Therapeutic modulation of tumor suppressors using exosomes

Abstract

Compositions of lipid-based nanoparticles, such as exosomes, are provided herein, the lipid-based nanoparticles comprising therapeutic substances that activate tumor suppressor factors. Methods of using such compositions to treat patients having cancer caused at least partially by loss of tumor suppressor are also provided. In particular, exosomes comprising siRNA targeting mutant p53 are provided along with methods of using such exosomes in the treatment of cancer.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 659,859, filed April 19, 2018, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. This ASCII copy was created on April 9, 2019, is named UTFCP1369WO_ST25.txt, and is 0.7 kilobytes in size.

[0003] 1. Field The present disclosure relates generally to the fields of medicine and oncology. More specifically, the present invention relates to compositions and methods for treating cancer by administering exosomes that carry cargo to activate wild-type tumor suppressors and / or inhibit oncogenic gain-of-function mutants of tumor suppressors. [Background technology]

[0004] 2. Description of Related Technology p53 is a tumor suppressor and is mutated or deleted in several different types of cancer. Several different studies have demonstrated that cancer progression and metastasis are promoted by mutations in the p53 gene. The central functional role of mutated p53 has been demonstrated for many different types of cancer, including pancreatic and breast cancer. Despite the central role of p53 in cancer biology, there is currently no drug that inhibits mutated p53. Using exosomes, the present inventors have developed a novel method for specifically inhibiting mutated p53.

[0005] Extracellular vesicles (EVs), including exosomes, are involved in several physiological processes and are nano-sized intercellular communication vehicles that contain DNA, RNA, and proteins. Exosomes have shown the ability to enter other cells and, in some cases, can deliver therapeutic substances to cancer cells. Summary of the Invention

[0006] overview Thus, provided herein are compositions and methods for specifically activating tumor suppressors, such as p53, using exosomes.

[0007] In one embodiment, a composition is provided that includes lipid-based nanoparticles that contain a therapeutic cargo that inactivates a dominant-negative tumor suppressor mutant or an oncogenic gain-of-function tumor suppressor mutant. In some aspects, the lipid-based nanoparticles include CD47 on their surface. In some aspects, the lipid-based nanoparticles include a growth factor on their surface. In some aspects, the lipid-based nanoparticles are liposomes or exosomes.

[0008] In some aspects, the therapeutic cargo is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug. In certain aspects, the therapeutic protein corresponds to a dominant negative version of an oncogenic gain-of-function tumor suppressor mutant. In certain aspects, the antibody binds to an intracellular antigen. In certain aspects, the antibody is a full-length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody. In certain aspects, the inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA. In certain aspects, the siRNA knocks down expression of the dominant negative tumor suppressor mutant or the oncogenic gain-of-function tumor suppressor mutant. In certain aspects, the gene editing system is a CRISPR system. In certain aspects, the CRISPR system includes an endonuclease and a guide RNA (gRNA). In certain aspects, the endonuclease and the gRNA are encoded on a single nucleic acid molecule within the exosome. In certain aspects, the CRISPR system targets an oncogenic mutation. In certain aspects, the dominant-negative tumor suppressor mutant or the oncogenic gain-of-function tumor suppressor mutant is one or more point mutations.

[0009] In some aspects, tumor suppressors include ACVR1B, APC, ARID1B, ARID2, ASXL1, ATM, ATRX, AXIN1, B2M, BAP1, BCOR, BLU(Beta*), BRCA1, BRCA2, CACNA2D2(Gene26), CASP8, C-CAM, CDKN1A(p21), CDKN1B(p27), CDKN1C(p57), CDKN2A( p16), CDKN2D(p19), CEBPA, CFTR, CIC, CHK2, CREBBP, CTS-1, CYB561D2, CYLD, DAXX, DCC, DPC4, EP300, F AM123B, FCC, FUBP1, FUS1, GATA1, GATA3, HIN-1, HNF1A, HYAL1(Luca-10, HYAL2(Luca-2), KDM5C, KDM6A, KRAS, KRAS2b, MADR2 / JV18, MAP3K1, MCC, MEN1, MEN2, MLH1, MLL2, MLL3, MMAC1, MSH2, MSH6, MTS1, NCOR1 , NF1, NF2, NOTCH1, NOTCH2, NPM1, NPRL2 (Gene21), PAX5, PBRM1, PHF6, PIK3R1, PL6, PLAGL1, PRDM1, PTCH 1, PTEN, RASSF1(123F2), RB1, RNF43, RUNX1, SCGB1A1, SEMA3A, SETD2, Skp2, SMAD2, SMAD4, SMARCA4, SMARCB1, SOCS1, SOX9, STAG2, STK11, TET2, TNPAIP3, TP53, TP73, TRAF7, TSC1, VHL, WRN, WT1, or WWOX.

[0010] In some aspects, the tumor suppressor is TP53. In certain aspects, the oncogenic gain-of-function tumor suppressor mutant is TP53R273H. In certain aspects, the therapeutic is an siRNA, and the siRNA has the sequence of SEQ ID NO:1.

[0011] In some aspects, the tumor suppressor is KRAS. In certain aspects, the oncogenic gain-of-function tumor suppressor mutant is KRASG12D. In certain aspects, the therapeutic is an siRNA, and the siRNA has the sequence of SEQ ID NO:2.

[0012] In some aspects, the composition comprises a first lipid-based nanoparticle comprising an siRNA having the sequence of SEQ ID NO:1, and a second lipid-based nanoparticle comprising an siRNA having the sequence of SEQ ID NO:2.

[0013] In one embodiment, a pharmaceutical composition is provided that includes any one of the lipid-based nanoparticles of the present embodiment. In some aspects, the composition is formulated for parenteral administration. In some aspects, the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. In some aspects, the composition further comprises an antibacterial agent. In certain aspects, the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethl alcohol, phenlymercuric nitrate, propylene glycol, or thimerosal.

[0014] In one embodiment, a method of treating cancer in a patient in need thereof is provided, comprising administering to the patient any one of the compositions of this embodiment, thereby treating the cancer in the patient.In some embodiments, the administration delivers a therapeutic cargo to the cancer cells of the patient.In some aspects, the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, 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 certain aspects, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0015] In some aspects, the cancer is metastatic. In some aspects, the cancer is homozygous for an oncogenic gain-of-function tumor suppressor mutant. In some aspects, the cancer cells are heterozygous for an oncogenic gain-of-function tumor suppressor mutant. In some aspects, the cancer cells are homozygous for a dominant-negative tumor suppressor mutant. In some aspects, the administration is systemic. In certain aspects, the systemic administration is intravenous.

[0016] In some aspects, the method further comprises administering at least a second therapy to the patient. In some aspects, the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy. In some aspects, the patient is a human. In certain aspects, the lipid-based nanoparticles are exosomes, and the exosomes are autologous to the patient. In certain aspects, the exosomes 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 aspirate, ocular exudate / tears, or serum. In some aspects, the method further comprises providing a growth factor gradient to the site of cancer to attract exosomes to the site and deliver a therapeutic substance to the site.

[0017] As used herein, "essentially free" is used herein to mean that, in terms of the named components, none of the named components are intentionally formulated into the composition and / or are present merely as contaminants or in trace amounts. Thus, the total amount of the named components resulting from unintentional contamination of the composition is significantly less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the named components is undetectable using standard analytical methods.

[0018] As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0019] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or to mutually exclusive alternatives, but this disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0020] 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 test subjects.

