Methods and compositions for treating cancer using exosome-related gene editing

Abstract

The present invention provides a composition comprising exosomes, wherein the exosomes comprise CD47 on their surface and further comprise a CRISPR system.Further provided are methods for using the exosomes for gene editing, and gene editing for the treatment of cancer.

Description

[Technical Field] 【0001】 Reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 599,340, filed on 15 December 2017, which is incorporated herein by reference in its entirety. 【0002】 1. Field This invention relates as a whole to medicine and oncology. More specifically, the invention relates to the use of exosomes for in vivo delivery of nuclease complexes for gene editing. [Background technology] 【0003】 2. Explanation of related technologies Gene editing is a technique that modifies target genes within living cells. Recently, the use of the CRISPR bacterial immune system to perform on-demand gene editing has revolutionized how scientists perform genome editing. The Cas9 protein in the CRISPR system, an RNA-guided DNA endonuclease, can be relatively easily manipulated to target new sites by altering its guide RNA sequence. This discovery has made sequence-specific gene editing functionally effective. Current CRISPR / Cas9 technology provides a reliable method for editing genes in cultured cells in vitro. However, there is a need for new methods to target specific cells in various organs in vivo. [Overview of the project] 【0004】 overview Therefore, exosomes engineered to efficiently deliver CRISPR-Cas9 to various organs and tumors are provided, thereby enabling the control of cancer and other genetic diseases by therapeutic gene editing. In one embodiment, a composition is provided comprising an exosome, wherein the exosome contains CD47 on its surface and the exosome comprises a CRISPR system. In some aspects, the CRISPR system comprises an endonuclease and a guide RNA (gRNA). In some aspects, the endonuclease is a Cas endonuclease. In some aspects, the endonuclease is a Cas9 endonuclease. In other aspects, the endonuclease is a Cpf1 endonuclease. In some aspects, the guide RNA is a single gRNA. In some aspects, the single gRNA is a CRISPR-RNA (crRNA). In some aspects, the single gRNA comprises a fusion of crRNA and trans-activated CRISPR RNA (tracrRNA). In some aspects, the guide RNA comprises crRNA and tracrRNA. In some cases, endonucleases and gRNAs are encoded on a single nucleic acid molecule within the exosome. In other cases, endonucleases and gRNAs are encoded on separate nucleic acid molecules within the exosome. 【0005】 In some aspects, the CRISPR system targets disease-causing mutations. In some aspects, disease-causing mutations are cancer-causing mutations. In some aspects, cancer-causing mutations are activating mutations in oncogenes. In some aspects, cancer-causing mutations are inhibitory mutations in tumor suppressor genes. In some aspects, the CRISPR system targets undruggable genes. In some aspects, cancer-causing mutations are Kras G12DIn some cases, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% (or any value that can be derived from among them) of the exosome contains endonucleases and gRNA. 【0006】 In one embodiment, a pharmaceutical composition is provided comprising an exosome and a pharmaceutically acceptable excipient, wherein the exosome contains CD47 on its surface and the exosome comprises a CRISPR system. In some aspects, the CRISPR system comprises an endonuclease and a guide RNA (gRNA). In some aspects, the endonuclease is a Cas endonuclease. In some aspects, the endonuclease is a Cas9 endonuclease. In other aspects, the endonuclease is a Cpf1 endonuclease. In some aspects, the guide RNA is a single gRNA. In some aspects, the single gRNA is a CRISPR-RNA (crRNA). In some aspects, the single gRNA comprises a fusion of crRNA and trans-activated CRISPR RNA (tracrRNA). In some aspects, the guide RNA comprises crRNA and tracrRNA. In some aspects, the endonuclease and gRNA are encoded on a single nucleic acid molecule within the exosome. In some aspects, endonucleases and gRNAs are encoded on separate nucleic acid molecules within exosomes. In some aspects, the CRISPR system targets disease-causing mutations. In some aspects, disease-causing mutations are cancer-causing mutations. In some aspects, cancer-causing mutations are activating mutations in oncogenes. In some aspects, cancer-causing mutations are inhibitory mutations in tumor suppressor genes. In some aspects, the CRISPR system targets undruggable genes. In some aspects, cancer-causing mutations are Kras G12DIn some aspects, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% (or any value that can be derived from there) of the exosomes contain endonucleases and gRNAs. 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 further aspects, the composition further contains an antimicrobial agent. In some cases, antimicrobial agents include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenlymercuric nitrate, propylene glycol, or thimerosal. 【0007】 In one embodiment, a method is provided for treating a disease in a patient in need thereof, the method comprising administering to the patient a composition comprising a pharmaceutical composition comprising an exosome and a pharmaceutically acceptable excipient, wherein the exosome comprises CD47 on its surface and the exosome comprises a CRISPR system, thereby treating the disease in the patient. In some aspects, the administration induces gene editing in the patient's diseased cells. In some aspects, the disease is cancer. In some aspects, the cancer is pancreatic ductal adenocarcinoma. In some aspects, the administration is systemic. In some aspects, the systemic administration is intravenous or intra-arterial. In some aspects, the method further comprises administering at least a second therapy to the patient. In some aspects, the second therapy includes surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, or immunotherapy. In some aspects, the patient is human. In some aspects, the exosomes are autologous to the patient. In some cases, administration of the pharmaceutical composition provides superior therapeutic benefits compared to administration of a CRISPR system that does not contain exosomes. In some cases, the pharmaceutical composition is administered to the patient only once. In some cases, the pharmaceutical composition is administered to the patient multiple times. In some cases, the pharmaceutical composition is administered to the patient a limited number of times. In some cases, the pharmaceutical composition is administered to the patient consecutively. In some cases, the pharmaceutical composition is administered to the patient at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 21, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 times (or any number that can be derived from there). 【0008】 In one embodiment, a composition comprising an exosome for use in the treatment of a disease in a patient is provided, wherein the exosome comprises CD47 on its surface and the exosome comprises a CRISPR system. In some aspects, the CRISPR system comprises an endonuclease and a guide RNA (gRNA). In some aspects, the endonuclease is a Cas endonuclease. In some aspects, the endonuclease is a Cas9 endonuclease. In other aspects, the endonuclease is a Cpf1 endonuclease. In some aspects, the guide RNA is a single gRNA. In some aspects, the single gRNA is a CRISPR-RNA (crRNA). In some aspects, the single gRNA comprises a fusion of crRNA and trans-activated CRISPR RNA (tracrRNA). In some aspects, the guide RNA comprises crRNA and tracrRNA. In some aspects, the endonuclease and gRNA are encoded on a single nucleic acid molecule within the exosome. In some aspects, endonucleases and gRNAs are encoded on separate nucleic acid molecules within exosomes. In some aspects, the CRISPR system targets disease-causing mutations. In some aspects, disease-causing mutations are cancer-causing mutations. In some aspects, cancer-causing mutations are activating mutations in oncogenes. In some aspects, cancer-causing mutations are inhibitory mutations in tumor suppressor genes. In some aspects, the CRISPR system targets undruggable genes. In some aspects, cancer-causing mutations are Kras G12DIn some aspects, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% (or any value that can be derived from there) of the exosomes contain endonucleases and gRNAs. In some aspects, administration induces gene editing in the patient's diseased cells. In some aspects, the disease is cancer. In some aspects, the cancer is pancreatic ductal adenocarcinoma. 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 further aspects, the composition further comprises an antimicrobial agent. In some aspects, the antimicrobial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, or thimerosal. In further aspects, the composition comprises at least a second therapy. In some aspects, the second therapy comprises surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, or immunotherapy. In some aspects, the patient is human. In some aspects, the exosomes are autologous to the patient. 【0009】 In one embodiment, a use is provided for the manufacture of a pharmaceutical product for treating a disease, wherein the exosome contains CD47 on its surface and the exosome contains a CRISPR system. In some aspects, the CRISPR system contains an endonuclease and a guide RNA (gRNA). In some aspects, the endonuclease is a Cas endonuclease. In some aspects, the endonuclease is a Cas9 endonuclease. In other aspects, the endonuclease is a Cpf1 endonuclease. In some aspects, the guide RNA is a single gRNA. In some aspects, the single gRNA is a CRISPR RNA (crRNA). In some aspects, the single gRNA contains a fusion of crRNA and trans-activated CRISPR RNA (tracrRNA). In some aspects, the guide RNA contains both crRNA and tracrRNA. In some aspects, the endonuclease and gRNA are encoded on a single nucleic acid molecule within the exosome. In some aspects, endonucleases and gRNAs are encoded on separate nucleic acid molecules within exosomes. In some aspects, the CRISPR system targets disease-causing mutations. In some aspects, disease-causing mutations are cancer-causing mutations. In some aspects, cancer-causing mutations are activating mutations in oncogenes. In some aspects, cancer-causing mutations are inhibitory mutations in tumor suppressor genes. In some aspects, the CRISPR system targets undruggable genes. In some aspects, cancer-causing mutations are Kras G12DIt is. In some aspects, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% (or any value derivable therefrom) of the exosomes contain an endonuclease and a gRNA. In some aspects, the disease is cancer. In some aspects, the cancer is pancreatic ductal adenocarcinoma. In some aspects, the medicament is formulated for parenteral administration. In some aspects, the medicament is formulated for intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection. In some aspects, the medicament contains an antibacterial agent. In some aspects, the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. 【0010】 As used herein, "essentially free of" in terms of a specified component means that none of the specified components are intentionally formulated in the composition and / or are present only as contaminants or in trace amounts for the purposes for which it is used herein. Thus, the total amount of the specified component due to 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 specified component cannot be detected using standard analytical methods. 【0011】 As used herein, "a" or "an" may mean one or more. When used in the claims herein, the words "a" or "an" may mean one or more when used in conjunction with the word "comprising". 【0012】 The use of the term "or" in the claims is used to mean "and / or" unless explicitly stated to refer only to alternatives or that the alternatives are mutually exclusive, but this disclosure supports definitions that refer only to alternatives and "and / or". As used herein, "another" may mean at least a second or more. 【0013】 [Invention 1001] A composition comprising an exosome, wherein the exosome contains CD47 on its surface, and the exosome contains a CRISPR system. <00001​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ The composition of the present invention 1011, wherein the mutation causing the aforementioned disease is a mutation causing cancer. [Invention 1013] The composition of the present invention 1012, wherein the mutation causing the aforementioned cancer is an activating mutation in an oncogene. [Invention 1014] The composition of the present invention 1012, wherein the mutation causing the aforementioned cancer is an inhibitory mutation in a tumor suppressor gene. [Invention 1015] The mutation that causes the aforementioned cancer is Kras G12D The composition of the present invention 1012. [Invention 1016] The composition of the present invention 1002, wherein at least 50% of the exosomes contain an endonuclease and gRNA. [Invention 1017] The composition of the present invention 1016, wherein at least 60% of the exosomes contain an endonuclease and gRNA. [Invention 1018] The composition of the present invention 1017, wherein at least 70% of the exosomes contain an endonuclease and gRNA. [Invention 1019] The composition of the present invention 1018, wherein at least 80% of the exosomes contain an endonuclease and gRNA. [Invention 1020] The composition of the present invention 1019, wherein at least 90% of the exosomes contain an endonuclease and gRNA. [Invention 1021] A pharmaceutical composition comprising any exosome according to invention 1001 to 1020 and an excipient. [Invention 1022] A composition of the present invention 1021, formulated for parenteral administration. [Invention 1023] A composition of the present invention 1022, formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [Invention 1024] A composition according to the present invention 1022, further comprising an antibacterial agent. [Invention 1025] The composition of Invention 1024, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, or thimerosal. [Invention 1026] A method for treating a disease in a patient in need thereof, comprising the step of administering to the patient any of the compositions of the present invention 1021 to 1025, thereby treating the disease in the patient. [Invention 1027] The method of the present invention 1026, wherein administration induces gene editing in the diseased cells of the patient. [Invention 1028] The method of the present invention 1026, wherein the disease is cancer. [Invention 1029] The method of the present invention 1028, wherein the cancer is pancreatic ductal adenocarcinoma. [Invention 1030] The method of the present invention 1026, wherein the administration is systemic administration. [Invention 1031] The method of the present invention 1030, wherein the systemic administration is intravenous administration. [Invention 1032] The method of the present invention 1026, further comprising the step of administering at least a second therapy to the patient. [Invention 1033] The method of the present invention 1032, wherein the second therapy comprises surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, or immunotherapy. [Invention 1034] The method of the present invention 1026, wherein the patient is a human. [Invention 1035] The method of the present invention 1034, wherein the exosome is of autologous origin to the patient. [Invention 1036] A composition comprising exosomes for use in the treatment of a disease in a patient, wherein the exosomes contain CD47 on their surface and the exosomes contain a CRISPR system. [Invention 1037] The composition of the present invention 1036, wherein the CRISPR system comprises an endonuclease and a guide RNA (gRNA). [Invention 1038] The composition of the present invention 1037, wherein the endonuclease is a Cas endonuclease. [Invention 1039] The composition of the present invention 1038, wherein the endonuclease is Cas9 endonuclease. [Invention 1040] The composition of the present invention 1037, wherein the endonuclease is Cpf1 endonuclease. [Invention 1041] The composition of the present invention 1037, wherein the guide RNA is a single gRNA. [Invention 1042] The composition of the present invention 1041, wherein the single gRNA is CRISPR-RNA (crRNA). [Invention 1043] The composition of the present invention 1041, wherein the single gRNA comprises a fusion of crRNA and trans-activated CRISPR RNA (tracrRNA). [Invention 1044] The composition of the present invention 1037, wherein the guide RNA comprises crRNA and tracrRNA. [Invention 1045] The composition of the present invention 1036, wherein the endonuclease and the gRNA are encoded on a single nucleic acid molecule within the exosome. [Invention 1046] The composition of the present invention 1036, wherein the CRISPR system targets disease-causing mutations. [Invention 1047] The composition of the present invention 1046, wherein the mutation causing the aforementioned disease is a mutation that causes cancer. [Invention 1048] The composition of the present invention 1047, wherein the mutation causing the aforementioned cancer is an activating mutation in an oncogene. [Invention 1049] The composition of the present invention 1047, wherein the mutation causing the aforementioned cancer is an inhibitory mutation in a tumor suppressor gene. [Invention 1050] The mutation that causes the aforementioned cancer is Kras G12D The composition of the present invention 1047. [Invention 1051] The composition of the present invention 1037, wherein at least 50% of the exosomes contain an endonuclease and gRNA. [Invention 1052] The composition of the present invention 1051, wherein at least 60% of the exosomes contain an endonuclease and gRNA. [Invention 1053] The composition