Method for producing MSC-derived exosomes

Abstract

Provided herein is a method for producing clinical-grade exosomes derived from mesenchymal stem cells (MSCs). Further provided is a method for loading therapeutic agents, such as siRNA, into exosomes. Also provided herein is a method for treating diseases by administering clinical-grade exosomes. [Selected Figure] Figure 1C

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 587,408, filed Nov. 16, 2017, which is incorporated herein by reference in its entirety.

[0002] [Technical Field] The present invention generally relates to the fields of molecular biology and medicine. More particularly, the present invention relates to methods for the large-scale production of GMP (good manufacturing practice) compliant exosomes.

Background Art

[0003] Extracellular vesicles (EVs), including exosomes and microvesicles, are nano-sized intercellular communication mediators involved in several physiological processes. Specifically, exosomes are nano-sized vesicles released by cells and constitute a mode of intercellular exchange of cellular components and products, which has prompted new interest in their usefulness as therapeutic delivery agents. Unlike their artificial counterparts, the features of these naturally produced, specialized shuttle services between cells can provide unique advantages for the efficient delivery of therapeutic payloads. Such features of exosome production and regulatory mechanisms associated with exosome uptake in cells remain to be further studied. Nevertheless, the use of exosomes for the therapeutic management of diseases, including cancer, has already shown promising results.

[0004] Due to their biological properties, exosomes are promising candidates for the treatment of immune disorders and for the systemic delivery of therapeutic compounds (e.g., cytokines, chemotherapeutic agents, nucleic acids, and viral vectors). However, their low production yields limit their potential clinical use. Thus, there remains an unmet need for an efficient method for producing exosomes that can be used in therapy.

Summary of the Invention

[0005] In a first embodiment, a method is provided for producing exosomes from MSCs, comprising: culturing mesenchymal stem cells (MSCs) in a medium containing human platelet lysates (PLT) in a functionally closed bioreactor until confluence (e.g., 75-95% or 80-90% confluence, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% confluence); further culturing the cells in a medium that is essentially free of PLT (e.g., PLT-free); collecting a conditioned medium fraction from the bioreactor; and isolating exosomes from the conditioned medium fraction.

[0006] In some embodiments, each conditioned medium fraction is stored at -80°C after collection. In certain embodiments, the conditioned medium fractions are thawed and pooled before isolation.

[0007] In certain embodiments, MSCs are further defined as bone marrow-derived MSCs. In certain embodiments, MSCs are further defined as adipose-derived MSCs.

[0008] In a further embodiment, the method involves adding at least 1 × 10⁶ units to the bioreactor before culturing the MSCs in the bioreactor. 7 This further includes sowing MSCs. In some embodiments, the MSCs are approximately 400-500 cells / cm². 2 It is sown at this concentration.

[0009] In certain embodiments, the sealed bioreactor is a hollow fiber bioreactor. In some embodiments, the hollow fiber bioreactor is a Terumo cell proliferation system.

[0010] In some embodiments, PLT is present at a concentration of 5% in the medium of the MSC culture. The concentration of PLT may be about 2–10%, for example, 3, 4, 5, or 6%. In certain embodiments, fresh medium is continuously added to the MSCs in the bioreactor. In certain embodiments, the cells are cultured in 5% oxygen. In some embodiments, the culture in step (a) is 5–10 days (for example, 6, 7, 8, or 9 days). In certain embodiments, the culture in step (a) is 8 days.

[0011] In certain embodiments, MSCs are cultured to 85%–90% confluence, e.g., 85%, 86%, 87%, 88%, 89%, or 90% confluence. Confluence can be measured by monitoring glucose and lactose levels. For example, lactose levels may be approximately 2–6 mmol / L, e.g., approximately 2, 3, 4, 5, or 6 mmol / L, and glucose levels may be approximately 80–140 mg / dL, e.g., approximately 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 mg / dL.

[0012] In some embodiments, the culture in a medium that is essentially PLT-free is for 24–72 hours, e.g., 24–48, 36–50, 48–60, or 50–72 hours, approximately 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 hours. In certain embodiments, the culture is in a medium that is essentially PLT-free and is for approximately 48 hours. In certain embodiments, the medium for the further culture step is PLT-free.

[0013] In certain embodiments, MSCs are washed between culture in PLT and culture in a medium that is essentially PLT-free or does not contain PLT. In some embodiments, MSCs are cultured in serum-free standard medium. In certain embodiments, the complete method is carried out under serum-free conditions.

[0014] In certain embodiments, the conditioned media fraction is collected in a sealed bag. In certain embodiments, the conditioned media fraction is collected every 24 to 72 hours. In some embodiments, the conditioned media fraction is collected every 40 to 50 hours. In one particular embodiment, the conditioned media fraction is collected every 48 hours.

[0015] In some embodiments, the conditioned media fraction is a volume of 200 - 300 mL, such as 200 - 250 or 250 - 300 mL. In certain embodiments, the conditioned media fraction is collected for 10 - 14 days (e.g., 10, 11, 12, 13, or 14 days). In a particular embodiment, the conditioned media fraction is collected for 12 days. In a particular embodiment, at least five conditioned media fractions are collected. In certain embodiments, this method is performed in less than three weeks.

[0016] In some embodiments, each conditioned media fraction contains exosomes from 9×10 11 to 50×10 11 In certain embodiments, at least 10×10 12 exosomes are isolated in the collected media fraction. In certain embodiments, at least 15×10 12 exosomes are isolated in the collected media fraction. At least 10×10 11 , 15×10 11 , 20×10 11 , 25×10 11 , 30×10 11 , 35×10 11 , 40×10 11 , 45×10 11 , 50×10 11 , 60×10 11 , 70×10 11 , 80×10 11 , 90×10 11 , 10×10 12 , 15×10 12 , 20×10 12 , or 25×10 12 exosomes can be isolated.

[0017] In certain embodiments, isolation involves filtering and ultracentrifugation of the pooled fraction to obtain an exosome-containing pellet, and then resuspending the exosome-containing pellet in a buffer. In some embodiments, isolation is performed in a functionally closed manner using a pump and heat-sealed tubing. In some embodiments, filtration is further defined as passing the pooled fraction through a filter such as a 0.2 μm filter. In further embodiments, isolation further includes a centrifugation step prior to filtration to remove large cell debris. In certain embodiments, isolation is performed at 4°C.

[0018] In certain embodiments, the buffer solution comprises about 0.01–0.1 M, e.g., about 0.08, 0.09, or 0.1 M, particularly about 0.1–0.5 M, e.g., about 0.1, 0.2, or 0.3 M of sodium chloride; about 0.1–0.5 M, e.g., about 0.1, 0.2, or 0.3 M, particularly about 0.23 M of sodium gluconate; about 0.1–0.5 M, e.g., 0.1, 0.2, or 0.3 M, particularly about 0.27 M of sodium acetate trihydrate; 1–10 mM, e.g., about 6–7 mM, particularly about 5 mM of potassium chloride; and about 1–5 mM, e.g., about 2–4 mM, particularly about 3 mM of magnesium chloride. In some embodiments, the buffer solution has a pH of approximately 6 to 8, for example, approximately 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, particularly approximately 7.4. In certain embodiments, the buffer solution is PLASMALYTE-A®.

[0019] In a further embodiment, the method further comprises the step of loading a therapeutic agent onto exosomes. In some embodiments, the therapeutic agent comprises one or more cytokines, chemotherapeutic agents, nucleic acids, small molecules, or proteins. In certain embodiments, the nucleic acid comprises DNA and / or RNA. In some embodiments, the RNA is siRNA, miRNA, or shRNA. In certain embodiments, the RNA is siRNA. In some embodiments, loading comprises electroporation of the exosomes. In certain embodiments, electroporation is performed in PLASMALYTE-A®. In certain embodiments, the method does not include a step of washing the exosomes or a step of exchanging buffers between the step of exosome isolation and the step of electroporation. In particular, the number of exosomes from the step of exosome isolation to the number of loaded exosomes does not decrease by more than 20%.

[0020] In another embodiment, a pharmaceutical composition is provided comprising exosomes produced by the method of the embodiment (e.g., culturing MSCs in a functionally closed bioreactor to 80-90% confluence in a medium containing human platelet lysates (PLT); further culturing the cells in a medium that is essentially free of PLT (e.g., PLT-free); collecting a conditioned medium fraction from the bioreactor; and isolating exosomes from the conditioned medium fraction).

[0021] In yet another embodiment, a method for treating cancer is provided, which includes administering an effective amount of exosomes produced according to the method of the embodiment (e.g., culturing MSCs in a functionally closed bioreactor in a medium containing human platelet lysates (PLT) to 80-90% confluence; further culturing the cells in a medium essentially free of PLT (e.g., PLT-free); collecting a conditioned medium fraction from the bioreactor; and isolating exosomes from the conditioned medium fraction). In some embodiments, the subject is human.

[0022] In certain embodiments, electroporated exosomes are directly injected into a subject. In additional embodiments, the method further includes administering at least a second anticancer therapy. In some embodiments, at least a second anticancer therapy includes chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

[0023] In another embodiment, a method for delivering RNA to cells is provided, comprising the steps of: culturing MSCs in a functionally closed bioreactor in a medium containing human platelet lysates (PLT) to 80-90% confluence; further culturing the cells in a medium that is essentially PLT-free (e.g., PLT-free); collecting a conditioned medium fraction from the bioreactor; isolating exosomes from the conditioned medium fraction; and loading RNA onto the isolated exosomes to produce RNA-loaded exosomes). In some embodiments, the cells are human cells. In particular, the cells are cancer cells or T cells.

[0024] In further embodiments, a method is provided for treating a disease or disorder in a subject requiring it, which includes administering an effective amount of exosomes produced by the method of this embodiment (e.g., culturing MSCs in a functionally closed bioreactor to 80-90% confluence in a medium containing human platelet lysates (PLT); further culturing the cells in a medium essentially free of PLT (e.g., PLT-free); collecting a conditioned medium fraction from the bioreactor; and isolating exosomes from the conditioned medium fraction). In some embodiments, the exosomes are loaded with siRNA or miRNA. In certain embodiments, the exosomes are loaded with KRAS siRNA. In some embodiments, the subject is human.

[0025] In some embodiments, the disease or disorder is cancer, an inflammatory disorder, or an immune-related disorder. In certain embodiments, the cancer is lung cancer.

[0026] In certain embodiments, exosomes are administered orally, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, topically, or by direct injection. In some embodiments, exosomes are administered intravenously.

[0027] In additional embodiments, the method further comprises administering at least a second therapeutic agent. In some embodiments, at least the second therapeutic agent is an anticancer agent. In certain embodiments, the anticancer agent is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

[0028] In another embodiment, a method is provided for treating an immune-mediated inflammatory disease in a subject suffering from the disease, comprising administering to the subject a therapeutically effective amount of MSC-derived exosomes produced according to the present method (e.g., culturing MSCs in a functionally closed bioreactor to 80-90% confluence in a medium containing human platelet lysates (PLT); further culturing the cells in a medium essentially free of PLT (e.g., PLT-free); collecting a conditioned medium fraction from the bioreactor; and isolating exosomes from the conditioned medium fraction). In some embodiments, the immune-mediated inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and Crohn's disease. In certain embodiments, the MSCs are allogeneic. In some embodiments, the exosomes are administered systemically or topically. In certain embodiments, exosomes are administered via rectal, intranasal, oral, vaginal, subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intra-articular, synovial, intrasternal, intrathecal, intralesional, or intracranial pathways, or via implantable reservoirs. In some embodiments, exosomes are administered together with at least one further therapeutic agent.

[0029] Other objects, features, and advantages of the present invention will become apparent from the detailed description below. However, the detailed description, specific examples, and preferred examples of the present invention are illustrative, and it should be understood that various changes and modifications that will become apparent to those skilled in the art through this detailed description are within the spirit and scope of the invention. [Brief explanation of the drawing]

[0030] The following drawings are included to illustrate portions of this specification and to further illustrate certain aspects of the invention. The invention will be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

[0031] [Figure 1] Figures 1A-1D: (Figure 1A) Schematic procedure designed to produce extracellular organisms (EVs) from mesenchymal stem cells (MSCs) using the Turmo Cell Expansion System (Bioreactor). (Figure 1B) Schematic diagram detailing the procedure for culturing MSCs in a bioreactor for exosome collection. (Figure 1C) Schematic diagram of the procedure for isolating exosomes from MSC conditioned medium and electroporating them. (Figure 1D) Schematic diagram showing the generation of MSC-derived exosomes.

[0032] [Figure 2] Figure 2 is a schematic diagram of a strategy for producing acclimatized media containing EVs from MSCs cultured on a bioreactor.

[0033] [Figure 3] Figure 3: Schematic diagram of the strategy for isolating EVs from MSC-conditioned medium cultured on a bioreactor.

