[00110] Another immune checkpoint that can be targeted in the methods

provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as

CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession

number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when

bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of

the immunoglobulin superfamily that is expressed on the surface of Helper T cells and

transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory

protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2

respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells,

whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory

T cells and may be important to their function. T cell activation through the T cell receptor

and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[00111] In some embodiments, the immune checkpoint inhibitor is an anti-

CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an

antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[00112] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived

therefrom) suitable for use in the present methods can be generated using methods well

known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For

example, the anti-CTLA-4 antibodies disclosed in: US Patent No. 8,119,129, WO 01/14424,

WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA

95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505

(antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the

methods disclosed herein. The teachings of each of the aforementioned publications are

hereby incorporated by reference. Antibodies that compete with any of these art-recognized

antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4

antibody is described in International Patent Application No. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[00113] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as

10D1, MDX- 010, MDX- 101, and Yervoy oror Yervoy®) antigen binding antigen fragments binding and fragments variants and variants

thereof (see, e.g., WO 01/14424). In other embodiments, the antibody comprises the heavy

and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody

comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the

CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment,

the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the

above- mentioned antibodies. In another embodiment, the antibody has at least about 90%

variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at

least about 90%, 95%, or 99% variable region identity with ipilimumab).

[00114] Other molecules for modulating CTLA-4 include CTLA-4 ligands and

receptors such as described in U.S. Patent Nos. 5844905, 5885796 and International Patent

Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference,

and immunoadhesins such as described in U.S. Patent No. 8329867, incorporated herein by

reference.

[00115] In some embodiment, the immune therapy could be adoptive

immunotherapy, which involves the transfer of autologous antigen-specific T cells generated

ex vivo. The T cells used for adoptive immunotherapy can be generated either by expansion

of antigen-specific T cells or redirection of T cells through genetic engineering (Park,

WO wo 2019/191444 PCT/US2019/024603

Rosenberg et al. 2011). Isolation and transfer of tumor specific T cells has been shown to be

successful in treating melanoma. Novel specificities in T cells have been successfully

generated through the genetic transfer of transgenic T cell receptors or chimeric antigen

receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a

targeting moiety that is associated with one or more signaling domains in a single fusion

molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of

a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal

antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains

have also been used successfully. The signaling domains for first generation CARs are

derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. CARs

have successfully allowed T cells to be redirected against antigens expressed at the surface of

tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et

al. 2010).

[00116] In one embodiment, the present application provides for a combination

therapy for the treatment of cancer wherein the combination therapy comprises adoptive T-

cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T-cell therapy comprises

autologous and/or allogenic T cells. In another aspect, the autologous and/or allogenic T cells

are targeted against tumor antigens.

4. 4. Surgery

[00117] Approximately 60% of persons with cancer will undergo surgery of

some type, which includes preventative, diagnostic or staging, curative, and palliative

surgery. Curative surgery includes resection in which all or part of cancerous tissue is

physically removed, excised, and/or destroyed and may be used in conjunction with other

therapies, such as the treatment of the present invention, chemotherapy, radiotherapy,

hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor

resection refers to physical removal of at least part of a tumor. In addition to tumor resection,

treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and

microscopically-controlled surgery (Mohs' surgery).

[00118] Upon excision of part or all of cancerous cells, tissue, or tumor, a

cavity may be formed in the body. Treatment may be accomplished by perfusion, direct

injection, or local application of the area with an additional anti-cancer therapy. Such

treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4,

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be

of varying dosages as well.

5. Other Agents

[00119] It is contemplated that other agents may be used in combination with

certain aspects of the present invention to improve the therapeutic efficacy of treatment.

These additional agents include agents that affect the upregulation of cell surface receptors

and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents

that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other

biological agents. Increases in intercellular signaling by elevating the number of GAP

junctions would increase the anti-hyperproliferative effects on the neighboring

hyperproliferative cell hyperproliferative cell population. population. In other In other embodiments, embodiments, cytostatic cytostatic or differentiation or differentiation agents agents

can be used in combination with certain aspects of the present invention to improve the anti-

hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to to

improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal

adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other

agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the

antibody c225, could be used in combination with certain aspects of the present invention to

improve the treatment efficacy.

III. Pharmaceutical Compositions

[00120] It is contemplated that exosomes that express or comprise a therapeutic

protein, inhibitory RNA, and/or small molecule drug can be administered systemically or

locally to inhibit tumor cell growth and, most preferably, to kill cancer cells in cancer patients

with locally advanced or metastatic cancers. They can be administered intravenously,

intrathecally, and/or intraperitoneally. They can be administered alone or in combination

with anti-proliferative drugs. In one embodiment, they are administered to reduce the cancer

load in the patient prior to surgery or other procedures. Alternatively, they can be

administered after surgery to ensure that any remaining cancer (e.g., cancer that the surgery

failed to eliminate) does not survive.

[00121] It is not intended that the present invention be limited by the particular

nature of the therapeutic preparation. For example, such compositions can be provided in

formulations together with physiologically tolerable liquid, gel, solid carriers, diluents, or

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

excipients. These therapeutic preparations can be administered to mammals for veterinary

use, such as with domestic animals, and clinical use in humans in a manner similar to other

therapeutic agents. In general, the dosage required for therapeutic efficacy will vary

according to the type of use and mode of administration, as well as the particular

requirements of individual subjects.

[00122] Where clinical applications are contemplated, it may be necessary to

prepare pharmaceutical compositions comprising recombinant proteins and/or exosomes in a

form appropriate for the intended application. Generally, pharmaceutical compositions may

comprise an effective amount of one or more recombinant proteins and/or exosomes or

additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The

phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and

compositions that do not produce an adverse, allergic, or other untoward reaction when

administered to an animal, such as, for example, a human, as appropriate. The preparation of

a pharmaceutical composition comprising a recombinant protein and/or exosomes as

disclosed herein, or additional active ingredient will be known to those of skill in the art in

light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th

Ed., 1990, incorporated herein by reference. Moreover, for animal (e.g., human)

administration, it will be understood that preparations should meet sterility, pyrogenicity,

general safety, and purity standards as required by the FDA Office of Biological Standards.

[00123] Further in accordance with certain aspects of the present invention, the

composition suitable for administration may be provided in a pharmaceutically acceptable

carrier with or without an inert diluent. As used herein, "pharmaceutically acceptable

carrier" includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions,

ethanol, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose,

etc.), non-aqueous solvents (e.g., fats, oils, polyol (for example, glycerol, propylene glycol,

and liquid polyethylene glycol, and the like), vegetable oil, and injectable organic esters, such

as ethyloleate), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants,

antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating

agents, inert gases, parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,

sorbic acid, thimerosal or combinations thereof), isotonic agents (e.g., sugars and sodium

chloride), absorption delaying agents (e.g., aluminum monostearate and gelatin), salts, drugs,

drug stabilizers, gels, resins, fillers, binders, excipients, disintegration agents, lubricants,

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sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials

and combinations thereof, as would be known to one of ordinary skill in the art. The carrier

should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. In

addition, if desired, the compositions may contain minor amounts of auxiliary substances,

such as wetting or emulsifying agents, stabilizing agents, or pH buffering agents. The pH and

exact concentration of the various components in a pharmaceutical composition are adjusted

according to well-known parameters. The proper fluidity can be maintained, for example, by by

the use of a coating, such as lecithin, by the maintenance of the required particle size in the

case of dispersion, and by the use of surfactants.

[00124] A pharmaceutically acceptable carrier is particularly formulated for

administration to a human, although in certain embodiments it may be desirable to use a

pharmaceutically acceptable carrier that is formulated for administration to a non-human

animal but that would not be acceptable (e.g., due to governmental regulations) for

administration to a human. Except insofar as any conventional carrier is incompatible with

the active ingredient (e.g., detrimental to the recipient or to the therapeutic effectiveness of a

composition contained therein), its use in the therapeutic or pharmaceutical compositions is

contemplated. In accordance with certain aspects of the present invention, the composition is

combined with the carrier in any convenient and practical manner, i.e., by solution,

suspension, emulsification, admixture, encapsulation, absorption, and the like. Such

procedures are routine for those skilled in the art.

[00125] Certain embodiments of the present invention may comprise different

types of carriers depending on whether it is to be administered in solid, liquid, or aerosol

form, and whether it needs to be sterile for the route of administration, such as injection. The

compositions can be administered intravenously, intradermally, transdermally, intrathecally,

intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly,

subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation),

by injection, by injection,byby infusion, by continuous infusion, infusion, by continuous by localized infusion, perfusion perfusion by localized bathing target cellstarget cells bathing

directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other

methods or any combination of the forgoing as would be known to one of ordinary skill in the

art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated

herein by reference).

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[00126] The active compounds can be formulated for parenteral administration,

e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even

intraperitoneal routes. Typically, such compositions can be prepared as either liquid

solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions

upon the addition of a liquid prior to injection can also be prepared; and the preparations can

also be emulsified.

[00127] The pharmaceutical forms suitable for injectable use include sterile

aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous

propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable

solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent

that it may be easily injected. It also should be stable under the conditions of manufacture

and storage and must be preserved against the contaminating action of microorganisms, such

as bacteria and fungi.

[00128] The therapeutics may be formulated into a composition in a free base,

neutral, or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g.,

those formed with the free amino groups of a proteinaceous composition, or which are

formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such

organic acids as acetic, oxalic, tartaric, or mandelic acid and the like like.Salts Saltsformed formedwith withthe the

free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium,

potassium, ammonium, calcium, or ferric hydroxides; or such organic bases as

isopropylamine, trimethylamine, histidine, or procaine and the like. Upon formulation,

solutions will be administered in a manner compatible with the dosage formulation and in

such amount as is therapeutically effective. The formulations are easily administered in a a

variety of dosage forms, such as formulated for parenteral administrations, such as injectable

solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations,

such as drug release capsules and the like.

[00129] In a specific embodiment of the present invention, the composition is

combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried

out in any convenient manner, such as grinding. Stabilizing agents can be also added in the

mixing process in order to protect the composition from loss of therapeutic activity, i.e.,

denaturation in the stomach. Examples of stabilizers for use in a composition include buffers,

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amino acids, such as glycine and lysine, carbohydrates, such as dextrose, mannose, galactose,

fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[00130] In further embodiments, the present invention may concern the use of a

pharmaceutical lipid vehicle composition comprising one or more lipids and an aqueous

solvent. As used herein, the term "lipid" will be defined to include any of a broad range of

substances that is characteristically insoluble in water and extractable with an organic

solvent. This broad class of compounds is well known to those of skill in the art, and as the

term "lipid" is used herein, it is not limited to any particular structure. Examples include

compounds that contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may

be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is

usually a biological substance. Biological lipids are well known in the art, and include for

example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,

glycosphingolipids, glycolipids, sulphatides, lipids with ether- and ester-linked fatty acids,

polymerizable lipids, and combinations thereof. Of course, compounds other than those

specifically described herein that are understood by one of skill in the art as lipids are also

encompassed by the compositions and methods.

[00131] One of ordinary skill in the art would be familiar with the range of

techniques that can be employed for dispersing a composition in a lipid vehicle. For

example, the therapeutic agent may be dispersed in a solution containing a lipid, dissolved

with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently

bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle

or liposome, or otherwise associated with a lipid or lipid structure by any means known to

those of ordinary skill in the art. The dispersion may or may not result in the formation of

liposomes.

[00132] The term "unit dose" or "dosage" refers to physically discrete units

suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic

composition calculated to produce the desired responses discussed above in association with

its administration, i.e., the appropriate route and treatment regimen. The quantity to be

administered, both according to number of treatments and unit dose, depends on the effect

desired. The actual dosage amount of a composition of the present invention administered to

a patient or subject can be determined by physical and physiological factors, such as body

weight, the age, health, and sex of the subject, the type of disease being treated, the extent of

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disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient,

the route of administration, and the potency, stability, and toxicity of the particular

therapeutic substance. For example, a dose may also comprise from about 1 ug/kg/body µg/kg/body

weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or

more per administration, and any range derivable therein. In non-limiting examples of a

derivable range from the numbers listed herein, a range of about 5 ug/kg/body µg/kg/body weight to

about 100 mg/kg/body weight, about 5 ug/kg/body µg/kg/body weight to about 500 mg/kg/body weight,

etc., can be administered. The practitioner responsible for administration will, in any event,

determine the concentration of active ingredient(s) in a composition and appropriate dose(s)

for the individual subject.