of the present invention 1052, wherein at least 70% of the exosomes contain an endonuclease and gRNA. [Invention 1054] The composition of the present invention 1053, wherein at least 80% of the exosomes contain an endonuclease and gRNA. [Invention 1055] The composition of the present invention 1054, wherein at least 90% of the exosomes contain an endonuclease and gRNA. [Invention 1056] The composition of the present invention 1036, wherein administration induces gene editing in the diseased cells of the patient. [Invention 1057] The composition of the present invention 1036, wherein the disease is cancer. [Invention 1058] The composition of the present invention 1057, wherein the cancer is pancreatic ductal adenocarcinoma. [Invention 1059] A composition of the present invention 1036, formulated for parenteral administration. [Invention 1060] A composition of the present invention 1059, formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [Invention 1061] A composition according to the present invention 1059, further comprising an antibacterial agent. [Invention 1062] The composition of the present invention 1061, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, or thimerosal. [Invention 1063] A composition of the present invention 1036, further comprising at least a second therapy. [Invention 1064] The composition of the present invention 1063, wherein the second therapy comprises surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, or immunotherapy. [Invention 1065] The composition of the present invention 1036, wherein the patient is a human. [Invention 1066] The composition of the present invention 1065, wherein the exosomes are of autologous origin to the patient. [Invention 1067] The use of exosomes in the manufacture of a pharmaceutical product for treating a disease, wherein the exosomes contain CD47 on their surface and the exosomes contain a CRISPR system. [Invention 1068] The use of the present invention 1067, wherein the CRISPR system comprises an endonuclease and guide RNA (gRNA). [Invention 1069] Use of the invention 1068, wherein the endonuclease is a Cas endonuclease. [Invention 1070] Use of the present invention 1069, wherein the endonuclease is Cas9 endonuclease. [Invention 1071] Use of the present invention 1068, wherein the endonuclease is Cpf1 endonuclease. [Invention 1072] The use of the present invention 1068, wherein the guide RNA is a single gRNA. [Invention 1073] The use of Invention 1072, wherein the aforementioned single gRNA is CRISPR-RNA (crRNA). [Invention 1074] The use of Invention 1072, wherein the single gRNA comprises a fusion of crRNA and trans-activated CRISPR RNA (tracrRNA). [Invention 1075] Use of the present invention 1068, wherein the guide RNA comprises crRNA and tracrRNA. [Invention 1076] Use of the present invention 1068, wherein the endonuclease and the gRNA are encoded on a single nucleic acid molecule within the exosome. [Invention 1077] The use of the CRISPR system described above, which targets disease-causing mutations, according to the present invention 1067. [Invention 1078] The use of the present invention 1077, wherein the mutation causing the aforementioned disease is a mutation causing cancer. [Invention 1079] The use of the invention 1078, wherein the mutation causing the aforementioned cancer is an activating mutation in an oncogene. [Invention 1080] The use of the invention 1078, wherein the mutation causing the aforementioned cancer is an inhibitory mutation in a tumor suppressor gene. [Invention 1081] The mutation that causes the aforementioned cancer is Kras G12D The use of the present invention 1078. [Invention 1082] Use of the present invention 1068, wherein at least 50% of the exosome contains an endonuclease and gRNA. [Invention 1083] Use of the present invention 1082, wherein at least 60% of the exosome contains endonuclease and gRNA. [Invention 1084] Use of the present invention 1083, wherein at least 70% of the exosome contains endonuclease and gRNA. [Invention 1085] Use of the present invention 1084, wherein at least 80% of the exosomes contain endonuclease and gRNA. [Invention 1086] Use of the present invention 1085, wherein at least 90% of the exosomes contain endonuclease and gRNA. [Invention 1087] Use of the present invention 1067, wherein the disease is cancer. [Invention 1088] Use of the present invention 1087, wherein the aforementioned cancer is pancreatic ductal adenocarcinoma. [Invention 1089] Use of the present invention 1067, wherein the aforementioned pharmaceutical is formulated for parenteral administration. [Invention 1090] The use of the aforementioned pharmaceutical product in which it is formulated for systemic administration, according to the invention 1067. [Invention 1091] Use of the present invention 1089, wherein the pharmaceutical product is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [Invention 1092] Use of the aforementioned pharmaceutical product containing an antibacterial agent, as described in invention 1067. [Invention 1093] Use of Invention 1092, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, or thimerosal. Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in the errors of the apparatus or method used to determine that value, or variations present among the test subjects. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples illustrate certain aspects of the present invention, it should be understood that they are merely illustrative, as various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing] 【0014】 The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of the particular embodiments shown herein. [Figure 1]Figures 1a-h: HEK293T cells were transfected with CRISPR-Cas9 vector control and CRISPR-Cas9-sgRab27a-2 using lipofectamine for 72 hours, and then selected with 1 μg / ml puromycin for 10 days to obtain stable HEK293T CRISPR-Cas9 vector control and CRISPR-Cas9-sgRab27a-2 cells. Stable cells were cultured in a selection medium containing 1 μg / ml puromycin. (Figure 1a) DNA and RNA were extracted from the above cells, and Cas9 levels were determined using quantitative real-time PCR (qPCR). (Figure 1b) Exosomes were collected from HEK293T blank cells and stable HEK293T CRISPR-Cas9 vector control and CRISPR-Cas9-sgRab27a-2 cells, and then Nanosight validation was performed. (Figure 1c) Exosome DNA and RNA were extracted and qPCR was performed to detect Cas9 levels and sgRNA against Rab27a-2 in exosomes. (Figure 1d) Cas9 protein levels in both cells and exosomes were evaluated by Western blotting with anti-Flag antibody or Cas9 antibody and vinculin or CD9 as controls, respectively. (Figure 1e) and (Figure 1f) DNA editing in cells (Figure 1e) and exosomes (Figure 1f) was confirmed using the T7 / SURVEYOR assay. (Figure 1g) and (Figure 1h) 3E10 exosomes collected from HEK293T blank cells, HEK293T CRISPR-Cas9 vector control, and CRISPR-Cas9-sgRab27a-2 stable cells were processed and placed in BxPC-3 every 24 hours. + 1 processing, ++ 2 processing. DNA and RNA were extracted from recipient cells. Cas9 levels were detected at both the DNA (g) and mRNA (h) levels. The bars at each time point, from left to right, represent "blank control," "CRISPR-Cas9 vector control," and "CRISPR-Cas9-sgRab27a-2." [Figure 2]Figures 2a-c: 3E10 exosomes collected from HEK293T blank cells, HEK293T CRISPR-Cas9 vector control, and CRISPR-Cas9-sgRab27a-2 stable cells were processed and introduced into BxPC-3 twice at 24-hour intervals. DNA and RNA were extracted from recipient cells. (Figures 2a) and (Figures 2c) sgRNA for Rab27a-2 was detected by PCR at both the DNA (Figure 2a) and mRNA (Figure 2c) levels. (Figure 2b) DNA editing in recipient BxPC-3 cells was confirmed using the T7 / SURVEYOR assay. [Figure 3] Figures 3a-d: Exosomes were collected from BJ cells. (Figure 3a) Exosomes were validated using Nanosight. (Figure 3b) To further confirm the presence of exosomes, exosome markers CD9, CD81, filtilin, and TSG101 were detected by Western blotting. (Figure 3c) 1E10 BJ exosomes were electroporated with 15ug of CRISPR-Cas9-GFP plasmid and then treated with DNase or not. Exosome DNA was extracted and Cas9 levels were evaluated by qPCR. Furthermore, copy numbers were calculated by absolute qPCR using CRISPR-Cas9-GFP plasmid as a reference. (Figure 3d) Electroporated exosomes treated with DNase were treated for 24 hours and introduced into BJ cells. Cas9 levels were detected at both the DNA and mRNA levels. [Figure 4]Figures 4a-b: HEK293T cells were transfected with a packaging plasmid along with CRISPR-Cas9 Rab27b-1 / 2 or an empty control plasmid using lipofectamine 2000. Lentivirus-containing medium was collected, then transduced into BxPC-3 cells. Transduced cells were further selected with 0.4 μg / mL puromycin, and single clones of BxPC-3 / CRISPR-Cas9-sgRab27b cells were pecked, grown, and validated by Western blotting and T7 / SURVEYOR assay. (Figure 4a) Rab27b and Rab27a protein levels were evaluated in all single clones. β-actin was used as a loading control. Representative Western blotting results are shown in (Figure 4a). (Figure 4b) The T7 / SURVEYOR assay was also used to further validate all clones. Representative Western blotting results are shown in (Figure 4b). For further experiments, BxPC-3 / CRIPSR-Cas9-sgRab27b-1 clone 3 (C3) and BxPC-3 / CRISPR-Cas9-sgRab27b-2 clone 6 (C6) were used. [Figure 5]Figures 5a-f: BxPC-3 / CRISPR-Cas9 vector control stable cells and single clones BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3 and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 were cultured in selective medium containing 0.4 μg / ml puromycin. (Figure 5a) DNA and RNA were extracted from the above cells, and Cas9 levels were determined by qPCR. (Figure 5b) Exosomes were collected from the above cells and subsequently validated using Nanosight. The number of secreted exosomes was analyzed and compared using Nanosight. (Figure 5c) Exosome DNA and RNA were extracted, and qPCR was performed to detect exosome Cas9 levels and sgRNA for Rab27b-1 / 2. (Figure 5d) Cas9 and Rab27b protein levels were evaluated by Western blotting in both cells and exosomes. β-actin or CD9 was used as a control, respectively. (Figure 5e) and (Figure 5f) DNA editing was confirmed in both cells (Figure 5e) and exosomes (Figure 5f) using two different primers with the T7 / SURVEYOR assay. [Figure 6] Figures 6a-b: (Figure 6a) Exosomes collected from stable cells of BxPC-3 / CRISPR-Cas9 vector control and single clones BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3 and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 were lysed, and protein content was detected using a BCA kit according to the manufacturer's instructions. (Figure 6b) 100 μL of BxPC-3 blank, BxPC-3 / CRISPR-Cas9 empty control, BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3, and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 cells were seeded in 96-well plates at a concentration of 1E5 cells / ml. Cell proliferation was evaluated at different time points using the MTT assay. The bars at each time point, from left to right, represent "blank control," "CRISPR-Cas9 vector control," "CRISPR-Cas9-sgRab27b-1-C3," and "CRISPR-Cas9-sgRab27b-2-C6." [Figure 7] Figures 7a-g: (Figure 7a) To prepare in vitro transcription of sgRab27b, sgRab27b-1 / 2 was first PCR-amplified, and then the PCR product was purified using the Qiagen® PCR purification kit. The purified PCR product of sgRab27-1 / 2 was in vitro-transcribed using the MEGAshortscript® kit according to the manufacturer's instructions. Furthermore, RNA quality was evaluated using an 8M urea polyacrylamide gel. (Figure 7b) To prepare in vitro transcription of Cas9, Cas9 was PCR-amplified, and then the PCR product was purified using the Qiagen® PCR purification kit. The purified Cas9 PCR product was in vitro-transcribed using the mMESSAGE mMACHINE® T7 Ultra kit. Cas9 RNA quality was detected using a formaldehyde gel. (Figures 7c-e) HEK293T / CRISPR-Cas9 vector control cells were treated with 1 μg of IVT-sgRab27b RNA for 72 hours using lipofectamine 2000 (Figure 7c), Exo-Fect / exosome transfection reagent (Figure 7d), or electropermeable exosomes (Figure 7e). DNA was extracted and gene editing was examined using the T7 / SURVEYOR assay. HEK293T cells (Figure 7f) and BxPC-3 cells (Figure 7g) were transfected with Cas9 mRNA using lipofectamine 2000 or Exo-Fect / exosome transfection reagent, or treated with 1E9 MSC exosomes electropermeable with Cas9 mRNA for 48 hours. Cas9 protein levels were detected by Western blotting. [Figure 8] Figures 8a-c: RNA was extracted from HEK293T / CRISPRCas9 vector control and BxPC-3 / CRISPR-Cas9 vector control cells. Relative Cas9 expression levels (Figure 8a) and 1 / Ct values ​​(Figure 8b) were confirmed by qPCR. (Figure 8c) 1 μg of Cas9 RNA was used for reverse transcription along with RNA from HEK293T / CRISPRCas9 vector control and BxPC-3 / CRISPR-Cas9 vector control cells. 1 / Ct values ​​were detected by qPCR. [Figure 9] Figures 9a-g: HEK293T cells were treated with 10 μg of plasmids (CRISPR-Cas9-lenti-V2 vector control, CRISPR-Cas9-lenti-V2-sgRab27b-1, CRISPR-Cas9-GFP vector control) every 24 hours for four times (days 1, 2, 3, and 4) using the Exo-Fect / exosome transfection reagent. Cells were collected on day 5. DNA, RNA, and proteins were extracted. (Figure 9a) The image taken on day 5 shows the transfection efficiency of the Exo-Fect / exosome transfection reagent, using the CRISPR-Cas9-GFP vector control plasmid as a control. (Figure 9b) Relative Cas9 expression levels and 1 / Ct values ​​were confirmed by qPCR. (Figure 9c) Cas9 protein levels were evaluated using Western blotting. (Figure 9d) Gene editing was examined in HEK293T cells after treatment with the CRISPR-Cas9-lenchi-V2-sgRab27b-1 plasmid using the T7 / SURVEYOR assay. The same experiment was performed in BxPC-3 cells. BxPC-3 cells were treated with 10 μg of plasmid (CRISPRCas9-lenchi-V2 vector control, CRISPR-Cas9-lenchi-V2-sgRab27b-1) four times at 24-hour intervals (days 1, 2, 3, and 4) using the Exo-Fect / exosome transfection reagent. Cells were collected on day 5. (Figure 9e) Relative Cas9 expression levels were confirmed by qPCR. (Figure 9f) Cas9 protein levels were evaluated using Western blotting. (Figure 9g) Gene editing was examined in BxPC-3 cells using the T7 / SURVEYOR assay. [Figure 10]Figures 10a-h: KPC689 cells were transfected with lipofectamine 2000 for 48 hours with 5 μg of plasmid (CRISPR-Cas9-sgmKrasG12D with lenti-V2, GFP, and puromycin backbone, and a vector control). DNA, RNA, and protein were extracted. (Figure 10a) To represent the transfection efficiency of lipofectamine, a photograph was taken after 48 hours of transfection using the CRISPR-Cas9-GFP vector control plasmid as a control. Relative Cas9 expression levels (Figure 10b) and mKrasG12D levels (Figure 10c) were confirmed by qPCR. (Figure 10d) Gene editing in KPC689 cells after lipofectamine transfection was examined using the T7 / SURVEYOR assay. KPC689 cells were treated with 10 μg of plasmid (CRISPR-Cas9-sgmKrasG12D with a GFP backbone and its vector control) three times at 24-hour intervals (days 1, 2, and 3) using the Exo-Fect / exosome transfection reagent. Cells were collected on day 4. DNA, RNA, and proteins were extracted (Figure 10e). The image taken on day 5 was shown to represent the transfection efficiency of the Exo-Fect / exosome transfection reagent. Relative Cas9 expression levels (Figure 10f) and mKrasG12D levels (Figure 10g) were confirmed by qPCR (Figure 10h). Gene editing in KPC689 cells after treatment with the CRISPR-Cas9-GFP-mKrasG12D plasmid was examined using the T7 / SURVEYOUR assay. [Figure 11]Figures 11a-f: (Figure 11a) and (Figure 11b) HEK293T cells were transfected with a packaging plasmid along with a CRISPR-Cas9 doxycycline-inducible plasmid using lipofectamine 2000. Lentivirus-containing medium was collected and then transduced into Panc1 cells. Transduced cells were further selected with 1 μg / ml puromycin. Panc1-inducible Cas9-stable cells were maintained with 1 μg / ml doxycycline. Exosomes were collected from doxycycline-treated or untreated Panc1-inducible cells. Cas9 protein levels in cells (Figure 11a) and exosomes (Figure 11b) were examined by Western blotting. (Figure 11c) Panc1-inducible cells were treated with 2 μg of IVT-sgRNA against hKrasG12D and 1 μg of hKrasG12D plasmid for 72 hours using lipofectamine, Fugene, or Exo-Fect. Gene editing in Panc1-inducible cells was examined using the T7 / SURVEYOR assay. (Figure 11d) Panc1 Cas9-stable cells were established using a lentivirus-based method. Cas9 protein levels were confirmed by Western blotting (Figure 11e). Panc1 cells were treated with lenti-V2, GFP, or CRISPR-Cas9-sghKrasG12D with a puromycin backbone using lipofectamine, Exo-Fect, or electroporated exosomes. Panc1 Cas9-stable cells were treated with sghKrasG12D plasmid using lipofectamine, Exo-Fect, or electroporated exosomes. Gene editing was investigated in Panc1 cells and Panc1 Cas9-stabilized cells using the T7 / SURVEYOR assay (Figure 11f). Panc1 sghKrasG12D T1-stabilized cells were established using a lentivirus-based method. Panc1 sghKrasG12D T1-stabilized cells were transfected for 24 hours with 10 μg or 20 μg of Cas9 plasmid containing GFP or puromycin backbone.The T7 / SURVEYOR assay was performed to examine gene editing in Panc1 sghKrasG12D T1 stable cells. [Figure 12] Figures 12a - b: KPC689 cells were subcutaneously transplanted onto the backs of mice. The mice were divided into four groups, with one or two mice per group. Group 1: Treated with 1E9 exosomes and 10 μl of Exo-Fect (n = 1, K504); Group 2: Treated with 10 μg of Cas9-GFP-sgmKrasG12D-mK1 plasmid (n = 1, K509); Group 3: Treated with 1E9 exosomes, 10 μg of Cas9-GFP vector control plasmid, and 10 μl of Exo-Fect (n = 2, #1: K501, #2: K510. K510 was registered 3 days later. Compared with all other mice); Group 4: Treated with 1E9 exosomes, 10 μg of Cas9-GFP-sgmKrasG12D-mK1 plasmid, and 10 μl of Exo-Fect (n = 2, #1: K502, #2: K505). Daily for two weeks, the mice in each group were injected intravenously (I.V.) and intratumorally (I.T.). (Figure 12a) The length (a, mm) and width (b, mm) of the tumors as well as the body weight (Figure 12b) were measured. The tumor volume (Figure 12a) was calculated as V(mm3)=0.52*a*b^2. 