[0021] [The present invention 1001] A composition comprising lipid-based nanoparticles containing a therapeutic cargo that inactivates a dominant-negative tumor suppressor mutant or an oncogenic gain-of-function tumor suppressor mutant. [The present invention 1002] The composition of claim 1001, wherein the lipid-based nanoparticles comprise CD47 on their surface. [The present invention 1003] The composition of claim 1001, wherein the lipid-based nanoparticles comprise a growth factor on their surface. [The present invention 1004] The composition of claim 1001, wherein the lipid-based nanoparticle is a liposome or an exosome. [The present invention 1005] The composition of the present invention 1001, wherein the therapeutic substance cargo is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug. [The present invention 1006] The composition of the present invention 1005, wherein said therapeutic protein corresponds to a dominant negative version of said oncogenic gain-of-function tumor suppressor mutant. [The present invention 1007] The composition of the present invention 1005, wherein said antibody binds to an intracellular antigen. [The present invention 1008] The composition of the present invention 1005, wherein said antibody is a full-length antibody, an scFv, a Fab fragment, (Fab)2, a diabody, a triabody, or a minibody. [The present invention 1009] The composition of the present invention 1005, wherein said inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA. [The present invention 1010] The composition of the present invention, wherein said siRNA knocks down expression of said dominant-negative tumor suppressor mutant or oncogenic gain-of-function tumor suppressor mutant. [The present invention 1011] The composition of the present invention 1009, wherein the gene editing system is a CRISPR system. [The present invention 1012] The composition of claim 1011, wherein the CRISPR system comprises an endonuclease and a guide RNA (gRNA). [The present invention 1013] The composition of claim 1012, wherein the endonuclease and the gRNA are encoded on a single nucleic acid molecule within the exosome. [The present invention 1014] The composition of the present invention, wherein the CRISPR system targets an oncogenic mutation. [The present invention 1015] The composition of the present invention 1014, wherein said dominant negative tumor suppressor mutant or oncogenic gain-of-function tumor suppressor mutant is one or more point mutations. [The present invention 1016] The tumor suppressor is selected from the group consisting of ACVR1B, APC, ARID1B, ARID2, ASXL1, ATM, ATRX, AXIN1, B2M, BAP1, BCOR, BLU(Beta*), BRCA1, BRCA2, CACNA2D2(Gene26), CASP8, C-CAM, CDKN1A(p21), CDKN1B(p27), CDKN1C(p57), CDKN2A(p16), CDK N2D(p19), CEBPA, CFTR, CIC, CHK2, CREBBP, CTS-1, CYB561D2, CYLD, DAXX, DCC, DPC4, EP300, FAM123B, FC C, FUBP1, FUS1, GATA1, GATA3, HIN-1, HNF1A, HYAL1(Luca-10, HYAL2(Luca-2), KDM5C, KDM6A, KRAS, KRAS 2b, MADR2 / JV18, MAP3K1, MCC, MEN1, MEN2, MLH1, MLL2, MLL3, MMAC1, MSH2, MSH6, MTS1, NCOR1, NF1, NF2, N OTCH1, NOTCH2, NPM1, NPRL2 (Gene21), PAX5, PBRM1, PHF6, PIK3R1, PL6, PLAGL1, PRDM1, PTCH1, PTEN, RAS The composition of the present invention 1001, which is SF1(123F2), RB1, RNF43, RUNX1, SCGB1A1, SEMA3A, SETD2, Skp2, SMAD2, SMAD4, SMARCA4, SMARCB1, SOCS1, SOX9, STAG2, STK11, TET2, TNPAIP3, TP53, TP73, TRAF7, TSC1, VHL, WRN, WT1, or WWOX. [The present invention 1017] The composition of the present invention, wherein said tumor suppressor is TP53. [The present invention 1018] The composition of the present invention 1017, wherein said oncogenic gain-of-function tumor suppressor mutant is TP53R273H. [The present invention 1019] The composition of claim 1018, wherein the therapeutic agent is an siRNA, and the siRNA has the sequence of SEQ ID NO:1. [The present invention 1020] The composition of the present invention, wherein said tumor suppressor is KRAS. [The present invention 1021] The composition of the present invention 1020, wherein said oncogenic gain-of-function tumor suppressor mutant is KRASG12D. [The present invention 1022] The composition of the present invention 1021, wherein the therapeutic agent is an siRNA, and the siRNA has the sequence of SEQ ID NO:2. [The present invention 1023] 1001. The composition of the present invention, comprising a first lipid-based nanoparticle comprising an siRNA having the sequence of SEQ ID NO:1, and a second lipid-based nanoparticle comprising an siRNA having the sequence of SEQ ID NO:2. [The present invention 1024] A pharmaceutical composition comprising the lipid-based nanoparticle of any one of claims 1001 to 1023 and an excipient. [The present invention 1025] A composition of the present invention 1024 formulated for parenteral administration. [The present invention 1026] A composition of the present invention 1025 formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [The present invention 1027] The composition of the present invention 1025 further comprising an antibacterial agent. [The present invention 1028] The composition of the present invention 1027, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. [The present invention 1029] A method for treating cancer in a patient in need thereof, comprising the step of administering to the patient any of the compositions of the present inventions 1024 to 1028, thereby treating the cancer in the patient. [The present invention 1030] The method of claim 1029, wherein administering results in delivery of said therapeutic substance cargo to cancer cells in said patient. [The present invention 1031] The method of claim 1029, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, 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. [The present invention 1032] The method of claim 1031, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma. [The present invention 1033] The method of claim 1029, wherein the cancer is metastatic. [The present invention 1034] The method of claim 1029, wherein said cancer is homozygous for said oncogenic gain-of-function tumor suppressor mutation. [The present invention 1035] The method of claim 1029, wherein said cancer cells are heterozygous for said oncogenic gain-of-function tumor suppressor mutant. [The present invention 1036] The method of claim 1029, wherein said cancer cells are homozygous for said dominant negative tumor suppressor mutant. [The present invention 1037] The method of claim 1029, wherein said administering is systemic. [The present invention 1038] The method of claim 1037, wherein said systemic administration is intravenous administration. [The present invention 1039] The method of claim 1029, further comprising administering at least a second therapy to said patient. [The present invention 1040] The method of claim 1039, wherein said second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy. [The present invention 1041] The method of claim 1029, wherein the patient is a human. [The present invention 1042] The method of claim 1041, wherein said lipid-based nanoparticles are exosomes, and said exosomes are autologous to said patient. [The present invention 1043] The method of claim 1042, wherein said exosomes are obtained from a body fluid sample obtained from said patient. [The present invention 1044] The method of claim 1043, wherein said body fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum. [The present invention 1045] The method of claim 1029, further comprising the step of providing a growth factor gradient to the site of the cancer to attract the exosomes to the site and deliver the therapeutic substance to the site. 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 specific examples, while indicating preferred embodiments of the present invention, are given by way of example only, since various modifications and changes within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0022] 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. [Figure 1] Figure 1A-C. Figure 1A - TP53 expression in Panc-1 cells after treatment with TP53R273H-targeting siRNA. Panc-1 cell line is homozygous mutant for TP53R273H. *p<0.05. Figure 1B-C - Nude mice were orthotopically injected with Panc-1 GFP / Luc cells. Tumor growth was monitored by IVIS imaging and expressed as total flux (p / s). Mice were treated ip with control exosomes (CE), iExosomes containing siRNA suppressing TP53R273H (P53 iexo), or a combination of iExosomes targeting KrasG12D and TP53R273H (P53 iexo and kras iexo). All mice exhibit tumors before treatment (Figure 1B). After treatment and during disease progression (Figure 1C), iexo and combination treatment against TP53R273H reduced tumor burden. [Diagram 2] Adult rhesus monkeys were administered intravenously with unlabeled exosomes (control) or exosomes labeled with PKH membrane dye (PKH-exosomes). The monkeys' livers and pancreases were frozen, sectioned, and mounted on slides. Microscopic evaluation of sections counterstained with DAPI to define nuclear contours revealed strong and specific accumulation of exosomes in the liver and pancreas. [Diagram 3]Figure 3A-B. Figure 3A - Adult rhesus monkeys were administered intravenously with unlabeled exosomes (control) or exosomes labeled with PKH membrane dye (PKH-exosomes). Monkey livers and pancreases were frozen, sectioned, and mounted on slides. Confocal microscopy of sections counterstained with DAPI to define nuclear contours revealed colocalization of exosomes with pancreatic cell nuclei, with strong and specific accumulation of exosomes in the liver and pancreas. Figure 3B - Quantitative analysis of exosome foci size observed in liver and pancreas, with larger foci size and higher exosome accumulation per cell in the pancreas compared to the liver. [Figure 4] Quantification of siRNA payload (in exosomes) in the listed organs after administration to adult rhesus monkeys, as determined by q-PCR. Two monkeys received iExosomes intravenously (iv) and the third monkey received iExosomes intraperitoneally (ip). "0" indicates no siRNA was detected. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description Exosomes loaded with siRNA specific to oncogenic p53 mutants attenuated tumor growth in mice bearing pancreatic tumors. This attenuation was further enhanced by the combination of exosomes loaded with siRNA specific to oncogenic p53 mutants and exosomes loaded with siRNA specific to oncogenic Kras mutants. Thus, provided herein is a method for targeting oncogenic mutations in tumor suppressors such as p53 and / or Kras, and dominant-negative mutations in tumor suppressors in cancer cells.

[0024] I. Lipid-Based Nanoparticles In some embodiments, the lipid-based nanoparticle is a liposome, an exosome, a lipid preparation, or another lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP:cholesterol vesicle). The lipid-based nanoparticle may be positively charged, negatively charged, or neutral. The lipid-based nanoparticle may include components necessary for transcription and translation, signal transduction, chemotaxis, or other cellular functions.

