Use of Exosomes for the Treatment of Disease

Abstract

To provide use of exosomes for treatment of disease.SOLUTION: The present invention provides lipid-based nanoparticles (e.g., liposomes or exosomes) having CD47 on their surface and comprising a therapeutic agent (e.g., a therapeutic protein, an antibody, an inhibitory RNA, and / or a small molecule drug). Furthermore, the present invention provides use of such lipid-based nanoparticles in therapy. In one embodiment, pharmaceutical compositions are provided that comprise a lipid-based nanoparticle and an excipient, where the lipid-based nanoparticle comprises CD47 on its surface and comprises a therapeutic agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 173,838, filed Jun. 10, 2015, the entire contents of which are incorporated herein by reference.

[0002] BACKGROUND OF THEINVENTION 1. FIELD OF THEINVENTION The present invention relates generally to the fields of medicine and oncology. More particularly, the present invention relates to the use of exosomes in methods of treatment. [Background technology]

[0003] 2. Description of the Prior Art Exosomes are small (40–150 nm) membrane vesicles with a lipid bilayer of endosomal origin that are released by all cells in the body (Kowal et al., 2014; El-Andaloussi et al., 2013; Thery et al., 2002). Exosomes contain proteins, lipids, They contain mRNA, microRNA (miRNA) and genomic DNA (Valadi et al., 2007; Peinado et al., 2012; Luga et al., 2012; Kahlert et al., 2014). Unlike liposomes and other synthetic drug nanoparticle carriers, exosomes contain many transmembrane and membrane anchor proteins that may enhance endocytosis and / or direct fusion with the plasma membrane of recipient cells and thus enhance cargo delivery (Marcus et al., 2013; van den Boorn et al., 2013; Johnsen et al., 2014). The natural plasma membrane-like phospholipid composition (including cytoplasmic phosphatidylserine and cholesterol) and membrane-associated protein composition of exosomes also provide superior stability in the systemic circulation compared to synthetic nanoparticles (e.g., liposomes) by reducing clearance from the circulation (in part due to lack of interactions with opsonins and coagulation and complement factors recognized by macrophages for phagocytosis) and minimizing immunogenic responses (Clayton et al., 2003; van der Meel et al., 2014). ; Gomes-da-Silva et al., 2012). These features may also minimize the cytotoxic effects observed when synthetic nanoparticles are used in vivo (Simoes et al., 2005). Finally, the endosomal and intercellular vesicular transport mechanisms involved in the generation of exosomes may also be used in exosomes taken up by recipient cells, possibly enhancing the release (and incorporation into the RNAi gene silencing machinery) of the cargo, thus enhancing the efficacy of any therapeutic agent (e.g., gene-targeting). Recent studies have evaluated the efficacy of exosomes as RNAi carriers for therapy, and have shown that systemic injection of exosomes allows the delivery of siRNA into the brain, leading to robust downregulation of target genes (Cooper et al., 2014; Alvarez-Erviti et al., 2011). Furthermore, it has been reported that human plasma-derived exosomes also enable RNAi delivery to recipient cells (Wahlgren et al., 2012), thereby supporting their potential therapeutic utility in delivering RNAi to modify gene expression in target cells. Single nucleotide variation in KRAS G12D / R / V Kras mutations have been found in up to 96% of pancreatic tumors (Chang et al., 2014), and Kras mutations are a major driver of pancreatic malignancies. It is believed to be an early neoplastic event that drives and maintains sex reversal (Ying et al., 2012; Collin et al., 2012; Collins et al., 2012; Smakman et al., 2005). RNAi-based targeting of Kras expression and downstream signaling using nanoparticles was recently reported to reduce tumor burden in lung and colorectal cancer models (Pecot et al., Unlike efforts focused on the specific targeting of oncogenic Kras, these approaches may induce cytotoxic effects that require careful dosing and monitoring. Specific targeting of oncogenic Kras has been limited to delivery by electroporation (Rejiba et al., 2007) or biopolymer implantation (Zorde Khvalevsky et al., 2013) in xenograft models of pancreatic cancer. There is a need for improved techniques for delivering therapeutic or diagnostic agents. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kowal et al., Biogenesis and secretion of exosomes. Current Opinion in Cell Biology, 29:116-125, 2014. [Non-Patent Document 2] El-Andaloussi et al., Extracellular vesicles: biology and emerging therapeutic opportunities. Nature Reviews Drug Discovery, 12:347-357, 2013. [Non-Patent Document 3] Thery et al., Exosomes: composition, biogenesis and function. Nature Reviews Immunology, 2:569-579, 2002. [Non-Patent Document 4] Valadi et al., Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature Cell Biology, 9:654-659, 2007. [Non-Patent Document 5] Peinado et al., Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nature Medicine, 18:883-891, 2012. [Non-Patent Document 6] Luga et al., Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell, 151:1542-1556, 2012. [Non-Patent Document 7] Kahlert et al., Identification of Double Stranded Genomic DNA Spanning all Chromosomes with Mutated KRAS and p53 DNA in the Serum Exosomes of Patients with Pancreatic Cancer. The Journal of biological chemistry 2014. [Non-Patent Document 8] Marcus and Leonard, FedExosomes: Engineering Therapeutic Biological Nanoparticles that Truly Deliver. Pharmaceuticals (Basel), 6:659-680, 2013. [Non-Patent Document 9] van den Boorn et al., Exosomes as nucleic acid nanocarriers. Advanced Drug Delivery Reviews, 65:331-335, 2013. [Non-Patent Document 10] Johnsen et al., A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy. Biochimica et Biophysica Acta, 1846:75-87, 2014. [Non-Patent Document 11] Clayton et al., Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59. European Journal of Immunology, 33:522-531, 2003. [Non-Patent Document 12] van der Meel et al., Extracellular vesicles as drug delivery systems: Lessons from the liposome field. Journal of Controlled Release, 195:72-85, 2014. [Non-Patent Document 13] Gomes-da-Silva et al., Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges. Accounts of Chemical Research, 45:1163-1171, 2012. [Non-Patent Document 14] Simoes et al., Cationic liposomes for gene delivery. Expert Opinion on Drug Delivery, 2:237-254, 2005. [Non-Patent Document 15] Cooper et al., Systemic exosomal siRNA delivery reduced alpha-synuclein aggregates in brains of transgenic mice. Movement Disorders, 29:1476-1485, 2014. [Non-Patent Document 16] Alvarez-Erviti et al., Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotechnology, 29:341-345, 2011. [Non-Patent Document 17] Wahlgren et al., Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic Acids Research, 40:e130, 2012. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention Methods and drugs are provided that use engineered liposomes and exosomes as delivery systems for treating cancer.

[0006] In one embodiment, a pharmaceutical composition is provided that includes a lipid-based nanoparticle and an excipient, the lipid-based nanoparticle comprising CD47 on its surface, and the lipid-based nanoparticle comprising a therapeutic agent. In some aspects, the lipid-based nanoparticle is a liposome or an exosome. In certain aspects, the exosome is isolated from a cell that overexpresses CD47. In some aspects, the exosome is isolated from a patient in need of treatment. In some aspects, the exosome is isolated from a fibroblast. In some aspects, the liposome is a single lamellar liposome. In some aspects, the liposome is a multilamellar liposome.

[0007] In various embodiments, the therapeutic agent is a therapeutic protein, an antibody (e.g., a full-length antibody, a monoclonal antibody, a scFv, a Fab fragment, a F(ab')2, a diabody, a triabody, or a minibody), an inhibitory RNA, or a small molecule drug. In some embodiments, the therapeutic protein is a protein whose loss or inactivation is known to be associated with the disease being treated, such as, for example, a tumor suppressor, a kinase, a phosphatase, or a transcription factor. In some embodiments, the antibody binds to an intracellular antigen. Such an intracellular antigen can be a protein whose activity is necessary for cell growth and / or survival, such as an oncogene. In some cases, the antibody prevents the function of the antigen. In some cases, the antibody disrupts a protein-protein interaction. In some embodiments, the inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA. In various embodiments, the inhibitory RNA prevents the expression of a protein whose activity is necessary for the maintenance of a certain pathology, such as, for example, an oncogene. When the oncogene is a mutant form of a gene, the inhibitory RNA can preferentially block the expression of the mutant oncogene and not the wild-type protein.In some embodiments, the small molecule drug is an imaging agent.In some embodiments, the small molecule drug is a chemotherapeutic agent.

[0008] In some aspects, the compositions are formulated for parenteral administration, such as, for example, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0009] In some embodiments, the composition includes an antibacterial agent, such as benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine. , imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenlymercuric nitrate, propylene glycol, or thimerosal It may be.

[0010] In some embodiments, a single lipid-based nanoparticle comprises more than one agent, such as a therapeutic agent and a diagnostic agent, more than one therapeutic agent, or more than one diagnostic agent.

[0011] In one embodiment, a method is provided for treating a disease in a patient in need thereof, comprising administering to the patient any of the compositions of the present embodiments, thereby treating the disease in the patient. In some aspects, the disease is cancer. In some aspects, the patient is a human. In some aspects, the patient has previously had a tumor surgically removed.

[0012] In some embodiments, the therapeutic agent is an inhibitory RNA that targets an oncogene. In certain embodiments, the inhibitory RNA targets Kras G12D In some aspects, the therapeutic agent is a tumor suppressor protein.

[0013] In some embodiments, the method further comprises administering at least a second therapy to the patient. In various embodiments, the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.

[0014] In one embodiment, a method is provided for treating a disease in a patient in need thereof, comprising electroporating liposomes or exosomes with a therapeutic agent (e.g., a monoclonal antibody) and providing the electroporated liposomes or exosomes to the patient, thereby treating the disease in the patient. In some aspects, the liposomes or exosomes comprise CD47 on their surface. In some aspects, the disease is cancer. In some aspects, the monoclonal antibody specifically or selectively binds to an intracellular antigen.

[0015] In one embodiment, a method is provided for administering a therapeutic protein to a patient in need thereof, the method comprising transfecting exosomes with nucleic acid (e.g., DNA or RNA) encoding the therapeutic protein (e.g., a monoclonal antibody or antigen-binding fragment thereof), incubating the transfected exosomes under conditions that allow the therapeutic protein to be expressed in the exosomes, and providing the incubated exosomes to the patient, thereby administering the therapeutic protein to the patient.

[0016] In one embodiment, a method is provided for administering a therapeutic antibody to a cell, the method comprising contacting the cell with a lipid-based nanoparticle comprising an antibody that specifically or selectively binds to an intracellular antigen. In some cases, the cell is contained within a patient, and the method comprises administering the lipid-based nanoparticle to the patient.

[0017] In one embodiment, a method for treating cancer in a patient includes administering to the patient a therapeutically effective amount of a lipid-based nanoparticle, the nanoparticle inhibiting a mutant Kras (e.g., a Kras G12DIn some embodiments, the cancer is lung cancer, colorectal cancer, or pancreatic cancer. In some embodiments, the cancer is pancreatic ductal adenocarcinoma. In some embodiments, the lipid-based nanoparticle is a liposome or an exosome. In certain embodiments, the exosome is derived from the patient's own cells. In some embodiments, the lipid-based nanoparticle comprises CD47 on its surface. In some embodiments, the inhibitory RNA is an siRNA or shRNA. In some embodiments, the inhibitory RNA is an inhibitor of Kras G12D To facilitate specific targeting of mRNA, the inhibitory RNA sequence is designed to contain a specific G to A nucleotide bias within the target region (e.g., as found in SEQ ID NO:1). In some embodiments, the inhibitory RNA comprises a target region having a sequence according to SEQ ID NO:2.

[0018] In one embodiment, a composition is provided that includes lipid-based nanoparticles and an excipient for use in treating a disease in a patient. In some aspects, the lipid-based nanoparticles include CD47 on their surface. In some aspects, the lipid-based nanoparticles include a therapeutic agent. In some aspects, the disease can be cancer. In some aspects, the therapeutic agent is an inhibitory RNA that targets an oncogene. In some aspects, the inhibitory RNA targets Kras G12D In some embodiments, the therapeutic agent is a tumor suppressor protein. In some embodiments, the composition further comprises at least a second therapy. In some embodiments, the second therapy is a surgical therapy, a chemotherapy, a radiation therapy, a cryotherapy, a hormone therapy, or an immunotherapy. In some embodiments, the patient is a human.

[0019] In some aspects, the compositions are formulated for parenteral administration, such as, for example, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0020] In some embodiments, the composition includes an antibacterial agent, which may be benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

[0021] In one embodiment, there is provided a use of lipid-based nanoparticles in the manufacture of a medicament for treating a disease. In some aspects, the lipid-based nanoparticles comprise CD47 on their surface. In some aspects, the lipid-based nanoparticles comprise a therapeutic agent. In some aspects, the disease is cancer. In some aspects, the therapeutic agent is an inhibitory RNA that targets an oncogene. In some aspects, the inhibitory RNA targets Kras G12D In some aspects, the therapeutic agent is a tumor suppressor protein.