[00133] The actual dosage amount of a composition administered to an animal

patient can be determined by physical and physiological factors, such as body weight,

severity of condition, the type of disease being treated, previous or concurrent therapeutic

interventions, idiopathy of the patient, and on the route of administration. Depending upon

the dosage and the route of administration, the number of administrations of a preferred

dosage and/or an effective amount may vary according to the response of the subject. The

practitioner responsible for administration will, in any event, determine the concentration of

active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[00134] In certain embodiments, pharmaceutical compositions may comprise,

for example, at least about 0.1% of an active compound compound.In Inother otherembodiments, embodiments,an anactive active

compound may comprise between about 2% to about 75% of the weight of the unit, or

between about 25% to about 60%, for example, and any range derivable therein. Naturally,

the amount of active compound(s) in each therapeutically useful composition may be

prepared in such a way that a suitable dosage will be obtained in any given unit dose of the

compound. Factors, such as solubility, bioavailability, biological half-life, route of

administration, product shelf life, as well as other pharmacological considerations, will be

contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as

such, a variety of dosages and treatment regimens may be desirable.

[00135] In other non-limiting examples, a dose may also comprise from about 1

microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 10

microgram/kg/body weight, about 50 microgram/kg/body weight, about about 100

microgram/kg/body microgram/kg/body weight, weight, about 200 about 200microgram/kg/body microgram/kg/bodyweight, about weight,about 350 350

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1 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body

weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200

milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 milligram/kg/body weight or more per

administration, and any range derivable therein. In non-limiting examples of a derivable

range from the numbers listed herein, a range of about 5 milligram/kg/body weight to about

100 milligram/kg/body 100 milligram/kg/body weight, weight, about about 5 microgram/kg/body 5 microgram/kg/body weight weight to500 to about about 500 milligram/kg/body milligram/kg/body weight, weight, etc., etc., can can be be administered, administered, based based on on the the numbers numbers described described above. above.

IV. Nucleic Acids and Vectors

[00136] In certain aspects of the invention, nucleic acid sequences encoding a

therapeutic protein or a fusion protein containing a therapeutic protein may be disclosed.

Depending on which expression system is used, nucleic acid sequences can be selected based

on conventional methods. For example, the respective genes or variants thereof may be

codon optimized for expression in a certain system. Various vectors may be also used to

express the protein of interest. Exemplary vectors include, but are not limited, plasmid

vectors, viral vectors, transposon, or liposome-based vectors.

V. Recombinant Proteins, Inhibitory RNAs, and Gene Editing Systems

A. Recombinant Proteins

[00137] Some embodiments concern recombinant proteins and polypeptides.

Particular embodiments concern a recombinant protein or polypeptide that exhibits at least

one therapeutic activity. In some embodiments, a recombinant protein or polypeptide may be

a therapeutic antibody. In some aspects, a therapeutic antibody may be an antibody that

specifically or selectively binds to an intracellular protein. In further aspects, the protein or

polypeptide may be modified to increase serum stability. Thus, when the present application

refers to the function or activity of "modified protein" or a "modified polypeptide," one ofof

ordinary skill in the art would understand that this includes, for example, a protein or

polypeptide that possesses an additional advantage over the unmodified protein or

polypeptide. It is specifically contemplated that embodiments concerning a "modified

protein" may be implemented with respect to a "modified polypeptide," and vice versa.

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[00138] Recombinant Recombinant proteins proteins may may possess possess deletions deletions and/or and/or substitutions substitutions of of

amino acids; thus, a protein with a deletion, a protein with a substitution, and a protein with a

deletion and a substitution are modified proteins. In some embodiments, these proteins may

further furtherinclude includeinsertions or added insertions amino amino or added acids, acids, such as such with as fusion withproteins fusion or proteins or proteins with proteins with

linkers, for example. A "modified deleted protein" lacks one or more residues of the native

protein, but may possess the specificity and/or activity of the native protein. A "modified

deleted protein" may also have reduced immunogenicity or antigenicity. An example of a a

modified deleted protein is one that has an amino acid residue deleted from at least one

antigenic region that is, a region of the protein determined to be antigenic in a particular

organism, such as the type of organism that may be administered the modified protein.

[00139]

[00139] Substitution or replacement variants typically contain the exchange of

one amino acid for another at one or more sites within the protein and may be designed to

modulate one or more properties of the polypeptide, particularly its effector functions and/or

bioavailability. Substitutions may or may not be conservative, that is, one amino acid is

replaced with one of similar shape and charge. Conservative substitutions are well known in

the art and include, for example, the changes of: alanine to serine; arginine to lysine;

asparagine asparagine totoglutamine glutamine or histidine; or histidine; aspartate aspartate to glutamate; to glutamate; cysteine cysteine to serine;to serine;toglutamine to glutamine

asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine;

isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine

to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to

threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or

phenylalanine; and valine to isoleucine or leucine.

[00140] In addition to a deletion or substitution, a modified protein may

possess an insertion of residues, which typically involves the addition of at least one residue

in the polypeptide. This may include the insertion of a targeting peptide or polypeptide or

simply a single residue. Terminal additions, called fusion proteins, are discussed below.

[00141] The term "biologically functional equivalent" is well understood in the

art and is further defined in detail herein. Accordingly, sequences that have between about

70% and about 80%, or between about 81% and about 90%, or even between about 91% and

about 99% of amino acids that are identical or functionally equivalent to the amino acids of a

control polypeptide are included, provided the biological activity of the protein is maintained.

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A A recombinant recombinant protein may may protein be biologically functionally be biologically equivalent functionally to its native equivalent counterpart to its in native counterpart in

certain aspects.

[00142] It also will be understood that amino acid and nucleic acid sequences

may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3'

sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, SO so

long as the sequence meets the criteria set forth above, including the maintenance of

biological protein activity where protein expression is concerned. The addition of terminal

sequences particularly applies to nucleic acid sequences that may, for example, include

various non-coding sequences flanking either of the 5' or 3' portions of the coding region or

may include various internal sequences, i.e., introns, which are known to occur within genes.

[00143] As used herein, a protein or peptide generally refers, but is not limited

to, a protein of greater than about 200 amino acids, up to a full length sequence translated

from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from

about about 33 to to about about 100 100 amino amino acids. acids. For For convenience, convenience, the the terms terms "protein," "protein," "polypeptide," "polypeptide," and and

"peptide are used interchangeably herein.

[00144] As used herein, an "amino acid residue" refers to any naturally

occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art.

In certain embodiments, the residues of the protein or peptide are sequential, without any

non-amino acids interrupting the sequence of amino acid residues. In other embodiments, the

sequence may comprise one or more non-amino acid moieties. In particular embodiments,

the sequence of residues of the protein or peptide may be interrupted by one or more non-

amino acid moieties.

[00145] Accordingly, the term "protein or peptide" encompasses amino acid

sequences comprising at least one of the 20 common amino acids found in naturally

occurring proteins, or at least one modified or unusual amino acid.

[00146] Certain embodiments of the present invention concern fusion proteins.

These molecules may have a therapeutic protein linked at the N- or C-terminus to a

heterologous domain. For example, fusions may also employ leader sequences from other

species to permit the recombinant expression of a protein in a heterologous host. Another

useful fusion includes the addition of a protein affinity tag, such as a serum albumin affinity

tag or six histidine residues, or an immunologically active domain, such as an antibody

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epitope, preferably cleavable, to facilitate purification of the fusion protein. Non-limiting

affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein

(MBP), and glutathione-S-transferase (GST).

[00147] In a particular embodiment, the therapeutic protein may be linked to a

peptide that increases the in vivo half-life, such as an XTEN polypeptide (Schellenberger et

al., 2009), IgG Fc domain, albumin, or albumin binding peptide.

[00148] Methods of generating fusion proteins are well known to those of skill

in the art. Such proteins can be produced, for example, by de novo synthesis of the complete

fusion protein, or by attachment of the DNA sequence encoding the heterologous domain,

followed by expression of the intact fusion protein.

[00149] Production of fusion proteins that recover the functional activities of

the parent proteins may be facilitated by connecting genes with a bridging DNA segment

encoding a peptide linker that is spliced between the polypeptides connected in tandem. The

linker would be of sufficient length to allow proper folding of the resulting fusion protein.

B. Inhibitory RNAs

[00150] siNA (e.g., siRNA) are well known in the art. For example, siRNA and

double-stranded RNA have been described in U.S. Pat. Nos. 6,506,559 and 6,573,099, as well

as in U.S. Patent Applications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707,

2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their

entirety.

[00151] Within a siNA, the components of a nucleic acid need not be of the

same type or homogenous throughout (e.g., a siNA may comprise a nucleotide and a nucleic

acid or nucleotide analog). Typically, siNA form a double-stranded structure; the double-

stranded structure may result from two separate nucleic acids that are partially or completely

complementary. In certain embodiments of the present invention, the siNA may comprise

only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded

structure by complementing with itself (e.g., forming a hairpin loop). The double-stranded

structure of the siNA may comprise 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90,

100, 150, 200, 250, 300, 350, 400, 450, 500 or more contiguous nucleobases, including all

ranges therein. The siNA may comprise 17 to 35 contiguous nucleobases, more preferably 18

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to 30 contiguous nucleobases, more preferably 19 to 25 nucleobases, more preferably 20 to

23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases

that hybridize with a complementary nucleic acid (which may be another part of the same

nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[00152] Agents of the present invention useful for practicing the methods of the

present invention include, but are not limited to siRNAs. Typically, introduction of double-

stranded RNA (dsRNA), which may alternatively be referred to herein as small interfering

RNA (siRNA), induces potent and specific gene silencing, a phenomena called RNA

interference or RNAi. RNA interference has been referred to as "cosuppression," "post-

transcriptional gene silencing," "sense suppression," and "quelling." RNAi is an attractive

biotechnological tool because it provides a means for knocking out the activity of specific

genes.

[00153] In designing RNAi there are several factors that need to be considered,

such as the nature of the siRNA, the durability of the silencing effect, and the choice of

delivery system. To produce an RNAi effect, the siRNA that is introduced into the organism

will typically contain exonic sequences sequences.Furthermore, Furthermore,the theRNAi RNAiprocess processis ishomology homology

dependent, SO so the sequences must be carefully selected SO so as to maximize gene specificity,

while minimizing the possibility of cross-interference between homologous, but not gene-

specific sequences. Preferably the siRNA exhibits greater than 80%, 85%, 90%, 95%, 98%,

or even 100% identity between the sequence of the siRNA and the gene to be inhibited.

Sequences less than about 80% identical to the target gene are substantially less effective.

Thus, the greater homology between the siRNA and the gene to be inhibited, the less likely

expression of unrelated genes will be affected.

[00154] In addition, the size of the siRNA is an important consideration. In

some embodiments, the present invention relates to siRNA molecules that include at least

about 19-25 nucleotides and are able to modulate gene expression. In the context of the

present invention, the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides inin

length. More preferably, the siRNA is from about 19 nucleotides to about 25 nucleotides in

length.

[00155] A target gene generally means a polynucleotide comprising a region

that encodes a polypeptide, or a polynucleotide region that regulates replication, transcription,

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or translation or other processes important to expression of the polypeptide, or a

polynucleotide comprising both a region that encodes a polypeptide and a region operably

linked thereto that regulates expression. Any gene being expressed in a cell can be targeted.

Preferably, a target gene is one involved in or associated with the progression of cellular

activities important to disease or of particular interest as a research object.

[00156] siRNA can be obtained from commercial sources, natural sources, or

can be synthesized using any of a number of techniques well-known to those of ordinary skill

in the art. For example, one commercial source of predesigned siRNA is Ambion®, Austin,

Tex. Another is Qiagen® (Valencia, Calif.). An inhibitory nucleic acid that can be applied in

the compositions and methods of the present invention may be any nucleic acid sequence that

has been found by any source to be a validated downregulator of a protein of interest.