[0034] [Figure 4]Figures 4A-4B: Identification of MSC-derived exosomes produced in the bioreactor. (Figure 4A) Flow cytometry of MSC-derived exosomes produced in the bioreactor at each collection time, showing the expression of exosome markers. (Figure 4B) Representative TEM from each collection, showing the typical morphology of exosomes.

[0035] [Figure 5] Figures 5A-5E: Quantification of exosomes produced in the bioreactor at each harvest time. (Figure 5A) Number of MSC-derived exosomes produced at quantum level as determined by microBCA and NanoSight. (Figure 5B) Particle size distribution for each harvest measure using NanoSight. (Figure 5C) Glucose and lactose levels in the bioreactor during exosome production. (Figure 5D) Number of exosomes produced per cell, isolated from MSC-conditioned medium and quantified by NanoSight at different time points. (Figure 5E) Representative flow cytometry of exosome markers on MSC-derived exosomes isolated at 24 and 48 hours.

[0036] [Figure 6] Figures 6A-6B: Quantification of MSC-derived exosomes produced in media supplemented with human platelet lysate (hPLT) or serum-free conditions. (Figure 6A) Number of exosomes per cell isolated from conditioned medium of MSCs cultured with or without hPLT, analyzed by NanoSight. (Figure 6B) Flow cytometry of MSC-derived exosomes produced using medium alone versus medium supplemented with human platelet lysate (hPLT), showing the purity of exosomes produced on serum-free medium.

[0037] [Figure 7]Figure 7: Evaluation of MSC-derived exosome electroporation using 16 different nucleofactor programs and three different nucleofactor solutions. The efficiency of electroporation was evaluated 48 hours later by apoptosis induced by siRNA delivery by MSC-derived exosomes in recipient cells.

[0038] [Figure 8] Figures 8A-8C: Evaluation of MSC-derived exosome electroporation using five different solutions. (Figure 8A) The efficiency of electroporation was evaluated by apoptosis induced by siRNA delivery by MSC-derived exosomes on recipient cells after 48 hours. (Figure 8B) Representative transmission electron micrographs of MSC exosomes after electroporation using either research buffer (RB) or clinical buffer (CB; i.e., platelet lysate (PLT)), demonstrating the maintenance of exosome integrity after electroporation. (Figure 8C) Silencing of gene transcription in recipient cells induced by siRNA delivery using MSC-derived exosomes electroporated using a Lonza apparatus and PLASMALYTE-A® solution.

[0039] [Figure 9] Figure 9: Number of exosomes before and after ultracentrifugation. Electroporation of exosomes using research buffers includes a second washing step, which can result in the loss of at least 50% of the sample.

[0040] [Figure 10] Figures 10A-10D: In vivo distribution of pre-labeled MSC-derived exosomes produced in a bioreactor and injected into mice. Fluorescence of DIR-labeled MSC exosomes 6 hours after intraperitoneal (Figures 10A, 10B) or intravenous (Figures 10C, 10D) administration of 8 × 10⁹ labeled exosomes in WT nude mice. (A, C) Dissected organs. (B, D) Dissected organs without spleen and liver. [Modes for carrying out the invention]

[0041] Formulating exosomes for human therapeutics requires careful consideration, including large-scale production of exosomes in accordance with GMP (good manufacturing practice) standards. Therefore, this study concerns innovative procedures and related systematic analyses for generating GMP-compliant exosomes.

[0042] Therefore, in certain embodiments, this disclosure provides an efficient and clinically relevant strategy for producing extracellular organisms (EVs), such as exosomes, from mesenchymal stromal cells (MSCs) using a functionally closed bioreactor, such as the Terumo Cell Growth System. The method provided herein produces at least 10 × 10¹⁶ EVs in a short period of time, such as about 3 weeks. 12 This method can produce extracellular viable cells (EVs). The present invention involves large-scale production and isolation of clinical-grade EVs in a functionally closed, serum-free system. EVs are produced from bone marrow-derived mesocellular cells (MSCs) or the like in clinically appropriate doses.

[0043] In preferred embodiments, the entire method is serum-free, and therefore, contamination of MSC-derived EVs from exosomes in serum is essentially absent. Specifically, this method may involve the use of human platelet lysate (PLT) in the initial culture of MSCs in a bioreactor. The inventors have found that efficient EV production is obtained when MSCs are cultured to about 80–90%, for example, about 85% confluence. Therefore, the MSCs may then be switched to a PLT-free medium for about 24–72 hours (e.g., about 24 or 48 hours) before collection of the acclimatized medium containing EVs. The acclimatized medium fraction may be collected every 48 hours and frozen at about -80°C until EV isolation. The acclimatized medium fraction can be collected about 4–10 times, for example, about 5, 6, 7, or 8 times, and especially about 6 times. Therefore, the period from seeding of MSCs in the bioreactor to final collection may be about 15–30 days, for example, about 20 days. Each conditioned medium fraction contains at least 1 × 1011 exosomes, for example, at least 9 × 10 11 , especially about 3 x 10 12 It can contain exosomes.

[0044] Preferably, the collected culture medium fraction is thawed and pooled before EV isolation. Exosome isolation may involve an initial centrifugation step (e.g., 1,000 g), followed by ultracentrifugation (e.g., about 100,000 g). Multiple rounds of ultracentrifugation, such as three rounds, may be performed to generate an exosome pellet. In some embodiments, the exosome pellet is resuspended in a GMP-compliant buffer such as PLASMALYTE-A®. The exosomes may be further subjected to filtration, such as through a 0.2 μm filter, before ultracentrifugation. This method is suitable for 9-10 × 10⁻⁶ cells. 12 or more exosomes, for example, 15 x 10 12 This can lead to the generation of total exosomes.

[0045] Furthermore, exosomes can be loaded with therapeutic agents (e.g., cytokines, chemotherapeutic agents, or nucleic acids). Therefore, a method is provided for loading exosomes, such as those produced by this method, by electroporation in an FDA-approved buffer such as PLASMALYTE-A®. Electroporation can be performed using a flow-through electroporation system (e.g., the 4D-Nucleofactor LV Large Scale Transfection System, Lonza). Each electroporation run should consist of at least 2 × 10⁻⁶ units. 12 It can contain exosomes. Exosomes can carry nucleic acids such as siRNA. The buffer is FDA approved for patient use, sterile, and non-pyrogenic, and can be injected directly into the patient, so there is no need for a washing step to change the buffer before administration to the patient. Therefore, there is no exosome loss from an additional washing step, which can result in the loss of approximately 50% of exosomes in conventional methods. This buffer can maintain the integrity of exosomes after electroporation.

[0046] Furthermore, this specification provides for the use of exosomes, such as siRNA-equipped exosomes, for the treatment of immune-related diseases and diseases such as cancer in patients.

[0047] [I.Definition] As used herein, “essentially absent” with respect to a particular component is used herein to mean that none of the particular component is intentionally included in the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from unintended contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is one in which the amount of the particular component cannot be detected by standard analytical methods.

[0048] In this context, "a" or "an" may mean one or multiple people, but in the context of claims, when used in conjunction with "includes," "a" or "an" may mean one or multiple people.

[0049] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated that it refers only to substitutes; however, this disclosure supports the definition that refers only to substitutes and “and / or”; however, as used herein, “another” may mean at least the latter or more; and the terms “about,” “substantially,” and “almost” generally mean plus or minus 5% of the stated value.

[0050] "Extracellular vesicles" and "EVs" are, as a class, cell-derived and cell-secreted microvesicles, including exosomes, exosome-like vesicles, ectosomes (resulting in vesicle budding directly from the cell membrane), microparticles, microvesicles, shedding microvesicles (SMVs), nanoparticles, and even (large) apoptotic vesicles or bodies (resulting in cell death) or membrane particles.

[0051] As used herein, the terms “microvesicle” and “MV” refer to larger extracellular vesicles or structures surrounded by a phospholipid bilayer, typically having a diameter of approximately 100 nm to 1,000 nm, or approximately 100 nm to 400 nm in plasma. Microvesicles / MVs are formed by release regulated by cell membrane budding or blebbing.

[0052] Within the class of extracellular vesicles, the important component is the “exosome” itself, which is a membranous vesicle (i.e., a vesicle surrounded by an endocytic phospholipid bilayer, arising from exocytic fusion or “exocytosis” of the multicellular endoplasmic reticulum (MVB)). Exosomes may be isolated from any suitable biological sample of mammalian origin, including, but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascites, bone marrow, and cultured mammalian cells (e.g., immature dendritic cells (wild-type or immortalized), induced and uninduced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and brown adipose tissue progenitor cells, etc.). As those skilled in the art will understand, the cultured cell sample is in a culture medium suitable for the cells (using serum that does not contain exosomes). Exosomes have surface markers such as tetraspanins, e.g., CD9, CD37, CD44, CD53, CD63, CD81, CD82, and CD151; Targeting markers or membrane fusion markers such as integrins, ICAM-1, EpCAM, Annexin, TSG101, and ALIX; and other exosomal transmembrane proteins such as Rab5b, HSP70, LAMP2 (lysosome-associated membrane protein), and LIMP (lysosomal integral membrane protein). It includes specific surface markers not found in other vesicles.

[0053] The term “mesenchymal stem cell” or “MSC,” as used herein, refers to pluripotent somatic stem cells derived from the mesoderm that possess the ability to regenerate and differentiate into progeny cells with great phenotypic diversity, particularly including connective tissue, bone marrow stroma, adipocytes, dermis, and muscle. MSCs generally have a cellular marker expression profile characterized by being negative for markers CD19, CD45, CD14, and HLA-DR, and positive for markers CD105, CD106, CD90, and CD73. MSCs can be isolated from any type of tissue. Generally, MSCs can be isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. In certain embodiments, MSCs are bone marrow-derived stem cells.

[0054] MSCs from which exosomes originate can be autosomal, homogeneous, or heterogeneous. As used herein, the term “autosomal” means that the donor of the MSC and the recipient of the exosomes (or isolated exosome population) derived from the MSC are of the same subject. The term “homogeneous” means that the donor of the MSC and the recipient of the exosomes (or isolated exosome population) derived from the MSC are of different subjects. The term “heterogeneous” means that the donor of the MSC and the recipient of the exosomes (or isolated exosome population) derived from the MSC are of different species. In certain embodiments, MSCs from which exosomes originate are homogeneous.

[0055] The term “adipose tissue-derived stem cells” or “ASC” as used herein refers to MSCs derived from adipose tissue. ASCs can be isolated from adipose tissue by methods known in the art (e.g., methods described below under “Isolation and Proliferation of Human Adipose-Derived Mesenchymal Stem Cells”). “Adipose tissue” means any adipose tissue. Adipose tissue may be brown or white adipose tissue derived from, for example, subcutaneous, omentum / visceral, mammary gland, gonad, periorgan, or other adipose tissue sites. Preferably, the adipose tissue is subcutaneous white adipose tissue. Adipose tissue may include primary cell cultures or immortalized cell lines. Adipose tissue may be derived from any organism that has adipose tissue. In some embodiments, the adipose tissue is mammalian, and in further embodiments, the adipose tissue is human. A convenient source of adipose tissue is liposuction. However, it will be understood that neither the source of adipose tissue nor the method of isolating adipose tissue is important to the present invention. In certain embodiments, ASCs are isolated from the liposuction product of the subject.

[0056] MSCs can be derived from any animal, preferably mammals including non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, or mice) and primates (e.g., monkeys or humans). In certain embodiments, the MSC is human.

[0057] The term "functionally closed" refers to a system that is sealed to ensure the sterility of a fluid, either by hermetically sealing the entire system or by providing sterile barrier filters at all connections to the collection system.

[0058] The term “bioreactor” refers to a large-scale cell culture system that provides nutrients to cells, removes metabolites, and provides a physiologically and chemically regulated environment for cell proliferation in a closed, sterile system. In certain embodiments, the biological and / or biochemical processes occur under monitored and controlled environmental and operational conditions (e.g., pH, temperature, pressure, nutrient supply, and waste removal). According to this disclosure, a basic class of bioreactors suitable for use with the method includes hollow fiber bioreactors.

[0059] The term “hollow fiber” is intended to include hollow structures (of any shape) containing pores of a specified size, shape, and density for use in delivering nutrients (in solution) to cells contained within a bioreactor and for removing waste matter (in solution) from cells contained within a bioreactor. For the purposes of this disclosure, hollow fibers may be constructed from reabsorbable or non-reabsorbable materials. Fibers include, but are not limited to, tubular structures.

[0060] As used herein, the terms “patient” or “subject” refer to living mammalian organisms, such as humans, monkeys, cattle, sheep, goats, dogs, cats, mice, rats, guinea pigs, or their transgenic species. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients include adults, adolescents, infants, and fetuses.

[0061] "Treatment" or "to treat" includes (1) inhibiting the disease in an object or patient experiencing or displaying the pathology or symptoms of the disease (e.g., halting further development of the pathology and / or symptoms), (2) improving the disease in an object or patient experiencing or displaying the pathology or symptoms of the disease (e.g., reversing the pathology and / or symptoms), and / or (3) resulting in any measurable reduction of the disease in an object or patient experiencing or displaying the pathology or symptoms of the disease.