[0022] In some aspects, the medicaments are formulated for parenteral administration, such as, for example, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0023] In some embodiments, the pharmaceutical agent comprises an antibacterial agent, which may be benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

[0024] As used herein, "essentially free" with respect to a specified component is used herein to mean that none of the specified components are intentionally formulated in the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the specified component resulting from any unintended contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which no amount of the specified component can be detected using standard analytical methods.

[0025] As used herein, the specification "a" or "an" may mean one or more. When used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0026] Although use of the term "or" in the claims is used to indicate the meaning of "and / or" unless expressly stated to refer to alternatives only or the alternatives are mutually exclusive, the present disclosure supports the definition to refer to alternatives only, as well as "and / or." As used herein, "another" can mean at least a second or more.

[0027] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the test subjects.

[0028] Other objects, features and advantages of the present invention will become apparent from the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and the specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description of the invention. In certain embodiments, for example, the following are provided: (Item 1) A pharmaceutical composition comprising a lipid-based nanoparticle and an excipient, wherein the lipid-based nanoparticle comprises CD47 on its surface, and the lipid-based nanoparticle comprises a therapeutic agent. (Item 2) 2. The composition of claim 1, wherein the lipid-based nanoparticle is a liposome or an exosome. (Item 3) 3. The composition of claim 2, wherein the exosomes are isolated from cells that overexpress CD47. (Item 4) 3. The composition of claim 2, wherein the exosomes are isolated from fibroblasts. (Item 5) 3. The composition according to item 2, wherein the liposome is a unilamellar liposome. (Item 6) 3. The composition according to item 2, wherein the liposome is a multilamellar liposome. (Item 7) 2. The composition of claim 1, wherein the therapeutic agent is a therapeutic protein, an antibody, an inhibitory RNA, or a small molecule drug. (Item 8) The composition of claim 7, wherein the antibody binds to an intracellular antigen. (Item 9) 8. The composition of claim 7, wherein the antibody is a full length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody. (Item 10) 8. The composition of claim 7, wherein the inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA. (Item 11) 8. The composition of claim 7, wherein the therapeutic protein is a kinase, phosphatase, or transcription factor. (Item 12) 8. The composition of claim 7, wherein the small molecule drug is an imaging agent. (Item 13) 2. The composition of claim 1, formulated for parenteral administration. (Item 14) 14. The composition of item 13, formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. (Item 15) 14. The composition of claim 13, further comprising an antibacterial agent. (Item 16) 16. The composition of claim 15, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. (Item 17) 17. A method for treating a disease in a patient in need thereof, comprising administering to said patient a composition according to any one of items 1 to 16, thereby treating said disease in said patient. (Item 18) 18. The method of claim 17, wherein the disease is cancer. (Item 19) The method according to item 18, wherein the therapeutic agent is an inhibitory RNA that targets an oncogene. The inhibitory RNA is a Kras G12D 20. The method according to item 19, which targets (Item 21) 19. The method of claim 18, wherein the therapeutic agent is a tumor suppressor protein. (Item 22) 18. The method of claim 17, further comprising administering at least a second therapy to the patient. (Item 23) 23. The method of claim 22, wherein the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy. (Item 24) 18. The method of claim 17, wherein the patient is a human. (Item 25) 1. A composition comprising lipid-based nanoparticles and an excipient for use in treating a disease in a patient, wherein the lipid-based nanoparticles comprise CD47 on their surface, and the lipid-based nanoparticles comprise a therapeutic agent. (Item 26) 26. The composition according to item 25, wherein the disease is cancer. (Item 27) 27. The composition of claim 26, wherein the therapeutic agent is an inhibitory RNA that targets an oncogene. (Item 28) The inhibitory RNA is a Kras G12D 28. The composition according to item 27, which targets (Item 29) 27. The composition of claim 26, wherein the therapeutic agent is a tumor suppressor protein. (Item 30) 26. The composition of item 25, formulated for parenteral administration. (Item 31) 31. The composition of item 30, formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. (Item 32) 31. The composition of claim 30, further comprising an antibacterial agent. (Item 33) 33. The composition of claim 32, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. (Item 34) 26. The composition of item 25, further comprising at least a second therapy. (Item 35) 35. The composition of claim 34, wherein the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy. (Item 36) 26. The composition of claim 25, wherein the patient is a human. (Item 37) 16. Use of lipid-based nanoparticles in the manufacture of a medicament for treating a disease, wherein the lipid-based nanoparticles comprise CD47 on their surface, and the lipid-based nanoparticles comprise a therapeutic agent. (Item 38) 38. The use according to item 37, wherein the disease is cancer. (Item 39) The use according to item 38, wherein the therapeutic agent is an inhibitory RNA that targets an oncogene. The inhibitory RNA is a Kras G12D 40. The use according to item 39, which targets (Item 41) 39. The use of item 38, wherein the therapeutic agent is a tumor suppressor protein. (Item 42) 38. The use according to item 37, wherein the medicament is formulated for parenteral administration. (Item 43) 43. The use according to item 42, wherein the medicament is formulated for intravenous, intramuscular, subcutaneous or intraperitoneal injection. (Item 44) 38. The use according to item 37, wherein the medicament comprises an antibacterial agent. (Item 45) 45. The use according to item 44, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

[0029] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0030] [Figure 1A]Figure 1A-F shows that targeting KrasG12D mediated by siRNA / shRNA packaged in exosomes induces cancer cell death. (Figure 1A) Quantification of confocal micrographs of Panc-1 cells stained with SYTOX® Green nuclear label and visualization of internalized exosomes (exos) and liposomes (lipos) containing Alexa Fluor® 647-tagged siRNA. Panc-1 cells were exposed to Alexa fluor 647-tagged siRNA containing exos or lipos after preincubation with and without proteinase K or trypsin. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. (Fig. 1B-C) Real-time PCR analysis of KRASG12D (Fig. 1B) or wild-type KRAS (Fig. 1C) transcript levels in Panc-1 cells treated for 3 h with siKrasG12D or shKrasG12D containing exos or lipos, siScrbl or shScrbl containing exos or lipos, or non-electroporated exos (empty cargo, control exos). Fold change relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1, is shown. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (Fig. 1D) Western blotting for phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK) and actin (loading control) of lysates from untreated Panc-1 (control) and Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos. (FIG. 1E) Relative number of Panc-1 cells over time after exposure to the listed treatments. (FIG. 1F) Quantification of immunostained micrographs performed for the apoptotic marker TUNEL in Panc-1 cells exposed to the listed treatments. Puromycin was used as a positive control. (0) indicates that no cells positive for TUNEL were detected.Control: untreated control exos: non-electroporated (no siRNA cargo) exos. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. Means are shown with + / - SEM. Statistical significance was determined using one-way ANOVA unless otherwise specified. ***p<0.01, ****p<0.001, ns: not significant. [Figure 1B]Figure 1A-F shows that targeting KrasG12D mediated by siRNA / shRNA packaged in exosomes induces cancer cell death. (Figure 1A) Quantification of confocal micrographs of Panc-1 cells stained with SYTOX® Green nuclear label and visualization of internalized exosomes (exos) and liposomes (lipos) containing Alexa Fluor® 647-tagged siRNA. Panc-1 cells were exposed to Alexa fluor 647-tagged siRNA containing exos or lipos after preincubation with and without proteinase K or trypsin. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. (Fig. 1B-C) Real-time PCR analysis of KRASG12D (Fig. 1B) or wild-type KRAS (Fig. 1C) transcript levels in Panc-1 cells treated for 3 h with siKrasG12D or shKrasG12D containing exos or lipos, siScrbl or shScrbl containing exos or lipos, or non-electroporated exos (empty cargo, control exos). Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (Fig. 1D) Western blotting for phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK) and actin (loading control) of lysates from untreated Panc-1 (control) and Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos. (FIG. 1E) Relative number of Panc-1 cells over time after exposure to the listed treatments. (FIG. 1F) Quantification of immunostained micrographs performed for the apoptotic marker TUNEL in Panc-1 cells exposed to the listed treatments. Puromycin was used as a positive control. (0) indicates that no cells positive for TUNEL were detected.Control: untreated control exos: non-electroporated (no siRNA cargo) exos. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. Means are shown with + / - SEM. Statistical significance was determined using one-way ANOVA unless otherwise specified. ***p<0.01, ****p<0.001, ns: not significant. [Figure 1C]Figure 1A-F shows that targeting KrasG12D mediated by siRNA / shRNA packaged in exosomes induces cancer cell death. (Figure 1A) Quantification of confocal micrographs of Panc-1 cells stained with SYTOX® Green nuclear label and visualization of internalized exosomes (exos) and liposomes (lipos) containing Alexa Fluor® 647-tagged siRNA. Panc-1 cells were exposed to Alexa fluor 647-tagged siRNA containing exos or lipos after preincubation with and without proteinase K or trypsin. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. (Fig. 1B-C) Real-time PCR analysis of KRASG12D (Fig. 1B) or wild-type KRAS (Fig. 1C) transcript levels in Panc-1 cells treated for 3 h with siKrasG12D or shKrasG12D containing exos or lipos, siScrbl or shScrbl containing exos or lipos, or non-electroporated exos (empty cargo, control exos). Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (Fig. 1D) Western blotting for phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK) and actin (loading control) of lysates from untreated Panc-1 (control) and Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos. (FIG. 1E) Relative number of Panc-1 cells over time after exposure to the listed treatments. (FIG. 1F) Quantification of immunostained micrographs performed for the apoptotic marker TUNEL in Panc-1 cells exposed to the listed treatments. Puromycin was used as a positive control. (0) indicates that no cells positive for TUNEL were detected.Control: untreated control exos: non-electroporated (no siRNA cargo) exos. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. Means are shown with + / - SEM. Statistical significance was determined using one-way ANOVA unless otherwise specified. ***p<0.01, ****p<0.001, ns: not significant. [Figure 1D]Figure 1A-F shows that targeting KrasG12D mediated by siRNA / shRNA packaged in exosomes induces cancer cell death. (Figure 1A) Quantification of confocal micrographs of Panc-1 cells stained with SYTOX® Green nuclear label and visualization of internalized exosomes (exos) and liposomes (lipos) containing Alexa Fluor® 647-tagged siRNA. Panc-1 cells were exposed to Alexa fluor 647-tagged siRNA containing exos or lipos after preincubation with and without proteinase K or trypsin. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. (Fig. 1B-C) Real-time PCR analysis of KRASG12D (Fig. 1B) or wild-type KRAS (Fig. 1C) transcript levels in Panc-1 cells treated for 3 h with siKrasG12D or shKrasG12D containing exos or lipos, siScrbl or shScrbl containing exos or lipos, or non-electroporated exos (empty cargo, control exos). Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (Fig. 1D) Western blotting for phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK) and actin (loading control) of lysates from untreated Panc-1 (control) and Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos. (FIG. 1E) Relative number of Panc-1 cells over time after exposure to the listed treatments. (FIG. 1F) Quantification of immunostained micrographs performed for the apoptotic marker TUNEL in Panc-1 cells exposed to the listed treatments. Puromycin was used as a positive control. (0) indicates that no cells positive for TUNEL were detected.Control: untreated control exos: non-electroporated (no siRNA cargo) exos. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. Means are shown with + / - SEM. Statistical significance was determined using one-way ANOVA unless otherwise specified. ***p<0.01, ****p<0.001, ns: not significant. [Figure 1E]Figure 1A-F shows that targeting KrasG12D mediated by siRNA / shRNA packaged in exosomes induces cancer cell death. (Figure 1A) Quantification of confocal micrographs of Panc-1 cells stained with SYTOX® Green nuclear label and visualization of internalized exosomes (exos) and liposomes (lipos) containing Alexa Fluor® 647-tagged siRNA. Panc-1 cells were exposed to Alexa fluor 647-tagged siRNA containing exos or lipos after preincubation with and without proteinase K or trypsin. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. (Fig. 1B-C) Real-time PCR analysis of KRASG12D (Fig. 1B) or wild-type KRAS (Fig. 1C) transcript levels in Panc-1 cells treated for 3 h with siKrasG12D or shKrasG12D containing exos or lipos, siScrbl or shScrbl containing exos or lipos, or non-electroporated exos (empty cargo, control exos). Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (Fig. 1D) Western blotting for phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK) and actin (loading control) of lysates from untreated Panc-1 (control) and Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos. (FIG. 1E) Relative number of Panc-1 cells over time after exposure to the listed treatments. (FIG. 1F) Quantification of immunostained micrographs performed for the apoptotic marker TUNEL in Panc-1 cells exposed to the listed treatments. Puromycin was used as a positive control. (0) indicates that no cells positive for TUNEL were detected.Control: untreated control exos: non-electroporated (no siRNA cargo) exos. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. Means are shown with + / - SEM. Statistical significance was determined using one-way ANOVA unless otherwise specified. ***p<0.01, ****p<0.001, ns: not significant. [Figure 1F]Figure 1A-F shows that targeting KrasG12D mediated by siRNA / shRNA packaged in exosomes induces cancer cell death. (Figure 1A) Quantification of confocal micrographs of Panc-1 cells stained with SYTOX® Green nuclear label and visualization of internalized exosomes (exos) and liposomes (lipos) containing Alexa Fluor® 647-tagged siRNA. Panc-1 cells were exposed to Alexa fluor 647-tagged siRNA containing exos or lipos after preincubation with and without proteinase K or trypsin. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. (Fig. 1B-C) Real-time PCR analysis of KRASG12D (Fig. 1B) or wild-type KRAS (Fig. 1C) transcript levels in Panc-1 cells treated for 3 h with siKrasG12D or shKrasG12D containing exos or lipos, siScrbl or shScrbl containing exos or lipos, or non-electroporated exos (empty cargo, control exos). Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (Fig. 1D) Western blotting for phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK) and actin (loading control) of lysates from untreated Panc-1 (control) and Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos. (FIG. 1E) Relative number of Panc-1 cells over time after exposure to the listed treatments. (FIG. 1F) Quantification of immunostained micrographs performed for the apoptotic marker TUNEL in Panc-1 cells exposed to the listed treatments. Puromycin was used as a positive control. (0) indicates that no cells positive for TUNEL were detected.Control: untreated control exos: non-electroporated (no siRNA cargo) exos. Statistical significance between groups was determined using unpaired two-tailed Student's t-test. Means are shown with + / - SEM. Statistical significance was determined using one-way ANOVA unless otherwise specified. ***p<0.01, ****p<0.001, ns: not significant.