[0025] In some embodiments, the lipid-based nanoparticles contain CD47 on their surface. CD47 (integrin binding protein) is a transmembrane protein expressed in most tissues and cells. CD47 is a ligand for Signal Regulatory Protein Alpha (SIRP-α) expressed in phagocytes such as macrophages and dendritic cells. Activated CD47-SIRP-α initiates a signaling cascade that inhibits phagocytosis. Thus, without being bound by theory, CD47 expression on the surface of exosomes may block phagocytosis by macrophages (see WO2016 / 201323, which is incorporated herein by reference in its entirety).

[0026] A. Liposomes "Liposome" is a generic term that includes various unilamellar and multilamellar lipid vesicles formed by forming a closed lipid bilayer or aggregate. Liposomes may be characterized as having a vesicular structure with a bilayer membrane that generally comprises phospholipids and an internal medium that generally comprises an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged.

[0027] Multilamellar liposomes contain multiple lipid layers separated by aqueous medium. Such liposomes form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution. The lipid components self-reorganize before forming a closed structure that traps water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0028] In certain aspects, the polypeptide, nucleic acid, or small molecule drug may, for example, be placed in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that binds both the liposome and the polypeptide / nucleic acid, entrapped within the liposome, or complexed with the liposome.

[0029] The liposomes used according to this embodiment can be prepared by various methods as known to those skilled in the art. For example, phospholipids, such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC), are dissolved in tert-butanol. The lipids are then mixed with polypeptides, nucleic acids, and / or other components. Tween 20 is added to the lipid mixture so that Tween 20 is about 5% of the composition's weight. Excess tert-butanol is added to this mixture so that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized preparations can be stored at -20°C and used for up to 3 months. When necessary, the lyophilized liposomes are reconstituted by dissolving in 0.9% saline.

[0030] Alternatively, liposomes can be prepared by mixing lipids dissolved in a solvent in a container, e.g., a glass pear-shaped flask. The volume of the container should be 10 times the volume of the expected liposome suspension. The solvent is removed using a rotary evaporator under negative pressure at about 40°C. Typically, the solvent is removed within about 5 minutes to 2 hours, depending on the desired volume of liposomes. The composition can be placed in a desiccator and further dried under reduced pressure. Dried lipids tend to deteriorate over time, so they are generally discarded after about one week.

[0031] The dried lipids can be dissolved and hydrated in sterile pyrogen-free water at approximately 25-50 mM phospholipid by shaking until all of the lipid film is resuspended. The aqueous liposome solution can then be divided into aliquots, each placed into a vial, lyophilized, and sealed under vacuum.

[0032] The dried lipid or lyophilized liposomes prepared as described above may be dehydrated, dissolved in a solution of protein or peptide, reconstituted, and diluted to an appropriate concentration with a suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer. The unencapsulated additional material, such as agents including, but not limited to, hormones, drugs, nucleic acid constructs, etc., is removed by centrifugation at 29,000×g, and the liposome pellet is washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM. The amount of encapsulated additional material or active agent can be determined according to standard methods. After determining the amount of encapsulated additional material or active agent in the liposome preparation, the liposomes may be diluted to an appropriate concentration and stored at 4° C. until use. Pharmaceutical compositions containing liposomes usually contain a sterilized pharma- ceutical acceptable carrier or diluent, e.g., water or saline.

[0033] Additional liposomes that may be useful with this embodiment include cationic liposomes, such as those described in WO02 / 100435A1, U.S. Patent No. 5,962,016, U.S. Patent Application No. 2004 / 0208921, WO03 / 015757A1, WO04 / 029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068, all of which are incorporated by reference in their entirety without disclaimer.

[0034] In preparing such liposomes, any protocol described herein or known to those skilled in the art can be used.More non-limiting examples of preparing liposomes are described in U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706; International Application PCT / US85 / 01161 and PCT / US89 / 05040, each of which is incorporated herein by reference.

[0035] In certain embodiments, the lipid-based nanoparticle is a neutral liposome (e.g., DOPC liposome). As used herein, "neutral liposome" or "uncharged liposome" is defined as a liposome having one or more lipid components that result in an essentially neutral net charge (substantially uncharged). "Essentially neutral" or "essentially uncharged" means that within a particular population (e.g., a population of liposomes), some lipid components, if any, contain a charge that is not counterbalanced by the opposite charge of another component (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1% of the components contain an uncounterbalanced charge). In certain embodiments, neutral liposomes may contain a majority of lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., about pH 7).

[0036] The liposomes and / or lipid-based nanoparticles of this embodiment may include phospholipids. In certain embodiments, one type of phospholipid may be used in making liposomes (e.g., neutral phospholipids, such as DOPC, may be used to make neutral liposomes). In other embodiments, multiple types of phospholipids may be used to make liposomes. Phospholipids may be derived from neutral or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., about pH 7), so these compounds may be particularly useful for making neutral liposomes. In certain embodiments, the phospholipid DOPC is used to generate uncharged liposomes. In certain embodiments, non-phospholipid lipids (e.g., cholesterol) may be used.

[0037] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myris ... phosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoylsphingomyelitis ("Sphingomyelin"), and the like. dioleoylphosphatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), di Myristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristoyl phosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,These include, but are not limited to, 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyleoylphosphatidylcholine ("POPC"), palmitoyleoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0038] B. Exosomes As used herein, the terms "microvesicle" and "exosome" refer to membranous particles having a diameter (or largest dimension if the particle is not a spheroid) of about 10 nm to about 5000 nm, more typically 30 nm to 1000 nm, and most typically about 50 nm to 750 nm, where at least a portion of the exosome's membrane is obtained directly from a cell. Most commonly, the size (average diameter) of the exosome is up to 5% of the size of the donor cell. Thus, exosomes specifically contemplated include exosomes that have been shed from a cell.

[0039] Exosomes may be detected in or isolated from any suitable sample type, such as, for example, bodily fluids. As used herein, the term "isolated" refers to separation from the natural environment and is intended 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 contains exosomes suitable for detection or isolation. Sources of samples include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, joint fluid, breast milk, sweat, tears, synovial fluid, and bronchial lavage fluid. In one aspect, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof. Blood samples suitable for use with the present invention can be extracted from any known source that contains blood cells or components thereof, such as, for example, venous, arterial, peripheral, tissue, cord, etc. For example, samples can be obtained and processed using well-known and routine clinical methods (e.g., procedures for collecting and processing whole blood). In one aspect, an exemplary sample can be peripheral blood taken from a subject with cancer.

[0040] 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 obtain a single cell suspension and then homogenize the tissue to lyse the cells and release the exosomes.When isolating exosomes from tissue samples, it is important to select a homogenization and lysis technique that does not destroy the exosomes.The exosomes contemplated herein are preferably isolated from body fluids dissolved in a physiologically acceptable solution, such as buffered saline, growth medium, various aqueous media, etc.

[0041] 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 media. Although not necessary, exosomes of higher purity may be obtained if the fluid sample is clarified before precipitation with a volume-excluding polymer to remove debris from the sample. Clarification methods 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 supernatant. Exemplary methods of exosome isolation are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Alternatively, exosomes may also be isolated by flow cytometry as described in (Combes et al., 1997).

[0042] One commonly accepted protocol for isolating exosomes involves ultracentrifugation, which is often combined with a sucrose density gradient or sucrose cushion to float the relatively low density exosomes. Isolation of exosomes by sequential differential centrifugation is complicated by the potential for overlap in size distribution with other microvesicles or macromolecular complexes. In addition, centrifugation can provide an insufficient means of separating vesicles based on size. However, exosomes can be highly enriched when sequential centrifugation is combined with sucrose gradient ultracentrifugation.

[0043] Size-based exosome isolation using alternatives to ultracentrifugation routes is another option. Successful exosome purification has been reported using ultrafiltration, a method that takes less time than ultracentrifugation and does not require specialized equipment. Similarly, a commercial kit (EXOMIR™, Bioo Scientific) is available that uses positive fluid-moving pressure to remove cells, platelets, and cellular debris in one microfilter and capture vesicles larger than 30 nm in a second microfilter. However, in this process, exosomes are not recovered, and their RNA content is extracted directly from the material captured in the second microfilter and can then be used for PCR analysis. Using HPLC-based protocols, these processes require specialized equipment and are difficult to scale up, but could potentially yield high purity exosomes. A significant problem is that both blood and cell culture media contain numerous nanoparticles (some of which are non-vesicles) in the same size range as exosomes. For example, some miRNA may be contained in extracellular protein complexes rather than exosomes. However, treatment with a protease (eg, proteinase K) can be carried out to eliminate any contamination with "extraexosomal" proteins.