[0062] Where the term “effective” is used herein and / or in the claims, it means sufficient to achieve a desired, expected, or intended result. Where “effective dose,” “therapeutic effective dose,” or “pharmaceutical effective dose” is used in the context of treating a patient or subject with the compound, it means an amount of the compound that, when administered to a subject or patient to treat or prevent a disease, would have an effect of treating or preventing such disease.

[0063] As used herein, the term “cancer” may be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer may originate from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus.

[0064] The terms “contacted” and “exposed,” when applied to cells, are used herein to describe the process by which a therapeutic agent is delivered to or directly juxtaposed with target cells. To achieve cell toxicity, for example, one or more agents are delivered to cells in amounts effective to kill the cells or prevent them from dividing.

[0065] Patient response, or patient “responsiveness,” refers to the clinical or therapeutic benefit given to a patient who is at risk of or suffering from a disease or disorder. Such benefits include cellular or biological responses, complete responses, partial responses, stable disease (without progression or relapse), or responses with subsequent relapses. For example, an effective response may be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer.

[0066] As used herein, “therapeutic agent” refers to any agent that can be administered to a subject for the purpose of obtaining therapeutic benefits for a disease or health-related condition. For example, nanoparticles containing a therapeutic agent may be administered to a subject for the purpose of reducing tumor size, reducing or inhibiting the local invasiveness of a tumor, or reducing the risk of metastasis.

[0067] As used herein, “diagnostic agent” refers to any drug that may be administered to a subject for the purpose of diagnosing a disease or health-related condition in that subject. Diagnosis includes determining whether a disease is present, whether the disease is progressing, or whether there is any change in the condition.

[0068] The therapeutic or diagnostic agent may be a small molecule, peptide, protein, polypeptide, antibody, antibody fragment, DNA, or RNA. In certain embodiments, the therapeutic or diagnostic agent is siRNA.

[0069] As used herein, “nucleic acid” generally refers to a molecule (i.e., a strand) of DNA, RNA, or its derivatives or analogues that contains nucleic acid bases. Nucleic acid bases include, for example, naturally occurring purine or pyrimidine bases found in DNA (e.g., adenine “A”, guanine “g”, thymine “T”, or cytosine “C”) or naturally occurring purine or pyrimidine bases found in RNA (e.g., A, G, uracil “U”, or C). The term “nucleic acid” encompasses “oligonucleotide” and “polynucleotide,” which are subtypes of the term “nucleic acid.” The term “oligonucleotide” refers to a molecule with a length of 3 to about 100 nucleic acid bases. The term “polynucleotide” refers to at least one molecule with a length greater than about 100 nucleic acid bases.

[0070] These definitions refer to single-stranded or double-stranded nucleic acid molecules. Double-stranded nucleic acids are formed by fully complementary bonds, but in some embodiments, double-stranded nucleic acids may be formed by partially or substantially complementary bonds. Thus, nucleic acids can encompass double-stranded molecules that typically contain one or more complementary strands or “complements” of a particular sequence, including molecules. As used herein, single-stranded nucleic acids may be denoted by the prefix “ss”, and double-stranded nucleic acids by the prefix “ds”.

[0071] As used herein, "nucleotide" refers to a nucleoside that further includes a "backbone." The backbone generally covalently links a nucleotide to another molecule containing a nucleotide, or to another nucleotide that forms a nucleic acid. In naturally occurring nucleotides, the "backbone" typically includes a phosphorus moiety covalently attached to a pentose sugar. The bond of the backbone typically occurs at either the 3′- or 5′-position of the pentose sugar. However, other types of bondage are known in the art, particularly when the nucleotide includes a derivative or analogue of a naturally occurring pentose sugar or phosphorus moiety.

[0072] Nucleic acids may include, or may consist entirely of, derivatives or analogues of nucleic acid bases, nucleic acid base linker moieties, and / or skeletal moieties that may be present in naturally occurring nucleic acids. As used herein, “derivative” refers to a chemically modified or altered form of a naturally occurring molecule, and the terms “mimic” or “analog” refer to a molecule that may or may not be structurally similar to a naturally occurring molecule or moiety, but has similar function. As used herein, “moiety” generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure. Nucleic acid bases, nucleosides, and nucleotide analogs or derivatives are well known in the art.

[0073] The term "siRNA" (short interfering RNA) refers to a short double-stranded RNA complex (typically 19–28 base pairs long). In other words, siRNA is a double-stranded nucleic acid molecule containing two nucleotide strands, each having approximately 19–28 nucleotides (i.e., approximately 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). This complex often includes a 3′-overhang. siRNA can be prepared using techniques known to those skilled in the art, and a wide variety of siRNAs are commercially available from suppliers such as Integrated DNA Technologies, Inc. (Coralville, Iowa). In one embodiment, the 2′-O-methyl modified siRNA double helix for TNF-α described herein can be incorporated into nanoparticles, where the 2′-O-methyl modification on the antisense strand eliminates off-target effects, minimizes nonspecific immune responses, and improves siRNA stability.

[0074] MicroRNAs (miRNAs) are short non-coding RNAs that can target and effectively silence protein-coding genes via a 3′-UTR element. miRNAs are approximately 21–22 nucleotides long and arise from longer precursors transcribed from non-protein-coding genes.

[0075] "Immune disorders," "immune-related disorders," or "immune-mediated disorders" refer to disorders in which the immune response plays a role in the onset or progression of a disease. Immune-mediated diseases include autoimmune diseases, allograft rejection, graft-versus-host diseases, and inflammatory and allergic conditions.

[0076] An "autoimmune disease" is a disease in which the immune system produces an immune response (e.g., a B-cell or T-cell response) to an antigen that is part of a normal host (i.e., an autoantigen), resulting in tissue damage. Autoantigens can originate from host cells or from symbiotic organisms such as microorganisms that normally colonize mucosal surfaces (known as symbionts).

[0077] As used herein, the term “confluence” refers to the percentage of cells covering a surface such as a hollow fiber in a bioreactor. Confluence may be measured by the levels of glucose and / or lactose in the culture medium in which the cells are cultured.

[0078] [II. Exosome Production] Specific embodiments of this disclosure relate to methods for the large-scale production of clinical-grade extracellular molecules, particularly exosomes, by using bioreactors, especially functionally closed systems such as hollow fiber bioreactors. Importantly, the entire process of exosome production, including subsequent loading of therapeutic agents, can be serum-free.

[0079] (A. Mesenchymal stem cells) The cells for EV production may be MSCs, such as adipose-derived or bone marrow-derived MSCs. In certain embodiments, the MSCs are human MSCs, which may be autologous or allogeneic.

[0080] MSCs have approximately 100-1,000 cells / cm² 2 For example, about 150 cells / cm² 2 , about 200 cells / cm 2 , about 250 cells / cm 2 , about 300 cells / cm 2 For example, approximately 350 cells / cm² 2 For example, approximately 400 cells / cm² 2 For example, approximately 450 cells / cm² 2 For example, approximately 500 cells / cm² 2 For example, approximately 550 cells / cm² 2 For example, approximately 600 cells / cm² 2 For example, approximately 650 cells / cm² 2 For example, approximately 700 cells / cm² 2 For example, approximately 750 cells / cm² 2 For example, approximately 800 cells / cm² 2 For example, approximately 850 cells / cm² 2 For example, approximately 900 cells / cm² 2 For example, approximately 950 cells / cm² 2 , or approximately 1,000 cells / cm² 2The cells can be seeded into the bioreactor at a density of approximately 400-500 cells / cm³. 2 For example, approximately 450 cells / cm² 2 It can be seeded at this cell density.

[0081] The total number of cells seeded in the bioreactor is approximately 1.0 × 10⁻⁶. 6 ~Approx. 1.0×10 8 Cells, for example, about 1.0 × 10 6 ~5.0.0×10 6 , 5.0×10 6 ~1.0×10 7 , 1.0 × 10 7 ~5.0×10 7 , 5.0×10 7 ~1.0×10 8 These can be cells. In certain embodiments, the total number of cells seeded in the bioreactor is approximately 1.0 × 10⁶ 7 ~Approx. 3.0×10 7 For example, approximately 2.0 × 10 7 It is a cell.

[0082] Cells can be seeded in any suitable cell culture medium, many of which are commercially available. Exemplary media include DMEM, RPMI, MEM, Media 199, HAMS, etc. In one embodiment, the medium is αMEM medium, particularly αMEM supplemented with L-glutamine. The medium may be supplemented with growth factors, cytokines, hormones, or one or more of B27, antibiotics, vitamins, and / or small molecule drugs. In particular, the medium may be serum-free.

[0083] In some embodiments, cells can be incubated at room temperature. The incubator may be heated and have an atmosphere of about 5% CO2 and about 1% O2. In some embodiments, the CO2 concentration may be about 1-20%, 2-10%, or 3-5%. In some embodiments, the O2 concentration may be about 1-20%, 2-10%, or 3-5%.

[0084] In certain embodiments, cells are seeded and cultured in serum-free medium. The medium may be supplemented with platelet lysates, particularly human platelet lysates (PLT). PLT may be present in the medium at concentrations of about 1–10%, for example, about 1–4%, 2–5%, 3–6%, 4–7%, 5–8%, or 6–10%, for example, about 4%, 5%, or 6%, particularly about 5%.

[0085] MSCs can be initially cultured in a bioreactor for approximately 5–10 days after seeding, for example, approximately 5, 6, 7, 8, 9, or 10 days, and especially approximately 7, 8, or 9 days. Specifically, MSCs can be initially cultured in a medium containing PLT until they reach approximately 75–95% confluence, for example, approximately 75–80%, 80–85%, 85–90%, or 90–95% confluence, and especially until they reach approximately 85–90%, for example, 85%, 86%, 87%, 88%, 89%, or 90% confluence. Once the cells reach the intended confluence, they can be transferred to a medium without PLT. Before transferring the cells to a medium without PLT, they can be washed at least once with a buffer such as PBS.

[0086] Culturing cells in a medium that does not contain PLT is used to prevent contamination or dilution of MSC-derived exosomes by exosomes that may be present in the PLT. In some embodiments, the PLT can be centrifuged to remove exosomes and obtain exosome-free PLT.

[0087] Cells can be cultured in a PLT-free medium for approximately 8–100 hours, for example, 12–72 hours, for example, approximately 12–15, 15–20, 20–25, 25–30, 35–40, 40–45, 45–50, 50–55, 55–60, 65–70, or 70–72 hours. In particular, cells can be cultured in a PLT-free medium for approximately 24–48 hours, for example, approximately 48 hours, before exosome collection.

[0088] (B. Bioreactor) Bioreactors can be classified according to general categories, including static bioreactors, stirred flask bioreactors, rotating wall vessel bioreactors, hollow fiber bioreactors, and direct perfusion bioreactors. Within the bioreactor, cells may be free or immobilized and seeded on a porous three-dimensional scaffold (hydrogel).

[0089] Hollow fiber bioreactors can be used to facilitate mass transfer during cell culture. A hollow fiber bioreactor is a 3D cell culture system based on hollow fibers, which are small, semipermeable capillary membranes arranged in parallel within a typical molecular weight cutoff (MWCO) range of 10–30 kDa. These hollow fiber membranes are often bundled and housed within a tubular polycarbonate shell to form a hollow fiber bioreactor cartridge. The cartridge contains two compartments: an intracapillary (IC) space within the hollow fibers and an extracapillary (EC) space surrounding the hollow fibers, both of which are fitted with inlet and outlet ports.

[0090] Thus, in this disclosure, the bioreactor may be a hollow fiber bioreactor. In a hollow fiber bioreactor, cells are embedded in the lumen of the fiber and a culture medium can perfuse the extraluminal space, or alternatively, gas and culture medium perfusion is provided through the hollow fiber and cells can grow in the extraluminal space. Hollow fiber bioreactors suitable for this disclosure are known in the art and may include, but are not limited to, the Cardian(Terumo)BCT Quantum Cell Expansion System.

[0091] Hollow fibers should be suitable for nutrient delivery and waste removal in bioreactors. Hollow fibers may be of any shape, for example, circular and tubular, or in the form of concentric rings. Hollow fibers may consist of absorbent or non-absorbent membranes. Suitable components of hollow fibers, for example, include polydioxanone, polylactide, polyglucin, polyglycolic acid, polylactic acid, polyglycolic acid / trimethylene carbonate, cellulose, methylcellulose, cellulose polymers, cellulose esters, regenerated cellulose, Pluronic®, collagen, elastin, and mixtures thereof.

[0092] The bioreactor may be primed before seeding cells. Priming may include flushing with a buffer such as PBS. Priming may also include coating the bioreactor with an extracellular matrix protein such as fibronectin. The bioreactor may then be washed with PLT medium (e.g., αMEM).