【Mode for Carrying Out the Invention】 【0015】 Detailed Description Exosomes (e.g., iExosome) having an integrated CRISPR / Cas9 system that utilizes various guide RNA molecules with the ability to target cancer cells and induce a gene editing program to alter the cancer cell genome are provided herein. Gene editing assays have been used to show that gene editing is efficiently performed in the exosomes themselves, and thus, iExosome for the purpose of targeting cancer cells having mutations such as Kras, deleting the mutated gene, replacing the mutated gene with the wild-type KRAS gene, or removing the dominant mutant gene and allowing the normal gene to resume its function CRISPR / Cas9 ) is provided herein. Gene editing assays have been used to show that gene editing is efficiently performed in the exosomes themselves, and thus, Kras G12D such as iExosome for the purpose of targeting cancer cells having mutations, deleting the mutated gene, replacing the mutated gene with the wild-type KRAS gene, or removing the dominant mutant gene and allowing the normal gene to resume its functionCRISPR / Cas9 This provided an efficient method for quickly verifying its subsequent use. iExosome CRISPR / Cas9 This technology allows for the editing of any gene that is part of the genomic DNA of cancer cells and tumors as a whole, contributing to the initiation, progression, and / or metastasis, in order to obtain therapeutic benefits or to alter the biology of cancer cells and tumors. This technology overcomes the shortcomings of the in vivo applications of CRISPR / Cas9 technology, currently used for cancer-related gene editing, with therapeutic benefits. Using exosomes with CD47 on their surface, iExosomes can be used for therapeutic benefits. CRISPR / Cas9 It can be successfully delivered to the tumor. 【0016】 I. Lipid-based nanoparticles In some embodiments, the lipid-based nanoparticles are liposomes, exosomes, lipid preparations, or other lipid-based nanoparticles, such as lipid-based vesicles (e.g., DOTAP: cholesterol vesicles). The lipid-based nanoparticles may be positively charged, negatively charged, or neutral. 【0017】 A. Liposomes "Liposome" is a general term encompassing various single-membrane and multi-membrane lipid vehicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are often characterized by having a vesicular structure with a phospholipid-containing bilayer membrane and an internal medium, generally containing an aqueous composition. The liposomes provided herein include monolayer liposomes, multi-membrane liposomes, and multivesicular liposomes. The liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. 【0018】 Multilayer liposomes consist of multiple lipid layers separated by an aqueous medium. Such liposomes spontaneously form when lipids, including phospholipids, are suspended in an excess aqueous solution. After self-reorganization, the lipid components form a closed structure, trapping water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or bind to the lipid bilayers. 【0019】 In certain contexts, polypeptides, nucleic acids, or small molecule drugs may, for example, be placed inside the aqueous part of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that binds to both the liposome and the polypeptide / nucleic acid, confined within a liposome, or complexed with a liposome. 【0020】 Liposomes used according to this embodiment can be prepared by various methods known to those skilled in the art. For example, a phospholipid, such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC), is dissolved in tert-butanol. The lipid is then mixed with a polypeptide, nucleic acid, and / or other components. Tween20 is added to the lipid mixture so that it accounts for about 5% of the weight of the composition. An excess of tert-butanol is added to the mixture so that the volume of tert-butanol accounts for at least 95%. The mixture is vortexed and frozen in a dry ice / acetone bath and freeze-dried overnight. The freeze-dried preparation can be stored at -20°C and used for up to 3 months. When needed, the freeze-dried liposomes are reconstituted by dissolving them in 0.9% saline solution. 【0021】 Alternatively, liposomes can be prepared by dissolving the lipids in a solvent and mixing them in a container, such as 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 under negative pressure at approximately 40°C using a rotary evaporator. Typically, the solvent is removed within approximately 5 minutes to 2 hours, depending on the desired liposome volume. This composition can be further dried in a desiccator under reduced pressure. Since dried lipids tend to degrade over time, they are generally discarded after about a week. 【0022】 The dried lipids can be hydrated by shaking them until the lipid membrane is completely resuspended, then dissolving them in approximately 25-50 mM phospholipid in sterile water free of pyrogens. The liposome aqueous solution can then be divided into aliquots, each placed in a vial, freeze-dried, and sealed under reduced pressure. 【0023】 The dried lipid or lyophilized liposomes prepared as described above can be dehydrated, reconstituted by dissolving them in a protein or peptide solution, and diluted to a suitable concentration using a suitable solvent, such as DPBS. The mixture is then placed in a vortex mixer and vigorously shaken. Further unencapsulated material, including but not limited to hormones, drugs, and nucleic acid constructs, is removed by centrifugation at 29,000 × g to wash the liposome pellet. The washed liposomes are resuspended at a suitable total phospholipid concentration, for example, about 50–200 mM. The amount of encapsulated further material or active substance can be determined according to standard methods. After determining the amount of encapsulated further material or active substance in the liposome preparation, the liposomes can be diluted to a suitable concentration and stored at 4°C until use. Pharmaceutical compositions containing liposomes typically contain a pharmaceutically acceptable sterile carrier or diluent, such as water or saline solution. 【0024】 Further liposomes that may be useful in conjunction 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, WO04029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068. All of these are incorporated herein by reference without disclaimer. 【0025】 When preparing such liposomes, any protocol described herein or known to those skilled in the art may be used. Further non-limiting examples of preparing liposomes are described in U.S. Patents No. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; WO1986 / 000238 and WO1990 / 004943, which are incorporated herein by reference, respectively. 【0026】 In certain embodiments, lipid-based nanoparticles are neutral liposomes (e.g., DOPC liposomes). As used herein, “neutral liposome” or “uncharged liposome” is defined as a liposome having one or more lipid components that give rise to an effective charge that is essentially neutral (substantially uncharged). “Surprisingly neutral” or “surprisingly uncharged” means that in a particular population (e.g., a population of liposomes), some lipid components, if present, contain charges that are not offset 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 uncharged charges). In certain embodiments, neutral liposomes may contain mostly lipids and / or phospholipids that are neutral themselves under physiological conditions (i.e., about pH 7). 【0027】 The liposomes and / or lipid-based nanoparticles of this embodiment may contain phospholipids. In certain embodiments, one type of phospholipid may be used in the preparation of liposomes (for example, a neutral phospholipid, e.g., DOPC, may be used to prepare neutral liposomes). In other embodiments, multiple types of phospholipids may be used to prepare liposomes. The phospholipids may be derived from a neutral source or a synthetic source. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Since phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., about pH 7), these compounds may be particularly useful in preparing neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce uncharged liposomes. In certain embodiments, non-phospholipid lipids (e.g., cholesterol) may be used. 【0028】 Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilaurilloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), and 1-palmitoyl-2-myristoylphosphatidyl Lucoline ("PMPC"), 1-Palmitoyl-2-Stearoylphosphatidylcholine ("PSPC"), 1-Stearoyl-2-Palmitoylphosphatidylcholine ("SPPC"), Dilauryloylphosphatidylglycerol ("DLPG"), Dimyristoylphosphatidylglycerol ("DMPG"), Dipalmitoylphosphatidylglycerol ("DPPG"), Distearoylphosphatidylglycerol ("DSPG"), Distearoyl sphingomyelia Phosphorus ("DSSP"), distearoylphophatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), di Myristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), cerebral phosphatidylserine ("BPS"), cerebral 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,This includes, but is not limited to, 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoylphosphatidylcholine ("POPC"), palmitoyloeoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine. 【0029】 B. Exosome "Extracellular vesicles" and "EVs" are, as a class, microvesicles of cell origin and secreted from cells, including exosomes, exosome-like vesicles, ectosomes (resulting from budding of vesicles directly arising from the plasma membrane), microparticles, microvesicles, secreted microvesicles (SMVs), nanoparticles, and even (large) apoptotic blebs or apoptotic bodies (resulting from cell death) or membrane particles. 【0030】 As used herein, the terms “microvesicle” and “exosome” refer to membranous particles with a diameter (or maximum dimension if the particle is not a spheroid) of approximately 10 nm to 5000 nm, more typically 30 nm to 1000 nm, and most typically 50 nm to 750 nm, in which at least a portion of the exosome membrane is obtained directly from the cell. Most commonly, the size (average diameter) of an exosome is up to 5% of the size of the donor cell. Therefore, exosomes of particular interest include exosomes detached from cells. 【0031】 Exosomes may be detected in any suitable sample type, such as body fluids, or isolated from any suitable sample type. As used herein, “isolated” means separation from the natural environment and is intended to include at least partial purification, and may include extensive purification. As used herein, “sample” means any sample suitable for the methods provided by the present invention. The sample may be any sample containing exosomes suitable for detection or isolation. Sources of the sample include blood, bone marrow, pleural fluid, ascites, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, sweat, tears, synovial fluid, and bronchial lavage fluid. In one aspect, the sample is a blood sample, for example, containing whole blood or any fraction or component thereof. Blood samples suitable for use with the present invention can be extracted from any known source containing blood cells or components thereof, such as veins, arteries, periphery, 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 may be peripheral blood collected from a subject with cancer. 【0032】 Exosomes may also be isolated from tissue samples, such as surgical specimens, biopsy specimens, 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 homogenization and lysis procedures that do not destroy the exosomes. Exosomes as intended herein are preferably isolated from body fluids dissolved in physiologically acceptable solutions, such as buffered saline, growth medium, and various aqueous media. 【0033】 Exosomes may be isolated from freshly collected samples or from samples that have been frozen or refrigerated. In some embodiments, exosomes may be isolated from cell culture media. Although not required, higher purity exosomes 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 a great many methods known in the art. One preferred method is fractional centrifugation from body fluids or cell culture supernatants. Exemplary methods for 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). 【0034】 One commonly accepted protocol for isolating exosomes involves ultracentrifugation, often combined with a sucrose density gradient or sucrose cushion to suspend relatively low-density exosomes. Isolation of exosomes by serial fractionation centrifugation is complicated by the possibility of overlapping size distributions with other microvesicles or macromolecular complexes. Furthermore, centrifugation can provide an inadequate means of separating vesicles based on size. However, when serial centrifugation is combined with sucrose gradient ultracentrifugation, exosomes can be highly enriched. 【0035】 Size-based exosome isolation using an alternative to ultracentrifugation is another option. Successful exosome purification using ultrafiltration procedures has been reported, which are less time-consuming and require no special equipment than ultracentrifugation. Similarly, commercially available kits (EXOMIR®, Bioo Scientific) can be used, which use positive pressure to move a fluid to remove cells, platelets, and cell debris with one microfilter and capture vesicles larger than 30 nm with a second microfilter. However, in this process, the exosomes are not recovered, and their RNA contents are extracted directly from the material captured with the second microfilter and then used for PCR analysis. When using HPLC-based protocols, these processes require specialized equipment and are difficult to scale up, but potentially yield high-purity exosomes. A significant problem is that both blood and cell culture media contain numerous nanoparticles (some non-vesicles) in the same size range as exosomes. For example, some miRNAs may be present in extracellular protein complexes rather than exosomes. However, any contamination by "extraexosomal" proteins can be eliminated by treatment with a protease (e.g., proteinase K). 【0036】 In another embodiment, cancer cell-derived exosomes may be captured by techniques commonly used to enrich exosomes in a sample, such as techniques involving immune-specific 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 a particular cell type, to small paramagnetic beads. When the antibody-coated beads are mixed with a sample such as blood, the beads attach to and surround specific cells. The sample is then placed in a strong magnetic field, causing the beads to pellet on one side. After the blood is removed, the captured cells are retained with the beads. Many variations of this general method are well known in the art and are suitable for use in isolating 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 known to be found on cancer cell-derived exosomes include ATP-binding cassette subfamily A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin β-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. 【0037】 The analyses used herein include any method that enables direct or indirect visualization of exosomes, and may be in vivo or ex vivo. For example, analyses may include, but are not limited to, ex vivo detection and visualization of exosomes bound to a solid support by microscopy or cell counting, flow cytometry, fluorescence imaging, etc. In exemplary scenarios, cancer cell-derived exosomes contain ATP-binding cassette subfamily A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin β-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 β-1 (LAMB1), and tubulointerstitial nephritis antigen-like protein (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 protein 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 It can be detected using antibodies made against one or more of the following: 6B (PSMC4), elongation factor 1-γ (EEF1G), titin (TTN), tyrosine protein phosphatase type 13 (PTPN13), triose phosphate isomerase (TPI1), or carboxypeptidase E (CPE), and subsequently visualized using a solid support that can be conjugated and / or detected by microscopy or cell counting. 【0038】 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, one study found that 190 / 190 patients with pancreatic ductal adenocarcinoma had higher levels of GPC1+ exosomes than healthy controls (Melo et al., 2015; the entire study is incorporated herein by reference). Notably, on average, only 2.3% of healthy controls had GPC1+ exosomes. 【0039】 1. Exemplary protocol for collecting exosomes from cell cultures On day 1, seed a sufficient number of cells (e.g., about 5 million cells) into a T225 flask containing medium with 10% FBS so that the cells reach approximately 70% confluence the following day. On day 2, aspirate the medium from the cells, wash the cells twice with PBS, and then add 25-30 mL of basic medium (i.e., without PenStrep or FBS) to the cells. Incubate the cells for 24-48 hours. A 48-hour incubation is preferable, but some cell lines are highly sensitive to serum-free medium, and therefore the incubation time must be shortened to 24 hours. Note that FBS contains exosomes that strongly distort NanoSight results. 【0040】 On day 3 / 4, collect the culture medium and centrifuge at 800×g at room temperature for 5 minutes to pellet dead cells and large debris. Transfer the supernatant to a new conical tube and recentrifuge the medium at 2000×g for 10 minutes 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., 25×89 mm Beckman Ultra-Clear) using 35 mL / tube. If the volume of medium per tube is less than 35 mL, fill the rest of the tube with PBS until it reaches 35 mL. Ultracentrifuge the medium at 28,000 rpm at 4°C for 2–4 hours using an 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 transfer the remaining medium into an aspirator pipette. If desired, the exosome pellet can be resuspended in PBS and ultracentrifugation at 28,000 rpm for 1-2 hours can be repeated to further purify the exosome population. 