[0044] In another embodiment, cancer cell-derived exosomes may be captured by techniques commonly used to enrich exosomes in a sample, such as techniques involving immunospecific interactions (e.g., immunomagnetic capture). Immunomagnetic capture, also known as immunomagnetic cell separation, typically involves attaching antibodies made against proteins found on the surface of certain cell types to small paramagnetic beads. When the antibody-coated beads are mixed with a sample, such as blood, the beads attach to and surround the specific cells. The sample is then placed in a strong magnetic field, causing the beads to pellet on one side. After removing the blood, the captured cells are retained together with the beads. Many variations of this general method are known in the art and are suitable for use to isolate exosomes. In one example, exosomes may be attached to magnetic beads (e.g., aldehyde / sulfate beads), and then antibodies are added to the mixture to recognize epitopes on the surface of the exosomes attached to the beads. Exemplary proteins found 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 can be isolated, for example, using antibodies or aptamers against one or more of these proteins.

[0045] 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 is not limited to, ex vivo detection and visualization by microscopy or cytometry of exosomes bound to a solid support, flow cytometry, fluorescent imaging, and the like. In an exemplary aspect, the cancer cell-derived exosomes are selected from the group consisting 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 (HIST1H1AA), 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 α-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 β-2B chain (TUBB2B), endoribonuclease Dicer (DICER1), E3 ubiquitin-protein ligase TRIM71 (TRIM71), Katanin p60 ATPase-containing subunit A-like 2 (KATNAL2), protein S100-A6 (S100A6), 5'-nucleotidase domain-containingprotein 3 (NT5DC3), 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 then bound to a solid support and / or visualized using microscopic or cytometric detection.

[0046] It should be noted that not all proteins expressed in a cell are found in the exosomes secreted by that cell. For example, calnexin, GM130, and LAMP-2 are all proteins expressed in MCF-7 cells, but are not found in the exosomes secreted by MCF-7 cells (Baietti et al., 2012). As another example, a study found that 190 / 190 pancreatic ductal adenocarcinoma patients have higher levels of GPC1+ exosomes than healthy controls (Melo et al., 2015, the entirety of which is incorporated herein by reference). Notably, on average, only 2.3% of healthy controls had GPC1+ exosomes.

[0047] 1. Exemplary Protocol for Harvesting Exosomes from Cell Cultures On day 1, seed a sufficient number of cells (e.g., about 5 million cells) into a T225 flask in medium containing 10% FBS so that the cells are about 70% confluent the next day. On day 2, aspirate the medium on the cells, wash the cells twice with PBS, and then add 25-30 mL of basal medium (i.e., no PenStrep or FBS) to the cells. Incubate the cells for 24-48 hours. Although a 48 hour incubation is preferred, some cell lines are sensitive to serum-free medium, and therefore the incubation time must be reduced to 24 hours. Note that FBS contains exosomes that strongly skew the NanoSight results.

[0048] On day 3 / 4, collect the medium and centrifuge at 800 x g for 5 min at room temperature to pellet dead cells and large debris. Transfer the supernatant to a new conical tube and recentrifuge the medium at 2000 x g for 10 min to remove other large debris and large vesicles. Pass the medium through a 0.2 μm filter and then aliquot it into ultracentrifuge tubes (e.g., 25 x 89 mm Beckman Ultra-Clear) using 35 mL / tube. If the volume of medium per tube is less than 35 mL, fill the remainder of the tube with PBS to make 35 mL. Ultracentrifuge the medium at 28,000 rpm for 2-4 hours at 4°C using a SW 32 Ti rotor (k-factor 266.7, RCF max 133,907). Carefully aspirate the supernatant until approximately 1 inch of liquid remains. Tilt the tube and slowly dispense the remaining medium into an aspirator pipette. If desired, the exosome pellet can be resuspended in PBS and ultracentrifuged at 28,000 rpm for 1-2 h repeatedly to further purify the exosome population.

[0049] Finally, resuspend the exosome pellet in 210 µL of PBS. If there are multiple ultracentrifuge tubes for each sample, use the same 210 µL PBS to successively resuspend each exosome pellet. For each sample, take 10 µL and add to 990 µL of H2O for use in nanoparticle tracking analysis. Use the remaining 200 µL exosome-containing suspension for downstream processes or store immediately at -80 °C.

[0050] 2. Exemplary Protocol for Extracting Exosomes from Serum Samples First, thaw the serum sample on ice. Then, dilute 250 μL of cell-free serum sample with 11 mL of PBS. Filter through a 0.2 μm pore filter. Ultracentrifuge the diluted sample at 150,000 × g at 4 ° C overnight. The next day, carefully discard the supernatant and wash the exosome pellet with 11 mL PBS. Ultracentrifuge a second time at 150,000 × g at 4 ° C for 2 hours. Finally, carefully discard the supernatant and resuspend the exosome pellet in 100 μL PBS for analysis.

[0051] C. Exemplary Protocols for Electroporating Exosomes and Liposomes 1×10 8 100 nm exosomes (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula Nano Sciences) are mixed with 1 μg siRNA (Qiagen) or shRNA in 400 μL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). Exosomes or liposomes are electroporated using a 4 mm cuvette (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After electroporation, exosomes or liposomes are treated with protease-free RNase followed by the addition of 10× concentrated RNase inhibitor. Finally, exosomes or liposomes are washed with PBS under ultracentrifugation as described above.

[0052] II. Treatment of Disease Certain aspects of the present invention provide for treating patients with exosomes that express or contain therapeutic agents that inactivate the oncogenic gain-of-function activity of mutant tumor suppressors in cancer cells or inactivate the dominant-negative activity of mutant tumor suppressors in cancer cells, thereby allowing wild-type alleles of tumor suppressors to function.As used herein, "therapeutic agents" are atoms, molecules, or compounds that are 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, gene editing systems, chelating agents, boron compounds, photoactivators, and dyes.

[0053] Since exosomes are known to contain DICER and active RNA processing RISC complexes (see PCT Publication WO2014 / 152622, which is incorporated herein by reference in its entirety), shRNAs introduced into exosomes by transfection can be matured into RISC complex-bound siRNAs in exosomes, or mature siRNAs themselves can be introduced into exosomes or liposomes by transfection.Thus, by way of example, tumor suppressors (e.g., ACVR1B, APC, ARID1B, ARID2, ASXL1, ATM, ATRX, AXIN1, B2M, BAP1, BCOR, BLU(Beta*), BRCA1, BRCA2, CACNA2D2(Gene26), CASP8, C-CAM, CDKN1A(p21), CDKN1B(p27), CDKN1C(p57), CDKN2A(p16), CDKN2D(p19), CEBPA, CFTR, CIC, CHK2, CREBBP, CTS-1, CYB561D2, CYLD, DAXX, DCC, D PC4, EP300, FAM123B, FCC, FUBP1, FUS1, GATA1, GATA3, HIN-1, HNF1A, HYAL1(Luca-10, HYAL2(Luca-2), KDM5C, KDM6A, KRAS, KRAS2b, MADR2 / JV18, MAP3 K1, MCC, MEN1, MEN2, MLH1, MLL2, MLL3, MMAC1, MSH2, MSH6, MTS1, NCOR1, NF1, NF2, NOTCH1, NOTCH2, NPM1, NPRL2 (Gene21), PAX5, PBRM1, PHF6, PIK3R1, PL 6, dominant-negative or oncogenic gain-of-function mutations (e.g., TP53R273H; TP53R175H; KRASG12R175H; KRASG12R275H; KRASG12 ...275H; KRASG1 Inhibitory RNA can be used in the method of the present invention to regulate or attenuate the expression of D). In some cases, sh / siRNA can be designed to specifically target the mutant version of a gene expressed in cancer cells without affecting the expression of the corresponding wild-type version. In fact, any inhibitory nucleic acid can be applied in the composition and method of the present invention, provided that such inhibitory nucleic acid is found by any supplier to be a verified downregulator of the protein of interest.