Without undue experimentation and using the disclosure of this invention, it is understood

that additional siRNAs can be designed and used to practice the methods of the invention.

[00157] The siRNA may also comprise an alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to

the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the

RNA). In certain aspects, the RNA molecule contains a 3'-hydroxyl group. Nucleotides in the

RNA molecules of the present invention can also comprise non-standard nucleotides,

including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded

oligonucleotide may contain a modified backbone, for example, phosphorothioate,

phosphorodithioate, or other modified backbones known in the art, or may contain non-

natural internucleoside linkages. Additional modifications of siRNAs (e.g., 2'-O-methyl

ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl

nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic

residue incorporation) can be found in U.S. Application Publication 2004/0019001 and U.S.

Pat. No. 6,673,611 (each of which is incorporated by reference in its entirety). Collectively,

all such altered nucleic acids or RNAs described above are referred to as modified siRNAs.

C. Gene Editing Systems

[00158] In general, "CRISPR system" refers collectively to transcripts and

other elements involved in the expression of or directing the activity of CRISPR-associated

("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR)

sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing

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a "direct repeat" and a racrRNA-processed tracrRNA-processedpartial partialdirect directrepeat repeatin inthe thecontext contextof ofan an

endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context

of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR

locus.

[00159] The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to

DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease

domains). One or more elements of a CRISPR system can derive from a type I, type II, or

type III CRISPR system, e.g., derived from a particular organism comprising an endogenous

CRISPR system, such as Streptococcus pyogenes.

[00160] In some aspects, a Cas nuclease and gRNA (including a fusion of

crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In

general, target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g.,

the gene, using complementary base pairing. The target site may be selected based on its

location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically

NGG, NGG, or or NAG. NAG. In In this this respect, respect, the the gRNA gRNA is is targeted targeted to to the the desired desired sequence sequence by by modifying modifying

the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to

correspond to the target DNA sequence. In general, a CRISPR system is characterized by

elements that promote the formation of a CRISPR complex at the site of a target sequence.

Typically, "target sequence" generally refers to a sequence to which a guide sequence is

designed to have complementarity, where hybridization between the target sequence and a

guide sequence promotes the formation of a CRISPR complex. Full complementarity is not

necessarily required, provided there is sufficient complementarity to cause hybridization and

promote formation of a CRISPR complex.

[00161] The CRISPR system can induce double stranded breaks (DSBs) at the

target site, target site,followed by disruptions followed as discussed by disruptions herein.herein. as discussed In other In embodiments, Cas9 variants, other embodiments, Cas9 variants,

deemed "nickases," are used to nick a single strand at the target site. Paired nickases can be

used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting

sequences such that upon introduction of the nicks simultaneously, a 5' overhang is

introduced. introduced. In In other other embodiments, embodiments, catalytically catalytically inactive inactive Cas9 Cas9 is is fused fused to to aa heterologous heterologous

effector domain such as a transcriptional repressor or activator, to affect gene expression.

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[00162] The target sequence may comprise any polynucleotide, such as DNA

or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of

the cell, such as within an organelle of the cell. Generally, a sequence or template that may be

used for recombination into the targeted locus comprising the target sequences is referred to

as an "editing template" or "editing polynucleotide" or "editing sequence" sequence".In Insome someaspects, aspects,

an exogenous template polynucleotide may be referred to as an editing template. In some

aspects, the recombination is homologous recombination.

[00163] Typically, in the context of an endogenous CRISPR system, formation

of the CRISPR complex (comprising the guide sequence hybridized to the target sequence

and complexed with one or more Cas proteins) results in cleavage of one or both strands in or

near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target

sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild-

type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more

nucleotides of a wild-type tracr sequence), may also form part of the CRISPR complex, such

as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr

mate sequence that is operably linked to the guide sequence. The traer tracr sequence has sufficient

complementarity to a tracr mate sequence to hybridize and participate in formation of the

CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence

complementarity along the length of the tracr mate sequence when optimally aligned aligned.

[00164] One or more vectors driving expression of one or more elements of the

CRISPR system can be introduced into the cell such that expression of the elements of the

CRISPR system direct formation of the CRISPR complex at one or more target sites.

Components can also be delivered to cells as proteins and/or RNA. For example, a Cas

enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be

operably linked to separate regulatory elements on separate vectors. Alternatively, two or

more of the elements expressed from the same or different regulatory elements, may be

combined in a single vector, with one or more additional vectors providing any components

of the CRISPR system not included in the first vector. The vector may comprise one or more

insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a

"cloning site"). In some embodiments, one or more insertion sites are located upstream

and/or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target

CRISPR activity to multiple different, corresponding target sequences within a cell.

[00165] A vector may comprise a regulatory element operably linked to an

enzyme-coding enzyme-coding sequence sequence encoding encoding the the CRISPR CRISPR enzyme, enzyme, such such as as aa Cas Cas protein. protein. Non-limiting Non-limiting

examples of Cas proteins include Casl, Cas1B Cas1B,Cas2, Cas2,Cas3, Cas3,Cas4, Cas4,Cas5, Cas5,Cas6, Cas6,Cas7, Cas7,Cas8, Cas8,

Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc1, Csc2,

Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,

Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4,

homologs thereof, or modified versions thereof. These enzymes are known; for example, the

amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database

under accession number Q99ZW2.

[00166] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S.

pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location

of a target sequence, such as within the target sequence and/or within the complement of the

target sequence. The vector can encode a CRISPR enzyme that is mutated with respect to a

corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to

cleave one or both strands of a target polynucleotide containing a target sequence. For

example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9

from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase

(cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination

with guide sequence(s), e.g., two guide sequences, which target respectively sense and

antisense strands of the DNA target. This combination allows both strands to be nicked and

used to induce used to induceNHEJ NHEJ or or HDR. HDR.

[00167] In some embodiments, an enzyme coding sequence encoding the

CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic

cells. The eukaryotic cells may be those of or derived from a particular organism, such as a

mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.

In general, codon optimization refers to a process of modifying a nucleic acid sequence for

enhanced expression in the host cells of interest by replacing at least one codon of the native

sequence with codons that are more frequently or most frequently used in the genes of that

host cell while maintaining the native amino acid sequence. Various species exhibit particular

bias for certain codons of a particular amino acid. Codon bias (differences in codon usage

WO wo 2019/191444 PCT/US2019/024603

between organisms) often correlates with the efficiency of translation of messenger RNA

(mRNA), which is in turn believed to be dependent on, among other things, the properties of

the codons being translated and the availability of particular transfer RNA (tRNA) molecules.

The predominance of selected tRNAs in a cell is generally a reflection of the codons used

most frequently in peptide synthesis synthesis.Accordingly, Accordingly,genes genescan canbe betailored tailoredfor foroptimal optimalgene gene

expression in a given organism based on codon optimization.

[00168] In general, a guide sequence is any polynucleotide sequence having

sufficient complementarity with a target polynucleotide sequence to hybridize with the target

sequence and direct sequence-specific binding of the CRISPR complex to the target

sequence. In some embodiments, the degree of complementarity between a guide sequence

and its corresponding target sequence, when optimally aligned using a suitable alignment

algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%,

or more.

[00169] Optimal alignment may be determined with the use of any suitable

algorithm for aligning sequences, non-limiting example of which include the Smith-

Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-

Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT,

Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available

at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net) maq.sourceforge.net).

[00170] The CRISPR enzyme may be part of a fusion protein comprising one

or more heterologous protein domains. A CRISPR enzyme fusion protein may comprise any

additional protein sequence, and optionally a linker sequence between any two domains.

Examples of protein domains that may be fused to a CRISPR enzyme include, without

limitation, epitope tags, reporter gene sequences, and protein domains having one or more of

the following activities: methylase activity, demethylase activity, transcription activation

activity, transcription repression activity, transcription release factor activity, histone

modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting

examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza

hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of

reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish

peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-

glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue

fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a

protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules,

including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding

domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus

(HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein

comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by

reference.

VI. Kits and Diagnostics

[00171] In various aspects of the invention, a kit is envisioned containing the

necessary components to purify exosomes from a body fluid or tissue culture medium. In

other aspects, a kit is envisioned containing the necessary components to isolate exosomes

and transfect them with a therapeutic nucleic acid, therapeutic protein, or a nucleic acid

encoding a therapeutic protein therein. The kit may comprise one or more sealed vials

containing any of such components. In some embodiments, the kit may also comprise a

suitable container means, which is a container that will not react with components of the kit,

such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container may be

made from sterilizable materials such as plastic or glass. The kit may further include an

instruction sheet that outlines the procedural steps of the methods set forth herein, and will

follow substantially the same procedures as described herein or are known to those of

ordinary skill. The instruction information may be in a computer readable media containing

machine-readable instructions that, when executed using a computer, cause the display of a

real or virtual procedure of purifying exosomes from a sample and transfecting a therapeutic

nucleic acid therein, expressing a recombinant protein therein, or electroporating a

recombinant protein therein.

VII. VII. Examples Examples

[00172] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the

techniques disclosed in the examples which follow represent techniques discovered by the

inventor to function well in the practice of the invention, and thus can be considered to

constitute preferred modes for its practice. However, those of skill in the art should, in light

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

of the present disclosure, appreciate that many changes can be made in the specific

embodiments which are disclosed and still obtain a like or similar result without departing

from the spirit and scope of the invention.

Materials and Methods

[00173] Cell Culture. MCF7, MDA-MB231, E10, HDF, and BJ human cell

lines, as well as the NIH 3T3 murine cell line were cultured in DMEM with 10% FBS. The

4T1 and 67NR murine cell lines were cultured in RPMI with 10% FBS. MCF10A human

mammary epithelial cell line was cultured in DMEM/F12 media with 5% Horse Serum, 20

ng/ml EGF, 0.5 mg/ml Hydrocortisone, 100 ng/ml Cholera Toxin, and 10 ug/ml µg/ml Insulin. All

cells originated from the American Type Culture Collection ---- ATCC.

[00174] Isolation and purification of exosomes. Exosomes were purified by

differential centrifugation as described previously (Luga et al., 2012; Thery et al., 2006).

Supernatant from cells cultured for 48 h were subjected to sequential centrifugation steps of

800g and 2000g. The resulting supernatant was filtered using a 0.2 um µm filter. A pellet was

recovered after ultracentrifugation in an SW40Ti swinging bucket rotor at 100,000g for 3 h

(Beckman-Coulter). Supernatant was removed and the pellet was re-suspended in PBS,

followed by a second ultracentrifugation at 100,000g for 3 h. The resulting pellet was

analyzed for exosomes content. Exosomes used for RNA extraction were resuspended in 500

uL µL of Trizol; exosomes used for protein extraction were resuspended in Urea/SDS lysis

buffer (8M Urea, 2.5% SDS, 5 ug/mL µg/mL leupeptin, 1 ug/mL µg/mL pepstatin, and 1 mM phenylmethylsulphonyl fluoride); and exosomes used for delivery to cells were re-suspended

in serum-free DMEM culture medium. For other applications, isolated exosomes were

processed as described in the remaining experimental procedures.

[00175] Imaging Flow Cytometry analysis (ImageStream). Exosomes were

attached to 4 um µm aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) in NaCl 0.9%

saline solution (B. Braun Medical Inc, Bethlehem, PA, USA). The reaction was stopped with

100 mM glycine and 2% BSA in saline and blocked with 10% BSA with rotation at room

temperature for 30 min. After washing in saline/2% BSA, bead-bound exosomes were

centrifuged for 2 min at 10,000 rpm and incubated with 1:200 anti-CD63 (Santa Cruz), anti-

CD9 (Abcam), anti-CD81 (Abcam), anti-CD82 (Abcam), and anti-FLOT1 (Santa Cruz) for

30 min rotating at 4°C. Beads were centrifuged for 2 min at 10,000 rpm, washed in saline/2%

WO wo 2019/191444 PCT/US2019/024603

BSA and incubated with 1:400 Alexa-488 secondary antibodies (Life Technologies, NY

14072) for 30 min rotating at 4°C. After three washes the beads were resuspended in saline

solution and analyzed on the ImageStream® (Merck Millipore). The image acquisition gain

(%) was set using a positive sample, in order to avoid pixel saturation. Image processing was

done using the IDEAS® (Merck Millipore) software. Gates were defined to exclude out-of-

focus beads and select single beads. Alexa-488 positive bead gates were defined on the

negative control sample. Percentage of positive beads is relative to the number of events

analyzed per sample.