【0041】 Finally, resuspend the exosome pellet in 210 μL of PBS. If multiple ultracentrifuge tubes are available for each sample, sequentially resuspend each exosome pellet using the same 210 μL of PBS. Take 10 μL of each sample and add it to 990 μL of H2O for use in nanoparticle tracking analysis. Use the remaining 200 μL of exosome-containing suspension for downstream processes or store immediately at -80°C. 【0042】 2. Exemplary protocol for extracting exosomes from serum samples First, thaw the serum sample on ice. Next, 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 overnight at 150,000 × g at 4°C. The next day, carefully discard the supernatant and wash the exosome pellet with 11 mL of 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 of PBS for analysis. 【0043】 Exemplary protocols for electroporation of C. exosomes and liposomes 1 x 10 8 Mix 1 exosome (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula Nano Sciences) with 1 μg of siRNA (Qiagen) or shRNA in 400 μL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). Electroporate the exosomes or liposomes using a 4 mm cuvette (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After electroporation, treat the exosomes or liposomes with protease-free RNase, followed by the addition of a 10× concentrated RNase inhibitor. Finally, wash the exosomes or liposomes with PBS under ultracentrifugation as described above. 【0044】 II. CRISPR / Cas Systems Generally, the “CRISPR system” refers collectively to transcripts and other elements involved in the expression or induction of activity of CRISPR-related ("Cas") genes, including sequences encoding the Cas gene; tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA); tracr-mate sequences (including “direct repeats” and partial direct repeats processed by tracrRNA in the context of the endogenous CRISPR system); guide sequences (also called “spacers” in the context of the endogenous CRISPR system); and / or other sequences and transcripts derived from the CRISPR locus. 【0045】 A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide) RNA that sequence-specifically binds to DNA, 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, or from a specific organism containing an endogenous CRISPR system, such as Streptococcus pyogenes. 【0046】 In some cases, a Cas nuclease and gRNA (a fusion of a target sequence-specific crRNA and a specific tracrRNA) are introduced into the cell. Generally, the target site at the 5' end of the gRNA is targeted by the Cas nuclease using complementary base pairing, for example, a gene. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, for example, typically immediately 5' of an 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, the CRISPR system is characterized by elements that facilitate CRISPR complex formation at the target sequence site. Typically, a "target sequence" refers to a sequence that is generally designed to have complementarity with a guide sequence, in which case hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex. 【0047】 The CRISPR system can induce double-strand breaks (DSBs) at a target site, and subsequently induce disruption as disclosed herein. In other embodiments, a Cas9 variant, thought to be a "nickase," is used to create a cleavage at a single-strand target site. Paired nickases can be used, for example, to improve specificity, with each nickase directed by a pair of different gRNAs targeting the sequence, such that the nicks are introduced simultaneously and a 5' overhang is introduced. In other embodiments, a catalytically inactive Cas9 is fused with a heterogeneous effector domain, such as a transcriptional repressor or activator, to influence gene expression. 【0048】 The target sequence may contain any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of a cell, for example, in a cell organelle. Generally, a sequence or template that can be used for recombination to a target locus containing the target sequence is called an “editing template,” “edited polynucleotide,” or “editing sequence.” In some contexts, an exogenous template polynucleotide is sometimes referred to as an editing template. In some contexts, the recombination is homologous recombination. 【0049】 Typically, in the context of the endogenous CRISPR system, when a CRISPR complex (containing a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) is formed, one or both strands are cleaved within or near the target sequence (for example, within a range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). The tracr sequence may also contain, or consist of, all or part of the wild-type tracr sequence (for example, about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence, or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides), and may become part of the CRISPR complex by hybridizing, for example, along at least part of the tracr sequence to all or part of a tracr mate sequence functionally linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of the CRISPR complex, for example, having sequence complementarity of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% along the length of the tracr mate sequence when optimally aligned. 【0050】 One or more vectors expressing elements of the CRISPR system can be introduced into cells such that a CRISPR complex is formed at one or more target sites by expressing one or more elements of the CRISPR system. The components can also be delivered to cells as proteins and / or RNA. For example, the Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be functionally linked to different regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in one vector with one or more further vectors supplying any components of the CRISPR system not contained in the first vector. This vector may contain one or more insertion sites, for example, restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, one expression construct may be used to target multiple different corresponding target sequences in the cell for CRISPR activity. 【0051】 The vector may include a regulatory element functionally linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-exclusive 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, their homologs, or modified versions thereof. These enzymes are well known. For example, the amino acid sequence of the S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. 【0052】 The CRISPR enzyme may be Cas9 (e.g., derived from S. pyogenes or S. pneumonia). The CRISPR enzyme can induce cleavage of one or both strands at the location of the target sequence, for example, within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a mutated CRISPR enzyme relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme loses the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 derived from S. pyogenes (D10A) converts Cas9, which is a nuclease that cleaves both strands, into a nickase (which cleaves one strand). In some embodiments, the Cas9 nickase may be used in combination with guide sequences, for example, two guide sequences that target the sense strand and antisense strand of the DNA target, respectively. This combination allows for the creation of breaks in both strands, which can then be used to induce NHEJ or HDR. 【0053】 In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon-optimized to be expressed in specific cells, such as eukaryotic cells. Eukaryotic cells include, but are not limited to, human, mouse, rat, rabbit, dog, or non-human primates, and may be eukaryotic cells of mammals, for example, or derived therefrom. Generally, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon in the native amino acid sequence with a codon that is frequently, or most frequently, used in the host cell's gene, while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species exhibit unique biases for certain codons of certain amino acids. Codon bias (differences in codon usage frequency between organisms) is often correlated with messenger RNA (mRNA) translation efficiency, and as a result, messenger RNA (mRNA) translation efficiency is thought to depend, in particular, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. In some cells, the dominance of a selected tRNA reflects that its codon is the most frequently used codon in peptide synthesis. Therefore, genes can be optimized based on codon optimization to achieve optimal gene expression in a particular organism. 【0054】 Generally, the guide sequence is any polynucleotide sequence that hybridizes with the target sequence and has sufficient complementarity to the target polynucleotide sequence to induce sequence-specific binding between the CRISPR complex and the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is approximately 50%, approximately 60%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 97.5%, approximately 99%, or greater, when properly aligned using a suitable alignment algorithm, or greater than approximately 50%, approximately 60%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 97.5%, approximately 99%, or greater. 【0055】 Optimal alignment can be verified using any suitable algorithm for aligning sequences. Non-restrictive examples include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the 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). 【0056】 A CRISPR enzyme may be part of a fusion protein containing one or more heterologous protein domains. A CRISPR enzyme fusion protein may contain any further protein sequences, and optionally a linker sequence, between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-exclusive examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, and autofluorescent proteins including green fluorescent protein (GFP), 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. Further domains that may be part of a fusion protein containing a CRISPR enzyme are described in US20110059502, incorporated herein by reference. 【0057】 III. Delivery using CRISPR systems In some cases, nucleic acids encoding a CRISPR-Cas9 target molecule, complex, or combination are administered or introduced into cells. In some cases, this system may already be present in the cell or may be present within the cell's exosomes. This nucleic acid is typically administered in the form of an expression vector, such as a viral expression vector. In some cases, the expression vector may be a retroviral expression vector, an adenovirus expression vector, a DNA plasmid expression vector, or an AAV expression vector. In some cases, disruption molecules or complexes, such as one or more polynucleotides encoding a DNA target molecule, are delivered to the cell. In some cases, delivery is by the delivery of one or more vectors, and one or more transcripts thereof, and / or one or more proteins transcribed therefrom, are delivered to the cell. 【0058】 In some embodiments, the polypeptide is synthesized in situ within a cell as a result of introducing a polynucleotide encoding the polypeptide into the cell. In some embodiments, the polypeptide can be produced extracellularly and then introduced into a cell. Methods for introducing polynucleotide constructs into animal cells are known and, in non-limiting examples, include stable transformation methods in which the polynucleotide construct is integrated into the cell genome, transient transformation methods in which the polynucleotide construct is not integrated into the cell genome, and viral-mediated methods. In some embodiments, the polypeptide may be introduced into the cell by, for example, a recombinant viral vector (e.g., retrovirus, adenovirus), liposomes, etc. For example, in some embodiments, transient transformation methods include microinjection, electroporation, or particle bombardment. In some embodiments, the polypeptide may be contained in a vector, more specifically a plasmid, or virus, considering that it will be expressed in the cell. 【0059】 In some embodiments, nucleic acids can be introduced into mammalian cells or target tissues using viral and nonviral gene transfer methods. Such methods can be used to administer nucleic acids encoding CRISPR system components to cells in culture or to cells within a host organism. Nonviral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA viruses and RNA viruses that, after delivery to cells, become episomal genomes or integrated genomes. For reviews of gene therapy procedures, see Anderson, 1992; Nabel & Feigner, 1993; Mitani & Caskey, 1993; Dillon, 1993; Miller, 1992; Van Brunt, 1988; Vigne, 1995; Kremer & Perricaudet, 1995; Haddada et al., 1995; and Yu et al., 1994. 【0060】 Nonviral methods of nucleic acid delivery include exosomes, lipofection, nucleofection, microinjection, bioristic, virosomes, liposomes, immunoliposomes, polycationic or lipid: nucleic acid conjugates, naked DNA, artificial virions, and DNA uptake enhanced by active agents. Lipofection is described (e.g., U.S. Patents 5,049,386, 4,946,787, and 4,897,355), and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Feigner, WO91117424; WO91116024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). 【0061】 In some embodiments, delivery is via the use of RNA virus or DNA virus-based systems for nucleic acid delivery. Viral vectors may, in some aspects, be delivered directly to the patient (in vivo), or they may be used to treat cells in vitro or ex vivo and then administer them to the patient. Virus-based systems in some embodiments include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene delivery. 【0062】 In some embodiments, a reporter gene, including but not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP), may be introduced into cells to encode a gene product that serves as a marker for measuring changes or modifications in gene product expression. In further embodiments, the DNA molecule encoding the gene product may be introduced into cells via a vector. In some embodiments, the gene product is luciferase. 【0063】 As will be understood by those skilled in the art, before or after cargo loading, the exosome may be further modified to enhance its usefulness as a cargo delivery vehicle by including a targeting portion. In this regard, the exosome may be engineered to incorporate an entity that specifically targets a particular cell of a tissue type. This target-specific entity, for example, a peptide having affinity for a receptor or ligand on a target cell or tissue, may be incorporated into the exosome membrane, for example, by fusing it with an exosome membrane marker using a method well established in the art. 【0064】 IV. Treatment of the disease A particular aspect of the present invention provides a method of treating a patient using exosomes that express or contain a gene editing system, such as the CRISPR system. The CRISPR system may induce gene editing in the patient's cancer cells. Since exosomes are known to possess the mechanisms necessary to complete mRNA transcription and protein translation (see WO2015 / 085096, which is incorporated herein by reference in its entirety), mRNA or DNA nucleic acids encoding a therapeutic protein may be introduced into the exosome by transfection. Alternatively, the therapeutic protein itself may be introduced into the exosome by electroporation or directly incorporated into liposomes. 【0065】 As used herein, “subject” refers to any individual or patient to whom this method is performed. Generally, the subject is human, but as will be understood by those skilled in the art, the subject may also be an animal. Accordingly, the definition of subject includes other animals, including mammals, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock (including cattle, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas). 【0066】 "Procedure" and "to treat" 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 from a disease or health-related condition. For example, a procedure may include the administration of exosomes, including CRISPR systems, chemotherapy, immunotherapy, or radiotherapy, the performance of surgery, or any combination thereof. 【0067】 As used herein, the terms “therapeutic benefit” or “therapeutic effect” refer to anything that promotes or improves the health of a subject with respect to a medical treatment of this condition. This term includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may include, for instance, a reduction in the invasiveness of the tumor, a decrease in the rate of cancer growth, or a halt to metastasis. Cancer treatment may also refer to an extension of the survival period of a subject with cancer. 【0068】 As used herein, the term “cancer” may be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer may occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidneys, liver, lungs, nasopharynx, neck, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus. 