[0031] [Figure 2A]Figures 2A-G are graphs showing that treatment with exosomes containing si / shKrasG12D cargo results in sustained Panc-1 orthotopic tumor regression. (Figure 2A) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. PBS: n=7, control exos: n=6, siKrasG12D lipos: n=3, shKrasG12D lipos: n=3, siKrasG12D exos: n=7, shKrasG12D exos: n=7. Statistical test results comparing treatment groups to the PBS control group at day 42 post-cancer cell injection are shown, with the exception of the siKrasG12D exos group compared to the PBS group at the experimental endpoint (day 28 post-cancer cell injection). The top line pair is PBS and control Exos; the middle line pair is lipos; the bottom line pair is exos. (Figure 2B) Relative radiance of bioluminescent BxPC3 orthotopic tumors over time. PBS: n=3, control exos: n=3, siKrasG12D exos: n=3, shKrasG12D exos: n=3. Statistical test results comparing treatment groups to the PBS control group at 77 days after cancer cell injection are shown. (Figure 2C) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. Experimental groups started with PBS: n=7, control exos: n=6, siKrasG12D exos: n=7, shKrasG12D exos: n=7 and gradually decreased as mice became moribund and were euthanized (PBS and control exos groups). Small foci of cancer cells were seen in pancreata treated with shKrasG12D exos, but the majority of the pancreas was histologically unremarkable. The top line pair is PBS and control Exos; the bottom line pair is si / shKrasG12D exos. (Figure 2D) Comparative analysis of measured bioluminescence radiance 77 days after cancer cell injection for orthotopic Panc-1 tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2E) Quantification of p-ERK immunolabeling in pancreatic tumors in experimental groups (scale bar: 50 μm) and percent p-ERK staining. n=6.Note that quantification was performed on measurably smaller tumor areas in the shKrasG12D exos-treated group. Statistical significance between both groups was determined using an unpaired two-tailed Student's t-test. (Figure 2F) Tumor burden (relative pancreas mass to body weight) at the time of euthanasia (PBS: days 62-130, control exos: days 30-132, shKrasG12D exos: day 200) in the indicated experimental groups. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2G) Kaplan-Meier curve comparisons and statistical differences for mouse survival in the indicated experimental groups were assessed using the log-rank Mantel-Cox test, PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Means + / - SEM are shown. Statistical significance was determined using one-way ANOVA unless otherwise specified. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 2B]Figures 2A-G are graphs showing that treatment with exosomes containing si / shKrasG12D cargo results in sustained Panc-1 orthotopic tumor regression. (Figure 2A) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. PBS: n=7, control exos: n=6, siKrasG12D lipos: n=3, shKrasG12D lipos: n=3, siKrasG12D exos: n=7, shKrasG12D exos: n=7. Statistical test results comparing treatment groups to the PBS control group at day 42 post-cancer cell injection are shown, with the exception of the siKrasG12D exos group compared to the PBS group at the experimental endpoint (day 28 post-cancer cell injection). The top line pair is PBS and control Exos; the middle line pair is lipos; the bottom line pair is exos. (Figure 2B) Relative radiance of bioluminescent BxPC3 orthotopic tumors over time. PBS: n=3, control exos: n=3, siKrasG12D exos: n=3, shKrasG12D exos: n=3. Statistical test results comparing treatment groups to the PBS control group at 77 days after cancer cell injection are shown. (Figure 2C) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. Experimental groups started with PBS: n=7, control exos: n=6, siKrasG12D exos: n=7, shKrasG12D exos: n=7 and gradually decreased as mice became moribund and were euthanized (PBS and control exos groups). Small foci of cancer cells were seen in pancreata treated with shKrasG12D exos, but the majority of the pancreas was histologically unremarkable. The top line pair is PBS and control Exos; the bottom line pair is si / shKrasG12D exos. (Figure 2D) Comparative analysis of measured bioluminescence radiance 77 days after cancer cell injection for orthotopic Panc-1 tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2E) Quantification of p-ERK immunolabeling in pancreatic tumors in experimental groups (scale bar: 50 μm) and percent p-ERK staining. n=6.Note that quantification was performed on measurably smaller tumor areas in the shKrasG12D exos-treated group. Statistical significance between both groups was determined using an unpaired two-tailed Student's t-test. (Figure 2F) Tumor burden (relative pancreas mass to body weight) at the time of euthanasia (PBS: days 62-130, control exos: days 30-132, shKrasG12D exos: day 200) in the indicated experimental groups. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2G) Kaplan-Meier curve comparisons and statistical differences for mouse survival in the indicated experimental groups were assessed using the log-rank Mantel-Cox test, PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Means + / - SEM are shown. Statistical significance was determined using one-way ANOVA unless otherwise specified. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 2C]Figures 2A-G are graphs showing that treatment with exosomes containing si / shKrasG12D cargo results in sustained Panc-1 orthotopic tumor regression. (Figure 2A) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. PBS: n=7, control exos: n=6, siKrasG12D lipos: n=3, shKrasG12D lipos: n=3, siKrasG12D exos: n=7, shKrasG12D exos: n=7. Statistical test results comparing treatment groups to the PBS control group at day 42 post-cancer cell injection are shown, with the exception of the siKrasG12D exos group compared to the PBS group at the experimental endpoint (day 28 post-cancer cell injection). The top line pair is PBS and control Exos; the middle line pair is lipos; the bottom line pair is exos. (Figure 2B) Relative radiance of bioluminescent BxPC3 orthotopic tumors over time. PBS: n=3, control exos: n=3, siKrasG12D exos: n=3, shKrasG12D exos: n=3. Statistical test results comparing treatment groups to the PBS control group at 77 days after cancer cell injection are shown. (Figure 2C) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. Experimental groups started with PBS: n=7, control exos: n=6, siKrasG12D exos: n=7, shKrasG12D exos: n=7 and gradually decreased as mice became moribund and were euthanized (PBS and control exos groups). Small foci of cancer cells were seen in pancreata treated with shKrasG12D exos, but the majority of the pancreas was histologically unremarkable. The top line pair is PBS and control Exos; the bottom line pair is si / shKrasG12D exos. (Figure 2D) Comparative analysis of measured bioluminescence radiance 77 days after cancer cell injection for orthotopic Panc-1 tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2E) Quantification of p-ERK immunolabeling in pancreatic tumors in experimental groups (scale bar: 50 μm) and percent p-ERK staining. n=6.Note that quantification was performed on measurably smaller tumor areas in the shKrasG12D exos-treated group. Statistical significance between both groups was determined using an unpaired two-tailed Student's t-test. (Figure 2F) Tumor burden (relative pancreas mass to body weight) at the time of euthanasia (PBS: days 62-130, control exos: days 30-132, shKrasG12D exos: day 200) in the indicated experimental groups. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2G) Kaplan-Meier curve comparisons and statistical differences for mouse survival in the indicated experimental groups were assessed using the log-rank Mantel-Cox test, PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Means + / - SEM are shown. Statistical significance was determined using one-way ANOVA unless otherwise specified. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 2D]Figures 2A-G are graphs showing that treatment with exosomes containing si / shKrasG12D cargo results in sustained Panc-1 orthotopic tumor regression. (Figure 2A) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. PBS: n=7, control exos: n=6, siKrasG12D lipos: n=3, shKrasG12D lipos: n=3, siKrasG12D exos: n=7, shKrasG12D exos: n=7. Statistical test results comparing treatment groups to the PBS control group at day 42 post-cancer cell injection are shown, with the exception of the siKrasG12D exos group compared to the PBS group at the experimental endpoint (day 28 post-cancer cell injection). The top line pair is PBS and control Exos; the middle line pair is lipos; the bottom line pair is exos. (Figure 2B) Relative radiance of bioluminescent BxPC3 orthotopic tumors over time. PBS: n=3, control exos: n=3, siKrasG12D exos: n=3, shKrasG12D exos: n=3. Statistical test results comparing treatment groups to the PBS control group at 77 days after cancer cell injection are shown. (Figure 2C) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. Experimental groups started with PBS: n=7, control exos: n=6, siKrasG12D exos: n=7, shKrasG12D exos: n=7 and gradually decreased as mice became moribund and were euthanized (PBS and control exos groups). Small foci of cancer cells were seen in pancreata treated with shKrasG12D exos, but the majority of the pancreas was histologically unremarkable. The top line pair is PBS and control Exos; the bottom line pair is si / shKrasG12D exos. (Figure 2D) Comparative analysis of measured bioluminescence radiance 77 days after cancer cell injection for orthotopic Panc-1 tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2E) Quantification of p-ERK immunolabeling in pancreatic tumors in experimental groups (scale bar: 50 μm) and percent p-ERK staining. n=6.Note that quantification was performed on measurably smaller tumor areas in the shKrasG12D exos-treated group. Statistical significance between both groups was determined using an unpaired two-tailed Student's t-test. (Figure 2F) Tumor burden (relative pancreas mass to body weight) at the time of euthanasia (PBS: days 62-130, control exos: days 30-132, shKrasG12D exos: day 200) in the indicated experimental groups. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2G) Kaplan-Meier curve comparisons and statistical differences for mouse survival in the indicated experimental groups were assessed using the log-rank Mantel-Cox test, PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Means + / - SEM are shown. Statistical significance was determined using one-way ANOVA unless otherwise specified. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 2E]Figures 2A-G are graphs showing that treatment with exosomes containing si / shKrasG12D cargo results in sustained Panc-1 orthotopic tumor regression. (Figure 2A) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. PBS: n=7, control exos: n=6, siKrasG12D lipos: n=3, shKrasG12D lipos: n=3, siKrasG12D exos: n=7, shKrasG12D exos: n=7. Statistical test results comparing treatment groups to the PBS control group at day 42 post-cancer cell injection are shown, with the exception of the siKrasG12D exos group compared to the PBS group at the experimental endpoint (day 28 post-cancer cell injection). The top line pair is PBS and control Exos; the middle line pair is lipos; the bottom line pair is exos. (Figure 2B) Relative radiance of bioluminescent BxPC3 orthotopic tumors over time. PBS: n=3, control exos: n=3, siKrasG12D exos: n=3, shKrasG12D exos: n=3. Statistical test results comparing treatment groups to the PBS control group at 77 days after cancer cell injection are shown. (Figure 2C) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. Experimental groups started with PBS: n=7, control exos: n=6, siKrasG12D exos: n=7, shKrasG12D exos: n=7 and gradually decreased as mice became moribund and were euthanized (PBS and control exos groups). Small foci of cancer cells were seen in pancreata treated with shKrasG12D exos, but the majority of the pancreas was histologically unremarkable. The top line pair is PBS and control Exos; the bottom line pair is si / shKrasG12D exos. (Figure 2D) Comparative analysis of measured bioluminescence radiance 77 days after cancer cell injection for orthotopic Panc-1 tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2E) Quantification of p-ERK immunolabeling in pancreatic tumors in experimental groups (scale bar: 50 μm) and percent p-ERK staining. n=6.Note that quantification was performed on measurably smaller tumor areas in the shKrasG12D exos-treated group. Statistical significance between both groups was determined using an unpaired two-tailed Student's t-test. (Figure 2F) Tumor burden (relative pancreas mass to body weight) at the time of euthanasia (PBS: days 62-130, control exos: days 30-132, shKrasG12D exos: day 200) in the indicated experimental groups. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2G) Kaplan-Meier curve comparisons and statistical differences for mouse survival in the indicated experimental groups were assessed using the log-rank Mantel-Cox test, PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Means + / - SEM are shown. Statistical significance was determined using one-way ANOVA unless otherwise specified. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 2F]Figures 2A-G are graphs showing that treatment with exosomes containing si / shKrasG12D cargo results in sustained Panc-1 orthotopic tumor regression. (Figure 2A) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. PBS: n=7, control exos: n=6, siKrasG12D lipos: n=3, shKrasG12D lipos: n=3, siKrasG12D exos: n=7, shKrasG12D exos: n=7. Statistical test results comparing treatment groups to the PBS control group at day 42 post-cancer cell injection are shown, with the exception of the siKrasG12D exos group compared to the PBS group at the experimental endpoint (day 28 post-cancer cell injection). The top line pair is PBS and control Exos; the middle line pair is lipos; the bottom line pair is exos. (Figure 2B) Relative radiance of bioluminescent BxPC3 orthotopic tumors over time. PBS: n=3, control exos: n=3, siKrasG12D exos: n=3, shKrasG12D exos: n=3. Statistical test results comparing treatment groups to the PBS control group at 77 days after cancer cell injection are shown. (Figure 2C) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. Experimental groups started with PBS: n=7, control exos: n=6, siKrasG12D exos: n=7, shKrasG12D exos: n=7 and gradually decreased as mice became moribund and were euthanized (PBS and control exos groups). Small foci of cancer cells were seen in pancreata treated with shKrasG12D exos, but the majority of the pancreas was histologically unremarkable. The top line pair is PBS and control Exos; the bottom line pair is si / shKrasG12D exos. (Figure 2D) Comparative analysis of measured bioluminescence radiance 77 days after cancer cell injection for orthotopic Panc-1 tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2E) Quantification of p-ERK immunolabeling in pancreatic tumors in experimental groups (scale bar: 50 μm) and percent p-ERK staining. n=6.Note that quantification was performed on measurably smaller tumor areas in the shKrasG12D exos-treated group. Statistical significance between both groups was determined using an unpaired two-tailed Student's t-test. (Figure 2F) Tumor burden (relative pancreas mass to body weight) at the time of euthanasia (PBS: days 62-130, control exos: days 30-132, shKrasG12D exos: day 200) in the indicated experimental groups. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2G) Kaplan-Meier curve comparisons and statistical differences for mouse survival in the indicated experimental groups were assessed using the log-rank Mantel-Cox test, PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Means + / - SEM are shown. Statistical significance was determined using one-way ANOVA unless otherwise specified. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 2G]Figures 2A-G are graphs showing that treatment with exosomes containing si / shKrasG12D cargo results in sustained Panc-1 orthotopic tumor regression. (Figure 2A) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. PBS: n=7, control exos: n=6, siKrasG12D lipos: n=3, shKrasG12D lipos: n=3, siKrasG12D exos: n=7, shKrasG12D exos: n=7. Statistical test results comparing treatment groups to the PBS control group at day 42 post-cancer cell injection are shown, with the exception of the siKrasG12D exos group compared to the PBS group at the experimental endpoint (day 28 post-cancer cell injection). The top line pair is PBS and control Exos; the middle line pair is lipos; the bottom line pair is exos. (Figure 2B) Relative radiance of bioluminescent BxPC3 orthotopic tumors over time. PBS: n=3, control exos: n=3, siKrasG12D exos: n=3, shKrasG12D exos: n=3. Statistical test results comparing treatment groups to the PBS control group at 77 days after cancer cell injection are shown. (Figure 2C) Relative radiance of bioluminescent Panc-1 orthotopic tumors over time. Experimental groups started with PBS: n=7, control exos: n=6, siKrasG12D exos: n=7, shKrasG12D exos: n=7 and gradually decreased as mice became moribund and were euthanized (PBS and control exos groups). Small foci of cancer cells were seen in pancreata treated with shKrasG12D exos, but the majority of the pancreas was histologically unremarkable. The top line pair is PBS and control Exos; the bottom line pair is si / shKrasG12D exos. (Figure 2D) Comparative analysis of measured bioluminescence radiance 77 days after cancer cell injection for orthotopic Panc-1 tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2E) Quantification of p-ERK immunolabeling in pancreatic tumors in experimental groups (scale bar: 50 μm) and percent p-ERK staining. n=6.Note that quantification was performed on measurably smaller tumor areas in the shKrasG12D exos-treated group. Statistical significance between both groups was determined using an unpaired two-tailed Student's t-test. (Figure 2F) Tumor burden (relative pancreas mass to body weight) at the time of euthanasia (PBS: days 62-130, control exos: days 30-132, shKrasG12D exos: day 200) in the indicated experimental groups. Statistical significance between groups was determined using an unpaired two-tailed Student's t-test. (Figure 2G) Kaplan-Meier curve comparisons and statistical differences for mouse survival in the indicated experimental groups were assessed using the log-rank Mantel-Cox test, PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Means + / - SEM are shown. Statistical significance was determined using one-way ANOVA unless otherwise specified. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0032] [Figure 3A]Figure 3A-G shows that injection of KrasG12D siRNA-packaged exosomes and KrasG12D shRNA-packaged exosomes induces slower tumor progression and increased survival in PKT mice. (Figure 3A) Schematic representation of the timeline of tumor progression with experimental treatment points in Ptf1acre / +;LSL-KrasG12D / +;Tgfbr2flox / flox(PKT) mice. Treatment with BJ fibroblast exosomes containing KrasG12D RNAi was started on day 33 and continued every 2 days thereafter until the mice reached experimental endpoint or became moribund and required euthanasia. The control group was treated with the same concentration of non-electroporated BJ exosomes (control exos). (Figure 3B) Comparison of Kaplan-Meier curves and statistical differences for survival of mice of the indicated experimental groups were evaluated using the log-rank Mantel-Cox test. n=5 for each group. (Fig. 3C) Tumor burden (relative pancreas mass to body weight) in the indicated experimental groups at 44 days of age. n=3 per group. (Fig. 3D) Quantification of relative percentages determined from photomicrographs of H&E stained tumors from 44-day-old PKT mice, n=3, treated with exos containing siKrasG12D or non-electroporated control exos. One-way ANOVA was used for statistical comparison. (Fig. 3E-F) Comparison of Kaplan-Meier curves for survival of mice in the indicated experimental groups (PKT-derived fibroblast-derived exosomes, n=5 per group; left line is control Exos and right line is siKrasG12D exos) (Fig. 3E), and (Fig. 3F) tumor burden, n=5. Log-rank Mantel-Cox test was performed for statistical analysis for comparison of Kaplan-Meier curves. (FIG. 3G) Quantification of photomicrographs of Masson's Trichrome staining (MTS) and immunolabeling for the apoptotic marker TUNEL, proliferation marker Ki-67, and phosphorylated ERK from 44-day-old PKT pancreatic tumors in the indicated experimental groups. n=3. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student's t-test unless otherwise indicated. **p<0.01, ****p<0.0001. [Figure 3B] Figure 3A-G shows that injection of KrasG12D siRNA-packaged exosomes and KrasG12D shRNA-packaged exosomes induces slower tumor progression and increased survival in PKT mice. (Figure 3A) Schematic representation of the timeline of tumor progression with experimental treatment points in Ptf1acre / +;LSL-KrasG12D / +;Tgfbr2flox / flox(PKT) mice. Treatment with BJ fibroblast exosomes containing KrasG12D RNAi was started on day 33 and continued every 2 days thereafter until the mice reached experimental endpoint or became moribund and required euthanasia. The control group was treated with the same concentration of non-electroporated BJ exosomes (control exos). (Figure 3B) Comparison of Kaplan-Meier curves and statistical differences for survival of mice of the indicated experimental groups were evaluated using the log-rank Mantel-Cox test. n=5 for each group. (Fig. 3C) Tumor burden (relative pancreas mass to body weight) in the indicated experimental groups at 44 days of age. n=3 per group. (Fig. 3D) Quantification of relative percentages determined from photomicrographs of H&E stained tumors from 44-day-old PKT mice, n=3, treated with exos containing siKrasG12D or non-electroporated control exos. One-way ANOVA was used for statistical comparison. (Fig. 3E-F) Comparison of Kaplan-Meier curves for survival of mice in the indicated experimental groups (PKT-derived fibroblast-derived exosomes, n=5 per group; left line is control Exos and right line is siKrasG12D exos) (Fig. 3E), and (Fig. 3F) tumor burden, n=5. Log-rank Mantel-Cox test was performed for statistical analysis for comparison of Kaplan-Meier curves. (FIG. 3G) Quantification of photomicrographs of Masson's Trichrome staining (MTS) and immunolabeling for the apoptotic marker TUNEL, proliferation marker Ki-67, and phosphorylated ERK from 44-day-old PKT pancreatic tumors in the indicated experimental groups. n=3. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student's t-test unless otherwise indicated. **p<0.01, ****p<0.0001. [Figure 3C] Figure 3A-G shows that injection of KrasG12D siRNA-packaged exosomes and KrasG12D shRNA-packaged exosomes induces slower tumor progression and increased survival in PKT mice. (Figure 3A) Schematic representation of the timeline of tumor progression with experimental treatment points in Ptf1acre / +;LSL-KrasG12D / +;Tgfbr2flox / flox(PKT) mice. Treatment with BJ fibroblast exosomes containing KrasG12D RNAi was started on day 33 and continued every 2 days thereafter until the mice reached experimental endpoint or became moribund and required euthanasia. The control group was treated with the same concentration of non-electroporated BJ exosomes (control exos). (Figure 3B) Comparison of Kaplan-Meier curves and statistical differences for survival of mice of the indicated experimental groups were evaluated using the log-rank Mantel-Cox test. n=5 for each group. (Fig. 3C) Tumor burden (relative pancreas mass to body weight) in the indicated experimental groups at 44 days of age. n=3 per group. (Fig. 3D) Quantification of relative percentages determined from photomicrographs of H&E stained tumors from 44-day-old PKT mice, n=3, treated with exos containing siKrasG12D or non-electroporated control exos. One-way ANOVA was used for statistical comparison. (Fig. 3E-F) Comparison of Kaplan-Meier curves for survival of mice in the indicated experimental groups (PKT-derived fibroblast-derived exosomes, n=5 per group; left line is control Exos and right line is siKrasG12D exos) (Fig. 3E), and (Fig. 3F) tumor burden, n=5. Log-rank Mantel-Cox test was performed for statistical analysis for comparison of Kaplan-Meier curves. (FIG. 3G) Quantification of photomicrographs of Masson's Trichrome staining (MTS) and immunolabeling for the apoptotic marker TUNEL, proliferation marker Ki-67, and phosphorylated ERK from 44-day-old PKT pancreatic tumors in the indicated experimental groups. n=3. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student's t-test unless otherwise indicated. **p<0.01, ****p<0.0001. [Figure 3D]Figure 3A-G shows that injection of KrasG12D siRNA-packaged exosomes and KrasG12D shRNA-packaged exosomes induces slower tumor progression and increased survival in PKT mice. (Figure 3A) Schematic representation of the timeline of tumor progression with experimental treatment points in Ptf1acre / +;LSL-KrasG12D / +;Tgfbr2flox / flox(PKT) mice. Treatment with BJ fibroblast exosomes containing KrasG12D RNAi was started on day 33 and continued every 2 days thereafter until the mice reached experimental endpoint or became moribund and required euthanasia. The control group was treated with the same concentration of non-electroporated BJ exosomes (control exos). (Figure 3B) Comparison of Kaplan-Meier curves and statistical differences for survival of mice of the indicated experimental groups were evaluated using the log-rank Mantel-Cox test. n=5 for each group. (Fig. 3C) Tumor burden (relative pancreas mass to body weight) in the indicated experimental groups at 44 days of age. n=3 per group. (Fig. 3D) Quantification of relative percentages determined from photomicrographs of H&E stained tumors from 44-day-old PKT mice, n=3, treated with exos containing siKrasG12D or non-electroporated control exos. One-way ANOVA was used for statistical comparison. (Fig. 3E-F) Comparison of Kaplan-Meier curves for survival of mice in the indicated experimental groups (PKT-derived fibroblast-derived exosomes, n=5 per group; left line is control Exos and right line is siKrasG12D exos) (Fig. 3E), and (Fig. 3F) tumor burden, n=5. Log-rank Mantel-Cox test was performed for statistical analysis for comparison of Kaplan-Meier curves. (FIG. 3G) Quantification of photomicrographs of Masson's Trichrome staining (MTS) and immunolabeling for the apoptotic marker TUNEL, proliferation marker Ki-67, and phosphorylated ERK from 44-day-old PKT pancreatic tumors in the indicated experimental groups. n=3. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student's t-test unless otherwise indicated. **p<0.01, ****p<0.0001. [Figure 3E] Figure 3A-G shows that injection of KrasG12D siRNA-packaged exosomes and KrasG12D shRNA-packaged exosomes induces slower tumor progression and increased survival in PKT mice. (Figure 3A) Schematic representation of the timeline of tumor progression with experimental treatment points in Ptf1acre / +;LSL-KrasG12D / +;Tgfbr2flox / flox(PKT) mice. Treatment with BJ fibroblast exosomes containing KrasG12D RNAi was started on day 33 and continued every 2 days thereafter until the mice reached experimental endpoint or became moribund and required euthanasia. The control group was treated with the same concentration of non-electroporated BJ exosomes (control exos). (Figure 3B) Comparison of Kaplan-Meier curves and statistical differences for survival of mice of the indicated experimental groups were evaluated using the log-rank Mantel-Cox test. n=5 for each group. (Fig. 3C) Tumor burden (relative pancreas mass to body weight) in the indicated experimental groups at 44 days of age. n=3 per group. (Fig. 3D) Quantification of relative percentages determined from photomicrographs of H&E stained tumors from 44-day-old PKT mice, n=3, treated with exos containing siKrasG12D or non-electroporated control exos. One-way ANOVA was used for statistical comparison. (Fig. 3E-F) Comparison of Kaplan-Meier curves for survival of mice in the indicated experimental groups (PKT-derived fibroblast-derived exosomes, n=5 per group; left line is control Exos and right line is siKrasG12D exos) (Fig. 3E), and (Fig. 3F) tumor burden, n=5. Log-rank Mantel-Cox test was performed for statistical analysis for comparison of Kaplan-Meier curves. (FIG. 3G) Quantification of photomicrographs of Masson's Trichrome staining (MTS) and immunolabeling for the apoptotic marker TUNEL, proliferation marker Ki-67, and phosphorylated ERK from 44-day-old PKT pancreatic tumors in the indicated experimental groups. n=3. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student's t-test unless otherwise indicated. **p<0.01, ****p<0.0001. [Figure 3F] Figure 3A-G shows that injection of KrasG12D siRNA-packaged exosomes and KrasG12D shRNA-packaged exosomes induces slower tumor progression and increased survival in PKT mice. (Figure 3A) Schematic representation of the timeline of tumor progression with experimental treatment points in Ptf1acre / +;LSL-KrasG12D / +;Tgfbr2flox / flox(PKT) mice. Treatment with BJ fibroblast exosomes containing KrasG12D RNAi was started on day 33 and continued every 2 days thereafter until the mice reached experimental endpoint or became moribund and required euthanasia. The control group was treated with the same concentration of non-electroporated BJ exosomes (control exos). (Figure 3B) Comparison of Kaplan-Meier curves and statistical differences for survival of mice of the indicated experimental groups were evaluated using the log-rank Mantel-Cox test. n=5 for each group. (Fig. 3C) Tumor burden (relative pancreas mass to body weight) in the indicated experimental groups at 44 days of age. n=3 per group. (Fig. 3D) Quantification of relative percentages determined from photomicrographs of H&E stained tumors from 44-day-old PKT mice, n=3, treated with exos containing siKrasG12D or non-electroporated control exos. One-way ANOVA was used for statistical comparison. (Fig. 3E-F) Comparison of Kaplan-Meier curves for survival of mice in the indicated experimental groups (PKT-derived fibroblast-derived exosomes, n=5 per group; left line is control Exos and right line is siKrasG12D exos) (Fig. 3E), and (Fig. 3F) tumor burden, n=5. Log-rank Mantel-Cox test was performed for statistical analysis for comparison of Kaplan-Meier curves. (FIG. 3G) Quantification of photomicrographs of Masson's Trichrome staining (MTS) and immunolabeling for the apoptotic marker TUNEL, proliferation marker Ki-67, and phosphorylated ERK from 44-day-old PKT pancreatic tumors in the indicated experimental groups. n=3. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student's t-test unless otherwise indicated. **p<0.01, ****p<0.0001. [Figure 3G]Figure 3A-G shows that injection of KrasG12D siRNA-packaged exosomes and KrasG12D shRNA-packaged exosomes induces slower tumor progression and increased survival in PKT mice. (Figure 3A) Schematic representation of the timeline of tumor progression with experimental treatment points in Ptf1acre / +;LSL-KrasG12D / +;Tgfbr2flox / flox(PKT) mice. Treatment with BJ fibroblast exosomes containing KrasG12D RNAi was started on day 33 and continued every 2 days thereafter until the mice reached experimental endpoint or became moribund and required euthanasia. The control group was treated with the same concentration of non-electroporated BJ exosomes (control exos). (Figure 3B) Comparison of Kaplan-Meier curves and statistical differences for survival of mice of the indicated experimental groups were evaluated using the log-rank Mantel-Cox test. n=5 for each group. (Fig. 3C) Tumor burden (relative pancreas mass to body weight) in the indicated experimental groups at 44 days of age. n=3 per group. (Fig. 3D) Quantification of relative percentages determined from photomicrographs of H&E stained tumors from 44-day-old PKT mice, n=3, treated with exos containing siKrasG12D or non-electroporated control exos. One-way ANOVA was used for statistical comparison. (Fig. 3E-F) Comparison of Kaplan-Meier curves for survival of mice in the indicated experimental groups (PKT-derived fibroblast-derived exosomes, n=5 per group; left line is control Exos and right line is siKrasG12D exos) (Fig. 3E), and (Fig. 3F) tumor burden, n=5. Log-rank Mantel-Cox test was performed for statistical analysis for comparison of Kaplan-Meier curves. (FIG. 3G) Quantification of photomicrographs of Masson's Trichrome staining (MTS) and immunolabeling for the apoptotic marker TUNEL, proliferation marker Ki-67, and phosphorylated ERK from 44-day-old PKT pancreatic tumors in the indicated experimental groups. n=3. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student's t-test unless otherwise indicated. **p<0.01, ****p<0.0001.