[0054] There are several factors that need to be considered when designing RNAi, such as the nature of the siRNA, the permanence of the silencing effect, and the choice of delivery system. To produce the RNAi effect, the siRNA introduced into the organism typically contains exon sequences. Furthermore, the RNAi process depends on homology. Therefore, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA shows more than 80%, more than 85%, more than 90%, more than 95%, more than 98% identity, or even 100% identity between the siRNA sequence and the gene to be inhibited. Sequences with less than about 80% identity with the target gene have a much smaller effect. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0055] Since exosomes are known to contain the machinery required to complete mRNA transcription and protein translation (see PCT / US2014 / 068630, the entirety of which is incorporated herein by reference), mRNA or DNA nucleic acid encoding therapeutic proteins, such as therapeutic antibodies, may be introduced into exosomes by transfection. Alternatively, therapeutic proteins themselves may be electroporated into exosomes or directly incorporated into liposomes. Exemplary therapeutic proteins include, but are not limited to, tumor suppressor proteins, peptides, wild-type protein counterparts of mutant proteins, DNA repair proteins, proteolytic enzymes, protein toxins, proteins that can inhibit the activity of intracellular proteins, proteins that can activate the activity of intracellular proteins, or any protein that needs to reconstitute loss of function.

[0056] One particular type of protein that may be desirable to introduce into the intracellular space of diseased cells is an antibody (e.g., a monoclonal antibody) that can specifically or selectively bind to an intracellular antigen. Such an antibody can disrupt the function of an intracellular protein and / or disrupt intracellular protein-protein interactions. Exemplary targets of such monoclonal antibodies include, but are not limited to, oncogenic gain-of-function mutants of tumor suppressors. In addition to monoclonal antibodies, any antigen-binding fragment thereof, such as scFv, Fab fragment, Fab', F(ab')2, Fv, peptibody, diabody, triabody, or minibody, is also contemplated. Any such antibody or antibody fragment may be glycosylated or aglycosylated.

[0057] Exosomes may also be engineered to contain gene editing systems, such as CRISPR / Cas systems, that correct oncogenic gain-of-function or dominant-negative mutations of tumor suppressors in cancer genes. In general, "CRISPR system" collectively refers to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also called "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts derived from CRISPR loci. In some aspects, Cas nuclease and gRNA (including a fusion of a crRNA specific for a target sequence with a given tracrRNA) are introduced into cells. Generally, the target site at the 5' end of the gRNA targets the Cas nuclease to the target site, e.g., to a gene, using complementary base pairing. The target site may be selected based on the position immediately 5' of the protospacer adjacent motif (PAM) sequence, e.g., typically NGG, or NAG. In this regard, the gRNA targets 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. Generally, CRISPR systems feature elements that promote CRISPR complex formation at the target sequence site. Typically, "target sequence" generally refers to a sequence that the guide sequence is designed to have complementarity with, and hybridization between the target sequence and the guide sequence promotes CRISPR formation. Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote CRISPR complex formation.The CRISPR system in exosomes engineered to contain such a system can function to edit genomic DNA in target cells, or the system can edit DNA within the exosome itself. Further aspects relate to the use of exosomes as a delivery vehicle for gene editing systems, see U.S. Patent Application No. 62 / 599,340, the entirety of which is incorporated herein by reference.

[0058] In addition to protein- and nucleic acid-based therapeutic agents, exosomes may be used to deliver small molecule drugs alone or in combination with any protein- or nucleic acid-based therapeutic agent. Exemplary small molecule drugs contemplated for use in this embodiment include, but are not limited to, toxins, chemotherapeutic agents, and agents that inhibit the activity of oncogenic gain-of-function mutants of tumor suppressors.

[0059] In some embodiments, exosomes can be induced to undergo chemotaxis toward serum factors. Thus, exosomes can be induced to preferentially accumulate in tumors by providing a growth factor gradient that attracts exosomes to tumors. In aspects involving the expression of protein therapeutics from nucleic acids within exosomes, growth factor receptors such as EGFR on the surface of exosomes can be stimulated to enhance transcription and / or translation. For further aspects related to the use of exosomes as minicells for targeting delivery to tumor tissues and for delivering therapeutic substances, see U.S. Patent Application No. 62 / 649,057, which is incorporated herein by reference in its entirety.

[0060] "Subject" as used herein refers to any individual or patient that the method is carried out.Generally, subject is human, but as understood by those skilled in the art, subject can also be animal.Thus, other animals, including mammals, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.

[0061] "Treatment" and "treating" refer to the administration or application of a therapeutic substance to a subject, or the performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment may include the administration of exosomes carrying a cargo, the administration of chemotherapy, immunotherapy, or radiation therapy, the performance of surgery, or any combination thereof.

[0062] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that enhances or improves the health of a subject with respect to the medical treatment of the condition. This term includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer may include, for example, reducing the invasiveness of a tumor, reducing the rate of cancer growth, or preventing metastasis. Treating cancer may also refer to extending the survival of a subject with cancer.

[0063] As used herein, the term "cancer" may be used to describe a solid tumor, a metastatic cancer, or a non-metastatic cancer. In certain aspects, the cancer may occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testicles, tongue, or uterus.

[0064] Cancers include, specifically, the following histological types of cancer: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell 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; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma adenocarcinoma;basophilic carcinoma;clear cell adenocarcinoma;granular cell carcinoma;follicular adenocarcinoma;papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma;adrenal cortical carcinoma;endometrioid carcinoma;skin adnexal carcinoma;apocrine gland carcinoma;sebaceous gland carcinoma;ceruminous adenocarcinoma);mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell tumor;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extra-mammary paraganglioma, malignant;pheochromocytoma;angioangiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus melanoma in giant pigmented nevus);epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Interstitial sarcoma;Mixed tumor, malignant;Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parocytoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor tumor, malignant;ameloblastic odontosarcoma;ameloblastoma, malignant;ameloblastic fibrosarcoma;pinealoma, malignant;chordoma;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrous astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;oligodendroglioblastoma;anaplastic neuroectodermal;cerebellar sarcoma;ganglionuclear ganglioblastoma;neuroblastoma;retinoblastoma;olfactory nerve tumor;meningioma, malignant;neurofibrosarcoma;schwannoma, malignant;granular cell tumor, malignant;malignant lymphoma;Hodgkin's disease disease); Hodgkin's disease; lateral granuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, diffuse large cell; malignant lymphoma, follicular; mycosis fungoides; other non-Hodgkin's lymphomas specified; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic 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.;

[0065] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic agent is delivered to or placed in direct juxtaposition with a target cell. To kill a cell, for example, one or more agents are delivered to the cell in an amount effective to kill the cell or prevent the cell from dividing.

[0066] An effective patient response or a patient's "responsiveness" to a treatment refers to a clinical or therapeutic benefit conferred on a patient at risk of or suffering from a disease or disorder. Such benefits may include a cellular or biological response, a complete response, a partial response, stable disease (no progression or recurrence), or a response that subsequently recurs. For example, an effective response may be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer. Treatment outcomes may be predicted and monitored, and / or patients who will benefit from such treatment may be identified or selected by the methods described herein.

[0067] Regarding the treatment of neoplastic conditions, depending on the stage of the neoplastic condition, the treatment of the neoplastic condition involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiation therapy, and chemotherapy.Other therapeutic regimens may be combined with the administration of anti-cancer agents, such as therapeutic compositions and chemotherapeutic agents.For example, the patient who is to be treated with such anti-cancer agents may also undergo radiation therapy and / or surgery.

[0068] When treating a disease, the appropriate dosage of the therapeutic composition will depend on the type of disease being treated, as defined above, the severity and course of the disease, the patient's medical history and response to the agent, and the judgment of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments.

[0069] The therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect.Tissue, tumor, or cell can be contacted with one or more compositions or pharmacological preparations that contain one or more of the active substances, or the tissue, tumor, and / or cell can be contacted by contacting with two or more separate compositions or preparations.It is also contemplated that such combination therapy can be used with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

[0070] Coadministration may include coadministration of two or more agents in the same dosage form, coadministration in separate dosage forms, and separate administration. That is, the therapeutic composition and another therapeutic agent can be formulated together in the same dosage form and coadministered. Or, the therapeutic composition and another therapeutic agent can be coadministered, where both agents are in separate formulations. In another option, a therapeutic agent can be administered immediately after the other therapeutic agent, or vice versa. In another administration protocol, the therapeutic composition and another therapeutic agent can be administered minutes apart, hours apart, or days apart.

[0071] The first anti-cancer treatment (e.g., exosomes containing a therapeutic agent) may be administered before, during, after, or in various combinations relative to the second anti-cancer treatment. Administration may be simultaneous or at intervals ranging from minutes to days to weeks. In embodiments in which the first treatment is provided to the patient separately from the second treatment, the effective period will generally not expire between the respective deliveries so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is contemplated that the first and second therapies may be provided to the patient within about 12-24 hours or 72 hours of administering either therapy, more particularly within about 6-12 hours of administering either therapy. In some circumstances, it may be desirable to extend the period for treatment significantly. In this case, the period between each administration can be from a few days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0072] In certain embodiments, the course of treatment lasts for 1 day to 90 days or longer (such ranges are inclusive). It is contemplated that one agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof, and another agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof. Within a day (24 hour period), the patient may receive one or more administrations of the agent. It is further contemplated that after the course of treatment, there will be a period during which no anti-cancer treatment is administered. This period may last for 1 day to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or more (such ranges are inclusive), depending on the patient's condition, e.g., the patient's prognosis, strength, health, etc. It is anticipated that the treatment cycle will be repeated as necessary.