[00176] Immunogold Labeling and Electron Microscopy. Pelleted exosomes

were fixed by re-suspending in 2.5% Glutaraldehyde in 0.1 M Phosphate buffer. Fixed

specimens at an optimal concentration were placed onto a 300 mesh carbon/formvar coated

grids and allowed to absorb to the formvar for a minimum of 1 minute. For immunogold

staining the grids were placed into a blocking buffer for a block/permeabilization step for 1 h.

Without rinsing, the grids were immediately placed into the primary antibody at the

appropriate dilution overnight at 4°C (polyclonal anti-GFP 1:10, Abcam). As controls, some

grids were not exposed to the primary antibody. The next day all grids were rinsed with PBS

and floated on drops of the appropriate secondary antibody attached with 10 nm gold

particles (AURION, Hatfield, PA) for 2 h at room temperature. Grids were rinsed with PBS

and placed in 2.5% Glutaraldehyde in 0.1 M Phosphate buffer for 15 minutes. After rinsing in

PBS and distilled water, the grids were allowed to dry and stained for contrast using uranyl

acetate. The samples were viewed with a Tecnai Bio Twin transmission electron microscope

(FEI, Hillsboro, OR) and images were taken with an AMT CCD Camera (Advanced Microscopy Techniques, Danvers, MA).

[00177] EGF stimulation of exosomes. Exosomes were collected from MBA-

MB-231 MB-231 cells cellsasasdescribed above. described 1-3 X1-3 above. 10°X exosomes were resuspended 10 exosomes in 1 mL in were resuspended PBS1and mL PBS and

different concentrations of rEGF were added. Exosomes suspensions, with or without EGF,

were incubated at 37°C with 5% CO2 for 15 minutes and then placed on ice. Three replicates

were pooled and PBS was added to a total volume of 11 mL and stimulated exosomes were

collected through ultracentrifugation in an SW40Ti swinging bucket rotor at 100,000g for 3 3

h, as before. Protein extracts were collected from the pelleted stimulated exosomes in in

Urea/SDS lysis buffer (with 100 mM NaF and 1 mM NaOV4) for immunoblot analysis or a

Triton X-100 buffer (150 mM NaCl, 1% (v/v) Triton X-100, 10 mM Na2HPO4, 2 mM

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

KH2PO4, pH 7.4, 50 mM 6-aminohexanoic acid, 10 mM EDTA, 5 mM N-ethylmaleimide, 5

mM benzamidine, 5 ug/mL µg/mL leupeptin, 1 ug/mL µg/mL pepstatin, 1 mM phenylmethylsulphonyl

fluoride, 100 mM NaF, and 1 mM NaOV4) for immunoprecipitation assays.

[00178] Protein Western Blot and Antibodies. Exosomes protein extracts were

loaded according to a Bicinchoninic Acid (BCA) protein assay kit (Pierce, Thermo Fisher

Scientific) onto acrylamide gels and transferred onto PVDF membranes (ImmobilonP) by wet

electrophoretic transfer. Blots were blocked for 1 h at RT with 5% non-fat dry milk in

TBS/0.05% Tween20 and incubated overnight at 4°C with the following primary antibodies:

1:300 anti-CD9 ab92726 (Abcam); 1:300 anti-TSG101 ab83 (Abcam); 1:1000 anti-EGFR

4267S (CST); 1:300 anti-CD63 sc-365604, (Santa Cruz); 1:200 anti-CD81 cs-166029 (Santa

Cruz); 1:1000 anti-pEGFR Tyr1068 3777S (CST); 1:2000 anti-GRB2 610111 (BD Biosciences); 1:1000 anti-Shc 06-203 (Millipore); 1:5000 anti-GFP ab13970 (Abcam);

1:1000 GAPDH ab9483 (Abcam); 1:10,000 HRPconjugated B-actin ß-actin a3854 (Sigma); 1:400

anti-RNA Pol II cat#39097 (Active Motif); 1:500 anti-Hsp90 ab1429 (Abcam); 1:500 anti-

eIF3A ab86146 (Abcam); 1:500 anti-eIF4A1 ab31217 (Abcam). HRP-conjugated secondary

antibodies (Sigma, 1:2000) were incubated for 1 h at room temperature. Washes after

antibody incubations were done on an orbital shaker, four times at 10 min intervals, with 1x

TBS 0.05% Tween20. Blots were developed with chemiluminescent reagents from Pierce.

[00179] Immunoprecipitation. Exosomes and cell protein extracts gently rocked

at 4°C for 2 h. The lysates were centrifuged at 14,000g in a pre-cooled centrifuge for 15

minutes and the pellet was discarded. Protein A or G agarose/sepharose beads were washed

twice with PBS and restored to a 50% slurry with PBS. A bead/slurry mix (100 ul) µl) was added

to 100 ug µg of exosomes protein extracts or 20 ug µg of cells protein extracts and incubated at 4°C

for 10 min. Beads were removed by centrifugation at 14,000g at 4°C for 10 minutes and

pellets discarded. 10 ug µg of anti-eIF4A1 antibody was added to 100 uL µL of exosomal lysate and

incubated overnight at 4°C on an orbital shaker. 100 uL µL of Protein A or G agarose/sepharose

bead slurry were added and left at 4°C overnight overnight.After Aftercentrifugation centrifugationthe thesupernatant supernatantwas was

discarded and beads washed 3 times with ice-cold Urea/SDS buffer. The agarose/sepharose

beads were boiled for 5 minutes to dissociate the immunocomplexes from the beads. The

beads were collected by centrifugation and immunoblot was performed on the supernatant.

[00180] Identification of amino acids using UPLC-MS. Exosomes were mixed

with 200 uL µL of methanol spiked with the Internal Standard tryptophan-d5 and incubated for

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an hour at -20 °C. After centrifugation at 16,000g for 15 min at 4°C, 190 UL µL of the

supernatants was collected and the solvent removed. Dried extracts were reconstituted in 15

UL µL of methanol, of which 10 uL µL were transferred to microtubes and derivatized.

Chromatographic separation and mass spectrometric detection conditions employed are

summarized in Table 1. The mass range, 50 - 1000 m/z, was calibrated with cluster ions of

sodium formate. An appropriate test mixture of standard compounds was analyzed before and

after the entire set of randomized duplicated sample injections, in order to examine the

retention time stability and sensitivity of the LC/MS system throughout the course of the run.

Table 1. Chromatographic conditions for the amino acids platform.

System SQD Column type UPLC BEH UPLC BEH C18, C18,1.0 X 100 mm, 1.0x100 mm, 1.7 1.7umum Flow rate 0.14 ml/min Solvent A H2O H20 + 10mM Ammonium Bicarbonate (+NH4OH until pH: 8.8) Solvent B CAN (%B), time 2%, 0 min (%B), time 8%, 6.5 min (%B), time 20%, 10 min (%B), time 30%, 11 min (%B), time 99.9%, 12 min (%B), time 2%, 14 min Column temperature 40 °C Injection volume 1 1 ul µl Ionisation Ionisation ES+ Source temperature 120 °C Nebulisation N2 flow 600 1 / hour

Nebulisation N2 temperature 350 °C Cone N2 flow 101 // hour 10 hour Capillary voltage 3.2 3.2 kV kV Cone voltage 30 V

[00181] Data were processed using the TargetLynx application manager for

MassLynx 4.1 software (Waters Corp., Milford, USA). A set of predefined retention time,

mass-to-charge ratio pairs, Rt-m/z, corresponding to metabolites included in the analysis are

fed into the program. Associated extracted ion chromatograms (mass tolerance window =

0.05 Da) are then peak-detected and noise-reduced in both the LC and MS domains such that

only true metabolite related features are processed by the software. A list of chromatographic

peak areas is then generated for each sample injection, using the Rt-m/z data pairs (retention

time tolerance = 6 s) as identifiers. Normalization factors were calculated for each metabolite by dividing their intensities in each sample by the recorded intensity of the internal standard in that same sample.

[00182] Digital qPCR. Digital RNA reaction was performed using 3 ng of

cDNA, TaqMan® Universal Master mix and QuantStudio 3D Digital PCR Master Mix vl v1

(Applied Biosystems) according to manufacture recommendations. Using QuantStudio 3D

Digital PCR Chip Loader (Applied Biosystems) a total of 14.5 uL µL of the mix were loaded to

a QuantStudio 3D Digital PCR 20K Chip Kit vl v1 (Applied Biosystems). PCR reaction was

performed on GeneAmp® 9700 (Applied Biosystems) following manufacture protocol. The

chips were imaged using QuantStudio 3D Digital PCR Instrument (Applied Biosystems).

Table 2. Digital PCR Primers.

Name Sequence SEQ ID NO 1 Met AGTAGGTAGCGCGTCAGTCTCATAATCTGAAGGTCGTGAGT TCGATCCTCACACGGGGCA TCGATCCTCACACGGGGCA Leu AGGCGCTGGATTAAGGCTCCAGTCTCTTCGGAGGCGTGGGT 2 TCGAATCCCACCGCTGCCA Val 3 AGTGGTTATCACGTTCGCCTAACACGCGAAAGGTCCCCGGT AGTGGTTATCACGTTCGCCTAACACGCGAAAGGTCCCCGGT TCGAAACCGGGCGGAAACA Ser 4 GGCGATGGACTAGAAATCCATTGGGGTTTCCCCGCGCAGGT GGCGATGGACTAGAAATCCATTGGGGTTTCCCCGCGCAGGT TCGAATCCTGCCGACTACG

[00183] [³S]methionine labeling labeling of of exosomes. exosomes. Exosomes Exosomes were were isolated isolated as as

previously described and resuspended in methionine-free culture medium without FBS with

0.1 - 1.0 --- mCi/ml 1.0 trans mCi/ml label trans [35S]-L-methionine label (Amersham

[³³S]-L-methionine Biosciences) (Amersham and Biosciences) incubated and incubated

overnight. Alternatively exosomes were incubated in the presence of cycloheximide (Sigma,

100 ug/mL). µg/mL). Exosomes were pelleted, washed in ice-cold PBS and resuspended in Urea/SDS

lysis buffer as previously described. Protein extracts were quantified using a BCA protein

assay kit, run on acrylamide gels and transferred onto PVDF membranes (ImmobilonP) by

wet electrophoretic transfer, after which the membranes were analyzed by autoradiography

using using the theEN3HANCE® EN3HANCEautoradiography autoradiographyenhancer according enhancer to the according tomanufacturer's the manufacturer's instructions (Perkin-Elmer).

[00184] Real-time PCR Analysis. DNase treated RNA was retro-transcribed

with MultiScribe Reverse Transcriptase (Applied Biosystems) and oligo-d(T) primers

following total exosomes RNA purification with Trizol (Invitrogen). Real-time PCR was

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performed on an ABI PRISM 7300HT Sequence Detection System Instrument using SYBR

Green Master Mix (Applied Biosystems) and B-actin ß-actin as the control. 28S rRNA primer pairs

(QF00318857) and 18S rRNA primer pairs (QF00530467) were purchased as ready specific

primer pairs from Qiagen. Other primers are listed below. Each measurement was performed

in triplicate. Threshold cycle (Rothstein et al.), the fractional cycle number at which the

amount of amplified target reached a fixed threshold, was determined and expression was

measured using the 2-ACt formula, as previously reported (Livak and Schmittgen, 2001).

Table 3. qPCR Probes.