[0033] [Figure 4A]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4B]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4C]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4D]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4E]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4F]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4G]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4H]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 4I]4A-I: Specific KrasG12D targeting using exosomes. (FIG. 4A) Schematic of electroporation of RNAi into exosomes. RNAi shown is tagged with Alexa Fluor® 647. (FIG. 4B) Number and size distribution of exosomes - using NanoSight. (FIG. 4C-D) Transmission electron micrographs of exosomes purified from BJ fibroblasts (FIG. 4C) and stained for CD9 by immunogold (FIG. 4D). (FIG. 4E) Northern blot of sucrose gradient of BJ fibroblast exosomes containing Alexa Fluor® 647 tagged siRNA. Detection of fluorescence of Alexa Fluor® 647 fluorophore is shown on the blot. (FIG. 4F) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with increasing concentrations of lipos (1×, 10×, 100×) for 3 hours with siKrasG12D lipos, shKrasG12D lipos, siScrbl lipos, or shScrbl lipos, as well as increasing treatment times (24 hours) of Panc-1 cells. Ratios are shown relative to expression in untreated Panc-1 cells (control), arbitrarily set to 1. Statistical significance compared to transcript levels in untreated Panc-1 cells was determined using an unpaired two-tailed Student's t-test. (FIG. 4G) Real-time PCR analysis of KRASG12D transcript levels in Panc-1 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours, as well as with increasing concentrations of exos (approximately 700 exos per cell instead of approximately 400 exos per cell) as shown in FIG. 1B. Unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated Panc-1 cells. (FIG. 4H) Real-time PCR analysis of wild-type KRAS transcript levels in BxPC-3 cells treated with siKrasG12D exos or shKrasG12D exos for 3 hours.An unpaired two-tailed Student's t-test was used to determine statistical significance compared to transcript levels in untreated BxPC3 cells. (FIG. 4I) Relative numbers of BXPC-3 cells over time after exposure to the listed treatments. An unpaired two-tailed Student's t-test was used for the final time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0034] [Figure 5A] 5A-D show that exosomes containing KrasG12D RNAi suppress Panc-1 orthotopic tumor growth. (Fig. 5A) Flow cytometry analysis of exosomes isolated from serum of mice treated with siKrasG12D exos 24 hours after ip injection. Labeled exosomes were detected using Alexa Fluor® 647-tagged RNAi contained after binding to 0.4 μm beads. (Fig. 5B) Flow cytometry analysis and quantification of percentage of Alexa Fluor® 647+ / CD11b+ macrophages in blood of mice 12 hours after ip injection of exos or lipos containing Alexa fluor 647-tagged RNAi. (Fig. 5C) Quantification of percent p-AKT stained area in photomicrographs of pancreatic tumors immunolabeled for phosphorylated AKT (p-AKT). n=6. Note that quantification was performed on the relatively small tumor area in the group treated with shKrasG12D exos. (FIG. 5D) Comparison of Kaplan-Meier curves for survival of mice bearing BxPC-3 orthotopic tumors in the indicated experimental groups: PBS: n=3, control exos: n=3, shKrasG12D exos: n=3, siKrasG12D exos: n=3 (Log-rank (Mantel-Cox) test was used for this analysis). Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tailed Student's t-test unless otherwise noted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 5B]5A-D show that exosomes containing KrasG12D RNAi suppress Panc-1 orthotopic tumor growth. (Fig. 5A) Flow cytometry analysis of exosomes isolated from serum of mice treated with siKrasG12D exos 24 hours after ip injection. Labeled exosomes were detected using Alexa Fluor® 647-tagged RNAi contained after binding to 0.4 μm beads. (Fig. 5B) Flow cytometry analysis and quantification of percentage of Alexa Fluor® 647+ / CD11b+ macrophages in blood of mice 12 hours after ip injection of exos or lipos containing Alexa fluor 647-tagged RNAi. (Fig. 5C) Quantification of percent p-AKT stained area in photomicrographs of pancreatic tumors immunolabeled for phosphorylated AKT (p-AKT). n=6. Note that quantification was performed on the relatively small tumor area in the group treated with shKrasG12D exos. (FIG. 5D) Comparison of Kaplan-Meier curves for survival of mice bearing BxPC-3 orthotopic tumors in the indicated experimental groups: PBS: n=3, control exos: n=3, shKrasG12D exos: n=3, siKrasG12D exos: n=3 (Log-rank (Mantel-Cox) test was used for this analysis). Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tailed Student's t-test unless otherwise noted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 5C]5A-D show that exosomes containing KrasG12D RNAi suppress Panc-1 orthotopic tumor growth. (Fig. 5A) Flow cytometry analysis of exosomes isolated from serum of mice treated with siKrasG12D exos 24 hours after ip injection. Labeled exosomes were detected using Alexa Fluor® 647-tagged RNAi contained after binding to 0.4 μm beads. (Fig. 5B) Flow cytometry analysis and quantification of percentage of Alexa Fluor® 647+ / CD11b+ macrophages in blood of mice 12 hours after ip injection of exos or lipos containing Alexa fluor 647-tagged RNAi. (Fig. 5C) Quantification of percent p-AKT stained area in photomicrographs of pancreatic tumors immunolabeled for phosphorylated AKT (p-AKT). n=6. Note that quantification was performed on the relatively small tumor area in the group treated with shKrasG12D exos. (FIG. 5D) Comparison of Kaplan-Meier curves for survival of mice bearing BxPC-3 orthotopic tumors in the indicated experimental groups: PBS: n=3, control exos: n=3, shKrasG12D exos: n=3, siKrasG12D exos: n=3 (Log-rank (Mantel-Cox) test was used for this analysis). Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tailed Student's t-test unless otherwise noted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. [Figure 5D]5A-D show that exosomes containing KrasG12D RNAi suppress Panc-1 orthotopic tumor growth. (Fig. 5A) Flow cytometry analysis of exosomes isolated from serum of mice treated with siKrasG12D exos 24 hours after ip injection. Labeled exosomes were detected using Alexa Fluor® 647-tagged RNAi contained after binding to 0.4 μm beads. (Fig. 5B) Flow cytometry analysis and quantification of percentage of Alexa Fluor® 647+ / CD11b+ macrophages in blood of mice 12 hours after ip injection of exos or lipos containing Alexa fluor 647-tagged RNAi. (Fig. 5C) Quantification of percent p-AKT stained area in photomicrographs of pancreatic tumors immunolabeled for phosphorylated AKT (p-AKT). n=6. Note that quantification was performed on the relatively small tumor area in the group treated with shKrasG12D exos. (FIG. 5D) Comparison of Kaplan-Meier curves for survival of mice bearing BxPC-3 orthotopic tumors in the indicated experimental groups: PBS: n=3, control exos: n=3, shKrasG12D exos: n=3, siKrasG12D exos: n=3 (Log-rank (Mantel-Cox) test was used for this analysis). Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tailed Student's t-test unless otherwise noted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0035] [Figure 6A]Figures 6A-B are graphs showing the progression of Panc-1 tumors. (Figure 6A) Comparative analysis of bioluminescence radiance measured at day 77. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance was determined using an unpaired two-tailed Student's t-test at day 77. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. (Figure 6B) Spider plot showing individual tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. [Figure 6B] Figures 6A-B are graphs showing the progression of Panc-1 tumors. (Figure 6A) Comparative analysis of bioluminescence radiance measured at day 77. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7. Statistical significance was determined using an unpaired two-tailed Student's t-test at day 77. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. (Figure 6B) Spider plot showing individual tumors. PBS: n=7, control exos: n=6, shKrasG12D exos: n=7.