[0093] (C. Collection of acclimatization medium) Acclimatized culture medium from cells cultured in PLT-free medium can be collected every 8 to 100 hours, for example, every 12 to 72 hours, for example, every 12 to 15, 15 to 20, 20 to 25, 25 to 30, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 65 to 70, or every 70 to 72 hours. In particular, the acclimatized culture medium fraction can be collected every 24 to 48 hours.

[0094] The conditioned medium fraction may contain volumes of approximately 100–500 mL, for example, approximately 100–150, 150–200, 250–300, 350–400, 400–450, or 450–500 mL, particularly around 250 mL. The fraction can be collected in a sealed bag and frozen at approximately -70°C to approximately -90°C, for example, approximately -80°C, until the exosomes are isolated.

[0095] In certain embodiments, the acclimatized medium is collected at least four times, for example, four to ten times, particularly five, six, seven, eight, or nine times, particularly six times, for each run. Individual fractions can be thawed, for example, overnight at 4°C, or for example, about 10 to 20 hours at 2 to 6°C.

[0096] (D. Isolation of exosomes) The pooled conditioned medium fraction can then be subjected to exosome isolation, particularly at 4°C. The conditioned medium can be centrifuged at temperatures of approximately 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, or 26°C, and in one embodiment, the conditioned medium is centrifuged at approximately 4°C. Exosome isolation can be carried out by methods known in the art for exosome isolation. Preferably, exosome isolation is also carried out in a closed system, similar to the exosome production described above. This can be achieved by using a pump and sealed tubing.

[0097] Isolation may include a centrifugation step to remove large fragments, followed by filtration, and one or more ultracentrifugation steps. The isolation method may be performed multiple times (e.g., three times) to process all of the pooled culture medium fraction.

[0098] The centrifugation step may involve centrifugation of approximately 500–2,000 g (e.g., approximately 1,000 g) for approximately 5–25 minutes (e.g., approximately 15 minutes). The acclimatization medium may be centrifuged at approximately 1,000 g, 2,000 g, 4,000 g, 6,000 g, 8,000 g; 10,000 g; 12,000 g, 14,000 g, 16,000 g, or 18,000 g. Centrifugation may be performed for periods such as 10–30 minutes, 12–28 minutes, 14–24 minutes, or 15–20 minutes. As those skilled in the art will understand, a suitable commercially available laboratory centrifuge, such as THERMO-SCIENTIFIC® or COLE-PARMER®, is used to carry out this centrifugation step. In particular, centrifugation can be performed in a closed system such as the Cobe 2991 Cell Processor (Terumo). Slow centrifugation may be performed two or more times to remove living cells, dead cells, and larger cellular debris.

[0099] The filtration step may include the use of a filtration bag having a submicron filter, such as a 0.1-0.3 micron filter, e.g., a 0.2 micron filter, to remove larger fragments like microvesicles. The supernatant can then be transferred to a tube (e.g., a polycarbonate tube) using a line directly connected to the tube. Filtration can be repeated two or more times. Filtration can be performed by passing the material through a filter of the same size, e.g., a 0.2 micron filter, one or more times. Alternatively, filtration can be performed using two or more filters of the same or decreasing size (e.g., one or more passes through a 40-50 micron filter, one or more passes through a 20-30 micron filter, one or more passes through a 10-20 micron filter, one or more passes through a 0.2-10 micron filter, etc.). Suitable filters for use in this step include 0.45 and 0.22 micron filters.

[0100] Ultracentrifugation can be performed at 75,000–150,000 g, for example, 100,000–170,000 g, for example, about 100,000 g, for about 2–6 hours, for example, 1–3 hours, for example, about 4 or 5 hours. A commercially available ultracentrifuge such as THERMO-SCIENTIFIC or Beckman can be used to perform this step. Specifically, ultracentrifugation can be performed using any closed-system centrifugation method, such as a Type 45 Ti rotor (Beckman-Coulter), but is not limited to this. This ultracentrifugation step may optionally be repeated, for example, two or more times, to improve the results. The exosome-containing pellet is removed from the supernatant using established techniques and resuspended in a suitable physiological solution.

[0101] As those skilled in the art will understand, the exosome pellet from either the centrifugation or ultracentrifugation step can be washed during the centrifugation step using a suitable physiological solution (e.g., sterile PBS, sterile 0.9% saline, or sterile carbohydrate-containing 0.9% saline buffer).

[0102] After centrifugation, the solution is removed and the exosomes are resuspended in a suitable buffer such as PBS. The pH of the buffer may be any pH suitable for the sample, but a typical range is 6–8. The buffer may have a pH of 4–10, 4–6, 4–8, 6–10, 6–8, or 8–10. In particular, the exosome pellet may be resuspended in a clinical-grade buffer (e.g., PLASMALYTE-A®) at a physiological pH of approximately 7.4. The volume of the buffer may be a precipitate solution of approximately 0.01 to 0.09 volumes, 0.02 to 0.08 volumes, or 0.03 to 0.07 volumes. The collected exosomes can be used immediately for electroporation or therapy, or they can be frozen and stored for later use, for example, at -20°C.

[0103] As used herein, analysis includes any method that enables direct or indirect visualization of exosomes and may be in vivo or ex vivo. For example, analysis may include, but is not limited to, ex vivo microscopy or cytometry detection and visualization of exosomes bound to a solid substrate, flow cytometry, fluorescence imaging, etc. In an exemplary embodiment, cancer cell-derived exosomes are detected using an antibody against glypican 1, subsequently bound to a solid substrate, and visualized using microscopy or cytometry detection. Exosomes may be analyzed by flow cytometry expression of exosome surface markers: CD63, CD47, CD9, and CD81, and / or by transmission electron microscopy (TEM). Furthermore, exosomes may be quantified using nanoparticle tracking analysis and micro-BCA assays.

[0104] Therefore, the period from seeding of MSCs into the bioreactor to final harvesting may be approximately 15–30 days, for example 16, 17, 18, 19, 20, 21, 22, 23, or 24 days, for example approximately 19 or 20 days. Each acclimatized medium fraction should contain at least 1 × 10⁶ 12 exosomes, for example, at least 2 × 10 12 exosomes, especially those approximately 3 × 10⁻⁶ 12 This method may include exosomes of at least 10 × 10 12 exosomes, for example, at least 11 × 10 12 exosomes, for example, at least 12 × 10 13 exosomes, for example, at least 13 × 10⁶ 12 exosomes, for example, at least 14 × 10 12 exosomes, for example, at least 15 × 10 12 exosomes, for example, at least 16 × 10 12 exosomes, for example, at least 17 × 10⁶ 12 exosomes, for example, at least 18 × 10 12 exosomes, for example, at least 19 × 10 12 exosomes, or for example, at least 17 × 10⁶ 12This can lead to the production of exosomes, or even the entire body of exosomes.

[0105] (E. exosome loading) Exosomes produced by the method of the present invention may carry cargo such as therapeutic or diagnostic agents. Examples of cargo that can be delivered using the exosomes of the present invention include, but are not limited to, exogenous substances that are not naturally present in the exosome (derived from external sources), such as nucleic acid molecules like DNA (both nuclear DNA and mitochondrial DNA), RNA such as mRNA, tRNA, miRNA, and siRNA, aptamers and other nucleic acid-containing molecules, peptides, proteins, ribozymes, carbohydrates, polymers, therapeutic agents, and small molecules.

[0106] In one embodiment, the isolated exosomes of the present invention are particularly useful for the delivery of compounds having a secondary structure (e.g., miRNA, mRNA, proteins / peptides), as well as large compounds (e.g., nucleic acid molecules including base pairs, peptides, proteins, etc., that exceed 20 base pairs, e.g., exceed 50 base pairs, or exceed 100 base pairs).

[0107] Cargo can be introduced into the exosome using methods established in the field for introducing cargo into cells. For example, cargo can be introduced into the exosome using electroporation with a voltage in the range of approximately 20 to 1000 V / cm. Transfection using cationic lipid-based transfection reagents can also be used to introduce cargo into exosomes. Examples of suitable transfection reagents include, but are not limited to, Lipofectamine MessengerMAX™ Transfection Reagent, Lipofectamine RNAiMAX Transfection Reagent, Lipofectamine 3000 Transfection Reagent, or Lipofectamine LTX Reagent with PLUSTM Reagent. An appropriate amount of transfection reagent is used for cargo loading, but this amount may vary depending on the reagent, sample, and cargo. For example, when using Lipofectamine MessengerMAX™ Transfection Reagent, an amount ranging from approximately 0.15 μL to 10 μL can be used to load 100 ng to 2500 ng of mRNA or protein into the exosome. Other methods, such as the use of cell-penetrating peptides for protein delivery, can also be used to introduce cargo into exosomes.

[0108] In certain embodiments, cargo such as nucleic acids, including siRNA, is loaded into exosomes using electroporation (e.g., electroporation in an FDA-approved buffer (e.g., PLASMALYTE-A)). Electroporation is performed using a flow-through electroporation system (e.g., a 4D-Nucleofactor LV Large Scale Transfection System, Lonza). Each electroporation run consists of at least 2 × 10⁻¹⁶ units. 12 It can contain exosomes. The buffer is FDA approved for patient use, sterile, and non-pyrogenic, and can be injected directly into the patient, so there is no need for the washing step required to change the buffer before administration to the patient. Therefore, there is no exosome loss due to the additional washing step, as in conventional methods, which can result in the loss of approximately 50% of the exosomes.

[0109] In certain embodiments, the therapeutic agent may be RNA such as siRNA, shRNA, plasmid, mRNA, miRNA, or ncRNA, particularly siRNA or miRNA therapeutic agents. The miRNA may be a miRNA mimetic or a miRNA precursor. The size of the RNA loaded into the exosome may be less than 100 nucleotides in length, for example less than 75 nucleotides, and particularly less than 50 nucleotides. For example, RNA may have a length of about 10 to 100 nucleotides (for example 20 to 50 nucleotides, particularly 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, or 45 to 50 nucleotides).

[0110] RNA may be modified or unmodified. RNA may include modifications of one or more nucleotides. Such modifications may include the addition of non-nucleotide substances (e.g., terminal or internal (in one or more nucleotides of RNA)). In certain embodiments, the RNA molecule includes a 3′-hydroxyl group. The nucleotides in the RNA molecules of this disclosure may also include non-standard nucleotides, including unnatural nucleotides or deoxyribonucleotides. Double-stranded oligonucleotides may contain a modified backbone, e.g., phosphorothioates, phosphorodithioates, or other modified backbones known in the art, or may contain unnatural nucleoside bonds. Further modifications of siRNA (e.g., 2′-O-methylribonucleotide, 2′-deoxy-2′-fluororibonucleotide, "universal base" nucleotide, 5-C-methylnucleotide, one or more phosphorothioate nucleotide interlinks, and inverted deoxyabasic residue incorporation) can be found in U.S. Publication No. 20040019001 and U.S. Patent No. 6,673,611 (each of which is incorporated in whole for reference). Collectively, all such modified nucleic acids or RNAs described above are referred to as modified siRNA.

[0111] Preferably, RNAi can reduce protein expression by at least 10%, 20%, 30%, or 40%, more preferably at least 50%, 60%, or 70%, and even more preferably at least 75%, 80%, 90%, 95%, or more.

[0112] The siRNA used in the methods or compositions described herein may include a portion complementary to the mRNA sequence encoded by the NCBI reference sequence of the described gene / protein. In one embodiment, the siRNA includes a double-stranded portion (double helix). In one embodiment, the siRNA is 20–25 nucleotides long. In one embodiment, the siRNA includes a 19–21 core RNA double helix with one or two nucleotide 3′ overhangs independently on one or both strands. In one embodiment, the overhangs are UU. The siRNA may be 5′ phosphorylated or unphosphorylated and may be modified with any of the modifications known in the art to improve efficacy and / or resistance to nuclease degradation. In non-limiting embodiments, the siRNA may be administered to be transfected into one or more cells. In one embodiment, the siRNA may include a double-stranded RNA comprising a first and second strand, where one strand of RNA is 80, 85, 90, 95, or 100% complementary to a portion of the RNA transcript of the gene.

[0113] In one embodiment, the single-stranded component of the siRNA of this disclosure is 14 to 50 nucleotides long. In another embodiment, the single-stranded component of the siRNA is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides long. In yet another embodiment, the single-stranded component of the siRNA of this disclosure is 21 nucleotides long. In yet another embodiment, the single-stranded component of the siRNA of this disclosure is 22 nucleotides long. In yet another embodiment, the single-stranded component of the siRNA of this disclosure is 23 nucleotides long. In one embodiment, the siRNA of this disclosure is 28 to 56 nucleotides long.

[0114] A target gene generally refers to a polynucleotide containing a polypeptide-coding region, or a polynucleotide region that modulates replication, transcription, translation, or other processes critical to polypeptide expression, or a polynucleotide containing both a polypeptide-coding region and a region operably linked to the polypeptide that modulates expression. A target gene can be chromosomal (genome) or extrachromosomal. It may be endogenous to the cell or exogenous (transgene). Exogenous genes may be integrated into the host genome or reside on extrachromosomal genetic constructs such as plasmids or cosmids. A target gene can also originate from a pathogen, such as a virus, bacterium, fungus, or protozoan, which can infect an organism or cell. Target genes can be viral and proviral genes that do not induce an interferon response, such as retroviral genes. A target gene may be a protein-coding gene, or a non-protein-coding gene, such as a gene encoding ribosomal RNA, splicesosomal RNA, tRNA, etc.