[0073] Various combinations may be used. For the example below, the first anti-cancer therapy is "A" and the second anti-cancer therapy is "B". TIFF0007602246000001.tif17128

[0074] Administration of any compound of the invention or administration of any therapy of the invention to a patient will follow standard protocols for administering such compounds, taking into account any toxicity of the agents, and thus in some embodiments, there will be a step of monitoring for toxicity resulting from the combination therapy.

[0075] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used according to the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, for example, whether or at what stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, to intercalate into DNA, or to affect nucleic acid synthesis to induce chromosomal and mitotic abnormalities.

[0076] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ethylenimines and methylamelamines, including ylolomelamime; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and biceresin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; Duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichi n), phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosurea, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γlI and calicheamicin ωI1); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxo rubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dino statins, or zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, propionate, Dromostanolone onate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents, such as mitotane and trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids, such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidanmol;Nitraerine;Pentostatin;Phenamet;Pirarubicin;Losoxantrone;Podophyllinic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Rhizoxin n); sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinosides ("Ara-C" );cyclophosphamide;taxoids, such as 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., CP T-11); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; cisplatin, carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the foregoing;

[0077] 2. Radiation therapy Other factors that cause DNA damage and have been used extensively include gamma radiation, X-rays, and / or what are commonly known as specific delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam radiation (U.S. Patents. 5,760,395 and 4,870,287), and UV radiation are also contemplated. All of these factors likely affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0078] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy may be used in combination with or together with the method of the present invention. In the context of cancer therapy, immunotherapy generally relies on the use of immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (Rituxan®) is one such example. The immune effector may be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody alone may act as the effector of therapy, or may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) or may simply act as a targeting agent. Alternatively, the effector may be a lymphocyte with a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0079] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., not present on the majority of other cells. There are many tumor markers, any of which 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, erbB, and p155. Another aspect of immunotherapy is to combine anti-cancer effects with immune stimulating effects. There are also immune stimulating molecules, including cytokines, e.g., IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, e.g., MIP-1, MCP-1, IL-8, and growth factors, e.g., FLT3 ligand.

[0080] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more types of anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0081] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints can either strengthen (e.g., costimulatory molecules) or weaken signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibition include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte antigen 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, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0082] The immune checkpoint inhibitor may be a drug, e.g., a small molecule, a recombinant ligand or receptor, or may be, in particular, an antibody, e.g., a human antibody (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both of which are incorporated herein by reference). Known immune checkpoint protein inhibitors or analogs thereof may be used, in particular chimeric, humanized, or human antibodies. As known by those skilled in the art, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by alternative and / or equivalent names, MK-3475 and pembrolizumab.

[0083] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its ligand binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its ligand binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Publication Nos. 20140294898, 2014022021, and 20110008369, all of which are incorporated herein by reference.

[0084] 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 portion or a 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 is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, 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.

[0085] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds 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 inhibitory signals to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to CD80, also known as B7-1, and CD86, also known as B7-2, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0086] 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 an oligopeptide.

[0087] 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, or art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly known as 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 disclosure of each of the aforementioned publications is incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0088] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, 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 to the same epitope on CTLA-4 as the aforementioned antibodies and / or binds to the same epitope on CTLA-4 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to an antibody described above (e.g., at least about 90%, at least about 95%, or at least about 99% variable region identity to ipilimumab).

[0089] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins, such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0090] In some embodiments, immunotherapy may be adoptive immunotherapy, involving the transfer of autoantigen-specific T cells generated ex vivo. T cells used for adoptive immunotherapy can be generated by expanding antigen-specific T cells or by redirecting T cells by genetic engineering (Park, Rosenberg et al. 2011). Isolation and transfer of tumor-specific T cells has been shown to be successful in melanoma treatment. Novel specificities in T cells have been successfully generated by gene 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 linked to one or more signaling domains in the form of a fusion molecule. In general, the binding moiety of a CAR consists of the light chain fragment of a monoclonal antibody and the antigen-binding domain of a single chain antibody (scFv), in which the variable fragment is connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains of first generation CARs are derived from the cytoplasmic region of CD3ζ or the Fc receptor γ chain. CARs have been used successfully to redirect T cells to antigens expressed on the surface of tumor cells from a variety of neoplasms, including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0091] In one embodiment, the present application provides a combination therapy for treating cancer, comprising adoptive T cell therapy and checkpoint inhibitors. In one aspect, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another aspect, the autologous and / or allogeneic T cells are targeted against tumor antigens.

[0092] 4.Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive, diagnostic, or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the 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, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs surgery).

[0093] Removal of part or all of cancer cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment may be performed by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be of various dosages.

[0094] 5. Other agents It is contemplated that other agents may be used in combination with certain aspects of the invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the invention to improve the efficacy of the treatment.

[0095] III. Pharmaceutical Compositions It is contemplated that exosomes expressing or containing therapeutic agents can be administered systemically or locally to inhibit tumor cell growth, most preferably to kill cancer cells in cancer patients with locally advanced or metastatic cancer. Exosomes expressing or containing CRISPR systems can be administered intravenously, intrathecally, and / or intraperitoneally. Exosomes expressing or containing CRISPR systems can be administered alone or in combination with anti-proliferative drugs. In one embodiment, exosomes expressing or containing CRISPR systems are administered to reduce the cancer load of patients before surgery or other procedures. Alternatively, exosomes expressing or containing CRISPR systems can be administered after surgery to ensure that residual cancer (e.g., cancer not removed by surgery) does not survive.

[0096] It is not intended that the present invention be limited by the specific nature of the therapeutic preparation.For example, such compositions can be provided in the form of a formulation together with physiologically acceptable liquid, gel, solid carrier, diluent or excipient.These therapeutic preparations can be administered to mammals for veterinary use, such as veterinary use with livestock, and clinical use in humans, as with other therapeutic substances.In general, the dosage required for therapeutic effectiveness varies according to the type of use and method of administration, and the specific requirements of each individual subject.

[0097] When clinical applications are intended, it may be necessary to prepare pharmaceutical compositions comprising exosomes in a form suitable for the intended use. In general, pharmaceutical compositions may comprise an effective amount of one or more exosomes and / or additional agents dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as, for example, a human, as appropriate. Preparation of pharmaceutical compositions comprising exosomes disclosed herein, or additional active ingredients, will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference. Furthermore, it is understood that for administration to animals (e.g., humans), preparations must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biological Standards.

[0098] Furthermore, according to certain aspects of the present invention, the composition suitable for administration may be provided dissolved in a pharma- ceutically acceptable carrier with or without an inert diluent. As used herein, a "pharma- ceutically acceptable carrier" refers to any and all aqueous solvents known to those skilled in the art (e.g., water, alcoholic / aqueous solutions, ethanol, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), vegetable oils, and injectable organic esters such as ethyl oleate), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants, and the like. Agents, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, noble gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof), isotonicity agents (e.g., sugars and sodium chloride), absorption retarders (e.g., aluminum monostearate and gelatin), salts, drugs, drug stabilizers, gels, resins, bulking agents, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquids and nutrient replenishers, such similar materials and combinations thereof. The carrier must be assimilable and includes liquid, semi-solid, i.e., paste, or solid carriers. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffering agents. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0099] Although pharma- ceutically acceptable carriers are specifically formulated for administration to humans, in certain embodiments, it may be desirable to use pharma- ceutically acceptable carriers that are formulated for administration to non-human animals but are not acceptable for administration to humans (e.g., due to government regulations).Unless a conventional carrier is incompatible with the active ingredient (e.g., harmful to the recipient or harmful to the therapeutic effect of the composition contained in the carrier), its use in 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 dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc.Such techniques are routine for those skilled in the art.

[0100] Certain embodiments of the invention may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, whether they need to be sterile for the route of administration, such as injection. The compositions may 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 infusion, by continuous infusion, by localized perfusion bathing the target cells directly, via a catheter, via lavage, in lipid compositions (e.g., liposomes), or by other methods known to those of skill in the art or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).

[0101] Exosomes can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes.Typically, such compositions can be prepared in either liquid solutions or suspensions.Solid dosage forms can also be prepared, suitable for use in preparing solutions or suspensions by adding liquid prior to injection.The preparation can also be emulsified.