[0036] [Figure 7A]Figures 7A-D are graphs showing histological analysis of PKT mice treated with KrasG12D RNAi exos. (Figure 7A) Tumor burden (relative pancreas mass to body weight) at experimental endpoints (control exos: median survival 43 days, siKrasG12D exos: median survival 60 days). n=5 per group. (Figure 7B-C) Quantification of relative percentages of histological phenotypes in H&E stained tumor photomicrographs of PKT mice treated with (Figure 7B) siKrasG12D exos derived from BJ fibroblasts and (Figure 7C) siKrasG12D exos derived from PKT fibroblasts or non-electroporated exos (control exos) at the indicated experimental endpoints. n=5. Two-way ANOVA was used for statistical comparisons. (Figure 7D) Quantification of tumor photomicrographs immunolabeled for phosphorylated AKT from 44-day-old PKT mice in the indicated experimental groups. Data are shown as mean ± SEM. Unless otherwise specified, statistical significance was determined using an unpaired two-tailed Student's t-test. **p<0.01, ****p<0.0001. [Figure 7B]Figures 7A-D are graphs showing histological analysis of PKT mice treated with KrasG12D RNAi exos. (Figure 7A) Tumor burden (relative pancreas mass to body weight) at experimental endpoints (control exos: median survival 43 days, siKrasG12D exos: median survival 60 days). n=5 per group. (Figure 7B-C) Quantification of relative percentages of histological phenotypes in H&E stained tumor photomicrographs of PKT mice treated with (Figure 7B) siKrasG12D exos derived from BJ fibroblasts and (Figure 7C) siKrasG12D exos derived from PKT fibroblasts or non-electroporated exos (control exos) at the indicated experimental endpoints. n=5. Two-way ANOVA was used for statistical comparisons. (Figure 7D) Quantification of tumor photomicrographs immunolabeled for phosphorylated AKT from 44-day-old PKT mice in the indicated experimental groups. Data are shown as mean ± SEM. Unless otherwise specified, statistical significance was determined using an unpaired two-tailed Student's t-test. **p<0.01, ****p<0.0001. [Figure 7C]Figures 7A-D are graphs showing histological analysis of PKT mice treated with KrasG12D RNAi exos. (Figure 7A) Tumor burden (relative pancreas mass to body weight) at experimental endpoints (control exos: median survival 43 days, siKrasG12D exos: median survival 60 days). n=5 per group. (Figure 7B-C) Quantification of relative percentages of histological phenotypes in H&E stained tumor photomicrographs of PKT mice treated with (Figure 7B) siKrasG12D exos derived from BJ fibroblasts and (Figure 7C) siKrasG12D exos derived from PKT fibroblasts or non-electroporated exos (control exos) at the indicated experimental endpoints. n=5. Two-way ANOVA was used for statistical comparisons. (Figure 7D) Quantification of tumor photomicrographs immunolabeled for phosphorylated AKT from 44-day-old PKT mice in the indicated experimental groups. Data are shown as mean ± SEM. Unless otherwise specified, statistical significance was determined using an unpaired two-tailed Student's t-test. **p<0.01, ****p<0.0001. [Figure 7D]Figures 7A-D are graphs showing histological analysis of PKT mice treated with KrasG12D RNAi exos. (Figure 7A) Tumor burden (relative pancreas mass to body weight) at experimental endpoints (control exos: median survival 43 days, siKrasG12D exos: median survival 60 days). n=5 per group. (Figure 7B-C) Quantification of relative percentages of histological phenotypes in H&E stained tumor photomicrographs of PKT mice treated with (Figure 7B) siKrasG12D exos derived from BJ fibroblasts and (Figure 7C) siKrasG12D exos derived from PKT fibroblasts or non-electroporated exos (control exos) at the indicated experimental endpoints. n=5. Two-way ANOVA was used for statistical comparisons. (Figure 7D) Quantification of tumor photomicrographs immunolabeled for phosphorylated AKT from 44-day-old PKT mice in the indicated experimental groups. Data are shown as mean ± SEM. Unless otherwise specified, statistical significance was determined using an unpaired two-tailed Student's t-test. **p<0.01, ****p<0.0001.