【0069】 Cancer specifically includes the following histological types: neoplasms, malignant; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinomas; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; calcifying epithelioma (pilomatrix carcinoma); transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma within adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid tumors; carcinoid tumors, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; pigmentaphobic carcinoma; eosinophil carcinoma; eosinophilic adenocarcinoma adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary adenocarcinoma and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ear canal 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; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal mammary Melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Müller's mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated germ cell tumor; embryonic carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; perivascular cell tumor, malignant; lymphangiosarcoma; osteosarcoma; paraosteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor malignant tumors; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastoma; pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; gliablastoma; oligodendroglioma; oligodendroglioma; undifferentiated neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurotumor; meningioma; neurofibrosarcoma; schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease This may include, but is not limited to, Hodgkin's disease; lateral granuloma; malignant lymphoma, microlymphocytic; malignant lymphoma, diffuse macrocytic; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; and pilocytic cell leukemia. Nevertheless, the present invention may also be used to treat non-cancerous diseases (e.g., fungal infections, bacterial infections, viral infections, neurodegenerative diseases, and / or hereditary disorders). 【0070】 The terms “contacted” and “exposed” are used herein to describe the process by which a therapeutic substance is delivered to or positioned directly alongside target cells when applied to cells. For example, to kill cells, one or more active ingredients are delivered to the cells in an amount effective enough to kill the cells or inhibit cell division. 【0071】 A patient's “responsiveness” to an effective response or treatment refers to the clinical or therapeutic benefit provided to a patient who is at risk of or suffering from a disease or disability. Such benefits may include cellular or biological responses, complete responses, partial responses, stable conditions (no progression or recurrence), or responses that later recur. For example, an effective response may be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer. 【0072】 Treatment outcomes can be predicted and monitored, and / or patients who will benefit from such treatment can be identified or selected by the methods described herein. 【0073】 Regarding the treatment of neoplasms, depending on the stage of the neoplasm, treatment involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiotherapy, and chemotherapy. Other treatment regimens may be combined with the administration of anticancer agents, such as therapeutic compositions and chemotherapeutic agents. For example, a patient to be treated with such anticancer agents may also receive radiotherapy and / or surgery. 【0074】 When treating a disease, the appropriate dosage of the therapeutic composition depends on the type of disease to be treated, 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 administered to the patient appropriately, either in a single dose or over a series of treatments. 【0075】 Therapeutic and prophylactic methods and compositions can be provided in combination amounts effective in achieving the desired effect. Tissues, tumors, or cells may be brought into contact with one or more compositions or pharmacological preparations containing one or more active substances, or by contacting tissues, tumors, and / or cells with two or more distinct compositions or preparations. Such combination therapies are also intended to be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy. 【0076】 Concomitant administration may include administering two or more agents simultaneously in the same dosage form, simultaneously in separate dosage forms, or separately. That is, this therapeutic composition and another therapeutic substance can be formulated together in the same dosage form and administered simultaneously. Alternatively, this therapeutic composition and another therapeutic substance can be administered simultaneously, in which case both substances exist in separate formulations. Another option is to administer one therapeutic substance immediately followed by another, and vice versa. In a different administration protocol, this therapeutic composition and another therapeutic substance may be administered with a gap of several minutes, several hours, or several days between administrations. 【0077】 The first anticancer treatment (e.g., an exosome expressing a recombinant protein, or a recombinant protein isolated from an exosome) may be administered before, during, or after the second anticancer treatment, or in various combinations with respect to the second anticancer treatment. Administration may be simultaneous or at intervals ranging from minutes to days to weeks. In a configuration where the first treatment is administered separately from the second treatment, the effective period will generally not end between deliveries, so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is intended that the first and second therapies may be administered to the patient within approximately 12–24 hours or 72 hours after either therapy, or more specifically, within approximately 6–12 hours after either therapy. In some circumstances, it may be desirable to significantly extend the time between treatments. In this case, the period between each implementation is 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). 【0078】 In certain embodiments, the course of treatment lasts 1 to 90 days or longer (including days in between). One agent may be administered on any day between day 1 and day 90 (including days in between) or in any combination thereof, and another agent may be administered on any day between day 1 and day 90 (including days in between) or in any combination thereof. Within a single day (24-hour period), the patient may receive one or more doses of the agent. Furthermore, it is intended that there will be a period after the course of treatment during which no anti-cancer treatment is performed. This period may last 1 to 7 days and / or 1 to 5 weeks and / or 1 to 12 months or more (including days in between), depending on the patient's condition, e.g., prognosis, physical strength, health status, etc. The treatment cycle is expected to be repeated as needed. 【0079】 Various combinations can be used. In the example below, the first anti-cancer therapy is "A" and the second anti-cancer therapy is "B". TIFF0007872660000001.tif17128 【0080】 The administration of any compound of the present invention to a patient or the implementation of any therapy of the present invention shall follow general protocols for the administration of such compounds, taking into consideration any toxicity of the agent. Accordingly, in some embodiments, there shall be a step of monitoring toxicity resulting from the combination therapy. 【0081】 1.Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with 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 the mode of their activity within cells, for example, whether they affect the cell cycle, or at what stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intercalate DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. 【0082】 Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclosphosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocon, meturedopa, and uredopa; altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomellamine. Ethyleneimine and methylamelamine (including ylolomelamime); acetogenins (especially bullatacin and bullatacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adzeresin, carzelesin, and biceresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; Duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembicin n) Phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosureas, e.g., carmustine, chlorozotocin, photemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., engine antibiotics (e.g., calitiamycin, in particular calitiamycin γ1I and calitiamycin ω1); dynemicins, including dynemicin A; bisphosphonates, e.g., clodronate; esperamicin;Furthermore, neocardinostatin chromophore and related pigment protein enediin antibiotics (antiobiotics) chromophore, acrasinomycin, actinomycin, authrarnnycin, azaserin, bleomycin, kactinomycin, carabicin, kaminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxo Rubicin (including cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, yubenimex, dino Statins, or zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, propio. Dromostanorone onate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents, e.g., mitotane and trilostane; folic acid supplements, e.g., folinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecoltin; diaziquan; elformithine; eriptinium acetate; eposylone; etogluside; gallium nitrate;Hydroxyureas; lentinan; lonidynin; mytansinoids, e.g., mytansin and ansamitocin; mitogwazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin n); sizofiran; spirogermanium; tenuazonic acid; triadiquan; 2,2',2''-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gacytosine; arabinoside ("Ara-C") ); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum-coordinated complexes, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CP This includes T-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; cisplatin, carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and any pharmaceutically acceptable salts, acids, or derivatives thereof. 【0083】 2. Radiation therapy Other factors that cause DNA damage and have been widely used include those commonly known as gamma rays, X-rays, and / or specific delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also intended, such as microwaves, proton beam irradiation (US Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors are likely to affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens for long-term use (3–4 weeks) to a single-dose dose of 2000–6000 roentgens. The dose range for radioisotopes is diverse and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by newly formed cells. 【0084】 3. Immunotherapy Those skilled in the art will understand that further immunotherapies may be used in combination with or in conjunction with the methods of the present invention. In the context of cancer treatment, 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. This antibody may act alone as the effector of the therapy, or it may mobilize other cells to actually influence cell death. This antibody may also be conjugated to a drug or toxin (such as a chemotherapeutic agent, radionuclide, lysine A chain, cholera toxin, pertussis toxin, etc.) or simply act as a targeting agent. Alternatively, the effector may be a lymphocyte having a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells. 【0085】 In one aspect of immunotherapy, tumor cells must possess some marker that is a target for targeting, i.e., one that is not present in the majority of other cells. Many tumor markers exist, and any of them may be suitable for targeting in the context of this 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 combining anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, 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. 【0086】 Examples of immunotherapies currently under investigation or in use include: immunoadjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998); U.S. Patents No. 5,830,880 and 5,846,945; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2013; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is intended that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein. 【0087】 In some embodiments, immunotherapy may involve immune checkpoint inhibitors. Immune checkpoints either enhance (e.g., co-stimulatory molecules) or de-enhance (weaken) signaling. 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. 【0088】 Immune checkpoint inhibitors may be drugs, such as small molecules, recombinant ligands or receptors, or in particular antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known immune checkpoint protein inhibitors or analogues may be used, in particular chimeric, humanized, or human-type antibodies. As those skilled in the art will know, certain antibodies referred to in this disclosure may be known by other names and / or equivalent names. Such other names and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is known to be also known by the other names and / or equivalent names MK-3475 and pembrolizumab. 【0089】 In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain contexts, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In other embodiments, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its ligand-binding partner. In certain contexts, the PDL1 binding partner is PD-1 and / or B7-1. In other embodiments, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its ligand-binding partner. In certain contexts, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents 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 as described in U.S. Patent Publications No. 20140294898, 2014022021, and 20110008369, all of which are incorporated herein by reference. 【0090】 In some embodiments, the PD-1 conjugated 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 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing the extracellular portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence) or a PD-1 binding moiety). In some embodiments, the PD-1 conjugated 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 WO2006 / 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. 【0091】 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 analogous to the T cell co-stimulatory protein CD28, 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, while CD28 transmits stimulating signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. When T cells are activated via the T cell receptor and CD28, the expression of CTLA-4, an inhibitory receptor for the B7 molecule, increases. 【0092】 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), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. 【0093】 An anti-human CTLA-4 antibody (or a VH domain and / or VL domain derived therefrom) suitable for use in this method can be prepared using methods well known in the art. Alternatively, an anti-CTLA-4 antibody approved in the art can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly also 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 disclosures of each of the aforementioned publications are incorporated herein by reference. In binding to CTLA-4, antibodies that compete with any of these antibodies recognized in the art may 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. 【0094】 Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDR or VR 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 epitopes on CTLA-4 as the aforementioned antibodies, and / or binds to the same epitopes on CTLA-4 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (for example, at least about 90%, at least about 95%, or at least about 99% variable region identity with ipilimumab). 【0095】 Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent No. 5,844,905, U.S. Patent No. 5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins, such as the immunoadhesin described in U.S. Patent No. 8,329,867, all of which are incorporated herein by reference. 【0096】 In some embodiments, immunotherapy may involve adoptive immunotherapy with the transfer of ex vivo-generated autoantigen-specific T cells. T cells used in adoptive immunotherapy can be generated by expanding antigen-specific T cells or by redirecting T cells through genetic engineering (Park, Rosenberg et al. 2011). Isolation and transfer of tumor-specific T cells have been shown to be successful in treating melanoma. Novel specificity in T cells has been successfully achieved 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 bound to one or more signaling domains in the form of a single fusion molecule. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv) in which a light chain fragment and a variable fragment of a monoclonal antibody are linked by a mobile linker. Binding moieties based on receptor or ligand domains have also been successfully utilized. The signaling domain of first-generation CARs is derived from the cytoplasmic region of CD3ζ or the Fc receptor γ chain. Using CARs, T cells are successfully redirected to antigens expressed on the surface of tumor cells from various neoplasms, including lymphomas and solid tumors (Jena, Dotti et al. 2010). 【0097】 In one aspect, the present invention provides a combination therapy for treating cancer comprising adoptive T cell therapy and a checkpoint inhibitor. 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 a tumor antigen. 【0098】 4.Surgery Approximately 60% of people with cancer undergo some form of surgery, including prophylactic surgery, surgery for diagnosis or staging, curative surgery, and palliative surgery. Curative surgery involves excision 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 procedures of the present invention, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor excision refers to the physical removal of at least part of the tumor. In addition to tumor excision, surgical procedures include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs procedure). 【0099】 When cancer cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment may be carried out by perfusing, directly injecting, or topically applying further anticancer therapies to the area. Such treatments 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 administered in various doses. 【0100】 5. Other active substances To improve the therapeutic efficacy of the treatment, other active agents may be used in combination with certain aspects of the present invention. These further active agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell division arrest agents and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing the number of gap junctions thereby increasing intercellular signaling enhances the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cell division arrest agents or differentiation agents may be used in combination with certain aspects of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are intended to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are local adhesion kinase (FAK) inhibitors and lovastatin. To further improve the efficacy of the treatment, other active agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present invention. 