[0102] 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 dosage form must be sterile and must be fluid to the extent that easy syringability exists. The dosage form must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0103] Once formulated, 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 variety of dosage forms, for example, formulated for parenteral administration, such as injections or aerosols, for delivery to the lungs, or formulated for edible administration, such as drug release capsules.

[0104] The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response, i.e., the desired response discussed above in connection with the appropriate route and treatment regimen. The amount to be administered depends on the desired effect, depending on the number of treatments and the unit dose. The actual dosage of the composition of the present invention administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health, and sex, the type of disease being treated, the extent of disease invasion, previous or concurrent therapeutic interventions, idiopathic diseases of the patient, the route of administration, and the efficacy, stability, and toxicity of the particular therapeutic agent. For example, the dosage may also include a dosage of about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (such ranges include doses therebetween) or more, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include ranges of about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. The administering physician will, in any given situation, determine the concentration of active ingredient(s) in the composition and the dose appropriate for the individual subject.

[0105] The actual dosage of the composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the frequency of administration of an effective amount may vary according to the subject's response. The physician responsible for administration will, in any given situation, determine the concentration of active ingredient in the composition and the dosage appropriate for the individual subject.

[0106] In certain embodiments, the pharmaceutical composition may, for example, contain at least about 0.1% of the active compound. In other embodiments, the active compound may, for example, comprise about 2% to about 75% or about 25% to about 60% of the weight of the unit, and any range derivable therein. Naturally, in each therapeutically useful composition, the amount of active compound may be prepared in such a way that a suitable dosage is obtained in a particular unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are considered by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimes may be desirable.

[0107] In other non-limiting examples, dosages may also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1000 milligrams / kg / body weight, or more, per administration, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numbers recited herein include ranges based on the above numbers, such as about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc.

[0108] IV. Exosomal Cargo A. Nucleic Acids and Vectors In certain aspects of the present invention, nucleic acid sequences encoding therapeutic proteins or antibodies can be disclosed. Depending on which expression system is used, nucleic acid sequences can be selected based on conventional methods. For example, each gene or its variant can be codon-optimized to express in a certain system. Various vectors can also be used to express proteins of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0109] B. Recombinant Proteins Some embodiments relate to recombinant proteins and polypeptides, such as, for example, therapeutic antibodies. In some embodiments, the therapeutic antibody may be an antibody that specifically or selectively binds to an intracellular protein. In a further aspect, the protein or polypeptide may be modified to increase serum stability. Thus, when the present application refers to a function or activity of a "modified protein" or "modified polypeptide," it will be understood by the skilled artisan that this includes, for example, a protein or polypeptide that has additional advantages over a non-modified protein or polypeptide. It is specifically intended that embodiments relating to a "modified protein" may also be implemented with respect to a "modified polypeptide," and vice versa.

[0110] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids up to the full-length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0111] As used herein, "amino acid residue" refers to any natural amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous and do not interrupt the amino acid residue sequence with non-amino acids. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0112] Thus, the term "protein or peptide" includes an amino acid sequence that includes at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0113] C. Inhibitory RNA siRNA (for example, siNA) is well known in the art.For example, siRNA and double-stranded RNA are described in U.S. Patent No. 6,506,559 and U.S. Patent No. 6,573,099 and U.S. Patent Application No. 2003 / 0051263, No. 2003 / 0055020, No. 2004 / 0265839, No. 2002 / 0168707, No. 2003 / 0159161 and No. 2004 / 0064842.All of these are incorporated herein by reference in their entirety.

[0114] Nucleic acid components in siRNA do not have to be the same type or uniform throughout (e.g., siRNA may comprise nucleotides and nucleic acids or nucleotide analogues). Typically, siRNA forms 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, siRNA may comprise only one nucleic acid (polynucleotide) or nucleic acid analogue, and form a double-stranded structure by complementing itself (e.g., forming a hairpin loop). The double-stranded structure of siRNA 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 consecutive nucleic acid bases, including all ranges therein. The siRNA may comprise 17-35 contiguous nucleobases, more preferably 18-30 contiguous nucleobases, more preferably 19-25 nucleobases, more preferably 20-23 contiguous nucleobases, or 20-22 contiguous nucleobases, or 21 contiguous nucleobases, that hybridize with a complementary nucleic acid (which may be another portion of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[0115] The agent of the present invention useful for carrying out the method of the present invention includes, but is not limited to, siRNA.Typically, the introduction of double-stranded RNA (dsRNA), which may be alternatively referred to herein as small interfering RNA (siRNA), induces strong and specific gene silencing, a phenomenon called RNA interference or RNAi.RNA interference has been called "cosuppression", "post-transcriptional gene silencing", "sense suppression", and "quelling".RNAi is an attractive biotechnology tool because it provides a means to knock out the activity of specific genes.

[0116] There are several factors that need to be considered when designing RNAi, such as the nature of the siRNA, the permanence of the silencing effect, and the choice of delivery system. To produce the RNAi effect, the siRNA introduced into the organism typically contains exon sequences. Furthermore, the RNAi process depends on homology. Therefore, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of mutual interference between homologous but non-gene-specific sequences. Preferably, the siRNA shows more than 80%, more than 85%, more than 90%, more than 95%, more than 98% identity, or even 100% identity between the siRNA sequence and the gene to be inhibited. Sequences with less than about 80% identity with the target gene have a much smaller effect. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0117] Furthermore, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules that contain at least about 19-25 nucleotides and can regulate gene expression. In the context of the present invention, the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

[0118] Target gene generally refers to a polynucleotide that includes a region that codes for a polypeptide, or a region that regulates the replication, transcription, or translation, or other processes important for the expression of a polypeptide, or a polynucleotide that includes both a region that codes for a polypeptide and a region that is functionally linked to the region that regulates expression. Any gene that is expressed in a cell can be targeted. Preferably, the target gene is a gene that is involved in or associated with the progression of a cellular activity that is important for disease, or a gene that is of particular interest as a research subject.

[0119] siRNA can be obtained from commercial suppliers, natural sources, or can be synthesized by any of the many techniques known to those skilled in the art.For example, one commercial supplier of pre-designed siRNA is Ambion®, Austin, Tex. Another commercial supplier is Qiagen® (Valencia, Calif.).The inhibitory nucleic acid applicable in the compositions and methods of the present invention can be any nucleic acid sequence that is found by any supplier to be a verified down-regulator of the protein of interest.It is understood that without undue experimentation and using the disclosure of the present invention, further siRNA can be designed and used to carry out the method of the present invention.

[0120] The siRNA may also contain one or more nucleotide changes. Such changes may include, for example, the addition of non-nucleotide material to the end of the 19-25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3'-hydroxyl group. The nucleotides in the RNA molecules of the invention may also contain non-standard nucleotides, including non-natural nucleotides or deoxyribonucleotides. The double-stranded oligonucleotides may contain modified backbones, such as phosphorothioates, phosphorodithioates, or other modified backbones known in the art, and may contain non-natural internucleoside linkages. Further modifications of siRNA (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleoside linkages, and inverted deoxyabasic residue incorporation) can be found in US Patent Application Publication No. 2004 / 0019001 and US Patent No. 6,673,611 (each of which is incorporated herein by reference in its entirety). Collectively, all such modified nucleic acids or RNAs described above are referred to as modified siRNAs.

[0121] D. Gene Editing Systems In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression or induction of activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and / or other sequences and transcripts derived from the CRISPR locus.

[0122] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) that has nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a type I, type II, or type III CRISPR system, and may be derived from a particular organism that contains an endogenous CRISPR system, such as, for example, Streptococcus pyogenes.

[0123] In some aspects, Cas nuclease and gRNA (comprising a fusion of a crRNA specific for a target sequence and a certain tracrRNA) are introduced into cells. In general, the target site at the 5' end of gRNA uses complementary base pairing to target the Cas nuclease to the target site, e.g., gene. The target site can be selected based on its location immediately 5' to a protospacer adjacent motif (PAM) sequence, e.g., typically immediately 5' to NGG or NAG. In this regard, the gRNA targets 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. In general, CRISPR systems are characterized by elements that promote CRISPR complex formation at the target sequence site. Typically, a "target sequence" generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex.

[0124] CRISPR system can induce double-strand break (DSB) at target site, and then induce destruction as disclosed herein.In other embodiments, Cas9 variants, considered as "nickases", are used to nick single-stranded target sites.Paired nickases can be used, for example, to improve specificity, and each nickase is directed by a pair of different gRNAs targeting sequences, so that nicks are introduced simultaneously to introduce 5' overhangs.In other embodiments, catalytically inactive Cas9 is fused with heterologous effector domains, such as transcriptional repressors or activators, to affect gene expression.