[0037] [Figure 8] Figures 8A-B are graphs showing that circulating monocytes phagocytose iLiposomes (i.e., liposomes containing drug substances such as inhibitory RNA) more efficiently than iExosomes (i.e., exosomes containing drug substances such as inhibitory RNA). (Figure 8A) The top plot shows the % of CD11b positive cells from the total live cell population. The bottom plot shows the % of A647 and CD11b double positive cells from the total live cell population. (Figure 8B) Quantification of the FACS plots presented in Figure 8A.

[0038] [Figure 9A]9A-C show that CD47 is detected in exosomes but not in exosomes. (FIG. 9A) Exosomes isolated from BJ fibroblasts. The top plot shows staining with secondary antibody only, and the bottom plot shows staining for either CD63 or CD47. (FIG. 9B) Liposomes (100 nm) stained with secondary antibody only or with antibodies against CD63 or CD47. (FIG. 9C) Expression of CD47 by exosomes isolated from three different cell lines by two different methods. [Figure 9B] 9A-C show that CD47 is detected in exosomes but not in exosomes. (FIG. 9A) Exosomes isolated from BJ fibroblasts. The top plot shows staining with secondary antibody only, and the bottom plot shows staining for either CD63 or CD47. (FIG. 9B) Liposomes (100 nm) stained with secondary antibody only or with antibodies against CD63 or CD47. (FIG. 9C) Expression of CD47 by exosomes isolated from three different cell lines by two different methods. [Figure 9C] 9A-C show that CD47 is detected in exosomes but not in exosomes. (FIG. 9A) Exosomes isolated from BJ fibroblasts. The top plot shows staining with secondary antibody only, and the bottom plot shows staining for either CD63 or CD47. (FIG. 9B) Liposomes (100 nm) stained with secondary antibody only or with antibodies against CD63 or CD47. (FIG. 9C) Expression of CD47 by exosomes isolated from three different cell lines by two different methods.

[0039] [Figure 10] 10 is a graph showing that anti-CD47 antibodies stimulate exosome uptake by circulating monocytes in vivo. Exosomes were treated with anti-CD47 antibodies, thereby enabling exosome uptake by circulating monocytes in vivo.

[0040] [Figure 11] Figure 11 is a Venn diagram showing the proteins present in subpopulations of pancreatic cancer cells and their respective exosomes. Proteins were extracted from subpopulations of pancreatic cancer cell line T3M4 and their respective exosomes. Mass spectrometry was used to identify the proteins expressed in cells and their respective exosomes. A list of proteins was identified for each sample. Comparison of cells and their respective exosomes identified proteins present in cells but not in exosomes (left side of each pair of circles), proteins present in cells and their respective exosomes (cross between circles), and proteins enriched in exosomes compared to cells (right side of each pair of circles). Cells express proteins that do not appear in exosomes. Exosomes also contain enriched proteins compared to cells of origin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Description of exemplary embodiments Despite current standard treatments, the median survival time for pancreatic ductal adenocarcinoma (PDAC) is 6 months for metastatic patients, with only 6.7% surviving after 5 years (Siegel et al., 2014; Howlader et al., 2013). Thus, effective new therapies are urgently needed for PDAC. Genetic analysis of PDAC has revealed that mutations in the RAS family of small GTPases (KrasG12 / D / R / V) occur in 70%–96% of patients (Biankin et al., 2012; Hruban et al., 1993; Almoguera et al., 1988; Chang et al., 2013). 14), which is a key driver of tumor growth and metastasis (Ying et al., 2012; Hingorani et al., 2005; Collins et al., 2012a; Collins et al., 2012b; Eser et al., 2014). Genetic manipulation in mice has been shown to reverse tumor progression by attenuating oncogenic KRAS (Ying et al., 2012; Collins et al. Oncogenic KRAS signaling and increased RAS activity have emerged as initiating drivers of pancreatic neoplasia (Collins et al., 2012a; Eser et al., 2014; Ji et al., 2009); however, RAS is a difficult target and remains a therapeutic challenge (Gysin et al., 2011). In the present invention, exosomes derived from normal fibroblasts are targeted to Oncogenic KRAS G12D The plasmid is engineered to carry an RNA interference (RNAi) payload against Kras G12D Exosomes containing TT-linked siRNA or shRNA against Kras efficiently entered cancer cells, specifically suppressed oncogenic Ras, reduced ERK activation, inhibited proliferation, and induced cancer cell apoptosis. G12D Systemic delivery of exosomes with targeted cargo shows robust pancreatic localization and tumor suppression in previously established orthotopic human pancreatic tumors and genetically engineered mouse models (GEMMs) of pancreatic cancer, along with improved survival, compared with liposomes containing si / shRNA and exosomes with scrambled RNAi constructs. Tumors from mice treated with exosomes containing si / shRNA showed significant reduction of downstream RAS signaling mediators and improved histopathological findings using routine pancreatic histology. Human fibroblast-derived exosomes improved the pathogenesis of PDAC. Similar efficacy was demonstrated as exosomes derived from mouse fibroblasts in GEMMs, suggesting that patient-specific exosomes may not be necessary to enable efficient RNAi delivery while minimizing potential toxic side effects. Such a strategy would provide a novel and efficient means to suppress oncogenic gene expression and downstream signaling while minimizing off-target effects.

[0042] CD47 (integrin-associated protein) is a transmembrane protein expressed in the majority of tissues and cells. CD47 is a receptor for signal regulatory protein alpha (SIRP-α) expressed in phagocytes such as macrophages and dendritic cells. Activated CD47-SIRP-α initiates a signaling cascade that inhibits phagocytosis. Injecting mice with a monoclonal antibody against CD47 prior to exosome injection or treating exosomes with CD47-neutralizing antibodies prior to injection both block CD47 and allow phagocytosis of exosomes by macrophages or monocytes. Thus, expression of CD47 on the surface of exosomes prevents phagocytosis by macrophages.

[0043] I. Lipid-Based Nanoparticles In some embodiments, the lipid-based nanoparticle is a liposome, an exosome, a lipid preparation, or another lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP:cholesterol vesicle). The lipid-based nanoparticle may be positively charged, negatively charged, or neutral.

[0044] A. Liposomes "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a bilayer membrane that generally comprises phospholipids and an internal medium that generally comprises an aqueous composition. The liposomes presented herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. The liposomes presented herein can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the charge of the liposome is neutral.

[0045] Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Such liposomes form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution. The lipid components self-rearrange before a closed structure is formed, trapping water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions are also dissolved in or associated with the lipid bilayer.

[0046] In certain embodiments, a polypeptide, nucleic acid, or small molecule drug may be, for example, encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome by being linked to a molecule that associates with the liposome and the polypeptide / nucleic acid, entrapped within the liposome, or complexed with a liposome.

[0047] The liposomes used according to the present embodiment can be produced by various methods, as known to those skilled in the art. For example, phospholipids, such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC), are dissolved in tert-butanol. The lipid(s) are then mixed with the polypeptide, nucleic acid, and / or other component(s). Tween 20 is added to the lipid mixture such that Tween 20 is about 5% of the composition's weight. An excess of tert-butanol is added to the mixture such that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized preparations can be stored at -20°C and used for up to 3 months. If necessary, the lyophilized liposomes are reconstituted in 0.9% saline.

[0048] Alternatively, liposomes can be prepared by mixing the solvent and lipid in a container, such as a glass pear-shaped flask. The volume of the container should be 10 times the volume of the expected liposome suspension. The solvent is removed using a rotary evaporator at approximately 40°C under negative pressure. Typically, the solvent is removed within about 5 minutes to 2 hours, depending on the desired liposome volume. The composition can be further dried under reduced pressure in a desiccator. Generally, dried lipids are discarded after about one week, as they tend to deteriorate over time.

[0049] The dried lipids can be hydrated to approximately 25-50 mM phospholipid in sterile pyrogen-free water by shaking until all of the lipid film is resuspended. The aqueous liposomes can then be separated into aliquots, each placed in a vial, lyophilized, and sealed under vacuum.

[0050] The dried lipid or lyophilized liposomes prepared as described above can be dehydrated, reconstituted in a solution of protein or peptide, and diluted to an appropriate concentration with a suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer. Any unencapsulated additional material of the agent, including but not limited to hormones, drugs, nucleic acid constructs, etc., is removed by centrifugation at 29,000×g, and the liposome pellet is washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM. The amount of additional material or active agent to be encapsulated can be determined according to standard methods. After determining the amount of additional material or active agent to be encapsulated in the liposome preparation, the liposomes can be diluted to an appropriate concentration and stored at 4° C. until use. Pharmaceutical compositions containing liposomes usually contain a sterilized, pharma- ceutical acceptable carrier or diluent, such as water or saline solution.