[0115] Any gene expressed within a cell can be targeted. Preferably, the target gene is involved in or associated with the progression of cellular activity that is important to the disease or of particular interest as a research subject. Accordingly, as an example, the following is a class of possible target genes that may be used in the methods of this disclosure to modulate or attenuate target gene expression: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Winged Helix family members, Hox family members, cytokines / lymphokines and their receptors, growth factors or differentiation factors and their receptors, neurotransmitters and their receptors), tumor suppressor genes (e.g., APC, CYLD, HIN-1, KRAS2b, p16, p19, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CHKN2A, DCC, DPC4, MADR2 / JV18, MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-1, zac1, ras, MMAC1, FCC, MCC, FUS1, Gene 26(CACNA2D2), PL6, Beta*(BLU), Luca-1(HYAL1), Luca-2(HYAL2), 123F2(RASSF1), 101F6, Gene 21(NPRL2) or SEMGenes encoding A3 polypeptides, pro-apoptotic genes (e.g., CD95, caspase-3, Bax, Bag-1, CRADD, TSSC3, bax, hid, Bak, MKP-7, PARP, bad, bcl-2, MST1, bbc3, Sax, BIK, and BID), cytokines (e.g., GM-CSF, G-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15) , IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IFN-α, IFN-β, IFN- γ, MIP-1α, MIP-1β, TGF-β, TNF-α, TNF-β, PDGF, and mda7), oncogenes (e.g., ABLI, BLC1, BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRBB2, ETS1, ETS1, ETV6, F GR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and YES), as well as enzymes (ACP-desaturase and hydroxylase, ADP-glucose pyrophorase, ATPase, alcohol dehydrogenase, amylase, amyloglucosidase, catalase, cellulase, cyclooxygenase, decarboxylase, dextrinase, esterase, DNA and Examples include RNA polymerase, galactosidase, glucanase, glucose oxidase, GTPase, helicase, hemicellulase, integrase, invertase, isomerase, kinase, lactase, lipase, lipoxygenase, lysozyme, pectin esterase, peroxidase, phosphatase, phospholipase, phosphorylase, polygalacturonase, proteinase and peptidase, pullanases, recombinase, reverse transcriptase, topoisomerase, and xylanase.

[0116] As those skilled in the art will understand, the exosomes of the present invention may be further modified by including a targeting moiety to enhance their usefulness as a vehicle for cargo delivery, either before or after cargo loading. In this regard, the exosomes may be engineered to incorporate entities that specifically target particular cells to specific tissue types. These target-specific entities (e.g., peptides having affinity for receptors or ligands on target cells or tissues) may be incorporated into the exosome membrane, for example, by fusion to an exosome membrane marker using methods well established in the art.

[0117] [III. How to use] In some embodiments, the present disclosure provides methods for using exosomes provided herein for the delivery of therapeutic agents, such as RNAi, to cells. Additional immune cells that can be targeted by exosomes for delivery include dendritic cells, NK cells, and / or B cells. In further embodiments, the therapeutic agent delivered by the exosomes of the present disclosure may be a small molecule, a peptide, a vaccine, or an antigen. The cells may be in vivo or ex vivo. In one embodiment, a method is provided for delivering RNA to cells, comprising administering an effective amount of exosomes containing RNAi to the cells. The cells may be immune cells such as T cells, or cancer cells such as KRAS-positive cancer cells.

[0118] In a further embodiment, a method is provided for immunostimulating an organism, comprising administering an effective amount of RNA-encapsulating exosomes to the subject. The RNA may be immunomodulatory RNA. In another embodiment, a method is provided for treating a subject with a disease or disorder, comprising administering an effective amount of the exosomes of the Disclosure. In some embodiments, the use of the exosomes of the Disclosure is provided for the treatment of a disease or disorder, or for immunostimulating a subject.

[0119] In vivo cells can be present in any subject, such as mammals. For example, the subject may be a human, mouse, rat, rabbit, dog, cat, cow, horse, pig, goat, sheep, primate, or bird. In certain embodiments, the subject refers to a human. For example, a human may be a subject with a disease. The disease may be any disease that afflicts the subject, such as an inflammatory disease, a hyperproliferative disease, an infectious disease, or a degenerative disease. In certain embodiments, the disease is a hyperproliferative disease such as cancer. For example, cancer may be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, kidney cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, malignant lymphoma, stomach cancer, pancreatic cancer, testicular cancer, intestinal cancer, lymphoma, or leukemia. In certain embodiments, cancer is ovarian cancer.

[0120] In another embodiment, a method is provided for treating an immune-mediated inflammatory disease in a subject suffering from the disease, comprising administering a therapeutically effective amount of the exosomes of the present disclosure to the subject.

[0121] Cancer is not limited to these, but can specifically be of the following histological types: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combination of hepatocellular carcinoma and cholangiocarcinoma; cord-like carcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis; solid carcinoma; carcinoid tumor, malignant; acinar cell adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; Eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; unencapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; testicular 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; adenosquamous cell carcinoma; adenocarcinoma with squamous epithelialization; thymoma, malignant; ovarian stromal tumor, malignant; thecoma; granulosa cell tumor, malignant; androblastoma, malignant Sertoli cell carcinoma; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extramammary paraganglioma, pheochromocytoma; Malignant melanoma; Melanin-deficient melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevi; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Fetal Childhood cancer; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangioectoderma, malignant; lymphangiosarcoma; osteosarcoma; paraosteal osteosarcoma; chondrosarcoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; supraamelosarcoma; ameloblastoma, malignant; ameloblastoma; pineal glandoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; protofibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma; ganglioblastoma; neuroblastoma;Olfactory neurogenic tumors; meningiomas, malignant; neurofibrosarcomas; schwannomas, malignant; granulomas, malignant; malignant lymphomas; Hodgkin's disease; Hodgkin's granuloma; small lymphocytic lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other certain 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 hairy cell leukemia.

[0122] In some embodiments, a method is provided for treating a disease or disorder in a subject, comprising administering an effective amount of exosomes carrying a therapeutic agent to a subject in need. This disease may be an immune-related disorder, such as an autoimmune disorder. Non-specific examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune adrenal gland disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré disease, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, and erythema infectiosum. Thematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (minimal change disease, focal segmental glomerulosclerosis, or membranous nephropathy, etc.), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis Examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (such as polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis or herpetiform cutaneous vasculitis), vitiligo, and Wegener's granulomatosis. Therefore, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subjects may also have allergic diseases such as asthma.

[0123] Treatment outcomes can be predicted and monitored, and / or patients who would benefit from such treatment can be identified or selected through the methods described herein.

[0124] Regarding the treatment of neoplastic conditions, depending on the stage of the neoplastic disease, treatment involves one or more therapies such as surgery to remove tumorous tissue, radiotherapy, and chemotherapy. Other treatment regimens can be combined with the administration of anticancer agents, such as therapeutic compositions and chemotherapeutic agents. For example, patients treated with such anticancer agents may also undergo radiotherapy and / or surgery.

[0125] For the treatment of a disease, the appropriate dosage of the therapeutic composition will depend, as defined above, on the type of disease being treated, the severity and course of the disease, the patient's medical history and response to the medication, and the judgment of the attending physician. The medication should be administered to the patient appropriately, either as a single dose or over a series of treatments.

[0126] Therapeutic and prophylactic methods and compositions may be provided in combination amounts effective in achieving the desired effect. Tissues, tumors, or cells can be brought into contact with one or more compositions or pharmacological preparations containing one or more agents, or by bringing tissues, tumors, and / or cells into contact with two or more distinct compositions or preparations. Such combination therapies can also be considered to be used in combination with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0127] Concomitant administration may include simultaneous administration of two or more drugs in the same dosage form, simultaneous administration in different dosage forms, and separate administration. That is, the therapeutic composition of the present invention and another therapeutic agent may be prescribed together in the same dosage form and administered simultaneously. Alternatively, the therapeutic composition of the present invention and another therapeutic agent may be administered simultaneously, where both drugs are present in separate formulations. In another alternative, the therapeutic agent may be administered immediately after the other therapeutic agent, or vice versa. In a separate administration protocol, the therapeutic composition of the present invention and another therapeutic agent may be administered at intervals of several minutes, several hours, or several days.

[0128] The exosomes described herein may be used in therapeutic, research, and diagnostic applications. For example, the exosomes described below may be added to cell cultures to enhance one or more phenotypic properties of cells. The exosomes of the present invention may be added to cell cultures to inhibit one or more phenotypic properties of cells. The exosomes of the present invention may be added to cell cultures to provide novel phenotypic properties of cells.

[0129] (A. Pharmaceutical composition) The specific methods described herein involve administering a pharmaceutically effective amount of the exosome-containing composition of this disclosure.

[0130] As used herein, “pharmaceutically acceptable carrier” includes any and all materials and combinations thereof, such as any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc., as known to those skilled in the art (Remington's, 1990). It is intended for use in therapeutic or pharmaceutical compositions, except insofar as any conventional carrier is incompatible with the active ingredient. The compositions used in this disclosure may include different types of carriers, depending on whether they are to be administered in solid, liquid, or aerosol form and whether they need to be sterilized for a route of administration such as injection.

[0131] The use of such media and agents for pharmaceutically active substances is well known in the field. Their use in therapeutic compositions is intended unless any conventional media or agent is incompatible with the active ingredient. Co-active ingredients may also be incorporated into the composition, which will be discussed in more detail below. For administration to humans, preparations should preferably meet standards of sterility, pyrogenicity, general safety, and purity, as required by FDA Biomedical Bureau standards.

[0132] Compositions containing exosomes may, if necessary, be extensively dialyzed to remove undesirable low molecular weight molecules and / or lyophilized for easier formulation into the desired vehicle. In particular, the compositions constituting the exosomes of this disclosure (e.g., PLASMALYTE-A) can be injected directly into the target without any processing. The active compound is then generally formulated for administration by any known route (e.g., parenteral administration). The method of administration will be discussed in more detail below.

[0133] This disclosure is intended to describe a method of using a composition that is a sterile solution for intravascular injection or for application by any other route, as will be discussed in more detail below. Those skilled in the art are familiar with the techniques for producing sterile solutions for injection or application by any other route. Sterile injection solutions are prepared by incorporating the required amount of the active compound in a suitable solvent together with various other components well known to those skilled in the art.

[0134] The formulation of the composition may vary depending on the route of administration. For parenteral administration in aqueous solution, for example, the solution should be adequately buffered, and the liquid diluent should first be isotonic with sufficient saline or glucose. In this regard, suitable sterile aqueous media will be known to those skilled in the art in light of this disclosure.

[0135] Other pharmaceutically acceptable forms include compounds formulated for parenteral administration, such as intravenous or intramuscular injection, formulations for administration via implantable drug delivery devices, and any other forms. The disclosure may also include nasal sprays or inhalants, aerosols or inhalants.

[0136] Oral formulations typically include commonly used excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. Those skilled in the art will be familiar with the well-known techniques for preparing oral formulations.

[0137] In certain embodiments, the pharmaceutical composition contains at least about 0.1% by weight of the active agent. The composition may contain, for example, about 0.01%. In other embodiments, the pharmaceutical composition may contain, for example, about 2% to about 75% by weight of the composition, or about 25% to about 60% by weight of the composition, and any range within which this can be derived.

[0138] The pharmaceutical composition may contain various antioxidants to slow the oxidation of one or more components. Furthermore, prevention of microbial action may be achieved by preservatives such as various antimicrobial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof. The composition may be stable under manufacturing and storage conditions and may be preserved against microbial (e.g., bacterial and fungal) contamination. It is understood that exotoxin contamination should be kept to a safe level, e.g., less than 0.5 ng / mg protein.

[0139] In embodiments where the composition is in liquid form, the carrier may be a solvent or dispersion medium that includes, but is not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. In many cases, it is preferable to include an isotonic agent such as sugar, sodium chloride, or combinations thereof.

[0140] In other embodiments, the disclosure may use nasal drops or sprays, aerosols or inhalants. The nasal drops may be aqueous solutions designed to be administered intranasally by drip or spray.

[0141] Sterile injection solutions are prepared by incorporating the required amount of nanoparticles, as needed, along with the various other components listed above, into a suitable solvent, followed by sterilization.

[0142] When prescribed, exosomes are administered in a manner compatible with the medication prescription and in a therapeutically effective amount.