【0101】 V. Pharmaceutical Compositions Exosomes expressing or containing the CRISPR system are intended to be administered systemically or locally to inhibit tumor cell proliferation, most preferably to kill cancer cells in cancer patients with locally advanced or metastatic cancer. Exosomes expressing or containing the CRISPR system can be administered intravenously, subarachnoidally, and / or intraperitoneally. Exosomes expressing or containing the CRISPR system may be administered alone or in combination with antiproliferative agents. In one embodiment, exosomes expressing or containing the CRISPR system are administered before surgery or other procedures to reduce the patient's cancer load. Alternatively, exosomes expressing or containing the CRISPR system may be administered after surgery to ensure that any remaining cancer (e.g., cancer not removed surgically) does not survive. 【0102】 The present invention is not intended to be limited by the specific properties of the therapeutic preparations. For example, such compositions may be provided in the form of formulations together with physiologically acceptable liquids, gels, solid carriers, diluents, or excipients. These therapeutic preparations, like other therapeutic substances, can be administered to mammals for veterinary use, e.g., veterinary use in livestock, and clinical use in humans. Generally, the dosage required for therapeutic efficacy varies according to the type of use and method of administration, as well as the specific requirements of the individual subject. 【0103】 When clinical application is intended, it may be necessary to prepare a pharmaceutical composition containing recombinant proteins and / or exosomes in a form suitable for the intended use. Generally, the pharmaceutical composition may be a parenteral formulation and may contain an effective amount of one or more recombinant proteins and / or exosomes and / or further active ingredients dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" means, where appropriate, molecular entities and compositions that do not produce harmful, allergic, or other undesirable reactions when administered to animals, such as humans. The preparation of pharmaceutical compositions containing recombinant proteins and / or exosomes, or further active ingredients, disclosed herein are exemplified in Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference in its entirety for all purposes. Furthermore, it is understood that, in the case of administration to animals (e.g., humans), the preparation must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biological Standards. 【0104】 Furthermore, according to certain aspects of the present invention, a composition suitable for administration may be provided dissolved in a pharmaceutically acceptable carrier with or without an inert diluent. “pharmaceutically acceptable carrier” as used herein includes any and all aqueous solvents known to those skilled in the art (e.g., water, alcohol / aqueous solutions, ethanol, saline solution, parenteral vehicles, e.g., sodium chloride, Ringer's dextrose), non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and organic esters for injection, e.g., ethyl oleate), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants, and antimicrobial agents. This includes oxidizing agents, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, noble gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof), isotonic agents (e.g., sugars and sodium chloride), absorption retarders (e.g., aluminum monostearate and gelatin), salts, drugs, drug stabilizers, gels, resins, fillers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, pigments, fluids and nutrient replenishers, and combinations thereof. The carrier must be assimilated and includes liquids, semi-solids, i.e., pastes or solid carriers. Furthermore, if desired, the composition may contain trace amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffers. The pH and precise concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters. Appropriate fluidity can be maintained, for example, by using a coating, such as lecithin, by maintaining the required particle size in the case of a dispersion, or by using a surfactant. 【0105】 While pharmaceutically acceptable carriers are formulated specifically for administration to humans, in certain embodiments, it may be desirable to use pharmaceutically acceptable carriers formulated for administration to non-human animals but not permitted for administration to humans (e.g., due to government regulations). Their use is intended in therapeutic or pharmaceutical compositions, except where conventional carriers are incompatible with the active ingredient (e.g., harmful to the recipient or detrimental to the therapeutic effect of the composition contained in the carrier). According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such procedures are routine for those skilled in the art. 【0106】 Certain embodiments of the present invention may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form and whether they need to be sterilized for administration routes such as injection. The compositions may be administered intravenously, intradermally, transdermally, subarachnoidally, intra-arterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, locally, or topically, and may be administered by inhalation (e.g., aerosol inhalation), injection, infusion, serial infusion, local perfusion directly immersing target cells, via catheter, via lavage, encapsulated in lipid compositions (e.g., liposomes), or by other methods or any combination thereof. These are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference. 【0107】 The active compound can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, or subcutaneous routes, or for injection via the intraperitoneal route. Therefore, the embodiments include parenteral formulations. Typically, such compositions can be prepared as either a liquid solution or a suspension. Solid dosage forms suitable for use in preparing a solution or suspension by adding liquid before injection can also be prepared. The preparations can also be emulsified. 【0108】 According to this embodiment, the parenteral formulation may contain the exosomes disclosed herein together with one or more solutes and / or solvents, one or more buffers and / or one or more antimicrobial agents, or any combination thereof. In some aspects, the solvent may include water, water-miscible solvents such as ethyl alcohol, liquid polyethylene glycol, and / or propylene glycol, and / or water-immiscible solvents such as non-volatile oils including, for example, corn oil, cottonseed oil, peanut oil, and / or sesame oil. In certain versions, the solute may include one or more antimicrobial agents, buffers, antioxidants, tonic substances, freeze-protective substances, and / or freeze-drying-protective substances (lyoprotectants). 【0109】 Antimicrobial agents pursuant to this disclosure may include antimicrobial agents provided elsewhere in this disclosure, as well as benzyl alcohol, phenol, mercury compounds, and / or parabens. Antimicrobial agents may include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and / or thimerosal, or any combination thereof. Antimicrobial agents may be present in various contexts at concentrations necessary to ensure sterility as required by the pharmaceutically active substance. For example, the active substance may be present in a preparation, for example, in a preparation contained in a multi-dose container, at bacteriostatic or fungistatic concentrations. In various embodiments, the active substance may be a preservative and / or present at a sufficient concentration at the time of use to prevent the growth of microorganisms, for example, microorganisms unintentionally introduced into the preparation, for example, while withdrawing a portion of the contents using a subcutaneous needle and syringe. In various aspects, the active substance has a maximum volume and / or concentration limit (e.g., phenylmercury nitrate and thimerosal 0.01%, benzethonium chloride and benzalkonium chloride 0.01%, phenol or cresol 0.5%, and chlorobutanol 0.5%). In various cases, the active substance, such as phenylmercury nitrate, is used at a concentration of 0.002%. A combination of 0.18% p-hydroxybenzoate methyl methyl benzoate and 0.02% p-hydroxybenzoate propyl propyl benzoate, and 2% benzyl alcohol can also be applied according to the embodiment. Antimicrobial agents may also include 0.5% hexylresorcinol, 0.1% phenylmercury benzoate, and / or therapeutic compounds. 【0110】 Antioxidants according to this disclosure may include ascorbic acid and / or salts thereof, and / or sodium salts of ethylenediaminetetraacetic acid (EDTA). Tonic substances described herein may include electrolytes and / or monosaccharides or disaccharides. Freeze-protective substances and / or freeze-drying-protective substances are additives that protect biopharmaceuticals from adverse effects of freezing and / or drying during freeze-drying. Freeze-protective substances and / or freeze-drying-protective substances may include sugars (non-reducing), e.g., sucrose or trehalose, amino acids, e.g., glycine or lysine, polymers, e.g., liquid polyethylene glycol or dextran, and polyols, e.g., mannitol or sorbitol. These are all possible freeze-protective or freeze-drying-protective substances. This embodiment may also include antifungal agents, e.g., butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid, or any combination thereof. Further solutes and antimicrobial agents, buffers, antioxidants, tonic substances, freeze-protective and / or freeze-drying-protective substances that may be used in accordance with this disclosure, as well as aspects of the methods for preparing the parenteral formulations, are described, for example, in Chapter 41 of Remington's Pharmaceutical Sciences, 21st Ed., 2005, which is incorporated herein by reference in its entirety for all purposes. 【0111】 Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or propylene glycol aqueous solutions; and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the dosage form must be sterile and liquid enough to be easily injected. The dosage form must also be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. 【0112】 Therapeutic agents may be formulated into compositions in the form of free bases, neutrals, or salts. Pharmacopoecitable salts include acid-added salts, such as those formed with free amino groups of protein compositions, or acid-added salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be obtained from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is administered in a manner compatible with the administered formulation and in an effective therapeutic amount. The formulations are readily administered in various dosage forms, for example, as injections or aerosols for parenteral administration when delivered to the lungs, or as drug-release capsules for oral administration. 【0113】 In certain embodiments of the present invention, the composition is combined with a semi-solid or solid carrier, or thoroughly mixed with a semi-solid or solid carrier. Mixing can be carried out by any convenient method, such as grinding. Stabilizers can also be added in the mixing process to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids, such as glycine and lysine, and carbohydrates, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, and mannitol. 【0114】 In further embodiments, the present invention may relate to the use of a pharmaceutically acceptable lipid vehicle composition comprising one or more lipids and an aqueous solvent. As used herein, the term “lipid” is defined to include a broad range of any substance characterized by being insoluble in water and extractable using an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term “lipid” as used herein is not limited to any particular structure. Examples include compounds containing long-chain aliphatic hydrocarbons and their derivatives. Lipids may be natural or synthetic (i.e., designed or produced by humans). However, lipids are usually biological substances. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingoglycolipids, glycolipids, sulfatides, lipids having ether-linked and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that those skilled in the art would understand to be lipids are also included in the compositions and methods described herein. 【0115】 Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in lipid vehicles. For example, a therapeutic substance may be dispersed in a lipid-containing solution, dissolved with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bonded with lipids, contained as a lipid-dissolved suspension, contained using micelles or liposomes, or complexed, or otherwise bonded to lipids or lipid structures by any means known to those skilled in the art. Liposomes may or may not be formed by the dispersion. 【0116】 The term “unit dose” or “dosage” refers to a physically distinct unit suitable for use in a subject, each unit containing a predetermined amount of the therapeutic composition calculated to produce the desired response discussed above in relation to the administration, i.e., 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 dose of the composition of the present invention administered to a patient or subject may be determined by physical and physiological factors, such as the subject’s weight, age, health status, and sex, the type of disease being treated, the extent of disease entry, previous or concurrent therapeutic interventions, the patient’s idiopathic disease, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, doses may also include doses of about 1 μg / kg / body weight to about 1000 mg / kg / body weight (such a range includes doses in between) or greater doses per single administration, and any range that can be derived from there. In non-limiting examples derived from the numbers listed herein, doses can be administered in ranges such as approximately 5 μg / kg / body weight to approximately 100 mg / kg / body weight, or approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight. The physician administering the medication will, under any circumstances, determine the concentration of the active ingredient in the composition and the appropriate dose for each individual patient. 【0117】 The actual dose of a composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and route of administration. Depending on the dose and route of administration, the preferred dose and / or number of effective doses may vary according to the subject's response. The physician administering the treatment will, under any circumstances, determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject. 【0118】 In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may contain, for example, about 2% to about 75% or about 25% to about 60% of the unit weight, or any range that can be derived therefrom. In nature, the aforementioned amounts of the active compound can be prepared in each therapeutically useful composition in such a manner that an appropriate dose is obtained at a particular unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations are taken into account by those skilled in the art who prepare such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable. 【0119】 In other non-limiting examples, the dose may also include approximately 1 microgram / kg / body weight, approximately 5 micrograms / kg / body weight, approximately 10 micrograms / kg / body weight, approximately 50 micrograms / kg / body weight, approximately 100 micrograms / kg / body weight, approximately 200 micrograms / kg / body weight, approximately 350 micrograms / kg / body weight, approximately 500 micrograms / kg / body weight, approximately 1 milligram / kg / body weight, approximately 5 milligrams / kg / body weight, approximately 10 milligrams / kg / body weight, approximately 50 milligrams / kg / body weight, approximately 100 milligrams / kg / body weight, approximately 200 milligrams / kg / body weight, approximately 350 milligrams / kg / body weight, approximately 500 milligrams / kg / body weight to approximately 1000 milligrams / kg / body weight, or more, and any range that can be derived from there. In non-limiting examples of the ranges that can be derived from the numbers listed herein, based on the above numbers, doses can be administered in ranges such as approximately 5 milligrams / kg / body weight to approximately 100 milligrams / kg / body weight, and approximately 5 micrograms / kg / body weight to approximately 500 milligrams / kg / body weight. 【0120】 VI. Nucleic Acids and Vectors In certain aspects of the present invention, nucleic acid sequences encoding a therapeutic protein or a fusion protein containing a therapeutic protein may be disclosed. Depending on the expression system used, the nucleic acid sequence may be selected based on conventional methods. For example, each gene or variant may be codon-optimized for expression in a particular system. Various vectors can also be used to express the protein of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors. 