[0051] Additional liposomes that may be useful in this embodiment include, for example, cationic liposomes, as described in WO02 / 100435A1, U.S. Pat. No. 5,962,016, U.S. Patent Application No. 2004 / 0208921, WO03 / 015757A1, WO04029213A2, U.S. Pat. No. 5,030,453, and U.S. Pat. No. 6,680,068, all of which are hereby incorporated by reference in their entirety without disclaimer.

[0052] In preparing such liposomes, any protocol described herein or known to those skilled in the art can be used.Non-limiting further examples of preparing liposomes are described in U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706; International Application PCT / US85 / 01161 and PCT / US89 / 05040, each of which is incorporated herein by reference.

[0053] In certain embodiments, the lipid-based nanoparticle is a neutral liposome (e.g., DOPC liposome). "Neutral liposome" or "uncharged liposome" as used herein is defined as a liposome having one or more lipid components that result in an essentially neutral net charge (substantially uncharged). "Essentially neutral" or "essentially uncharged" means that a minority of lipid components, if any, within a given population (e.g., a population of liposomes) contain a charge that is not cancelled by the opposite charge of another component (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1% of the components contain a charge that is not cancelled). In certain embodiments, a neutral liposome may contain primarily lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., at about pH 7).

[0054] The liposomes and / or lipid-based nanoparticles of the present embodiment may include phospholipids. In certain embodiments, a single type of phospholipid may be used to create liposomes (e.g., a neutral phospholipid such as DOPC may be used to generate neutral liposomes). In other embodiments, more than one type of phospholipid may be used to create liposomes. Phospholipids may be derived from natural or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at about pH 7), so these compounds may be particularly useful for generating neutral liposomes. In certain embodiments, the phospholipid DOPC is used to create uncharged liposomes. In certain embodiments, lipids that are not phospholipids (e.g., cholesterol) may be used.

[0055] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol, ) ("DLPG"), dimyristoyl phosphatidylglycerol ("DMPG"), dipalmitoyl phosphatidylglycerol ("DPPG"), distearoyl phosphatidylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoyl phosphatidylethanolamine ("DSPE"), dioleoyl phosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ( "BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoyl phosphatidylethanolamine ("DOPE"), palmitoyloleoyl phosphatidylcholine ("POPC"), palmitoyleoyl phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoyl phosphatidylcholine.

[0056] B. Exosomes The terms "microvesicles" and "exosomes" as used herein refer to membrane-like particles with a diameter (or maximum dimension if the particle is not spherical) between about 10 nm and about 5000 nm, more commonly between 30 nm and 1000 nm, and most typically between about 50 nm and 750 nm, where at least a portion of the membrane of the exosome is obtained directly from a cell. Most commonly, the size (average diameter) of the exosome is up to 5% of the size of the donor cell. Thus, exosomes that are specifically contemplated include those that emanate from a cell.

[0057] Exosomes can be detected or isolated from any suitable sample type, such as, for example, bodily fluids. As used herein, the term "isolated" refers to separation from its natural environment, and includes at least partial purification, and may include substantial purification. As used herein, the term "sample" refers to any sample suitable for the methods provided by the present invention. The sample may be any sample that contains exosomes suitable for detection or isolation. Sources of samples include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, sweat, tears, joint fluid, and bronchial lavage fluid. In one aspect, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof. Blood samples suitable for use with the present invention can be extracted from any known source that contains blood cells or components thereof, such as, for example, venous, arterial, peripheral, tissue, umbilical cord, etc. For example, samples can be obtained and processed using well-known routine clinical methods (e.g., procedures for drawing and processing whole blood). In one aspect, an exemplary sample can be peripheral blood drawn from a subject with cancer.

[0058] Exosomes can also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells.When isolating exosomes from tissue sources, it may be necessary to homogenize the tissue to obtain a single cell suspension, and then dissolve the cells to release the exosomes.When isolating exosomes from tissue samples, it is important to select homogenization and dissolution procedures that do not cause the destruction of exosomes.The exosomes contemplated herein are preferably isolated from body fluids in physiologically acceptable solutions, such as buffered saline, growth medium, various aqueous media, etc.

[0059] Exosomes can be isolated from freshly collected samples or from samples stored frozen or refrigerated. In some embodiments, exosomes can be isolated from cell culture medium. Although not necessary, exosomes can be obtained with higher purity by clarification of the body fluid sample followed by precipitation with a volume-exclusion polymer to remove any debris from the sample. Clarification methods include centrifugation, ultracentrifugation, filtration, or ultrafiltration. Most typically, exosomes can be isolated by a number of methods well known in the art. One preferred method is differential centrifugation from body fluids or cell culture supernatant. Exemplary methods for isolating exosomes are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Alternatively, exosomes can be isolated by flow cytometry as described in (Combes et al., 1997).

[0060] One accepted protocol for isolating exosomes involves ultracentrifugation, often combined with a sucrose density gradient or sucrose cushion to suspend the relatively low density exosomes. Isolation of exosomes by sequential differential centrifugation is complicated by the possibility of overlapping size distribution with other microvesicles or macromolecular complexes. Furthermore, centrifugation may be a poor means to separate vesicles based on their size. However, sequential centrifugation can achieve high enrichment of exosomes when combined with sucrose gradient ultracentrifugation.

[0061] Another option is the isolation of exosomes based on size using alternative methods to the ultracentrifugation route. Successful exosome purification has been reported using an ultrafiltration procedure that is less time consuming than ultracentrifugation and does not require the use of special equipment. Similarly, a commercial kit is available that uses positive pressure to drive a fluid to allow the removal of cells, platelets, and cell debris in one microfilter and the capture of vesicles larger than 30 nm in a second microfilter (EXOMIR™, Bioo Scientific). However, for this process, exosomes are not recovered, but their RNA content is directly extracted from the material captured in the second microfilter, and the RNA content can then be used for PCR analysis. HPLC-based protocols can potentially allow for highly pure exosomes to be obtained, but these processes require dedicated equipment and are difficult to scale up. A significant problem is that both blood and cell culture media contain a large number of nanoparticles (some of which are non-vesicles) in the same size range as exosomes. For example, some miRNAs may be contained within extracellular protein complexes rather than exosomes, but treatment with a protease (e.g., proteinase K) can be performed to eliminate any possible contamination with "exo-exosomal" proteins.

[0062] In another embodiment, cancer cell-derived exosomes can be captured by techniques commonly used to enrich samples for exosomes, such as those involving immune-specific interactions (e.g., immunomagnetic capture). Immunomagnetic capture, also known as immunomagnetic cell separation, generally involves attaching antibodies directed to proteins found in specific cell types to small paramagnetic beads. When the antibody-coated beads are mixed with a sample, such as blood, they attach to and surround specific cells. The sample is then placed in a strong magnetic field, which causes the beads to pellet to one side. After removing the blood, the captured cells are retained with the beads. Many variations of this general method are known in the art and are suitable for use to isolate exosomes. In one example, exosomes can be attached to magnetic beads (e.g., aldehyde / sulfate beads), and then antibodies can be added to the mixture to recognize epitopes on the surface of the exosomes attached to the beads. Exemplary proteins known to be found in cancer cell-derived exosomes include ATP-binding cassette subfamily A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), and glypican-1 (GPC1).For example, antibodies or aptamers against one or more of these proteins can be used to isolate cancer cell-derived exosomes.

[0063] As used herein, analysis includes any method that allows direct or indirect visualization of exosomes, and may be in vivo or ex vivo. For example, analysis may include, but is not limited to, ex vivo microscopic or cytometric detection and visualization of exosomes bound to solid substrates, flow cytometry, fluorescence imaging, and the like. In an exemplary embodiment, cancer cell-derived exosomes are analyzed by analyzing the expression levels of ATP-binding cassette subfamily A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), glypican-1 (GPC1), histone H2A type 2-A (HIST1H2AA), histone H2A type 1-A (HIST1H1AA), histone H3.3 (H3F3A), histone H3.1 (HIST1H3A), zinc finger protein 37 homolog (ZFP37), laminin subunit beta-1 (LAMB1), tubulointerstitial nephritis antigen-like (TINAGL1), peroxiredoxin-4 (PRDX4), collagen alpha-2(IV) chain (COL4A2), putative protein C3P1 (C3P1), hemicentin-1 (HMCN1), putative rhophilin-2-like protein (RHPN2P1), ankyrin repeat domain-containing protein 62 (ANKRD62), tripartite motif-containing protein 42 (TRIM42), junction plakoglobin (JUP), tubulin beta 2B chain (TUBB2B), endoribonuclease Dicer (DICER1), E3 ubiquitin-protein ligase TRIM71 (TRIM71), katanin p60 ATPase-containing subunit A-like 2 (KATNAL2), protein S100-A6 (S100A6), 5'-nucleotidase domain-containing protein 3 (NT5DC3), valine-tRNA ligase (VARS), kazurin (KAZN), ELAV-like protein 4 (ELAVL4), ring finger protein 166 (RNF166), FERM and PDZ domain-containing protein 1 (FRMPD1), 78 kDa glucose-regulated protein (HSPA5), transport protein particle complex subunit 6A (TRAPPC6A), squalene monooxygenase (SQLE), tumor susceptibility gene 101 protein (TSG101), The cells are detected using antibodies directed to one or more of the following: vacuolar protein sorting 28 homolog (VPS28), prostaglandin F2 receptor negative regulator (PTGFRN), isobutyryl-CoA dehydrogenase, mitochondrial (ACAD8), 26S protease regulatory subunit 6B (PSMC4), elongation factor 1-oncogene (EEF1G), titin (TTN), tyrosine-protein phosphatase type 13 (PTPN13), triosephosphate isomerase (TPI1), or carboxypeptidase E (CPE), followed by binding to a solid substrate and / or visualization using microscopy or detection by cytometry.

[0064] It should be noted that not all proteins expressed in a cell are found in exosomes secreted from that cell (see FIG. 11). For example, calnexin, GM130, and LAMP-2 are all proteins expressed in MCF-7 cells, but are not found in exosomes secreted from MCF-7 cells (Baietti et al., 2012). As another example, one study found that in 19 patients with pancreatic ductal adenocarcinoma, calnexin was expressed in 19 patients with pancreatic ductal adenocarcinoma, but LAMP-2 ... not found in exosomes secreted from MCF-7 cells (Baietti et al., 2012). 0 out of 190 had higher levels of GPC1+ exosomes than healthy controls (Melo et al., 2015, incorporated herein by reference in its entirety). Notably, on average, only 2.3% of healthy controls had GPC1+ exosomes.

[0065] 1. Exemplary Protocol for Harvesting Exosomes from Cell Culture On day 1, seed enough cells (e.g., approximately 5 million cells) in a T225 flask in medium containing 10% FBS so that the cells are approximately 70% confluent the next day. On day 2, aspirate the medium on the cells, wash the cells twice with PBS, then add 25-30 mL of basal medium (i.e., without PenStrep or FBS) to the cells. Incubate the cells for 24-48 hours. Although incubating for 48 hours is preferred, some cell lines are more sensitive to serum-free medium and therefore the incubation time should be reduced to 24 hours. Note that FBS contains exosomes that severely skew the NanoSight results.

[0066] On day 3 / 4, collect the medium and centrifuge at 800 x g for 5 min at room temperature to pellet dead cells and large debris. Transfer the supernatant to a new conical tube and centrifuge the medium again at 2000 x g for 10 min to remove other large debris and large vesicles. Pass the medium through a 0.2 μm filter and then aliquot into ultracentrifuge tubes (e.g., 25 x 89 mm Beckman Ultra-Clear) using 35 mL per tube. If the volume of medium per tube is less than 35 mL, fill the remainder of the tube with PBS to reach 35 mL. Ultracentrifuge the medium at 28,000 rpm at 4 °C for 2-4 h using a SW 32 Ti rotor (k factor 266.7, max RCF 133,907). Carefully aspirate the supernatant until approximately 1 inch of liquid remains. Tilt the tube and slowly advance the remaining medium into an aspirator pipette. If desired, resuspend the exosome pellet in PBS and repeat ultracentrifugation at 28,000 rpm for 1-2 h to further purify the exosome population.

[0067] Finally, resuspend the exosome pellet in 210 µL of PBS. If there are multiple ultracentrifuge tubes for each sample, use the same 210 µL of PBS to resuspend each exosome pellet stepwise. Take 10 µL for each sample and add to 990 µL of HO to use for nanoparticle tracking analysis. Use the remaining 200 µL of exosome-containing suspension for downstream processes or immediately store at -80 °C.