Name Sequence SEQ ID NO p21 F 5'TACCCTTGTGCCTCGCTCAG3' 5 p21 R 5'GAGAAGATCAGCCGGCGTTT3 6 5'GAGAAGATCAGCCGGCGTTT3 hsa-Actin F 7 5'CATGTACGTTGCTATCCAGGC3 5'CATGTACGTTGCTATCCAGGC3 hsa-Actin R 8 5'CTCCTTAATGTCACGCACGAT3' 5'CTCCTTAATGTCACGCACGAT3 mmu-Actin F 5'GGCTGTATTCCCCTCCATCG3' 9 mmu-Actin R 5'CCAGTTGGTAACAATGCCATGT3' 10 5'CCAGTTGGTAACAATGCCATGT3'

[00185] Lysate preparation for in vitro transcription and translation.

Exosomes and cell pellets were washed once in ice-cold PBS and resuspended in an equal

volume of ice-cold 20 mM HEPES (pH 7.5), 100 mM potassium acetate, 1 mM magnesium

acetate, 2 mM dithiothreitol, and 100 ug/mL µg/mL lysolecithin. After 1 min on ice, they were again

pelleted and resuspended in an equal volume of ice-cold hypotonic extraction buffer. After 5

min on ice, the lysates were disrupted by passing 10 times through a 26-gauge needle

attached to a 1-mL syringe. The resulting homogenates were centrifuged at 1000g for 5 min

at 4°C. The supernatant was collected, and aliquots were frozen in liquid nitrogen and stored

at -80°C for use in the in vitro translation assay.

[00186] In vitro coupled transcription and translation. Lysates obtained from

cells and exosomes as previously described were used for in vitro translation in reaction

volumes of 12 uL. µL. Standard reaction conditions were as follows: cell lysate (final protein

ug) or exosomes lysate (final concentration 100 µg), concentration 10 µg) ug), 1 µg ug pEMT7-GFP

cDNA expression plasmid, 20 mM HEPES-KOH (pH 7.6), 80 mM potassium acetate, 1 mM

magnesium acetate, 1 mM ATP, 0.12 mM GTP, 17 mM creatine phosphate, 0.1 mg/mL

creatine phosphokinase, 2 mM dithiothreitol, 40 uM µM of each of the 20 amino acids, 0.15 mM

spermidine, and 400 U/mL RNAsin (Promega). Incubations were carried out for 3 h at 37°C.

WO wo 2019/191444 PCT/US2019/024603

[00187]

[00187] Electroporation Electroporation and and culture culture of of exosomes. exosomes. Exosomes Exosomes were were pelleted pelleted and and

resuspended in 400 uL µL of electroporation buffer (1.15 mM potassium phosphate pH 7.2, 25

mM potassium chloride, 21% Optiprep), with 20 ug µg of plasmid (pCMV-GFP, pEGFP-p53

Addgene plasmid 12091, pcDNA3-RLUC-POLIRES-FLUC and pcDNA-FLUC). Exosomes

were electroporated using a 4 mm cuvette using a Gene Pulser Xcell Electroporation System

(BioRad), as previously described (Alvarez-Erviti et al., 2011). When appropriate, exosomes

were electroporated in the presence of cycloheximide (Sigma, 100 ug/mL) µg/mL) or a-amanitin -amanitin

(Sigma, 30 ug/mL), µg/mL), for inhibition of translation and transcription, respectively.

Electroporated exosomes were cultured in serum-free DMEM at 37°C for the time points

indicated.

[00188] Flow cytometry analysis of electroporated exosomes. Exosomes

preparations (5 -10 ug) µg) were incubated with 5 uL µL of 4 um µm diameter aldehyde/sulfate latex

beads (Interfacial Dynamics, Portland, OR) and resuspended into 600 uL. µL. Exosomes-coated

beads were analyzed on a FACS Calibur flow cytometer (BD Biosciences) and analyzed for

green fluorescence.

[00189] Exosomes delivery and confocal microscopy. MCF10A cells were

plated at an appropriate confluency in 12-well plates on inserted coverslips and cultured

overnight. The following day cells were incubated with MDA-MB-231 exosomes resuspended in serum-free culture DMEM for 2 h, washed 2h, washed with with cold cold PBS PBS 11x X and fixed for 20

min at room temperature with 4% PFA/PBS. Slides were permeabilized for 10 min at RT

with PBS 0.5% Triton X-100 and counterstained with DAPI. Images were obtained using a

Zeiss LSM510 Upright Confocal System using the recycle tool to maintain identical settings.

For data analysis, images were selected from a pool drawn from at least two independent

experiments. Figures show representative fields.

[00190] Reverse transwell assay. Exosomes were isolated from MDA-MB-231

cells as previously described, and resuspended in PBS and quantified using Nanosight NTA.

10 10 XX 10° 109exosomes exosomesin in 150150 uL PBS were were µL PBS addedadded to each to bottom well of well each bottom a 96-well of a CorningTM 96-well Corning

HTSTranswell® system. PBS alone was added to bottom wells as a negative control. An

insert containing a polycarbonate membrane with 40 nm pores was added to each well, and

100 uL µL of PBS alone, PBS with 20% FBS, or PBS with 10,000 ng/ml of EGF were added to

the insert. Trasnswell plates were incubated at 37°C with 5% CO2, and at samples were

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collected from the upper inserts after 4 h and 24 h incubation for exosomes quantification

using Nanosight NTA.

[00191] Statistics. Error bars indicate + ± s.d. between biological replicates.

Technical as well as biological triplicates of each experiment were performed. Statistical

significance was calculated by Student's t-test, ANOVA or Mann-Whitney test, as

appropriate and specified in the description of the figures.

Example 1 - EGFR phosphorylation is detected in exosomes derived from MDA-MB-

231, triple negative human breast cancer cells

[00192] Exosomes were isolated from different murine and human cell lines

using established ultracentrifugation techniques (Melo et al., 2015; Melo et al., 2014). The

isolated exosomes represent a heterogeneous mix, with the same size distribution being

consistently observed between preparations. NanoSight nanoparticle tracking analysis (NTA)

as well as atomic force microscopy (AFM) revealed particles with a size distribution

averaging 104 I ± 1.5 nm in diameter, and ranging roughly between 30 and 200 nm. This was

confirmed by transmission electron microscopy (TEM) showing extracellular vesicles

surrounded by a lipid bilayer (FIGS. 8A-C). The isolated exosomes were further shown by

immunogold/TEM imaging, immunoblot analysis and imaging flow cytometry to possess

known markers of exosomes (Raposo and Stoorvogel, 2013) (FIGS. 8D-F). To further

confirm their purity, exosomes samples were inoculated onto solidified LB plates, showing

no colony formation when compared to bacterial controls obtained from mouth swabs. This

demonstrates the absence of bacterial contamination in the isolated exosomes (FIG. 8G).

[00193] Exosomes obtained from different cell lines were probed by

immunoblotting for their EGFR content. While exosomes from all cell lines show low levels

of EGFR expression, exosomes derived from the BJ fibroblast cell line and MDA-MB-231

triple negative breast cancer cell line showed strong expression of the receptor. The known

exosomes marker CD81 is shown as a loading control (FIG. 1A). Given the importance of

EGFR for the progression of triple negative breast cancer (Lim et al., 2016; Liu et al., 2012;

Nakai et al., 2016), its functional role in MDA-MB-231 derived exosomes was further

explored. Exosomes were derived from MDA-MB-231 and MCF10A cells, and 1 billion

exosomes were incubated with 500 ng/ml of recombinant human EGF (rhEGF) for 15

minutes at 37°C in serum-free culture media. Immunoblotting of protein extracts obtained

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from these exosomes with an antibody specific for the Tyr1068 residue of EGFR revealed an

increase in the phosphorylation levels of this receptor in exosomes derived from MDA-MB-

231, but not non-tumorigenic MCF10A breast epithelial cells (FIG. 1B). Baseline levels of of

EGFR did not change in any of the samples, confirming the specificity of the observed

increase in phosphorylation. Recombinant human EGF (rhEGF) stimulation also lead to an

increase in the levels of phosphorylated ERK, suggesting that the observed EGFR

phosphorylation triggers downstream signaling events within the exosomes (FIG. 1C).

Further probing of the protein content of MDA-MB-231 exosomes showed that they also

contained downstream effectors of EGFR, namely GRB2 and She (FIG. 1C).

[00194] It was then investigated whether upon rhEGF stimulation, exosomal

EGFR could engage its downstream adaptors. Exosomes were stimulated with rhEGF for 15

minutes at 37°C. The exosomal protein extracts were subjected to pull down assays using

specific antibodies for GRB2 and Shc, and it was detected that upon EGF stimulation they

showed increased co-immunoprecipitation with EGFR (FIGS. 1D,E). Isotype IgGs were used

as a negative control for the pull down, and did not reveal EGFR co-immunoprecipitation.

Additionally, by reversing the assay and pulling down EGFR, it was also possible to detect

coimmunoprecipitated GRB2 only in EGF stimulated exosomes (FIG. 8B). Taken together

these results demonstrate that exosomes from MDA-MB-231 cells contain EGFR that can be

phosphorylated by incubation with its ligand in cell-free conditions, leading to putative

downstream signaling events within the exosomes.

Example 2 - EGF stimulation of exosomes alters their protein content

[00195] Receptor tyrosine kinases require ATP as a substrate for their kinase

activity, and prostate-derived exosomes have been shown to have the capacity to generate

ATP (Ronquist et al., 2013a). To further confirm the existence of phosphorylation activity in

the absence of cells, an ATP quantification assay was performed on exosomes with or

without rhEGF stimulation. Using a luminescence based kit, ATP was detected in exosomes

from both MDA-MB-231 cells and MCF10A cells, albeit in smaller quantities in the latter.

Exosomes from MDA-MB-231 cells, but not MCF10A cells, demonstrated a slight decrease

in their ATP quantity upon stimulation with EGF (FIG. 2A). To further investigate the impact

of EGF stimulation on exosomes, they were incubated them in cell-free conditions for a

period of 48 h. The levels of GRB2 protein levels were consistently higher in exosomes

stimulated with EGF for 48 h, compared to their unstimulated counterparts (FIG. 2B). This raised the intriguing possibility that the protein content of exosomes might have changed upon growth factor stimulation. To further investigate this possibility, mass spectrometry analysis was performed on protein extracts obtained from rhEGF unstimulated or stimulated exosomes. Protein extracts were subjected to trypsin digestion and evaluated using an ESI-

TRAP mass spectrometer to obtain an MS/MS peptide spectrum for each sample. The

obtained spectra were then evaluated against a SwissProt database for peptide identification

to obtain a list of proteins for each exosomes sample. Using the open access FunRich

functional enrichment analysis tool (Pathan et al., 2015) it was observed that the majority of

identified hits in both the unstimulated and stimulated exosomes matched proteins previously

identified in exosomes (Vesiclepedia database) (FIG. 2C). A higher number of proteins were

identified in exosomes stimulated with rhEGF when compared to the unstimulated ones (491

VS. 371, FIG. 2D). While the majority of these proteins were common to both stimulated and

unstimulated exosomes, 224 out of 491 proteins were detected only upon rhEGF stimulation.

While EGFR was identified on both samples, GRB2 was only identified in rhEGF stimulated

exosomes (FIG. 2E). It should be stressed however that this does not mean that GRB2 is not

present in unstimulated exosomes, but it might be present at a level under the detectable

threshold for this type of analysis. The Exponentially Modified Protein Abundance Index

(emPAI), which allows for label-free quantification of relative changes in protein content

based on the observable peptide matches, was employed (Ishihama et al., 2005). The top 15

proteins that revealed a stronger increase in rhEGF stimulated exosomes when compared to

their unstimulated counterparts included several participants of actin remodeling and

membrane dynamics, such as a-actinin, MARCKS, ezrin, moesin, and integrin alpha-2

(Tables 4&5). A gene ontology (GO) analysis was then performed using the PANTHER

overrepresentation test. Interestingly, among the top GO biological processes enriched in the

rhEGF stimulated exosomes, several were related to actin remodeling and migration (5 out of

the 20 top pathways, Table 6).

Table 4. Top 15 proteins identified as upregulated in exosomes from MDA-MB-231 cells incubated with 500 ng/ml of EGF at 37°C for 48 h compared to control exosomes, based on the protein scores using the emPAI method (Ishihama et al., 2005).