[0125] The target sequence may comprise any polynucleotide, for example, DNA or RNA polynucleotide. The target sequence may be in the nucleus or cytoplasm of a cell, for example, in an organelle of a cell. In general, the sequence or template that can be used for recombination into a target locus that comprises a target sequence is called "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, the exogenous template polynucleotide may be called an editing template. In some aspects, the recombination is a homologous recombination.

[0126] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). The tracr sequence may also comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, about 26, about 32, about 45, about 48, about 54, about 63, about 67, about 85 or more nucleotides of the wild-type tracr sequence, or more than about 20, about 26, about 32, about 45, about 48, about 54, about 63, about 67, about 85 or more nucleotides of the wild-type tracr sequence), and may become part of a CRISPR complex, e.g., by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0127] One or more vectors expressing one or more elements of the CRISPR system can be introduced into a cell so that expression of said elements results in the formation of a CRISPR complex at one or more target sites. Components can also be delivered to a cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can 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 in one vector can be combined with one or more additional vectors that provide any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, the 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, one expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0128] The vector may include regulatory elements operably linked to a CRISPR enzyme, e.g., an enzyme coding sequence encoding a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0129] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme may induce cleavage of one or both strands at the location of the target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector may encode a CRISPR enzyme mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme is incapable of cleaving one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9, which is derived from a nuclease that cleaves both strands, into a nickase (cleaves one strand). In some embodiments, the Cas9 nickase may be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination allows for the creation of nicks in both strands and can be used to induce NHEJ or HDR.

[0130] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized to be expressed in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be or may be derived from a particular organism, such as a mammalian eukaryotic cell, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence with a codon that is frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species show a unique bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, and as a result, it is believed that the efficiency of messenger RNA (mRNA) translation depends, among other things, on the nature of the codon that is translated and the availability of a particular transfer RNA (tRNA) molecule. In a given cell, the predominance of a selected tRNA reflects that it is the codon most frequently used in peptide synthesis, and thus genes can be tailored based on codon optimization for optimal gene expression in a particular organism.

[0131] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and induce sequence-specific binding of CRISPR complex to the target sequence.In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99% or more, or more than about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99% or more, when properly aligned using a suitable alignment algorithm.

[0132] Optimal alignment can be determined using any suitable algorithm for aligning sequences.Non-limiting examples include Smith-Waterman algorithm, Needleman-Wunsch algorithm, algorithm based on Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available from soap.genomics.org.cn), and Maq (available from maq.sourceforge.net).

[0133] CRISPR enzyme can be part of a fusion protein that includes one or more heterologous protein domains. CRISPR enzyme fusion protein can include any additional protein sequence between any two domains, optionally a linker sequence. Examples of protein domains that can be fused to CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have 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) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes may be fused with gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tags, LexA DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can be part of fusion proteins containing CRISPR enzymes are described in US20110059502, which is incorporated herein by reference.

[0134] V. Kits and Diagnostics In various aspects of the present invention, kits are envisaged that contain the necessary components for purifying exosomes from body fluids or tissue culture media. In other aspects, kits are envisaged that contain the necessary components for isolating exosomes and transfecting exosomes with therapeutic nucleic acids, therapeutic proteins or inhibitory RNA. The kits may contain one or more sealed vials that contain any of these components. In some embodiments, the kits may also contain suitable container means, such as eppendorf tubes, assay plates, syringes, bottles or tubes, that are containers that do not react with the components of the kit. The containers may be made of sterilizable materials such as plastic or glass. The kits may further comprise instructions outlining the procedural steps of the methods described herein, following substantially the same procedures as those described herein or known to those skilled in the art. The information of the instructions may be present in a computer readable medium that comprises machine readable instructions that, when executed using a computer, display a real or virtual method of purifying exosomes from a sample and transfecting therapeutic cargo into the exosomes. EXAMPLES

[0135] VI. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples demonstrate techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred embodiments for practicing the invention. However, in light of this disclosure, it should be understood by those of skill in the art that many changes can be made in the specific embodiments disclosed and still obtain a similar or similar result without departing from the spirit and scope of the invention.

[0136] Example 1 –Delivery of TP53R273H siRNA to Panc-1 orthotopic tumors using exosomes siRNA targeting TP53R273H TIFF0007602246000002.tif4128 was tested for knockdown efficiency in Panc-1 cells, which are homozygous mutant for TP53R273H, using lipofectamine transfection (Figure 1A).

[0137] Kras G12D The siRNA construct was designed to specifically target the siRNA sequence. TIFF0007602246000003.tif4128 is a Kras protein found in cell lines and animal models. G12D It reflects a G → A nucleotide deviation from the wild-type Kras gene sequence (underlined and bolded) intended to specifically target the glycine → aspartate amino acid substitution of the mutation, and a TT nucleotide overhang (underlined) intended to promote silencing efficiency ( Rejiba et al., 2007 ; Ma et al., 2004 ; Du et al., 2005 ).

[0138] Mice bearing Panc-1 GFP / Luc orthotopic tumors were treated (i.p.) with either (1) control exosomes, (2) exosomes containing TP53R273H-targeting siRNA, or (3) exosomes containing TP53R273H-targeting siRNA and KrasG12D-containing siRNA. The control group showed the highest level of tumor growth (Figure 1B and C), and both treatment groups were found to reduce tumor burden (Figure 1B and C).

[0139] To evaluate exosome delivery to the liver and pancreas, adult rhesus monkeys were intravenously administered unlabeled exosomes (control) or exosomes labeled with PKH membrane dye (PKH-exosomes). Monkey livers and pancreases were frozen, sectioned, and mounted on slides. Microscopic evaluation of sections counterstained with DAPI to clarify nuclear contours revealed strong and specific accumulation of exosomes in the liver and pancreas (Figure 2). Furthermore, colocalization of exosomes with pancreatic cell nuclei was observed (Figure 3A). Quantitative analysis of exosome foci size observed in the liver and pancreas and larger size foci and higher exosome accumulation per cell in the pancreas compared to the liver (Figure 3B).

[0140] To assess delivery of exosomal cargo to various organs, two monkeys received iExosomes intravenously (iv) and a third monkey received iExosomes intraperitoneally (ip). Quantitation of siRNA payload (within exosomes) after administration to adult rhesus monkeys was confirmed by q-PCR analysis (Figure 4).

[0141] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain specific agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions 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.

[0142] References The following references, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007602246000004.tif183150TIFF0007602246000005.tif230150TIFF0007602246 000006.tif223151TIFF0007602246000007.tif231150TIFF0007602246000008.tif52150

Claims

1. Inhibitory RNA targeting TP53R273H and KRAS G12D A composition comprising lipid-based nanoparticles containing an inhibitory RNA targeting TP53R273H, wherein the inhibitory RNA targeting TP53R273H has the sequence of SEQ ID NO:1, and the inhibitory RNA targeting KRAS G12D has the sequence of SEQ ID NO:

2.

2. The composition of claim 1, wherein the lipid-based nanoparticles comprise CD47 on their surface.

3. The composition of claim 1 , wherein the lipid-based nanoparticles comprise a growth factor receptor on their surface.

4. The composition of claim 1 , wherein the lipid-based nanoparticle is a liposome or an exosome.

5. The composition of claim 1 , wherein the inhibitory RNA is a siRNA.

6. The composition of claim 1, comprising a first lipid-based nanoparticle comprising an siRNA having the sequence of SEQ ID NO:1, and a second lipid-based nanoparticle comprising an siRNA having the sequence of SEQ ID NO:

2.

7. A pharmaceutical composition comprising the lipid-based nanoparticles of any one of claims 1 to 6 and an excipient.

8. 8. The composition of claim 7, formulated for parenteral administration.

9. 10. The composition of claim 8, formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

10. 8. The composition of claim 7 for administration to a patient to treat cancer.

11. 11. The composition of claim 10, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, 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.

12. The composition of claim 11, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

13. the cancer is TP53R273H mutant and / or KRAS G12D The composition of claim 10, which is homozygous for the mutation.

14. The cancer cells are characterized in that the cancer cells are G12D The composition of claim 10, which is heterozygous for the variant.

15. 11. The composition of claim 10, administered to a patient in combination with at least a second therapy, wherein the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.

16. 11. The composition of claim 10, wherein the lipid-based nanoparticles are exosomes, and the exosomes are autologous to the patient.

17. 17. The composition of claim 16, wherein the exosomes are obtained from a bodily fluid sample obtained from the patient.

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