【0121】 VII. Recombinant Proteins and Inhibitory RNA Some embodiments relate to recombinant proteins and polypeptides. Certain embodiments relate to recombinant proteins or polypeptides having RNA-guided endonuclease activity. In further aspects, such proteins or polypeptides may be modified to enhance serum stability. Thus, when this application refers to the function or activity of a “modified protein” or “modified polypeptide,” it will be understood by those skilled in the art that this includes, for example, proteins or polypeptides that have further advantages compared to unmodified proteins or polypeptides. Embodiments relating to “modified proteins” may be carried out with respect to “modified polypeptides” and vice versa, and so on. 【0122】 Recombinant proteins may have amino acid deletions and / or substitutions. Therefore, deletion proteins, substitution proteins, and deletion and substitution proteins are modified proteins. In some embodiments, these proteins may further include inserted or added amino acids, e.g., proteins with fusion proteins or linkers. A “modified deletion protein” lacks one or more residues of a native protein but may possess the specificity and / or activity of the native protein. A “modified deletion protein” may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is one in which amino acid residues are deleted from at least one antigenic region, which is a protein region that has been proven to be antigenic in a specific organism, e.g., the type of organism to which the modified protein may be administered. 【0123】 Substitutional or exchange variants typically involve the exchange of one amino acid for another at one or more sites within a protein and may be designed to modulate one or more properties of a polypeptide, particularly its effector function and / or bioavailability. The substitution may be a conservative substitution, i.e., one amino acid is exchanged for an amino acid of similar shape or charge, and the substitution may not be a conservative substitution. Conservative substitutions are well known in the art and include, for example, the exchange of alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. 【0124】 In addition to deletions or substitutions, modified proteins may have residue insertions, which typically involve the addition of at least one residue to a polypeptide. This may include the insertion of a targeting peptide or targeting polypeptide, or it may simply involve the insertion of a single residue. Terminal additions, known as fusion proteins, are discussed below. 【0125】 The term “biologically functional equivalent” is well understood in the art and is further defined herein in detail. Therefore, if the biological activity of the protein is maintained, it may include approximately 70% to 80% of the amino acid sequence, or approximately 81% to 90%, or even 91% to 99%, of the amino acid sequence of the control polypeptide, if they are identical or functionally equivalent. Recombinant proteins may, in certain aspects, be biologically and functionally equivalent to their natural counterparts. 【0126】 The amino acid sequences and nucleic acid sequences may include additional residues, such as additional N-terminal or C-terminal amino acids or 5' or 3' sequences, as long as they satisfy the criteria set forth above, including the maintenance of biological protein activity related to protein expression, but are still essentially as shown in one of the sequences disclosed herein. The addition of terminal sequences applies in particular to nucleic acid sequences that may include, for example, various non-coding sequences adjacent to the 5' or 3' portion of the coding region, or various internal sequences, i.e., introns known to exist within genes. 【0127】 As used herein, protein or peptide generally refers to, but is not limited to, proteins translated from genes, ranging from more than about 200 amino acids to full-length sequences; polypeptides of more than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms “protein,” “polypeptide,” and “peptide” are used herein as synonymous. 【0128】 As used herein, “amino acid residue” refers to any natural amino acid, any amino acid derivative, or any amino acid mime known in the art. In certain embodiments, the residues of a protein or peptide are continuous, and the amino acid residue sequence is not interrupted by non-amino acids. In other embodiments, this sequence may contain one or more non-amino acid moieties. In certain embodiments, the sequence of residues in a protein or peptide may be interrupted by one or more non-amino acid moieties. 【0129】 Therefore, the term “protein or peptide” includes an amino acid sequence containing at least one of the 20 common amino acids found in natural proteins, or at least one modified or rare amino acid. 【0130】 A particular aspect of the present invention relates to fusion proteins. These molecules may have the N-terminus or C-terminus of a therapeutic protein linked to a heterologous domain. For example, the fusion may also utilize a leader sequence derived from another species to enable recombinant expression of the protein in a heterologous host. Other useful fusions include the addition of protein affinity tags, e.g., serum albumin affinity tags or six histidine residues, or immunologically active domains, e.g., antibody epitopes, preferably cleavable antibody epitopes to facilitate the purification of the fusion protein. Non-limiting affinity tags include polyhistidines, chitin-binding proteins (CBPs), maltose-binding proteins (MBPs), and glutathione-S-transferase (GST). 【0131】 Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by the novel synthesis of a complete fusion protein, or by attaching DNA sequences encoding heterologous domains and subsequently expressing an intact fusion protein. 【0132】 The production of fusion proteins that restore the functional activity of parent proteins may be facilitated by linking genes using cross-linking DNA segments that encode peptide linkers spliced ​​between polypeptides connected in series. The linkers would need to be long enough to allow for the correct folding of the resulting fusion protein. 【0133】 VIII. Kits and Diagnostic Agents In various aspects of the present invention, a kit is envisioned that contains the necessary components for purifying exosomes from body fluids or tissue culture media. In other aspects, a kit is envisioned that contains the necessary components for isolating exosomes and transfecting them with a CRISPR system. The kit may contain one or more sealed vials containing any such components. In some embodiments, the kit may also include suitable container means, such as Eppendorf tubes, assay plates, syringes, bottles, or tubing, which are containers that do not react with the components of the kit. The containers may be made from sterile materials such as plastic or glass. 【0134】 The kit may further include instructions outlining the procedures of the methods described herein, which are substantially the same as those described herein or are known to those skilled in the art. The information in the instructions may be in a computer-readable medium, which includes machine-readable instructions that, when performed using a computer, display the actual or hypothetical procedures for purifying exosomes from a sample and introducing a CRISPR system into the exosomes by transfection or electroporation. [Examples] 【0135】 IX. Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. It will be understood by those skilled in the art that the techniques disclosed in the following embodiments demonstrate that the techniques discovered by the inventors are fully functional in carrying out the present invention and that they may constitute preferred embodiments for carrying out the present invention. However, in consideration of this disclosure, it will be understood by those skilled in the art that many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and similar or equivalent results can still be obtained. 【0136】 Example 1 - Materials and Methods Exosome isolation and purification. Exosomes were purified by fractionation centrifugation as previously described (Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). The supernatant was collected from cells cultured for 48 hours in a medium containing exosome-depleted FBS and then subjected to continuous centrifugation at 800 g for 5 minutes and at 2000 g for 10 minutes. The resulting supernatant was then filtered through a 0.2 μm filter in a culture bottle and ultracentrifuged (Beckman) at 28,000 g in an SW 32 Ti rotor for 2 hours, after which the pellet was collected. The supernatant was aspirated, the pellet was resuspended in PBS, and then ultracentrifuged for another 2 hours. The purified exosomes were then analyzed and used for experimental procedures. 【0137】 Electroporation of exosomes and liposomes. 1 × 10⁻⁶ 8 ~3×10 8Individual exosomes (measured by nanosight analysis) were mixed with the indicated amount of RNA in 400 μl of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep™). As previously described (Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012), exosomes were electroporated using a Gene Pulser Xcell™ Electroporation System (BioRad) with a 4 mm cuvette. After electroporation, exosomes were treated with protease-free RNAse A (Sigma Aldrich), then 10× concentrated RNase inhibitor (Ambion) was added, and they were washed with PBS under ultracentrifugation as described above. 【0138】 Exosome transfection. For in vitro transfection using exosomes, exosomes were electroporated, washed with PBS as described above, and 200,000 cells in a 6-well plate were treated with exosomes for the time required as described for each assay, then washed with PBS and used for further analysis. 【0139】 Real-time PCR analysis. Total RNA was purified using TRIzol® (Invitrogen) according to the manufacturer's instructions, and then retro-transcribed using MultiScribe Reverse Transcriptase (Applied Biosystems) and oligo-d(T) primers. Real-time PCR analysis was performed on an ABI PRISM® 7300HT Sequence Detection System Instrument using SYBR® Green Master Mix (Applied Biosystems). Transcripts of interest were normalized to 18S transcript levels. Each measurement was repeated three times. The threshold cycle, which is the fractional cycle number at which the amount of amplified target reaches a fixed threshold, was determined, and expression was measured using the 2 -ΔCt formula. 【0140】 Western blot. After 24 hours, to assess protein expression in cells after treatment with exosomes, cells were harvested in RIPA buffer and protein lysates were normalized using Bradford quantification. Forty micrograms of lysate was loaded onto acrylamide gels for electrophoretic separation under denaturing conditions and transferred to a PVDF membrane (ImmobilonP) by wet electrophoresis transfer. The membrane was then blocked with 5% non-fat dry milk dissolved in PBS / 0.05% Tween-20 for 1 hour at room temperature and incubated overnight at 4°C with the appropriate primary antibody. The secondary antibody was incubated for 1 hour at room temperature. After antibody incubation, washing was performed three times at 15-minute intervals using 1× PBS 0.05% Tween®-20 on an orbital shaker. The membrane was developed using Pierce's chemiluminescent reagent according to the manufacturer's instructions, and chemiluminescence on the membrane was captured. 【0141】 Transfection and validation of CRISPR-Cas9-sgRab27a-2 cells. HEK293T cells were transfected with either a CRISPR-Cas9 vector control or CRISPR-Cas9-sgRab27a-2 by treatment with lipofectamine for 72 hours. The cells were then selected with 1 μg / ml puromycin for 10 days to obtain stable HEK293T CRISPR-Cas9 vector control and CRISPR-Cas9-sgRab27a-2 cells. The stable cells were then cultured in a selective medium containing 1 μg / ml puromycin. DNA and RNA were extracted from the stable cell lines as described above, and Cas9 levels were determined using qPCR and RT-qPCR. 【0142】 Exosome collection and validation. As described above, exosomes were collected from untransfected HEK293T cells, as well as from stable HEK293T CRISPR-Cas9 vector control and CRISPR-Cas9-sgRab27a-2 cells. Exosome quality was validated by Nanosight. 【0143】 CRISPR-Cas9 genome editing. To ensure the presence and quantity of the appropriate vector, exosomal DNA and RNA were extracted, and qPCR and RT-qPCR were performed to detect exosomal Cas9 vector control levels and sgRNA levels against Rab27a-2. Furthermore, Cas9 protein levels in both cells and exosomes were evaluated by Western blotting using anti-Flag antibody or Cas9 antibody with vinculin or CD9 as a control, respectively. The T7 / SURVEYOR assay was used to confirm that DNA editing had occurred in both cells and exosomes. 【0144】 Treatment of BxPC-3 adenocarcinoma cells using exosomes. 3 × 10⁶ cells were collected from HEK293T blank cells, HEK293T CRISPR-Cas9 vector control, and CRISPR-Cas9-sgRab27a-2 stable cells. 10Exosomes were introduced into BxPC-3 adenocarcinoma cells by treating them once or twice every 24 hours as described above. DNA and RNA were extracted from the recipient cells. Cas9 levels or sgRNA levels were detected from both DNA and RNA using qPCR and RT-qPCR. Editing in recipient BxPC-3 cells was then confirmed using the T7 / SURVEYOR assay. 【0145】 Treatment of BJ cells with CRISPR-Cas9 exosomes isolated from BJ cells. Exosomes were collected from BJ cells as described above. Exosomes were validated using Nanosight. To further confirm the exosomes, exosome markers CD9, CD81, flotillin, and TSG101 were detected by Western blotting. 1 × 10⁻⁶ 10 Individual isolated and validated BJ cell exosomes were electroporated with 15 μg of CRISPR-Cas9-GFP plasmid, and then treated with DNase or not. After DNase treatment, exosomal DNA was extracted and Cas9 levels were assessed by qPCR. Furthermore, the copy number was calculated by absolute qPCR using CRISPR-Cas9-GFP plasmid as a reference. Subsequently, the electroporated exosomes with DNase were introduced into BJ cells for 24 hours by transfection as described above. Then, Cas9 levels were detected from DNA and mRNA using qPCR or RT-qPCR. 【0146】 Transduction of BxPC-3 cells using HEK293T / CRISPR-Cas9 medium. HEK293T cells were transfected with a packaging plasmid along with CRISPR-Cas9 Rab27b-1 / 2 or an empty control plasmid using lipofectamine 2000 as described above. Medium containing lentivirus was collected and then transduced into BxPC-3 cells. Transduced cells were further selected using 0.4 μg / mL puromycin, and single clones of BxPC-3 / CRISPR-Cas9-sgRab27b cells were pricked, clonally grown, and validated by both Western blotting and T7 / SURVEYOR assay. Rab27b and Rab27a protein levels were then evaluated in all single clones. The T7 / SURVEYOR assay was also used to verify that gene editing had occurred in all clones. BxPC-3 / CRISPR-Cas9 vector control stable cells and single clones BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3 and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 were cultured in selective medium containing 0.4 μg / ml puromycin. Exosomes were collected from the aforementioned cells, similar to secreted exosomes, and then Nanosight validation was performed. Exosome DNA and RNA were extracted, and exosome Cas9 levels were detected by qPCR, and sgRNA for Rab27b-1 / 2 was detected by RT-qPCR. Cas9 and Rab27b protein levels were evaluated by Western blotting in both cells and exosomes, and the T7 / SURVEYOR assay was used to determine whether DNA editing occurred in both cells and exosomes. 【0147】 Evaluation of protein concentration from exosomes. BxPC-3 / CRISPR-Cas9 vector-controlled stable cells and single clones BxPC-3 / CRISPR-Cas9-sgRab27b-1 clone 3 (C3) and BxPC-3 / CRISPR-Cas9-sgRab27b-2 clone 6 (C6) were cultured, and exosomes were collected as described above. Exosomes collected from BxPC-3 / CRISPR-Cas9 vector-controlled stable cells and single clones BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3 and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 were lysed, and protein content was evaluated using a BCA kit according to the manufacturer's instructions. 【0148】 Cell proliferation assay. To ensure that cell proliferation is not affected by the presence of CRISPR-Cas9 or gene editing, control and CRISPR-Cas9-treated cells were evaluated. 100 μL of untreated BxPC-3 cells, BxPC-3 cells using a CRISPR-Cas9 empty vector control, BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3 cells, and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 cells were placed in 96-well plates at a rate of 1 × 10⁶ 5 Cells were seeded at a concentration of cells / mL. Cell proliferation was evaluated at various time points using the MTT assay. 【0149】 In vitro transcription of sgRab27b. To prepare in vitro transcription of sgRab27b, sgRab27b-1 / 2 was first amplified by PCR, and then the PCR product was purified using the Qiagen® PCR purification kit. The purified PCR product of sgRab27-1 / 2 was in vitro transcribed using the MEGAshortscript® kit (Thermo Fisher Scientific® catalog number 1354) according to the manufacturer's instructions. Furthermore, RNA quality was evaluated by electrophoresis using an 8M urea polyacrylamide gel. To prepare in vitro transcription of Cas9, Cas9 was amplified by PCR, and then the PCR product was purified using the Qiagen® PCR purification kit. The purified Cas9 PCR product was in vitro transcribed using the mMESSAGE mMACHINE® T7 Ultra kit. Cas9 RNA quality was detected using a formaldehyde gel. 【0150】 Cell treatment with in vitro transcription RNA. To evaluate transfection and CRISPR-Cas9 efficiency, HEK293T / CRISPR-Cas9 vector control cells were transfected with 1 μg of IVT-sgRab27b RNA for 72 hours using lipofectamine 2000, Exo-Fect / exosome transfection reagent, or electropermeable exosomes. After transfection, DNA was extracted and T7 / SURVEYOR assay was performed to confirm whether gene editing had occurred. HEK293T cells and BxPC-3 cells were either transfected with Cas9 mRNA using lipofectamine 2000 or Exo-Fect / exosome transfection reagent, or HEK293T cells and BxPC-3 cells were electropermeable with Cas9 mRNA (1 × 10⁶ cells). 