Protein Name Description Control EGF- Fold EGF- Treatment Change Change Charged multivesicular body 0.4 2.25 5.625 CHMP2A protein 2a OS=Homo spapiens GN=CHMP2A PE=1 SV=1 Charged multivesicular body 0.19 1.03 5.421052632 CHMP2B wo 2019/191444 WO PCT/US2019/024603

Protein Name Description Control EGF- Fold Treatment Change protein 2b OS=Homo sapiens GN=CHMP2B PE=1 GN=CHMP2B PE=1SV==1 SV=1 Long-chain-fatty-acid--CoA ligase 0.06 0.31 0.31 5.166666667 ACSL4 4 OS=Homo sapiens GN=ACSL4 PE=1 SV=2 L-lactate dehydrogenase B chain 0.12 0.59 4.916666667 LDHB OS=Homo sapiens GN=LDHB PE=1 PE=1 SV==2 SV=2 Alpha-actinin-1 OS=Homo 0.04 0.18 4.5 4.5 ACTN1 sapiens sapiens GN=ACTN11 GN=ACTN1 PE=1 PE=1SV=2 SV=2 ITGA2 Integrin alpha-2 OS=Homo 0.07 0.26 3.714285714 sapiens GN=ITGA2 PE=1 SV=1 Histone H2A type 2-A OS=Homo 6.9 6.9 24.85 3.601449275 HIST2H2AA3 sapiens GN=HIST2H2AA3 PE=1 SV=3 Myristoylated alanine-rich C- 0.14 0.5 0.5 3.571428571 MARCKS MARCKS kinase substrate OS=Homo sapiens GN=MARCKS PE=1 SV=4 Vacuolar protein sorting- 0.14 0.5 3.571428571 VPS37B associated protein 37B OS=Homo sapiens GN=VPS37B PE=1 SV=1 RPL5 60S ribosomal protein L5 0.13 0.45 3.461538462 OS=Homo sapiens GN=RPL5 PE=1 SV=3 EH domain-containing protein 2 0.07 0.07 0.23 3.285714286 EHD2 OS=Homo sapiens GN=EHD2 PE=1 SV=2 DnaJ homolog subfamily A 0,61 0.61 1.84 3.016393443 DNAJA1 member 1 OS=Homo sapiens GN=DNAJA1 PE=1 SV=2 Ezrin OS=Homo sapiens 1.23 3.39 2.756097561 EZR GN=EZR PE=1 SV=4 Moesin OS=Homo sapiens 1.74 4.48 2.574712644 MSN GN=MSN PE=1 SV=3 ADP-ribosylation factor 1 0.5 1.26 2.52 ARF1 OS=Homo sapiens GN=ARF1 PE=1 SV=2

Table 5. Top 15 proteins identified as downregulated in exosomes from MDA-MB-231 cells incubated with 500 ng/ml of EGF at 37°C for 48 h compared to control exosomes, based on the protein scores using the emPAI method (Ishihama et al., 2005).

Protein Name Description Control EGF- Fold Treatment Change "Keratin, type 1 cytoskeletal 9 6.86 1.28 0.186588921 KRT9 OS=Homo sapiens GN=KRT9

Protein Name Description Description Control EGF- Fold Treatment Change PE=1 SV=3" "Keratin, type II cytoskeletal 2 2.46 0.92 0.37398374 KRT2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2" Golgin subfamily A member 7 0.7 0,7 0.3 0.428571429 GOLGA7 OS=Homo sapiens GN=GOLGA7 PE=1 SV=2 CALM1 Calmodulin OS=Homo sapiens 0.64 0.28 0.4375 0.4375 CALM1 GN=CALM1 PE=1 SV=2 HRAS GTPase HRas OS=Homo sapiens 0.48 0.22 0.458333333 GN=HRAS PE=1 SV=1 Secretory carrier-associated 0,26 0.26 0.12 0.461538462 SCAMP2 membrane protein 2 OS=Homo sapiens GN=SCAMP2PE=1 GN=SCAMP2 PE=1 SV=2 RGS19 Regulator of G-protein signaling 0,41 0.41 0.19 0.463414634 19 OS=Homo sapiens GN=RGS19 PE=1 SV=1 Ras-related protein Rab-5B 0.43 0.2 0.465116279 RAB5B OS=Homo sapiens GN=RAB5B PE=1 SV=1 CORO1C Coronin-1C OS=Homo sapiens 0.17 0.08 0.470588235 COROIC GN=CORO1C PE=1 GN=CORO1C SV=1 PE=1SV=1 SLC7A11 Cystine/glutamate transporter 0.17 0.08 0.470588235 OS=Homo sapiens GN=SLC7A11 PE=1 SV=1 PACSIN3 Protein kinase C and casein kinase 0.19 0.09 0.473684211 substrate in neurons protein 3

OS=Homo sapiens GN=PACSIN3 PE=1 SV=2 VPS4B Vacuolar protein sorting- 0.19 0.09 0.473684211 associated protein 4B OS=Homo sapiens GN=VPS4B PE=1 SV=2 OR51E1 Olfactory receptor 51E1 0.27 0.13 0.481481481 OS=Homo sapiens GN=OR51E1 PE=2 SV=1

Table 6. Top 20 gene ontology (GO) pathways identified based on the differential protein scores between control exosomes and exosomes incubated with 500 ng/ml EGF at 37°C for 48 h. A list of differentially expressed proteins was obtained using the emPAI method and used as input for GO analysis using the PANTHER overrepresentation test.

upload_1 upload_1 1 upload_1 upload_1 upload_1 upload_1 GO Homo Homo biological sapiens - (113) (expected) (over/under) (fold (P-value)

process REFLIST Enrichment) complete (21002) actomyosin 3 3 0.02 + > 100 5.77E-03

Homo Homo upload_1 upload de1 upload_1 upload 11 upload_1 upload_ 11 upload_1 upload_1 upload_1a GO biological sapiens --- (113) (expected) (over/under) (fold ----

(P-value)

process process REFLIST Enrichment) complete (21002) contractile

ring ring organization (GO:0044837) actomyosin 3 3 0.02 > 100 5.77E-03 + contractile

ring assembly (GO:0000915) (GO:0000915) assembly of 3 3 3 0.02 > 100 5.77E-03 + actomyosin apparatus involved in cytokinesis (GO:0000912) positive 4 3 0.02 > 100 1.36E-03 + regulation of extracellular

exosome assembly (GO: 1903553) (GO:1903553) regulation of 6 4 0.03 > 100 3.58E-04 + extracellular

exosome assembly (GO. 1903551) (GO:1903551) viral release 6 3 0.03 92.93 4.56E-02 + from host cell

(GO:0019076) (GO:0019076) movement in 6 3 0.03 + 92.93 4.56E-02 environment of other

organism involved in symbiotic interaction

(GO:0052192) movement in 6 3 0.03 + 92.93 4.56E-02 host environment (GO:0052126) exit from host 6 3 0.03 + 92.93 4.56E-02 cell

(GO:0035891) exit from host 6 3 0.03 92.93 4.56E-02 + (GO:0035890) cell separation 17 7 0.09 76.53 6.98E-08 +

73

WO wo 2019/191444 PCT/US2019/024603

and and

upload_1 upload 11 upload_ 1 upload_1 upload_1 upload_ 11 upload_ upload_1 upload_1 GO Homo ----

biological sapiens --- (113) (expected) (over/under) (fold (P-value)

process REFLIST Enrichment) complete (21002) after

cytokinesis (GO:0000920) ESCRT III 10 4 0.05 + 74.34 2.72E-03 complex disassembly (GO: 1904903) (GO:1904903) ESCRT 10 4 0.05 + 74.34 2.72E-03 complex disassembly (GO: 1904896) (GO:1904896) positive 15 6 0.08 74.34 2.69E-06 + regulation of exosomal secretion

(GO: 1903543) (GO:1903543) regulation of 16 6 0.09 69.7 3.94E-06 + exosomal secretion

(GO :1903541) (GO:1903541) regulation of 15 5 0.08 + 61.95 2.09E-04 mitotic spindle assembly (GO: 1901673) (GO:1901673) positive 16 5 0.09 58.08 2.88E-04 + regulation of viral release from host cell

(GO :1902188) (GO:1902188) viral budding 23 7 0.12 + 56.57 5.64E-07 (GO:0046755) viral budding 20 6 0.11 0.11 + 55.76 1.48E-05 via host

ESCRT complex (GO:0039702)

[00196] Taken together, these mass spectrometry data suggest that MDA-MB-

231 exosomes can alter their protein content upon rhEGF stimulation. These data also suggest

that the same exosomes stimulated with rhEGF could undergo actomyosin remodeling and

migration, indicative of a motility phenotype in response to rhEGF stimulation. A

bicinchoninic acid (BCA) assay for protein quantification confirmed the increase in protein

WO wo 2019/191444 PCT/US2019/024603

content in exosomes stimulated with rhEGF, when compared with unstimulated controls

(FIG. 2F). Immunoblotting for B-actin ß-actin also shows an increase in the levels of polymerized

actin in exosomes stimulated with different amounts of rhEGF when compared with control

unstimulated exosomes (FIG. 2G). Collectively, these observations indicate an unexpected

degree of biological activity in exosomes. Therefore, the possibility that exosomes are

capable of synthesizing proteins de novo under permissive conditions was further

investigated and the potential induction of exosomes motility upon growth factor stimulation

was explored.

Example 3 - Exosomes derived from different cell types contain the functional

constituents required for transcription and translation

[00197] Analysis of proteomics data from exosomes of different cellular

origins revealed the presence of several constituents of the protein synthesis machinery, such

as eukaryotic initiation factors, ADP ribosylation factors, and ribosomal proteins (Choi et al.,

2012; Melo et al., 2015; Pisitkun et al., 2004; Valadi et al., 2007) (FIGS. 10, 11A,B). This

information, taken together with the knowledge that mRNAs and their corresponding proteins

are found in exosomes, further suggested that isolated exosomes could possess the capacity to

translate mRNA into proteins.

[00198] Using quantitative PCR (qPCR) analysis, the presence of both 18S and

28S rRNAs was confirmed, as well as tRNAs for methionine, glycine, leucine, serine, and

valine in all analyzed exosomes (FIGS. 12A,B). Additionally, Ultra Performance Liquid

Chromatography-Mass Spectrometry (UPLC-MS) analysis of exosomes revealed the

existence of all free amino acids (FIG. 3A). Immunoblotting analysis identified the presence

of different members of the translation initiation complex in exosomes, including eIF4A,

elF3A, eIF3A, and eIF1A (FIG. 3B), confirming the observations made through mass spectrometry

analysis. In addition, initiation factors eIF4A and elF3A eIF3A co-immunoprecipitate in protein

extracts obtained from exosomes (Morino et al., 2000) (FIG. 12C).

[00199] To functionally address the relevance of constituents for protein

production present in exosomes, total protein extracts of exosomes isolated from MCF10A

and MDA-MB-231 cells were incubated with a cDNA expression plasmid for green

fluorescent protein (GFP plasmid) and a coupled in vitro transcription and translation assay

was performed. Western Blot analysis of the extracts after incubation with the GFP encoding

WO wo 2019/191444 PCT/US2019/024603

plasmid revealed production of GFP protein (FIG. 3C). The fact that exosomes lysates from

both MDA-MB-231 and MCF10A cells allowed for the synthesis of protein from the GFP

expression plasmid confirmed that exosomes derived from different cells likely contain all

the necessary functional components for both DNA transcription and mRNA translation.

Consistent with the potential for DNA transcription, additional immunoblot analysis of

protein extracts from exosomes isolated from different cell sources identified the presence of

RNA polymerase II subunits, both in its phosphorylated and non-phosphorylated forms (FIG.

3D).