[0143] Nanoparticles can be administered to a subject by any method known to those skilled in the art. For example, a pharmaceutically effective amount of a composition containing exosomes can be administered intravenously, intracerebrally, intracranially, intrathecally, substantia nigra or region of substantia nigra, intradermally, intraarterially, intraperitoneally, intrafocally, intratracheally, intranasally, topically, intramuscularly, intraperitoneally, subcutaneously, orally, topically, partially, by inhalation (e.g., aerosol inhalation), injection, infusion, serial infusion, directly to target cells by topical perfusion bath, via catheter, via lavage, in cream, in a lipid composition (e.g., liposomes), or by any other method known to those skilled in the art or any combination of the above (Remington's, 1990). In certain embodiments, the composition is administered to a subject using a drug delivery device.

[0144] In other embodiments, exosomes are formulated for administration by routes including, but not limited to, oral, intranasal, enteral, topical, sublingual, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal, or rectal routes, and each includes an appropriate carrier. For example, an exosome composition for topical application may be prepared including an appropriate carrier. Creams, lotions, and ointments can be prepared for topical application using an appropriate base, such as a triglyceride base. Such creams, lotions, and ointments may also include a surfactant. Aerosol formulations may also be prepared so as to use an appropriate propellant adjuvant. Other adjuvants may also be added to the composition, regardless of how it is administered; for example, antimicrobial agents, antioxidants, and other preservatives may be added to the composition to prevent microbial growth and / or degradation over long storage periods.

[0145] The pharmaceutically effective amount of nanoparticles is determined based on the intended target, e.g., inhibition of cell death. The amount administered depends on the target being treated, the condition of the target, the desired protection, and the route of administration, as well as both the number of treatments and the dosage. The exact amount of therapeutic agent also depends on the practitioner's judgment and is specific to each individual.

[0146] For example, the dosage of the therapeutic agent may be about 0.0001 milligrams to about 1.0 milligram per dose, or about 0.001 milligrams to about 0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or even about 10 milligrams. Repeated administration is also possible. In some embodiments, the dosage is at least about 0.0001 milligrams. In further embodiments, the dosage is at least about 0.001 milligrams. In yet further embodiments, the dosage is at least 0.01 milligrams. In yet further embodiments, the dosage is at least about 0.1 milligrams. In more specific embodiments, the dosage may be at least 1.0 milligrams. In even more specific embodiments, the dosage may be at least 10 milligrams. In further embodiments, the dosage is at least 100 milligrams or more.

[0147] In other non-limiting examples, doses 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, approximately 1000 mg / kg body weight, or more / dose, and any range that can be derived therefrom. In non-limiting examples of ranges that can be derived from the numbers listed herein, ranges such as approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, approximately 5 micrograms / kg / body weight to approximately 500 milligrams / kg / body weight, etc., may be administered based on the above numbers.

[0148] Dosages can be repeated as determined by those skilled in the art. Accordingly, in some embodiments of the methods described herein, a single dose is intended. In other embodiments, two or more doses are intended. When two or more doses are administered to a subject, the time interval between doses may be any time interval determined by those skilled in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval that can be derived within any of these enumerated ranges.

[0149] In certain embodiments, this method can provide continuous delivery of a pharmaceutical composition to a patient. This can be achieved by a catheterization procedure followed by continuous administration of the therapeutic agent. Administration may be intraoperative or postoperative.

[0150] (B. Combination therapy) Certain embodiments of this disclosure provide one or more secondary forms of administration or application of a treatment for the treatment or prevention of a disease. For example, the disease may be a hyperproliferative disease such as cancer.

[0151] The secondary treatment may involve the administration of one or more secondary pharmacological preparations applicable to the treatment or prevention of cancer.

[0152] If the second-line treatment is a pharmacological formulation, it may be administered before, concurrently with, or after the administration of nanoparticles.

[0153] The interval between exosome administration and second-line treatment can be any interval determined by those skilled in the art. For example, the interval may range from a few minutes to several weeks. In embodiments where the drugs are administered separately, it is generally ensured that no long intervals elapse between each delivery so that each therapeutic agent can still exert a favorably combined effect on the target. For example, the interval between therapeutic agents may be about 12 to 24 hours from each other, more preferably within about 6 to 12 hours from each other. However, in some situations, the treatment period may be extended, in which case several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) may elapse between each administration. In some embodiments, the timing of administration of the second-line therapeutic agent is determined based on the target's response to the nanoparticles.

[0154] Various combinations can be used. In the following example, the exosome composition is "A" and the anticancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0155] The administration of any compound or treatment described herein to a patient shall follow general protocols for the administration of such compounds, taking into account the toxicity of the drug (if any). Therefore, in some embodiments, there is a step to monitor toxicity resulting from the combination therapy. It is expected that treatment cycles may be repeated. Furthermore, various standard therapies, as well as surgical interventions, are considered to be applicable in combination with the described therapies.

[0156] In certain embodiments, standard therapies include chemotherapy, radiotherapy, immunotherapy, surgical therapy, or gene therapy, and are intended to be used in combination with gene expression therapy inhibitors, anticancer therapies, or both gene expression therapy inhibitors and anticancer therapies, as described herein.

[0157] (1.Chemotherapy) According to this embodiment, a wide variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer, and "chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These drugs or agents are classified, for example, by the mode of their activity within cells, such as whether and at what stage they affect the cell cycle. Alternatively, drugs may be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0158] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and biposulfan; aziridines such as benzodopa, carbocone, metredopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethyloromelamamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); briostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodicin; spongistatin; chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembicin, fenestrine, prednimustine, trophosphamide, uracil mustard and other nitrogen mustards; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine; antibiotics such as enegyoin antibiotics (calicheamicin, especially calicheamicin gamma I and calicheamicin omega I1); dynemicins including dynemicin A; bisphosphonates such as clodronate; esperamycin;Similarly, neocardinostatin chromophores and related chromophores, neocardinostatin chromophores, actinomycin, actinomycin, autoralnicin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epil Mitomycins such as biscin, esorubicin, idarubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olibomycin, peplomycin, potfiromycin, buromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimethrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dronaron propionate, epithiostanol, mepitiostane, and testactone; anti-adrenal agents such as mitotane and trilostane; folic acid supplements such as floric acid; acegraton; aldofamide glycoside; aminolevriel Calcium sulfate, enyluracil; amsacrin; bestrabusil; bisanthren; edatrexate; dehofamine; demecolsin; elformithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; meitansinoids such as meitansin and anthamitocin; mitogluazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex;Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2"-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Beraclin A, Loridine A and Angidin); Urethane; Vindesine; Dacarbaidine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., Paclitaxel and D Examples include: cetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum-coordinate complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl hydroxylunitine (DMFO); retinoids (such as retinoic acid); capecitabine; carboplatin, procarbazine, pricomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0159] (2. Radiation therapy) Other widely used factors that cause DNA damage include those known as gamma rays, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors such as microwaves, proton beam irradiation (US Patents 5,760,395 and 4,870,287), and UV irradiation are also intended. All of these factors are likely to affect DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance, causing widespread damage. The dose range for X-rays ranges from a daily dose of 50–200 roentgens over long periods (3 to 4 weeks) to a single dose of 2,000–6,000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by tumor cells.

[0160] (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 embodiments. In relation to cancer treatment, immunotherapies may rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. Immune effectors may be antibodies specific to certain markers on the surface of tumor cells, for example. Antibodies alone may serve as therapeutic effectors or mobilize other cells to actually influence cell killing. Antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and serve as targeting agents. Alternatively, effectors may be lymphocytes carrying surface molecules that directly or indirectly interact with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0161] Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently bound to a cytotoxic drug. This approach combines the high specificity of the MAb to its antigenic target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells at abundant levels of antigen. Targeted drug delivery also minimizes its exposure to normal tissue, leading to reduced toxicity and improved therapeutic index. The validity of this approach was validated by the FDA's approval of two ADC therapies: ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013. This approach combines the high specificity of the MAb to the antigen. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment. As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly depend on the identification and confirmation of novel targets suitable for this approach, as well as the generation of target MAbs. Two criteria for ADC targets are upregulation / high levels of expression in tumor cells and robust internalization.

[0162] In one embodiment of immunotherapy, tumor cells are easily targeted, meaning they can carry several markers that are not present in most other cells. Many tumor markers exist, and any of them may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative embodiment of immunotherapy is to combine anticancer effects with immunostimulatory effects. Immunostimulatory molecules also include cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN, chemokines such as MIP-1, MCP-1, and IL-8, and growth factors such as FLT3 ligand.

[0163] Examples of immunotherapies currently under investigation or in use include immunoadjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapies, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapies, e.g., TNF, IL-1, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185. One or more anticancer therapies are intended to be used in conjunction with the antibody therapies described herein.

[0164] In some embodiments, immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints are molecules in the immune system that either increase or decrease signaling (e.g., co-stimulatory molecules). Suppressive checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activator gene-3 (LAG3), programmed DES 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and T cell activator V domain Ig inhibitor (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0165] Immune checkpoint inhibitors may be drugs such as small molecules, ligands, or recombinant receptor forms, or in particular antibodies such as human antibodies (e.g., International Patent Publication WO2015016718; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogues may be used, in particular chimeric, humanized, or human forms of antibodies. As those skilled in the art will know, alternative and / or equivalent names may be used for specific antibodies referred to herein. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0166] 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 embodiments, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain embodiments, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain embodiments, 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. Patent Nos. 8,735,553, 8,354,509 and 8,008,449 (all 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 Applications No. 20140294898, No. 2014022021, and No. 20110008369 (all incorporated herein by reference).

[0167] 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 an extracellular or PD-1 binding moiety of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 conjugated antagonist is AMP224. 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, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0168] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated 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 functions 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 and is expressed on the surface of helper T cells, transmitting inhibitory signals to T cells. CTLA4 is analogous to the T cell costimulatory protein CD28, both molecules binding to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CD28 transmits stimulatory signals, while CTLA4 transmits inhibitory signals to T cells. Intracellular CTLA4 is also found on regulatory T cells and is thought to be important for their function. T cell activation via T cell receptors and CD28 increases the expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0169] 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.

[0170] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be produced using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, International Publication No. 01 / 14424, International Publication No. 98 / 42752; International Publication No. 00 / 37504 (CP675,206, also known as tremelimumab; formerly tisilimunab), and U.S. Patent No. 6,207,156 can be used in the methods disclosed herein. Lessons learned from each of the above publications are incorporated here for reference only. Antibodies that compete with any of these technically recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0171] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX010, MDX101, and Yervoy®) or its antigen-binding fragments and variants. 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, as well as the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binding to the same epitope on CTLA-4 as the antibody described above. In yet another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0172] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. US5844905, US5885796, International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference; and immunoadhesion, such as that described in U.S. Patent No. US8329867 (all incorporated herein by reference).

[0173] (4.Surgery) Approximately 60% of people with cancer undergo some form of surgery, including prophylactic, diagnostic, or staging, curative, and palliative surgery. Curative surgery involves the physical removal, excision, and / or destruction of all or part of the cancerous tissue and may be used in conjunction with other therapies such as therapies of this embodiment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor excision means the physical removal of at least a portion of the tumor. In addition to tumor excision, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0174] When cancerous cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment can be achieved by perfusion, direct injection, or local application of the area with additional anticancer treatment. Such treatment can 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 can also be in various dosages.

[0175] (5. Other medications) To improve the therapeutic effect of the treatment, other agents may be used in combination with specific embodiments of this embodiment. Therefore, further examples can be considered. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferating cells to apoptosis inducers, or other biological agents. An increase in intercellular signaling by increasing the number of gap junctions would likely increase the anti-hyperproliferative effect on adjacent hyperproliferating cell populations. In other embodiments, cell proliferation inhibitors or differentiation agents may be used in combination with specific embodiments of this embodiment to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are intended to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are local adhesion kinase (FAK) inhibitors and lovastatin. Furthermore, it is further intended that other agents that increase the sensitivity of hyperproliferating cells to apoptosis, such as the antibody c225, may be used in combination with specific embodiments of this embodiment to improve therapeutic efficacy.

[0176] [kit] In various embodiments of the embodiment, a kit containing a therapeutic agent and / or other therapeutic agents and delivery agents is envisioned. In some embodiments, the embodiment intends a kit for preparing and / or administering the exosome composition of the embodiment. The kit may include one or more sealed vials containing any of the pharmaceutical compositions of the embodiment. The kit may include, for example, exosomes, as well as reagents for preparing, formulating and / or administering the components of the embodiment, or reagents for performing one or more steps of the method of the present invention. In some embodiments, the kit may also include a suitable container that does not react with the components of the kit, such as an Eppendorf tube, assay plate, syringe, bottle, or tube. The container may be made from a sterilizable material such as plastic or glass.

[0177] The kit may further include instructions outlining the steps of the procedure of the method described herein, and which follow substantially the same procedure as described herein or which are known to those skilled in the art. The instructions may be in computer-readable media containing machine-readable instructions that, when executed using a computer, cause a display of an actual or virtual procedure for delivering a pharmaceutically effective amount of therapeutic agent. [Examples]

[0178] [V. Examples] The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will understand that the methods disclosed in the following examples represent the techniques found by the inventors and therefore function well in the practice of the present invention. However, those skilled in the art will understand, considering the disclosures of the present invention, that many modifications are possible in the specific examples disclosed and yielding similar results without departing from the spirit and scope of the invention.