9 Each MSC exosome was treated for 48 hours. Western blotting was performed to detect Cas9 protein levels. 【0151】 Evaluation of Exo-Fect / exosome treatment. Hekt293T cells were treated with 10 μg of plasmids (CRISPR-Cas9-lenchi-V2 vector control, CRISPR-Cas9-lenchi-V2-sgRab27b-1, CRISPR-Cas9-GFP vector control) four times at 24-hour intervals (days 1, 2, 3, and 4) using Exo-Fect / exosome transfection reagent. CRISPR-Cas9-GFP cells were imaged on day 5 to detect GFP expression. Cells were also collected on day 5 to isolate nucleic acids and proteins. DNA, RNA, and proteins were extracted. Relative Cas9 expression levels and 1 / Ct values ​​were confirmed by qPCR for cells transfected with each plasmid, and Cas9 protein levels were detected by Western blotting. The T7 / SURVEYOR assay was performed to confirm the occurrence of gene editing in HEK293T cells after treatment with the CRISPR-Cas9-lente-V2-sgRab27b-1 plasmid. The same experiment was repeated using BxPC-3 cells. 【0152】 sgmKras editing of KPC689 cells. KPC689 cells were treated with 5 μg of control plasmid or CRISPR-Cas9-sgmKras by lipofectamine 2000. G12D The lenti-V2 plasmid was transfected for 48 hours. After transfection, CRISPR-Cas9-GFP vector control cells were imaged to confirm transfection efficiency. DNA, RNA, and proteins were extracted from the total culture as described above. Relative Cas9 and mKras G12D The expression levels were confirmed by qPCR and as described above. A T7 / SURVEYOR assay was performed to investigate whether gene editing occurred in KPC689 cells after transfection with lipofectamine. Fresh KPC689 cells were transfected using an Exo-Fect / exosome transfection reagent with CRISPR-Cas9-sgmKras, which has a GFP backbone. G12DCells were treated with 10 μg of the plasmid or its vector control every 24 hours for 3 days. GFP expression in the cells was imaged to confirm transfection efficiency, and cells were collected on day 4. DNA, RNA, and proteins were extracted, and relative Cas9 and mKras were analyzed. G12D The expression level was confirmed by qPCR. A T7 / SURVEYOR assay was performed to determine the expression level of CRISPR-Cas9-GFP-mKras. G12D Gene editing was confirmed in KPC689 cells after plasmid treatment. 【0153】 Transfection and validation of doxycycline-inducible CRISPR-Cas9 plasmid. HEK293T cells were transfected with a mixture of lentiviral packaging plasmid and CRISPR-Cas9 doxycycline-inducible plasmid using lipofectamine 2000. Lentivirus-containing medium was collected and then transfected into Panc1 cells. Transfected cells were further selected with 1 μg / ml puromycin. Stable Panc1 cells with inducible Cas9 were maintained by culturing with 1 μg / ml doxycycline. Exosomes were collected from doxycycline-treated or untreated Panc1-inducible cells. Cas9 protein levels were examined in both cells and exosomes using Western blotting. Panc1-inducible cells were treated with 2 μg hKras by lipofectamine, Fugene, or Exo-Fect. G12D IVT-sgRNA, 1 μg of hKras G12D The cells were treated with plasmids for 72 hours. Subsequently, a T7 / SURVEYOR assay was performed to confirm gene editing in Panc1-inducible cells. 【0154】 CRISPR-Cas9-sghKRas G12D Treatment of Panc1 cell lines using Panc1-Cas9 and Panc1 sghKras. G12DT1 stable cell lines were established using lentivirus-based methods. Cas9 protein expression in Panc1-Cas9 cells was confirmed by Western blotting. Untransfected Panc1 cells were transfected with lenti-V2, GFP, or CRISPR-Cas9-sghKras with a puromycin backbone using lipofectamine, Exo-Fect, or electropermeable exosomes. G12D Panc1-Cas9 stable cells were treated with lipofectamine, Exo-Fect, or electropermeable exosomes using sghKras. G12D Processed with plasmid, Panc1 sghKras G12D Stable T1 cells were transfected for 24 hours with 10 μg or 20 μg of Cas9 plasmid containing GFP or puromycin backbone. Gene editing was confirmed by T7 / SURVEYOR assay. 【0155】 In vivo treatment of transplanted KPC689 tumors. KPC689 cells were subcutaneously transplanted into the backs of each mouse. The mice were divided into four groups, with one or two mice per group. Group 1 was 1 × 10⁶ 9 Group 2 was treated with 10 μg of exosomes and 10 μL of Exo-Fect. Group 2 was treated with 10 μg of Cas9-GFP-sgmKras. G12D - Treated with mK1 plasmid. Group 3 was 1 × 10⁻⁶ 9 Each exosome was treated with 10 μg of Cas9-GFP vector control plasmid and 10 μL of Exo-Fect. Group 4 was treated with 1 × 10⁶ cells. 9 exosomes, 10 μg of Cas9-GFP-sgmKras G12D The mice were treated with the mK1 plasmid and 10 μL of Exo-Fect. Each group of mice received intravenous (IV) and intratumor (IT) injections daily for two weeks. Tumor length (a, mm) and width (b, mm), as well as body weight, were measured, and tumor volume was calculated. 【0156】 Example 2 - Establishment of CRISPR-Cas9 exosome DNA and RNA were extracted from HEK293T cells transfected with a CRISPR-Cas9 vector control and HEK293T cells transfected with CRISPR-Cas9-sgRab27a-2, and Cas9 levels were determined using quantitative real-time PCR (qPCR) (Figure 1a). Both vectors were efficiently transfected, and the transfected cells showed significantly higher Cas9 expression compared to β-actin controls. Exosomes were collected from HEK293T blank cells and stable HEK293T CRISPR-Cas9 vector control and CRISPR-Cas9-sgRab27a-2 cells. Nanosight validation of exosomes can be seen in Figure 1b. Exosome DNA and RNA were extracted, and qPCR was performed to detect exosome Cas9 levels and sgRNA against Rab27a-2. Similar to the above cells, both vector controls and vectors and guide RNAs were expressed in exosomes (Figure 1c). To confirm Cas9 expression, Cas9 protein levels in both cells and exosomes were assessed by Western blotting using anti-Flag antibody or Cas9 antibody, respectively, with vinculin or CD9 as controls (Figure 1d). DNA editing was confirmed in both cells and exosomes using the T7 / SURVEYOR assay. This can be seen in the squares in multiple parts of Figures 1e and 1f. BxPC-3 cells treated with exosomes were evaluated for the presence of Cas9 DNA and Cas9 expression (Figures 1g and 1h). As can be seen from Figure 1g, Cas9 DNA increased in cells treated twice. To confirm the presence of guide RNA, BxPC-3 cells treated with exosomes were tested for the presence of guide RNA (Figures 2a and 2c). DNA was clearly visible, but RNA expression was absent. This was confirmed by the lack of activity in the T7 / SURVEYOR assay (Figure 2b). 【0157】 Exosomes were collected from BJ cells and confirmed by nanosight analysis as shown in Figure 3a. To further confirm the presence of exosomes, exosome markers CD9, CD81, filtilin, and TSG101 were detected by Western blotting (Figure 3b). BJ exosomes were electroporated with 15 ug of CRISPR-Cas9-GFP plasmid and then treated with DNase or not. Cas9 DNA was strongly detected in samples not treated with DNase, and was detected even more efficiently in DNase-treated samples containing both exosomes and plasmid than in plasmid alone (Figure 3c). Plasmid copy number was determined using a calibration curve created from 1 / Ct values ​​(Figure 3c). Electroporated exosomes treated with DNase were introduced into BJ cells for 24 hours. Cas9 levels increased in both DNA and mRNA compared to blank exosomes (Figure 3d). 【0158】 Clonal BxPC-3 cells transduced using lentiviral medium containing the CRISPR-CAS9 Rab27b-1 / 2 plasmid were validated by both Western blotting (Figure 4a) and T7 / SURVEYOR assay (Figure 4b). Two clones were found to be active and are shown in the squares in Figure 4b. Clones 3 (C3) of BxPC-3 / CRIPSR-Cas9-sgRab27b-1 and 6 (C6) of BxPC-3 / CRISPR-Cas9-sgRab27b-2 were used for further experiments. BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3 and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 were cultured in selective medium containing 0.4 μg / ml puromycin, and DNA and RNA were extracted. While the presence of Cas9 DNA was confirmed by qPCR, Cas9 expression was confirmed by RT-qPCR and found to be significantly higher than in vector control cells (Figure 5a). Exosomes were collected from these cells and identified by nanosight analysis (Figure 5b). Exosome DNA and RNA were extracted, and Cas9 levels in exosomes were detected by qPCR. Cas9 expression in BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 was found to be significantly higher than in BxPC-3 / CRISPR-Cas9-sgRab27b-2 C3 (Figure 5c). This was confirmed by detecting sgRNA for Rab27b-1 / 2 (Figure 5c, bottom). Cas9 and Rab27b protein levels in both cells and exosomes were evaluated by Western blotting using β-actin or CD9 as controls, respectively. Rab27b was found to be knocked down in cells and exosomes with guide RNA (Figure 5d). DNA editing was confirmed in both cells and exosomes using two different primer sets with the T7 / SURVEYOR assay (Figures 5e and 5f). DNA editing can be seen as dark bands in the areas enclosed by squares. 【0159】 The exosomal protein content of BxPC-3 / CRISPR-Cas9 vector control stable cells and clonally expanded BxPC-3 / CRISPR-Cas9-sgRab27b-1 C3 and BxPC-3 / CRISPR-Cas9-sgRab27b-2 C6 cells was evaluated by BCA (Figure 6a). Cell proliferation was evaluated by MTT assay. The presence of CRISPR-Cas9 did not have a negative impact on proliferation (Figure 6b). 【0160】 Transfection using IVT RNA. sgRab27b-1 / 2 was PCR amplified and purified (Figure 7a). Next, the purified PCR products of sgRab27-1 / 2 were in vitro transcribed as described above and run on a denaturing gel to analyze quality (Figure 7a, right). Cas9 was PCR amplified and purified (Figure 7b). The purified Cas9 PCR product was in vitro transcribed and detected by electrophoresis on a formaldehyde gel (Figure 7b). HEK293T / CRISPR-Cas9 vector control cells were treated with 1 μg of IVT-sgRab27b RNA for 72 hours using Lipofectamine 2000 (Figure 7c), Exo-Fect / exosome transfection reagent (Figure 7d), or electroporated exosomes (Figure 7e). Gene editing was confirmed in cells treated with RNA and Lipofectamine or exosome transfection reagent (Figure 7c and 7d), but not in exosomes. HEK293T cells and BxPC-3 cells were transfected with Cas9 mRNA using Lipofectamine ..... 【0161】 HEK293T and BxPC-3 transfection and gene editing. HEK293T cells were treated with 10 μg of plasmid (CRISPR-Cas9-lenchi-V2 vector control, CRISPR-Cas9-lenchi-V2-sgRab27b-1, CRISPR-Cas9-GFP vector control) four times every 24 hours (days 1, 2, 3, and 4) using Exo-Fect / exosome transfection reagent, and the transfection efficiency of Cas9-GFP transfected cells was examined (Figure 9a). Relative Cas9 expression levels and 1 / Ct values ​​were confirmed by qPCR (Figure 9b), and the presence of Cas9 was confirmed by Western blotting (Figure 9C). Gene editing was confirmed by T7 / SURVEYOR assay (Figure 9d). The same experiment was performed on BxPC-3 cells, but gene editing could not be detected by T7 / SURVEYOR assay (Figures 9e-9g). 【0162】 KPC689 transfection and gene editing. Lipofectamine 2000 was used to deliver 5 μg of plasmid (Lenti-V2, GFP, CRISPR-Cas9-sgmKras with puromycin backbone) to KPC689 cells. G12D Transfection was confirmed by transfecting cells (and vector control) for 48 hours and imaging them with a GFP backbone (Figure 10a). Relative Cas9 levels (Figure 10b) and mKras G12D The level (Figure 10c) was confirmed by qPCR, and gene editing in KPC689 cells was examined using the T7 / SURVEYOR assay, but no gene editing was found (Figure 10d). Similarly, using the Exo-Fect / exosome transfection reagent, KPC689 cells were transfected with 10 μg of plasmid (CRISPR-Cas9-sgmKras with a GFP backbone). G12D Cells were processed with the vector control and then transfected with GFP to confirm the transfection efficiency of the Exo-Fect / exosome transfection reagent (Figure 10e). Relative Cas9 expression levels (Figure 10f) and mKras G12DThe levels (Figure 10g) were determined by qPCR. A T7 / SURVEYOR assay was performed to obtain CRISPR-Cas9-GFP-mKras G12D Gene editing was examined in KPC689 cells after plasmid treatment, but no editing was found (Figure 10h). 【0163】 Treatment of cells containing exosomes with inducible plasmids. HEK293T cells were transfected with a packaging plasmid along with a CRISPR-Cas9 doxycycline-inducible plasmid using lipofectamine 2000. Lentivirus-containing medium was collected and then transfected into Panc1 cells. Transfected cells were selected with puromycin and maintained with doxycycline. Exosomes were collected from doxycycline-treated or untreated Panc1-inducible cells, and Cas9 protein levels in cells and exosomes were confirmed by Western blotting (Figures 11a and 11b). Panc1-inducible cells were treated with 2 μg of hKras using lipofectamine, Fugene, or Exo-Fect. G12D IVT-sgRNA, 1 μg of hKras G12D Gene editing in Panc1-inducible cells was examined by treating them with plasmids for 72 hours and performing a T7 / SURVEYOR assay. Editing was detected only in cells transfected with a guide RNA plasmid in lipofectamine (Figure 11c). Cas9 protein levels in Pacn1 Cas9-stable cells were confirmed by Western blotting (Figure 11d). Panc1 cells were examined using lipofectamine, Exo-Fect, or electroporated exosomes for CRISPR-Cas9-sghKras with lenti-V2, GFP, and a puromycin backbone. G12D In contrast to the treatment with sghKras, Panc1 Cas9-stabilized cells were treated with lipofectamine, Exo-Fect, or electropermeable exosomes as shown. G12DThe cells were treated with plasmids. A T7 / SURVEYOR assay was performed to examine gene editing in Panc1 cells and Panc1 Cas9 stable cells (Figure 11e). Gene editing was found in Panc1 cells transformed with a puromycin backbone Cas9, as indicated by the boxed areas, and in Panc1-Cas9 stable cell lines transfected with guide RNA using lipofectamine or Exo-Fect (Figure 11e). Panc1 sghKras G12D T1 stable cells were established using a lentivirus-based method. Panc1 sghKras G12D T1 stable cells were transfected with 10 μg or 20 μg of Cas9 plasmid containing GFP or puromycin backbone for 24 hours, and the T7 / SURVEYOR assay was performed. Panc1 sghKras G12D Gene editing was observed in stable T1 cells (Figure 11f). 【0164】 Treatment of induced tumors using exosomes and CRISPR-Cas9. KPC689 cells were subcutaneously transplanted into the backs of mice. The mice were divided into four groups and treated as follows (Figures 12a and 12b). Each group of mice received intravenous (IV) and intratumor (IT) injections daily for two weeks, and tumor volume was assessed (Figure 12a). Treatment with exosomes and transfection agents did not slow tumor growth, but treatment with exosomes, Cas9 and guide RNA plasmids, and transfection agents inhibited tumor growth throughout the treatment period and even after the treatment period (Figure 12a). Mouse body weight was also assessed. No treatment adversely affected body weight in any group (Figure 12b). 【0165】 All methods disclosed and claimed herein can be prepared and carried out without excessive experimentation, given the present disclosure. While the compositions and methods of the present invention have been described in preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods herein and to the steps or order of steps thereof without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related active substances can be used in place of the active substances described herein, and that the same or similar results can be obtained at the same time. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims. 【0166】 References The following references are incorporated herein by reference to the extent that they provide details of exemplary procedures or other details that supplement those described herein. TIFF0007872660000002.tif189151TIFF0007872660000003.tif230150TIFF00078726600 00004.tif231151TIFF0007872660000005.tif231150TIFF0007872660000006.tif196150

Claims

[Claim 1] A composition comprising an exosome, wherein the exosome contains CD47 on its surface, the exosome contains a CRISPR system, and the CRISPR system targets the Kras G12D mutation, which causes cancer. [Claim 2] The composition according to claim 1, wherein the CRISPR system comprises an endonuclease and a guide RNA (gRNA). [Claim 3] The composition according to claim 2, wherein the endonuclease is a Cas endonuclease. [Claim 4] The composition according to claim 2, wherein the endonuclease is Cpf1 endonuclease. [Claim 5] The composition according to claim 2, wherein the guide RNA comprises crRNA and tracrRNA. [Claim 6] The composition according to claim 2, wherein the endonuclease and the gRNA are encoded on a single nucleic acid molecule within the exosome. [Claim 7] The composition according to claim 2, wherein at least 90% of the exosomes contain an endonuclease and gRNA. [Claim 8] A pharmaceutical composition for use in a method of treating cancer in a patient in need, comprising an exosome according to any one of claims 1 to 7 and an excipient, wherein the method comprises the step of administering the composition to the patient. [Claim 9] The composition according to claim 8, formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [Claim 10] The composition according to claim 8, wherein the method further comprises the step of administering an antibacterial agent. [Claim 11] The composition according to claim 10, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, or thimerosal. [Claim 12] The composition according to claim 8, wherein the cancer is pancreatic ductal adenocarcinoma. [Claim 13] The composition according to claim 8, wherein the administration is systemic administration. [Claim 14] The composition according to claim 8, wherein the method further comprises the step of administering at least a second therapy to the patient, the second therapy comprising surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, or immunotherapy. [Claim 15] The composition according to claim 8, wherein the exosome is autologous to the patient.

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