Example 4 - Exosomes are capable of cell-independent protein synthesis

[00200] To further validate the finding that exosomes have the autonomous

capability for de novo mRNA translation, isolated exosomes obtained from MDAMB-231

³S-methionineto cells as well as the murine lung cancer E10 cell line were incubated with S-methionine to

enable labeling of newly synthesized proteins by the exosomes. The assay was performed at

37°C in order to activate the putative biosynthetic processes and potential autocrine

stimulation. Autoradiography of protein extracts from exosomes incubated for 72 h in the

presence of 35 S-methionine ³S-methionine exhibited exhibited the the incorporation incorporation ofof the the radioactive radioactive amino amino acid acid into into

several proteins in the range of 40 to 300 kDa. This was largely inhibited when exosomes

were incubated with the protein translation inhibitor cycloheximide along with Sups 35S-

methionine (FIG. 3E). A distinct pattern of labeled proteins was observed when exosomes

derived from different cancer cells were incubated with 35-methionine. ³S-methionine. Additionally, total

protein content was quantified from freshly isolated exosomes incubated in cell-free culture

media. After 48 h of incubation, the total exosomal protein content was significantly

increased (FIG. 3F).

[00201] Next, whether transcription and translation can take place in intact

exosomes, rather than just their lysates, was confirmed by setting up a protocol of exosomes

in vitro translation. A pCMV-GFP expression plasmid was electroporated directly into

exosomes derived from MDA-MB-231 cells (Borges et al., 2013; El-Andaloussi et al., 2012;

Kamerkar et al., 2017) and the electroporated exosomes were incubated at 37°C in serum-free

culture media, for 48 h. qPCR analysis of isolated exosomal RNA after digestion with

DNAse revealed the presence of GFP mRNA in exosomes electroporated with the pCMV-

GFP expression plasmid (FIG. 4A). Transmission electron microscopy showed that the

structure of the exosomes electroporated with the pCMV-GFP plasmid was intact, and

WO wo 2019/191444 PCT/US2019/024603

immunogold labeling using an anti-GFP antibody showed that the protein could only be

detected in the GFP plasmid-containing exosomes (FIG. 4B). Immunoblot analysis of

exosomes protein extracts using a GFP antibody further confirmed the presence of GFP in

pCMV-GFP plasmid electroporated exosomes, observed as early as 12 h after electroporation

(FIG. 4C), 4C). GFP could be observed in exosomes electroporated with the expression plasmid

after 1 week and up to 1 month (FIGS. 4C,D), albeit without any increase over the levels

observed at 24 h. The same pattern was also observed in exosomes derived from MCF10A

cells, confirming that exosomes derived from different cells, not just tumorigenic, contain all

the required constituents and have the capacity for de novo protein synthesis (FIG. 13A).

[00202] Immunoblot analysis of exosomes electroporated with a GFP plasmid

showed a reduction of about 80% in GFP levels when incubated in the presence of the protein

translation inhibitor cycloheximide (FIG. 4E). GFP production was also decreased in the

presence presenceofofa-amanitin, -amanitin,a a transcription inhibitor transcription of RNAofpolymerase inhibitor II (FIGS. RNA polymerase II 4E,F). (FIGS. 4E,F).

NanoSight NTA of electroporated exosomes using a 488 nm laser also detected green

fluorescence in exosomes electroporated with the pCMV-GFP plasmid but not in mock-

electroporated exosomes or exosomes electroporated with the plasmid and cycloheximide or

a-amanitin -amanitin (FIG. (FIG.13B). 13B).Additionally, beads Additionally, basedbased beads flow cytometry analysis flow cytometry of plasmid- analysis of plasmid-

containing exosomes using different electroporation conditions detected the presence of GFP

(FIG. 13C). Next, exosomes were incubated for 24 h at 37°C to initiate biological processes,

before electroporation with pCMV-GFP plasmid. The GFP production, as detected by

immunoblotting, was impaired, suggesting an exhaustion of the required components for

transcription and translation in the exosomes that are pre-incubated at 37°C. (FIG. 4G).

[00203] To confirm that these results were not specific to just GFP, an

ovalbumin expression plasmid (pCMV-Ova), a protein that is also not expressed in

mammalian cells, was used. As with GFP, immunoblotting analysis of exosomes after

electroporation and incubation at 37°C for 48 h showed production of ovalbumin only in

exosomes electroporated with the pCMV-Ova plasmid (FIG. 13D).

[00204] Initiation of protein translation of most mRNAs in eukaryotes involves

recognition of the 5' cap structure by the eIF4F complex (Merrick, 2004). To determine

whether protein translation in exosomes is cap-dependent, a cDNA bicistronic construct was

employed consisting of two different luciferase cistrons separated by an internal ribosome

entry site (FIG. 4H) (Poulin et al., 1998). In this system, translation of Renilla luciferase is

WO wo 2019/191444 PCT/US2019/024603 PCT/US2019/024603

cap-dependent, whereas translation of firefly luciferase is directed by the poliovirus IRES,

and is therefore cap-independent (FIG. 4H). Electroporation of the plasmid directly into

exosomes led to an increase in Renilla Luciferase activity with no apparent change in Firefly

Luciferase activity (FIG. 4I) 41) (Poulin et al., 1998), suggesting that protein translation in

exosomes occurs in a cap-dependent manner. Since Firefly and Renilla Luciferase enzymes

have different activity requirements, this assay was repeated using a plasmid where Firefly

luciferase is expressed under the control of a CMV promoter promoter.The Theluciferase luciferaseactivity activitywas wasalso also

observed in the pCMV-Fluc electroporated exosomes (FIG. 4J).

Example 5 - mRNA translation in exosomes generates functional proteins and can be

stimulated by growth factors

[00205] MCF10A cells pretreated with cycloheximide were incubated with

MDA-MB-231 exosomes that were directly electroporated with the pCMV-GFP plasmid.

Imaging by confocal microscopy detected green fluorescence in the MCF10A cells, likely

contributed by the GFP protein (after transcription and translation) delivered by MDAMB-

231 exosomes (FIG. 5A, top and bottom panels). Interestingly, cells directly electroporated

with the pCMV-GFP plasmid show a GFP fluorescence pattern distinct from fluorescence

pattern observed in cells incubated with pCMV-GFP plasmid containing exosomes (FIG. 5A,

middle and bottom panels).

[00206] MDA-MB-231 cells overexpress an inactive mutant form of the tumor

suppressor protein p53, which is therefore unable to activate the p21 promoter (Gartel et al.,

2003). Wild-type (wt) p53 typically responds to DNA damage by direct induction of p21,

facilitating cell cycle arrest (Zilfou and Lowe, 2009). Exosomes isolated from MDA-MB-231

cells were electroporated with a plasmid encoding for wt p53 fused to GFP. The

electroporated exosomes were incubated in culture media for 48 h to allow transcription and

translation to generate wt p53 protein (FIG. 5B). Subsequently, exosomes containing the

newly formed wt p53 were incubated with recipient MDA-MB-231 cells under the influence

of cycloheximide. The recipient MDA-MB-231 cells revealed a substantial increase in

expression of p21 (FIG. 5C), confirming the functionality of wt p53 protein that was

exclusively synthetized by the exosomes (FIG. 5B). To additionally confirm that this increase

in p21 expression was indeed due to wt p53 protein newly translated by exosomes rather than

due to delivery of the plasmid, MDA-MB-231 derived exosomes were electroporated with

the p53-GFP plasmid and either allowed to incubate for 48 h to synthesize the wt p53 protein

WO wo 2019/191444 PCT/US2019/024603

prior to incubation with the recipient MDA-MB-231 cells (48 h), or delivered immediately to

the recipient MDA-MB-231 cells without allowing them to produce the wt p53 protein (0 h,

FIG. 13E). Exosomes that were allowed to actively synthesize wt p53 protein 48 h prior to

delivery induced p21 expression in the recipient MDA-MB-231 cells as early as 30 minutes

post exosomes incubation, higher than when compared to exosomes with just the plasmid,

which show the same baseline p21 expression observed in control MDA-MB-231 cells (FIG.

13E).

[00207] In order to further demonstrate that exosomes from MDA-MB-231

cells exhibit a baseline capacity for intrinsic protein synthesis, exosomes were incubated at

37°C, with and without the presence of cycloheximide. Immunoblotting of protein extracts

from these exosomes showed a consistent reduction in the expression levels of small

cytoplasmic proteins, B-actin ß-actin and GAPDH, upon incubation with cycloheximide, again

demonstrating the existence of a baseline level of protein synthesis in these exosomes (FIG.

5D).

[00208] In order to determine which proteins are produced by the MDA-MB-

231 exosomes in the absence of external stimuli, an adapted version of stable isotope labeling

with amino acids in culture (SILAC) was performed. Exosomes derived from MDA-MB-231

cells were incubated in SILAC medium supplemented with heavy labeled 13C-Lysine ¹³C-Lysine and

¹N-Arginine. MDA-MB-231 MDA-MB-231 exosomes exosomes were were incubated incubated in in heavy heavy labeled labeled SILAC SILAC medium medium for for

5 days and protein extracts were obtained, trypsin digested, and subjected to mass

spectrometry analysis. While only a small number of heavy-labeled peptides matched the

obtained MS/MS spectra, 11 proteins were able to be identified each matching 1 or 2 peptides

containing the heavy-labeled amino acids (Table 7). This confirms that, albeit at low levels,

baseline mRNA translation occurs in exosomes leading to the formation of very small

amounts of newly synthesized proteins.

wo 2019/191444 PCT/US2019/024603

at contains listed protein Each days. 5 for medium SILAC ¹N-Arginine and ¹³C-Lysine with incubated exosomes MDA-MB-231 of extracts protein of analysis spectrometry mass from obtained isotopes, heavy with spectra matching peptides containing proteins of List 7. Table protein of analysis spectrometry mass from obtained isotopes, heavy with spectra matching peptides containing proteins of List 7, Table at calc. PI calc. PI

contains listed protein Each days. 5 for medium SILAC N-Arginine and C-Lysine 13 with incubated exosomes MDA-MB-231 6.42 7.47 7.47 8.03 5.41 9.41 4.61 4.61

[kDa] 187.1 187.1 151.1

MW 29.6 29.6 24.4 68.4 68.4

32

# AAs

1674 1411 1411 276 253 212 585

## PSMs PSMs

4 2 4 3 2 2 Peptides Peptides

# # Unique 3 1 1 3 1 2 peptides spectra. heavy-labeled ¹N-Arginine or ¹³C-Lysine a matching peptide 1 least spectra. heavy-labeled 5N-Arginine or Superscript(1)-C-Lysine a matching peptide 1 least 1 1 1 2 1 1 Proteins Proteins

# 1 ] 1 1 a 1 1 1 Coverage Coverage

1,31% 1.31% 5.80% 5.80% 4.35% 4.35% 8.96% 1.71% 1.71% 1.49% 1.49% 8,96%

Score Score 51.34 51.34 32.14 57.22 57.22 26.41 26.41 40.81 40.81 40.51 OS=Homo Involucrin OS=Homo Involucrin sapiens GN=KIF21A sapiens GN=KIF21A 3 integrator Bridging 3 integrator Bridging protein Kinesin-like protein Kinesin-like KIF21A OS=Homo KIF21A OS=Homo

[KI21A HUMAN)

[KI21A HUMAN) OS=Homo sapiens OS=Homo sapiens sapiens OS=Homo sapiens OS=Homo sapiens Zinc finger Zinc finger protein protein

[SEGN HUMAN]

[SEGN HUMAN] OS=Homo

[EDNI HUMAN] [INVO HUMAN]

[EDN1 HUMANI

[INVO HUMAN

GN=SCGN PE=1 PE=1 [BIN3 HUMAN] GN=SCGN [BIN3 HUMAN GN=EDN1 PE=1 GN=EDN1 PE=1 sapiens GN=IVL sapiens GN=IVL

GN=BIN3 PE=1 GN=BIN3 PE=1 609 OS=Human 609 OS=Human

PE=1 SV=2 PE=1 SV=2-- PE=1 SV=2 PE=1 SV=2--

Secretagogin Secretagogin Endothelin-1 Endothelin-1 Description Description

SV=1 ren SV=2 - SV=1 ne SV== - SV=1 - SV=2-

of Acession Acession Q9NQY0 Q9NQY0

Q7Z4S6 Q7Z4S6 extracts O76038 O76038 O15014 P05305 P07476 P07476 P05305 wo 2019/191444 PCT/US2019/024603 calc. PI calc. PI

8.73 8.73 8.48 8.48 9.83 9.83 7.43 7.43 8.35 8.35

[kDa]