[0179] [Example 1 - Exosome Production and Characterization] To generate large quantities of exosomes from MSCs, bioreactor cultures of bone marrow-derived MSCs were adapted to allow for the collection of 250 mL samples in conditioned medium. The Terumo Quantum Cell Expansion system is an automated hollow fiber cell culture platform designed for GMP-compliant cell production.

[0180] MSCs are approximately 450 cells / cm². 2 At this concentration, approximately 2.0 × 10 7 The cells were seeded in a bioreactor along with MSCs. To promote MSC proliferation and adhesion, the cells were cultured for 8 days in 5% oxygen using αMEM medium supplemented with L-glutamine + human platelet lysate (PLT).

[0181] A unique step in the present invention's method, not reported by others, is the initial use of platelet lysates for optimal MSC confluence in the bioreactor, followed by the next step of replacing the growth medium with serum-free conditioned medium once the MSCs in the bioreactor have reached a confluence of 85% or more. This step avoids contamination of the final product with exosomes, which are found in large quantities in the platelet lysates and would otherwise dilute the MSC-derived exosomes. The conditioned medium was left in the bioreactor for 48 hours and then collected for EV purification (Figure 1). The bioreactor continuously produced EVs for 12 days, including 6 collections (one every 48 hours) (Figure 2).

[0182] Using this approach, the system was optimized by sequentially collecting extracellular viable cells (EVs) from approximately 600 million MSCs. After 21 days, fractions were collected every 48 hours, thawed, pooled, and filtered in a functionally closed system. These acclimatized medium fractions were stored at -80°C (Figure 3).

[0183] A pump (Baxter) was used for filtration after heat sealing of the bag tubes for this process, which was consistent with the GMP compliance required for clinical cell therapy procedures. EVs were isolated as exosomes from the conditioned medium by ultracentrifugation at 100,000 g for 4 hours at 4°C using an XE-90 Ultracentrifuge (Beckman Coulter). Exosomes were removed from the ultracentrifuge in a functionally closed manner for the highest quality clinical product using the Baxter pump and heat-sealed tubes.

[0184] Exosomes were concentrated by filtration and ultracentrifugation, with exosome counts ranging from 900 billion to 4.5 trillion per harvest. The total number of exosomes generated ranged from 9.8 to 15.1 trillion per bioreactor run. The mode size of exosomes from all six harvests in the bioreactor experiment showed a characteristic peak at approximately 170 nm. Measurements of exosome protein content approximated the exosome count determined by NanoSight analysis. In particular, metabolic harvests in the conditioned medium remained constant, supporting the survival of MSCs.

[0185] Purified exosomes were identified by flow cytometry expression of exosome surface markers: CD63, CD47, CD9, and CD81; and by transmission electron microscopy (TEM) (Figure 4). Furthermore, exosomes were quantified using nanoparticle tracking analysis and micro-BCA assay (Figure 5). The yield from a single bioreactor run was approximately equal to the exosome yield from a 100 T-225 flask, with a minimum yield of 10 × 10⁶. 12 These were exosomes. Therefore, the method of the present invention enables the efficient production of clinical-grade exosomes.

[0186] [Example 2 - Electroporation of exosomes] The 4D Nucleofactor system produces exosomes similar to those derived from Example 1, such as 2.5 × 10⁶ 10 ~2.5×10 12The LV unit was adapted to enable closed, efficient, and scalable in vitro transfection of large exosome numbers in the range of [specified range]. The 4D Nucleofactor device contains three different nuclear factor solutions, SE, SF, and SG, each of which was tested in combination with 16 different nuclear factor programs using MSC-derived exosomes and specific siRNAs. The efficiency of each condition for efficiently incorporating siRNA into MSC-derived exosomes was evaluated by apoptosis assays of recipient cells induced by MSC exosomes containing siRNA (Figure 7).

[0187] Previously, siRNA was introduced into exosomes using a prescribed electroporation buffer (research buffer, "RB"), because that buffer is not approved for human use. This required a wash step for exosomes before treating cells or mice. The wash step is associated with exosome loss, which can be mitigated by the use of a diluent that allows for successful electroporation of siRNA into exosomes and can be administered directly to cells or mice. Clinical buffer (PLASMALYTE-A®, "CB"), an FDA-approved diluent for human use (used for injecting MSCs and many other cell products into patients), was tested for electroporation. Following electroporation of MSC-derived exosomes, electron microscopy analysis confirmed the presence of complete exosomes using either the initially prescribed research buffer (RB) or PLASMALYTE-A® (Figure 8B).

[0188] [Example 3 - Optimization of conditions in exosome production] To determine the optimal time for exosome production from MSC cultures, acclimatization medium was collected at different time points (Figure 5D). Data obtained from nanocytometry (i.e., particle count) were combined with data obtained from flow cytometry (i.e., percentage of exosome markers). The results showed that the number of particles from MSCs reached its peak at 24 hours and was maintained until 48 hours. After that, the number of particles decreased significantly. Flow data showed that the percentage of exosome markers was concentrated at 48 hours compared to the percentage at 24 hours (Figure 5E). Since the number of particles did not differ significantly between 24 hours and 48 hours, 48 ​​hours was selected as the time point for collection.

[0189] Next, to determine the ideal conditions for exosome production from cultured MSCs, MSC-derived exosomes were isolated from conditioned medium after 24 hours of incubation, with and without the presence of PLT-containing medium. The results showed that a greater number of particles were isolated from the PLT-containing medium than from the serum-free medium (Figure 6A). However, flow cytometry of exosome markers showed a lower percentage of exosomes, likely due to the higher number of proteins and lipids in the PLT (Figure 6B).

[0190] Therefore, the process of electroporation of nucleic acid or protein subsequences into exosomes was optimized using a sterile solution (i.e., PLASMALYTE-A®) (Figure 8A), which can be directly injected in humans, reducing the cost of operation, material loss, and treatment (Figure 9). Five different research buffers were tested before demonstrating that PLASMALYTE-A® produced optimal electroporation results (Figure 8A). Furthermore, the ability of MSC-derived exosomes produced by this strategy to target several tissues was demonstrated in vivo (Figure 10).

[0191] Therefore, this study demonstrated the efficient large-scale production of MSC-exosomes containing KRAS siRNA capable of silencing the in vitro expression of target RNA in recipient cells (Figure 8C). This approach generates a greater number of EVs than previous methods, by more than one-logarithmic rate. Furthermore, the present invention allows for the direct injection of engineered exosomes without washing or other procedures.

[0192] [Example 4 - Materials and Methods] Cells: MSCs (passage 3) derived from bone marrow obtained from the Cell Therapy Laboratory at MD Anderson Cancer Center were cultured in αMEM (complete medium) supplemented with 1% L-glutamine, 5% human platelet lysate, and 1% penicillin-streptomycin. MSCs from three different donors were evaluated, and a single donor was selected based on its high exosome production yield. For in vitro transfection, 250,000 Panc-1 cells per well in a 6-well plate were seeded overnight. Prior to exosome processing, the monolayer was washed twice with 1 ml of PBS, and then processed with exosomes in 1 ml of serum-free medium (RPMI supplemented with 1% penicillin-streptomycin) at the indicated time points as described for each assay.

[0193] The clinical buffer solution (PLASMALYTE-A® pH 7.4 or "CB") consists of 0.09 M sodium chloride, 0.23 M sodium gluconate, 0.27 M sodium acetate trihydrate, 5 mM potassium chloride, and 3 mM magnesium chloride. It does not contain antibacterial agents. Adjust the pH to 7.4 with sodium hydroxide.

[0194] Clinical-grade exosomes were strictly prepared from MSCs cultured in the GMP facility of the Cell Therapy Laboratory at MD Anderson Cancer Center. Quantum bioreactor culture systems (Terumo BCT) were primed with 1 L of 1×PBS (automated process) and coated for 24 hours with 5 mg of human fibronectin (BD Biosciences, Germany) diluted in 250 ml of 1×PBS. The bioreactors were then washed with 500 ml of cultured αMEM supplemented with 1% L-glutamine, 5% human platelet lysate, and 1% penicillin-streptomycin (complete medium), and diluted in 25 ml of complete medium to produce 20×10⁶ exosomes. 6MSCs (passage 3) were loaded and grown for 9 days using complete medium. Fresh complete medium was continuously added to the cells, and the infusion rate was adjusted as determined by daily glucose and lactate measurements. After 9 days, when the cells reached approximately 80% confluence (confirmed by glucose and lactose measurements), the cells were washed with 2 L of 1×PBS and the complete medium was replaced with PLT-free medium (αMEM supplemented with 1% L-glutamine and 1% penicillin-streptomycin). Bioreactor-conditioned medium (250 ml) was then collected every 48 hours in a sealed bag (closed system), for a total of 6 collections. During these 12 days, the cultures did not expand based on a constant glucose level measured daily. Therefore, exosomes were continuously collected every 48 hours over the 12 days. The harvested material was stored at -80°C for further processing. Each harvested sample was tested for sterility (confirmed to be negative for anaerobic and aerobic bacteria), endotoxin (<1 EU / ml), and mycoplasma (PCR, negative). The collected samples were then thawed overnight at 4°C, pooled, and centrifuged at 1,000 g for 15 minutes in a closed system using a Cobe 2991 Cell Processor (Terumo BCT). After removing large cell debris by centrifugation (15 minutes at 4°C, 1,000 g), the acclimatized medium was filtered in a closed system using a filtration bag with a 0.2 μm filter (Terumo BCT). Next, 600 ml of the supernatant was transferred in a semi-closed system to six clear polycarbonate tubes (each with a capacity of 100 ml) using a syringe and a line directly connected to a polycarbonate tube (Beckman-Coulter). The tubes were sealed and centrifuged at 100,000 g for 3 hours in a 45 Ti rotor (Beckman-Coulter). This process was repeated three times until all samples had been centrifuged (totaling 1500 ml). The supernatant was then aspirated using a 16G syringe (BD Biosciences, catalog no. 14-826-18B) connected to a pump.The exosome pellet was manually resuspended in 4 ml (per tube) of clinical buffer using an 18G syringe (BD, catalog no. 408360) and transferred to a sterile glass container (APP Pharma, 30 ml capacity). This was maintained at 4°C for up to 72 hours until all centrifugation was complete. Upon completion of all centrifugation, the final pooled volume of the resuspended exosomes was 72 ml. The pooled MSC exosomes were analyzed by NanoSight™ (0.5 ml), flow cytometry (1 ml), and tested for endotoxin (using 0.5 ml of the pooled sample) and sterility (using 1 ml of the pooled sample, as detailed above). Finally, the exosomes (final volume 69 ml) were divided into cryoglass vials, each containing 2 ml, and stored at -80°C. For the manufacture of future clinical products, exosomes are directly processed for large-scale electroporation (see below for details), then aliquoted and stored at -80°C.

[0195] Measurement of particle size and concentration distribution by NTA: The isolated exosome suspension was measured using NanoSight TM Analysis was performed using an LM 10 instrument (NanoSight Ltd). Analysis settings were optimized and kept constant between samples, and each video was analyzed to obtain the mean, mode, median, and estimated concentration for each particle size.

[0196] Exosome quantification by microBCA assay: MSC exosomes resuspended in CB were washed with 1×PBS and centrifuged at 100,000 g for over 3 hours in a SW 41 Ti toter (Beckman Coulter). The washed MSC exosomes were then measured again using NanoSight™, and the total protein content was analyzed using the microBCA protein assay reagent kit (Thermo Scientific) according to the manufacturer's specifications.

[0197] Exosome electroporation: 0.5 × 10¹² in total 12MSC-derived exosomes and 0.5 mg of siRNA source 2 (Avecia) were mixed in 20 ml of clinical buffer. These exosomes were electroporated in a closed system using a 4D Nucleofator LV unit (Lonza). TM The cartridge is a new cuvette system that enables electroporation of up to 20 ml. The cartridge is connected to two reservoir bags (inlet and outlet) and a peristaltic pump that fills the cartridge at a rate of 1 ml per hour. The outlet bag is kept on ice throughout the procedure, which takes approximately 10 minutes to complete. After electroporation, the exosomes are transferred to NanoSight TM The exosomes were analyzed and tested for endotoxin and sterility (as detailed above), dispensed into frozen vials, and stored at -80°C. These exosomes were then thawed on ice and used for subsequent in vitro and in vivo experiments. For in vitro experiments, the exosomes were diluted for downstream applications as detailed below. For in vivo experiments, 10⁹ electroporated exosomes were diluted in 100 μL of research buffer or clinical buffer.

[0198] Electron microscopy: Specimens fixed at optimal concentration were placed on a 300-mesh carbon / formvar-coated grid and absorbed in formvar for at least 1 minute. The grid was rinsed with PBS and placed in 2.5% glutaraldehyde in 0.1M phosphate buffer for 15 minutes. After rinsing with PBS and distilled water, the grid was dried and contrast stained with uranyl acetate. Samples were observed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR), and images were captured with an AMT CCD camera (Advanced Microscopy Techniques, Danvers, MA).

[0199] Flow cytometry analysis of exosomes: Exosomes were isolated from MSCs as described above and resuspended in 200 μl of PBS. Aldehyde / sulfate beads (10 μl, Life Technologies) were added to the solution, and the beads and exosome mixture were mixed at room temperature for 15 minutes using a benchtop rotator. Then, PBS (600 μl) was added to the solution, and mixing continued overnight at 4°C. 1M glycine (400 μl) was added, and mixing continued at room temperature for 1 hour. Next, the mixture was spun down at 8,000 g for 1 minute. The precipitate was then resuspended in 100 μl of 10% BSA in PBS and mixed at room temperature for 45 minutes. The mixture was spun down at 8,000 g for 1 minute, and the supernatant was aspirated. Next, the beads (pellets) to which the exosomes were attached were resuspended in 20 μl of 2% BSA in PBS, and then immunolabeled with CD47, CD63, CD81, CD9, CD29, CD90, or isotype control. Exosomes bound to beads were incubated in 20 μl volumes with 1 μl of anti-CD47 antibody (eBiosciences, catalog no. 14-0479), 1 μl of anti-CD63 antibody (BD Biosciences, catalog no. 556019), 1 μl of anti-CD-81 antibody (BD Biosciences, catalog no. 555675), 1 μl of anti-CD9 antibody (Sigma, catalog no. SAB4700092), 1 μl of anti-CD29 immunization (Biolegend, catalog no. 303001), 1 μl of anti-CD90 immunization a (Biolegend, catalog no. 328101), or 1 μl of mouse IgG1, κ isotype control immunization (BD Biosciences, catalog no. 555746), and mixed at room temperature for 30 minutes. Next, the mixture was centrifuged at 8,000 g for 1 minute, the supernatant was aspirated, and the pellet was resuspended in 20 μl of 2% BSA in PBS. Then, 1 μl of secondary antibody (Invitrogen, catalog number A21202) was added to the sample and isotype control. Then, all samples were mixed at room temperature for 1 hour. Next, the samples were centrifuged at 8,000 g for 1 minute, the supernatant was aspirated, and the pellet was resuspended in 200 μl of 2% BSA in PBS. The exosomes bound to the beads were washed three times with 2% BSA in PBS.The expression of exosome markers (CD9, CD63, CD81, CD47) and mesenchymal markers (CD29, CD90) was analyzed using an LSR Fortessa X-20 cell analyzer. Data were analyzed using FlowJoR software (TreeStar Inc.). Flow cytometry data were acquired side-by-side for both isotype controls and samples from each experiment. Flow cytometry experiments were independently repeated twice using the same exosome preparation.

[0200] All methods disclosed and claimed herein can be prepared and performed in light of this disclosure without excessive experimentation. While the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods and steps or sequences of steps of the methods herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be understood that the same or similar results can be achieved by substituting the agents described herein with certain chemically and physiologically relevant agents. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to fall within the spirit, scope, and concept of the invention as defined by the appended claims.

[0201] [References] The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement those presented herein. International Publication WO 00 / 37504 International Publication WO 01 / 14424 International Publication WO 98 / 42752 International Publication No. WO1995001994 International Publication No. WO1998042752 International Publication No. WO2000037504 International Publication No. WO2001014424 International Publication No. WO2006 / 121168 International Publication No. WO2009 / 101611 International Publication No. WO2009 / 114335 International Publication No. WO2010 / 027827 International Publication No. WO2011 / 066342 International Publication No. WO2015016718 U.S. Patent No. 4,870,287 U.S. Patent No. 5,760,395 U.S. Patent No. 5,844,905 U.S. Patent No. 5,885,796 U.S. Patent No. 8,008,449 U.S. Patent No. 8,017,114 U.S. Patent No. 8,119,129 U.S. Patent No. 8,329,867 U.S. Patent No. 8,354,509 U.S. Patent No. 8,735,553 U.S. Patent Publication No. 20110008369 U.S. Published Patent Number 2014022021 U.S. Patent Publication No. 20140294898

Claims

1. A method for producing exosomes from mesenchymal stem cells (MSCs): (a) Culturing MSCs in a functionally closed hollow fiber bioreactor in a medium containing human platelet lysate (PLT) until 80–90% confluence; (b) Culturing the cells in a medium that is essentially free of PLT; (c) Collecting one or more conditioned medium fractions from the hollow fiber bioreactor (where the conditioned medium fractions are collected every 24 to 48 hours); and (d) Isolating exosomes from the conditioned medium fraction. Includes, The aforementioned MSCs are seeded at a density of 100 to 1000 cells / cm². A method comprising continuously adding fresh culture medium for step (a) to the MSC in the hollow fiber bioreactor.

2. The method according to claim 1, wherein each acclimatized culture medium fraction is stored at -80°C after collection.

3. The method according to claim 2, wherein the conditioned medium fraction is thawed and pooled before step (d).

4. The method according to claim 1, wherein the MSC is further defined as bone marrow-derived MSC.

5. The method according to claim 1, wherein the MSC is further defined as adipose-derived MSC.

6. Prior to step (a), at least 1 × 10 of the hollow fiber bioreactor is added. ７ The method according to any one of claims 1 to 5, further comprising sowing individual MSCs.

7. The method according to any one of claims 1 to 6, wherein the culture medium of step (a) has a concentration of 5% PLT.

8. The method according to any one of claims 1 to 7, wherein the cells are cultured in 5% oxygen.

9. The method according to any one of claims 1 to 8, wherein the culture in step (a) is performed for 5 to 10 days.

10. The method according to claim 9, wherein the culture in step (a) is performed for 8 days.

11. The method according to any one of claims 1 to 10, wherein the MSCs are cultured to 85-90% confluence.

12. The method according to any one of claims 1 to 11, wherein the culture in step (b) is 24 to 72 hours.

13. The method according to claim 12, wherein the culture in step (b) is 48 hours.

14. The method according to any one of claims 1 to 13, wherein the culture medium in step (b) does not contain PLT.

15. The method according to any one of claims 1 to 14, wherein the MSC is washed between steps (a) and (b).

16. The method according to any one of claims 1 to 15, wherein the MSCs are cultured in a serum-free standard medium.

17. The method according to any one of claims 1 to 16, wherein steps (a) to (d) are carried out under serum-free conditions.

18. The method according to any one of claims 1 to 17, wherein the conditioned culture medium fraction is collected in a sealed bag.

19. The method according to any one of claims 1 to 18, wherein the conditioned medium fraction is collected every 48 hours.

20. The method according to any one of claims 1 to 19, wherein each of the conditioned medium fractions has a volume of 200 to 300 mL.

21. The method according to any one of claims 1 to 20, wherein the acclimatized culture medium fraction is collected for 10 to 14 days.

22. The method according to any one of claims 1 to 21, wherein the acclimatized culture medium fraction is collected for 12 days.

23. The method according to any one of claims 1 to 22, wherein at least five conditioned medium fractions are collected.

24. The method according to any one of claims 1 to 23, wherein steps (a) to (d) are carried out in less than three weeks.

25. Each conditioned medium fraction is 9 × 10 １１ ~50 x 10 １１ The method according to any one of claims 1 to 24, comprising exosomes.

26. In step (c), at least 10 × 10 １２ The method according to any one of claims 1 to 25, wherein a number of exosomes are isolated.

27. In step (c), at least 15 × 10 １２ The method according to any one of claims 1 to 26, wherein individual exosomes are isolated.

28. The method according to any one of claims 1 to 27, wherein isolation comprises filtering and ultracentrifugation of a pooled fraction to obtain an exosome-containing pellet and resuspending the exosome-containing pellet in a buffer.

29. The method according to claim 28, wherein isolation is performed in a functionally closed manner using a pump and a heat-sealed tube.

30. The method according to claim 28, wherein filtration is further defined as passing the pooled fraction through a 0.2 μm filter.

31. The method according to claim 28, wherein isolation further comprises a centrifugation step prior to filtration to remove large cell debris.

32. The method according to any one of claims 28 to 31, wherein isolation is performed at 4°C.

33. The method according to claim 28, wherein the buffer solution comprises 0.09 M sodium chloride, 0.23 M sodium gluconate, 0.27 M sodium acetate trihydrate, 5 mM potassium chloride, and 3 mM magnesium chloride.

34. The method according to claim 28 or 33, wherein the pH of the buffer solution is 7.

4.

35. The method according to claim 34, wherein the buffer solution is PLASMA-LYTE A (registered trademark).

36. The method according to any one of claims 1 to 35, further comprising loading a therapeutic agent into the exosome.

37. The method according to claim 36, wherein the therapeutic agent comprises one or more cytokines, chemotherapeutic agents, nucleic acids, small molecules, or proteins.

38. The method according to claim 37, wherein the nucleic acid comprises DNA and / or RNA.

39. The method according to claim 38, wherein the RNA is siRNA, miRNA, or shRNA.

40. The method according to claim 38, wherein the RNA is siRNA.

41. The method according to any one of claims 36 to 40, wherein the loading includes electroporating the exosome.

42. The method according to claim 41, wherein electroporation is performed using PLASMA-LYTE A (registered trademark).

43. The method according to claim 42, wherein the method does not involve washing the exosomes or exchanging the buffer between step (d) and electroporation.

44. The method according to claim 43, wherein the number of loaded exosomes does not decrease by more than 20% relative to the number of exosomes in step (d).

45. A method for producing a pharmaceutical composition, comprising the step of producing exosomes by the method described in any one of claims 1 to 44.

46. A method for producing the pharmaceutical composition according to claim 45, for administering to a target to treat cancer.

47. A method for producing the pharmaceutical composition according to claim 46, wherein the subject is a human.

48. A method for producing a pharmaceutical composition according to claim 46 or 47, wherein electroporated exosomes are directly injected into the target.

49. A method for producing a pharmaceutical composition according to any one of claims 46 to 48, further comprising subjecting to at least a second anti-cancer therapy.

50. A method for producing the pharmaceutical composition according to claim 49, wherein the at least second anticancer therapy includes chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

51. A method for delivering RNA to cells in vitro, comprising administering to cells in vitro an exosome carrying an effective amount of RNA produced by the method described in any one of claims 36 to 44.

52. The method according to claim 51, wherein the cells are human cells.

53. The method according to claim 52, wherein the cells are cancer cells or T cells.

54. The process includes a step of producing an exosome by the method described in any one of claims 1 to 44, A method for producing a pharmaceutical composition containing an effective amount of the exosomes produced, for treating a disease or disorder by administering the exosomes to a subject in need.

55. A method for producing the pharmaceutical composition according to claim 54, wherein the exosome is loaded with siRNA or miRNA.

56. A method for producing the pharmaceutical composition according to claim 54 or 55, wherein the disease or disorder is cancer, an inflammatory disorder, or an immune-related disorder.

57. A method for producing the pharmaceutical composition according to claim 56, wherein the cancer is lung cancer.

58. A method for producing the pharmaceutical composition according to any one of claims 54 to 57, wherein KRAS siRNA is mounted in the exosome.

59. A method for producing the pharmaceutical composition according to any one of claims 54 to 58, wherein the subject is a human.

60. A method for producing the pharmaceutical composition according to any one of claims 54 to 59, administered orally, topically, intravenously, intraperitoneally, intramuscularly, endoscopically, percutaneously, subcutaneously, topically, or by direct injection.

61. A method for producing the pharmaceutical composition according to claim 60, which is administered intravenously.

62. A method for producing a pharmaceutical composition according to any one of claims 54 to 61, further comprising subjecting the subject to at least a second therapy.

63. A method for producing a pharmaceutical composition according to claim 62, wherein at least the second therapy is an anticancer therapy.

64. A method for producing the pharmaceutical composition according to claim 63, wherein the anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

65. A step comprising producing an exosome by the method described in any one of Claims 1 to 44, A method for producing a pharmaceutical composition containing a therapeutically effective amount of exosomes derived from MSCs, for administering the exosomes to a subject suffering from an immune-mediated inflammatory disease to treat the disease.

66. A method for producing the pharmaceutical composition according to claim 65, wherein the immune-mediated inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and Crohn's disease.

67. A method for producing the pharmaceutical composition according to claim 65 or 66, wherein the MSCs are of the same type but different in composition.

68. A method for producing the pharmaceutical composition according to any one of claims 65 to 67, which is administered systemically or locally.

69. A method for producing the pharmaceutical composition according to any one of claims 65 to 68, administered via the rectum, nose, cheek, vagina, subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intra-articular, synovial, intrasternal, intrathecal, intralesional, or intracranial pathway, or via an implantable reservoir.

70. A method for producing the pharmaceutical composition according to any one of claims 65 to 69, which is administered in combination with at least one further therapeutic agent.

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