[kDa] 112.7 112.7 301.5 301.5

MW 44.2 44.2 97.2 20.1 20.1

## AAs AAs

2839 2839

377 377 955 892 892 183 183

## PSMs PSMs

2 5 1 5 1 Peptides Peptides

# ## Unique Unique 2 3 1 4 1 peptides peptides

1 1 1 1 1 Proteins Proteins

# 1 1 1 1 1 Coverage Coverage

1.23% 1.62% 3.83% 4.51% 4.51% 3.87% 3.87% 1.23% 1.62% 3.83%

Score Score 57.08 57.08 81.48 81.48 24.02 24.02 40.91 40.91 23.93 23.93

GN=CCDC113 PE=2 GN=CCDC146PE=2 GN=CCDC146 PE=2 GN=CCDC113 PE=2 sapiens GN=ZNF609 sapiens GN=ZNF609 GN=ZNF512B PE=1 GN=ZNF512B1 PE=1 domain- Coiled-coil domain- Coiled-coil 2 protein containing 2 protein containing

[PDZD2 HUMAN

[PDZD2 HUMANI domain Coiled-coil domain Coiled-coil

[ZN609 HUMAN] [CC113 HUMAN] [CC146 HUMAN]

[ZN609 HUMAN [CC113 HUMAN [Z512B HUMAN]

[CC146 HUMANI [Z512B HUMANI Zincfinger Zinc fingerprotein protein OS=Homosapiens OS=Homo sapiens GN=PDZD2PE=1 GN=PDZD2 PE=1

containing protein containing protein containing protein containing protein

512B OS=Homo 512B OS=Homo

113OS= 113 OS=Homo Homo

146 OS=Homo 146 OS=Homo

PDZdomain- PDZ domain- PE=1 PE=1 SV=2 - SV=2-

Description Description Translocon- Translocon-

sapiens sapiens SV=1 -- SV=1 sapiens SV=2-- SV=2 sapiens sapiens SV=1 - SV=1 -- SV=1 SV=1-

Q96KM6 Acession Acession Q96KM6 Q8IYE0 Q9H0I3 Q8IYE0 O15018 Q9H013 015018 P43308 P43308 wo 2019/191444 PCT/US2019/024603 calc. calc. PIPI

[kDa]

[kDa]

MW MW

## AAs AAs

# # PSMs PSMs

Peptides Peptides

# # # Unique Unique

peptides peptides

Proteins Proteins

# Coverage Coverage

Score Score

los=Homo sapiens OS=Homo sapiens

associated protein [SSRB, HUMAN]

[SSRB HUMAN associated protein

GN=SSR2 PE=1 GN=SSR2 PE=1

Description Description subunit beta subunit beta

SV=1 - SV=1- -

Acession Acession

WO wo 2019/191444 PCT/US2019/024603

[00209] Next, the protein translation assay was repeated using exosomes

derived from MDA-MB-231 cells with electroporated pCMV-GFP plasmid. The exosomes

were incubated in serum-free culture media at 37°C for 48 h with or without stimulation with

rhEGF. While the unstimulated exosomes presented with a baseline level of GFP production,

the GFP levels increased upon incubation with different concentrations of rhEGF (FIG. 5E).

This again confirmed that, while all exosomes can synthetize proteins, growth factor

stimulation can alter their rate of production by leading to increased levels of protein

synthesis.

Example 6 - Exosomes actively migrate in response to stimulation by rhEGF and serum

factors

[00210] In order to determine whether growth factors can induce a motility

phonotype in exosomes, a reverse migration assay based on the Boyden chamber system was

designed including rhEGF and serum factors. Ten billion exosomes isolated from MDA-MB-

231 cells were placed in the culture wells of a 96-well plate, covered by a polycarbonate

surface insert containing 400 nm pores. The insert contained either PBS, PBS with 10 M µM

rhEGF, or PBS with 20% exosomes-depleted FBS (FIG. 6A). Because exosomes-depleted

FBS could still contain trace amounts of exosomes (data not shown), 20% FBS was placed on

the top insert with no exosomes in the bottom well, as a control. After incubation at 37°C,

samples were obtained from the top insert at different time points and exosomes quantified

using Nanosight NTA.

[00211] After a 4 h incubation, the levels of exosomes on the top insert were

comparable across all experimental groups (FIG. 6B). After a 24 h incubation, 20% FBS

significantly increased migration of the exosomes from the bottom to the top, suggesting a

sustained chemotactic influence on MDA-MB-231 exosomes towards the higher serum

growth factor gradient (FIG. 6C). The inserts with 20% FBS over wells with no exosomes

had significantly fewer exosomes after 24 h, confirming the identity of the migrating

exosomes as being from MDA-MB-231 cells (FIG. 6C). PBS resulted in negligible amounts

of exosomes migration but rhEGF alone also induced motility of exosomes, albeit at a lower

level when compared to complete serum associated growth factors (FIG. 6C). Taken together,

these results suggest that exosomes exhibit functional chemotactic capacity that can be

induced by growth factors.

WO wo 2019/191444 PCT/US2019/024603

Example 7 --- Exosomes specifically exhibit enhanced protein production in tumor

bearing mice

[00212] To address whether the capacity of exosomes to respond to the growth

factor gradient induced by tumors involves intrinsic production of new proteins with

functional consequences, a reference mouse model was generated. Mice with established 4T1

mammary tumors were injected with 5 billion MDA-MD 231/CD63-mCherry exosomes

electroporated with either GFP or ovalbumin expression plasmids. Control experimental arms

of this study included CD63-mCherry exosomes without the plasmids and CD63-mCherry

exosomes electroporated with plasmid and cyclohexamide. Twenty-four hours after the I.P.

injection of exosomes in tumor-bearing mice or non-tumor-bearing mice, the tumor, serum,

and several other organs were collected. Exosomes were FACS isolated using the CD63

mCherry tag and evaluated for GFP or ovalbumin protein. GFP and ovalbumin were

predominantly detected in the tumor, lung, bone, brain, and serum of mice with tumors, but

were found only at very low levels in the tissues of non-tumor bearing mice and in tumor

bearing mice that were injected with cyclohexamide-containing cyclohexamide-containing.exosomes. exosomes.These Theseresults results

demonstrate that while exosomes might be detected in the liver, lung, and brain of the non-

tumor bearing mice, the exosomes enter these organs and more robustly (presumably via

enhanced motility) including the tumor tissue, and biologically respond by generating de

novo proteins. Additionally, the serum-derived exosomes from tumor bearing mice exhibit

protein production, suggesting that tumors biologically influence exosomes at a systemic

level.

[00213] All of the methods disclosed and claimed herein can be made and executed

without undue experimentation in light of the present disclosure. While the compositions and

methods of this invention have been described in terms of preferred embodiments, it will be

apparent to those of skill in the art that variations may be applied to the methods and in the

steps or in the sequence of steps of the method described herein without departing from the

concept, spirit and scope of the invention. More specifically, it will be apparent that certain

agents which are both chemically and physiologically related may be substituted for the

agents described herein while the same or similar results would be achieved. All such similar

substitutes and modifications apparent to those skilled in the art are deemed to be within the

spirit, scope and concept of the invention as defined by the appended claims.

WO wo 2019/191444 PCT/US2019/024603

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Claims (30)

WHAT IS CLAIMED IS 29 Aug 2025

1. A method of treating cancer in a patient in need thereof, the method comprising: (a) obtaining exosomes having an epidermal growth factor receptor on their surface; (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; 5 (c) stimulating the exosomes with recombinant epidermal growth factor to induce protein expression by the exosomes; (d) administering the transfected exosomes to a patient; 2019243179

wherein the cancer provides an epidermal growth factor gradient at a site of the cancer to attract the exosomes to the site and deliver the therapeutic protein to the site, thereby treating the 10 disease in the patient.

2. Use of exosomes having an epidermal growth factor receptor on their surface in the manufacture of a medicament for treating cancer, wherein treating the cancer comprises: (a) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; (b) stimulating the exosomes with recombinant epidermal growth factor to induce protein 15 expression by the exosomes; (c) administering the transfected exosomes to a patient; wherein the cancer provides an epidermal growth factor gradient at a site of the cancer to attract the exosomes to the site and deliver the therapeutic protein to the site, thereby treating the disease in the patient.

20 3. A method of treating cancer in a patient in need thereof, the method comprising: (a) obtaining exosomes having an epidermal growth factor receptor on their surface; (b) incorporating a therapeutic agent into the exosomes; (c) stimulating the exosomes with recombinant epidermal growth factor to induce protein expression by the exosomes; 25 (d) administering exosomes obtained from step (c) to a patient; wherein the cancer provides an epidermal growth factor gradient at a site of the cancer to attract the exosomes to the site and deliver the therapeutic agent to the site, thereby treating the cancer in the patient.

4. Use of exosomes having an epidermal growth factor receptor on their surface in the 30 manufacture of a medicament for treating cancer, wherein treating the cancer comprises: (a) incorporating a therapeutic agent into the exosomes;

(b) stimulating the exosomes with recombinant epidermal growth factor to induce protein 29 Aug 2025

expression by the exosomes; (c) administering exosomes obtained from step (b) to a patient, wherein the cancer provides an epidermal growth factor gradient at a site of the cancer to attract 5 the exosomes to the site and deliver the therapeutic agent to the site, thereby treating the cancer in the patient.

5. The method of claim 1 or the use of claim 2, wherein the nucleic acid is an mRNA. 2019243179

6. The method of claim1 or the use of claim 2, wherein the nucleic acid is a plasmid.

7. The method of claim 1 or the use of claim 2, wherein the nucleic acid is a cDNA.

10 8. The method of any one of claims 1, 3 and 5 to 7, wherein the method is further defined as a method of administering a therapeutic protein or therapeutic agent to a cancer cell in a patient.

9. The method or the use of any one of claims 1 to 8, wherein the exosomes are obtained from a body fluid sample obtained from the patient.

15 10. The method or the use of claim 9, wherein the body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum.

11. The method or the use of any one of claims 3, 4, and 8-10, wherein the therapeutic agent is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug.

20 12. The method or the use of claim 11, wherein the antibody binds an intracellular antigen.

13. The method or the use of claim 12, wherein the antibody is a full-length antibody, an scFv, a Fab fragment, a (Fab)2, a diabody, a triabody, or a minibody.

14. The method or the use of claim 11, wherein the therapeutic agent is an inhibitory RNA targeting an oncogene.

25 15. The method or the use of claim 11 or claim 14, wherein the inhibitory RNA is a siRNA, shRNA, miRNA, or pre-miRNA.

16. The method or the use of claim 11, wherein the gene editing system is a CRISPR/Cas 29 Aug 2025

system.

17. The method or the use of any one of claims 1 to 16, wherein the therapeutic protein is a kinase, a phosphatase, or a transcription factor.

5

18. The method or the use of any one of claims 1 to 17, wherein the therapeutic protein corresponds to a wildtype version of a protein that is mutated or inactivated in a cell at the site 2019243179

of the cancer.

19. The method or the use of any one of claims 1 to 18, wherein the therapeutic protein corresponds to a dominant negative version of a protein that is hyperactive in a cell at the site 10 of the cancer.

20. The method or the use of claim 11 wherein the small molecule drug is an imaging agent.

21. The method or the use of any one of claims 1 to 20, wherein the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, 15 uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

22. The method or the use of any one of claims 1 to 21, wherein the cancer is a tumor.

23. The method or the use of any one of claims 1 to 22, wherein the cancer is metastatic.

24. The method or the use of claim 23, wherein the site of the cancer is a metastatic node.

25. The method or the use of any one of claims 1 to 24, wherein the therapeutic protein is 20 a tumor suppressor.

26. The method or the use of any one of claims 1 to 25, wherein the exosomes comprise CD47 on their surface.

27. The method or the use of any one of claims 1 to 26, wherein transfection comprises electroporation.

25 28. The method or the use of any one of claims 1 to 27, wherein the treating comprises administering at least a second therapy to the patient.

29. The method or the use of claim 28, wherein the second therapy comprises a surgical 29 Aug 2025

therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.

30. The method or the use of any one of claims 1 to 29, wherein said exosomes are comprised in tissue scaffold matrix. 2019243179