[0068] 2. Exemplary Protocol for Extracting Exosomes from Serum Samples First, the serum sample is thawed on ice. Then, 250 μL of the cell-free serum sample is diluted in 11 mL of PBS and filtered through a 0.2 μm pore filter. The diluted sample is ultracentrifuged overnight at 150,000 × g at 4 ° C. The following day, the supernatant is carefully discarded and the exosome pellet is washed with 11 mL of PBS. A second round of ultracentrifugation is performed at 150,000 × g at 4 ° C for 2 hours. Finally, the supernatant is carefully discarded and the exosome pellet is resuspended in 100 μL of PBS for analysis.

[0069] C. Exemplary Protocols for Electroporation of Exosomes and Liposomes 1×10 8 Mix 100 nm exosomes (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula Nano Sciences) with 1 µg of siRNA (Qiagen) or shRNA in 400 µL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). Electroporate the exosomes or liposomes using a 4 mm cuvette (e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2013). (See, e.g., 2012). After electroporation, the exosomes or liposomes are treated with protease-free RNAse, followed by the addition of 10x concentrated RNase inhibitor. Finally, the exosomes or liposomes are washed with PBS under ultracentrifugation as described above.

[0070] II. Disease Diagnosis, Prognosis, and Treatment The detection, isolation, and characterization of cancer cell-derived exosomes using the method of the present invention are useful in assessing cancer prognosis and in monitoring treatment efficacy to detect early treatment failure that may lead to disease recurrence.Furthermore, cancer cell-derived exosome analysis according to the present invention allows for the detection of early recurrence in presymptomatic patients who have completed a course of treatment.This is possible because the presence of cancer cell-derived exosomes may accompany and / or correlate with tumor progression and spread, poor response to therapy, disease recurrence, and / or reduced survival over a period of time.Therefore, the enumeration and characterization of cancer cell-derived exosomes provides a method for stratifying patients for baseline characteristics that predict initial and subsequent risk based on response to therapy.

[0071] The cancer cell-derived exosomes isolated according to the method disclosed in the present invention can be analyzed to diagnose or prognose cancer in a subject.As such, the method of the present invention can be used, for example, to evaluate cancer patients and those at risk of cancer.In any of the diagnostic or prognostic methods described herein, the presence or absence of one or more indicators of cancer, such as genomic mutations or cancer-specific exosome surface markers, or one or more indicators of any other disorder, can be used to diagnose or prognose.

[0072] In one embodiment, blood samples are drawn from patients, and cancer cell-derived exosomes are detected and / or isolated as described herein.For example, exosomes can be labeled with one or more antibodies or aptamers that bind to ATP-binding cassette subfamily A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), and / or glypican-1 (GPC1), and the antibodies can be covalently linked to fluorescent labels.Analysis can then be performed to determine the number and characterization of cancer cell-derived exosomes in the sample, and from this measurement, the number of cancer cell-derived exosomes present in the initial blood sample can be determined. Exosomes identified as cancer cell-derived exosomes may contain a second (or more) marker known to be selectively or specifically found in cancer cell-derived exosomes, such as histone H2A type 2-A (HIST1H2AA), histone H2A type 1-A (HIST1H1AA), histone H3.3 (H3F3A), histone H3.1 (HIST1H3A), zinc finger protein 37 homolog (ZFP37), laminin subunit beta-1 (LAMB1), tubulointerstitial nephritis antigen-like (TINAGL1), peroxiredoxin-4 (PRDX4), collagen alpha-2(IV) chain (COL4A2), putative protein C3P1 (C3P1), hemicentin-1 (HMCN1), putative rhophilin-2-like protein (RHPN2P1), ankyrin repeat domain-containing protein 62 (ANKRD62), tripartite motif motif-containing protein 42 (TRIM42), junction plakoglobin (JUP), tubulin beta 2 B chain (TUBB2B), endoribonuclease dicer (DICER1), E3 ubiquitin-protein ligase TRIM71 (TRIM71), katanin p60 ATPase-containing subunit A-like 2 (KATNAL2), protein S100-A6 (S100A6), 5'-nucleotidase domain-containing protein 3 (NT5DC3), valine-tRNA ligase (VARS), kazurin (KAZN), ELAV-like protein 4 (ELAVL4), ring finger protein 166 (RNF166), FERM and PDZ domain-containing protein 1 (FRMPD1), 78 kDa glucose-regulated protein (HSPA5), transport protein particle complex subunit 6A (TRAPPC6A), squalene monooxygenase (SQLE), tumor susceptibility This can be verified by detecting the following: sex gene 101 protein (TSG101), vacuolar protein sorting 28 homolog (VPS28), prostaglandin F2 receptor negative regulator (PTGFRN), isobutyryl-CoA dehydrogenase, mitochondrial (ACAD8), 26S protease regulatory subunit 6B (PSMC4), elongation factor 1-oncogene (EEF1G), titin (TTN), tyrosine-protein phosphatase type 13 (PTPN13), triosephosphate isomerase (TPI1), or carboxypeptidase E (CPE). The number of cancer cell-derived exosomes can be determined by hemocytometry or microscopic techniques to visually quantify and characterize exosomes. Cancer cell-derived exosomes can be detected and quantified by other methods known in the art (e.g., ELISA).

[0073] In various embodiments, the analysis of the number and characterization of the cancer cell-derived exosomes of a subject is carried out at various intervals over a certain time course to evaluate the progression and pathology of the subject.For example, the analysis can be carried out at regular intervals, such as 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year, to track the level and characterization of the cancer cell-derived exosomes over time.In the case of existing cancer patients, this provides a useful indication of disease progression, and helps medical practitioners make appropriate therapeutic choices based on the increase, decrease, or no change in the cancer cell-derived exosomes.Any increase of 2-fold, 5-fold, 10-fold, or more over time of cancer cell-derived exosomes reduces the patient's prognosis, which is an early indicator that the patient should change therapy.Similarly, any increase of 2-fold, 5-fold, 10-fold, or more indicates that the patient should undergo further examination, such as imaging, to further evaluate the prognosis and response to therapy. Any decrease in cancer cell-derived exosomes over time that is 2-fold, 5-fold, 10-fold or more indicates disease stabilization and patient response to therapy, which is an indication of not changing therapy.For those at risk of cancer, a sudden increase in the number of detected cancer cell-derived exosomes can provide an early warning that the patient is developing a tumor, thus providing an early diagnosis.In one embodiment, the detection of cancer cell-derived exosomes increases with cancer staging.

[0074] In any of the methods provided herein, additional analysis can also be carried out to characterize cancer cell-derived exosomes to provide additional clinical evaluation.For example, in addition to image analysis and bulk number measurement, PCR techniques such as multiplexing with primers specific for specific cancer markers can be used to obtain information such as the type of tumor from which cancer cell-derived exosomes are derived, metastatic status, and the degree of malignancy.In addition, DNA or RNA analysis, proteome analysis, or metabolome analysis can be carried out as a means of evaluating additional information regarding the characterization of patient's cancer.

[0075] For example, further analysis provides sufficient data to determine the responsiveness of a subject to a particular treatment regimen or to determine the effectiveness of a candidate drug in treating cancer. Thus, the present invention provides a method for determining the responsiveness of a subject to a particular treatment regimen or the effectiveness of a candidate drug in treating cancer by detecting / isolating a cancer cell-derived exosome of a subject as described herein and analyzing said cancer cell-derived exosome. For example, once a drug treatment is administered to a patient, the method of the present invention can be used to determine the effectiveness of the drug treatment. For example, a sample obtained from a patient before drug treatment, as well as one or more samples obtained from the patient simultaneously with or after drug treatment, can be processed using the method of the present invention. By comparing the analysis results of each of the processed samples, the effectiveness of the drug treatment or the patient's responsiveness to the drug can be determined. In this way, early identification of failed compounds can be performed, and early validation of promising compounds can be performed.

[0076] Certain aspects of the present invention provide for the treatment of patients with exosomes expressing or containing therapeutic or diagnostic agents. A "therapeutic agent," as used herein, is an atom, molecule, or compound useful in the treatment of cancer or other conditions. Examples of therapeutic agents include, but are not limited to, drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, chelators, boron compounds, photoactive agents, and dyes. A "diagnostic agent," as used herein, is an atom, molecule, or compound useful in the diagnosis, detection, or visualization of disease. According to embodiments described herein, diagnostic agents can include, but are not limited to, radioactive substances (e.g., radioisotopes, radionuclides, radiolabels, or radiotracers), dyes, imaging agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzymes, and enhancers (e.g., paramagnetic ions).

[0077] In some embodiments, the therapeutic recombinant protein may be a protein with an activity lost in the patient's cells, a protein with a desired enzymatic activity, a protein with a desired inhibitory activity, etc. For example, the protein may be a transcription factor, an enzyme, a protein toxin, an antibody, a monoclonal antibody, etc. The monoclonal antibody may specifically or selectively bind to an intracellular antigen. The monoclonal antibody may inhibit the function of the intracellular antigen and / or disrupt protein-protein interactions. Another embodiment of the present invention provides for the diagnosis of disease based on the presence of cancer cell-derived exosomes in a patient sample.

[0078] Since exosomes are known to contain the machinery required for complete mRNA transcription and protein translation (PCT / US2014 / 068630, the entirety of which is incorporated herein by reference), mRNA or DNA nucleic acid encoding therapeutic protein can be transfected into exosomes. Alternatively, therapeutic protein itself can be electroporated into exosomes or directly incorporated into liposomes. Exemplary therapeutic proteins include, but are not limited to, tumor suppressor proteins, peptides, wild-type protein counterparts of mutant proteins, DNA repair proteins, proteolytic enzymes, proteinaceous toxins, proteins that can inhibit the activity of intracellular proteins, proteins that can activate the activity of intracellular proteins, or any protein that needs to be reconstituted for loss of function. Specific examples of exemplary therapeutic proteins include 123F2, Abcb4, Abcc1, Abcg2, Actb, Ada, Ahr, Akt, Akt1, Akt2, Akt3, Amhr2, Anxa7, Apc, Ar, Atm, Axin2, B2m, Bard1, Bcl2l1, Becn1, Bhlhal5, Bin1, Blm, Braf, Brca1, Brca2, Brca3, Braf, Brcata, Brinp3, Brip1, Bub1b, Bwscr1a, Cadm3, Casc1, Casp3, Casp7, Casp8, Cav1, Ccam, Ccnd1, Ccr4, Ccs1, Cd28, Cdc25a, Cd95, Cdh1, Cdkn1a, Cdkn1b, Cdkn2a, Cd kn2b, Cdkn2c, Cftr, Chek1, Chek2, Crcs1, Crcs10, Crcs11, Crcs2, Crcs3, Crcs4, Crcs5, Crcs 6, Crcs7, Crcs8, Crcs9, Ctnnb1, Cts1, Cyp1a1, Cyp2a6, Cyp2b2, Cyld, Dcc, Dkc1, Dicer1, Dmtf 1, Dnmt1, Dpc4, E2f1, Eaf2, Eef1a1, Egfr, Egfr4, Erbb2, Erbb4, Ercc2, Ercc6, Ercc8, Errfi1, Esr1, Etv4, Faslg, Fbxo10, Fcc, Fgfr3, Fntb, Foxm1, Foxn1, Fus1, Fzd6, Fzd7, Fzr1, Gadd45a,<h2 style=";text-align:left;direction:ltr">Gast、Gnai2、Gpc1、Gpr124、Gpr87、Gprc5a、Gprc5d、Grb2、Gstm1、Gstm5、Gstp1、Gstt1、H19、H2afx、 Hck、Lims1、Hdac、Hexa、Hic1、Hin1、Hmmr、Hnpcc8、Hprt、Hras、Htatip2、Il1b、Il10、Il2、Il6、Il8rb Inha, Itgav, Jun, Jak3, Kit, Klf4, Kras, Kras2, Kras2b, Lig1, Lig4, Lkb1, Lmo7, Lncr1, Lncr2, Lncr3, Lncr4, Ltbp4, Luca1, Luca2, Lyz2, Lzts1, Mad1l1, Mad2l1, Madr2 / Jv18, Mapk14, Mcc, Mcm4, Men1, Men2, Met, Mgat5, Mif, Mlh1, Mlh3, Mmac1, Mmp8, M nt、Mpo、Msh2、Msh3、Msh6、Msmb、Mthfr、Mts1、Mutyh、Myh11、Na t2、Nbn、Ncoa3、Neil1、Nf1、Nf2、Nfe2l1、Nhej1、Nkx2-1、Nkx2- 9、Nkx3-1、Nprl2、Nqo1、Nras、Nudt1、Ogg1、Oxgr1、p16、p19、p2 1、p27、p27mt、p57、p14ARF、Palb2、Park2、Pggt1b、Pgr、Pi3k、P ik3ca、Piwil2、Pl6、Pla2g2a、Plg、Plk3、Pms1、Pms2、Pold1、P ole、Ppard、Pparg、Ppfia2、Ppm1d、Prdm2、Prdx1、Prkar1a、Ptc h、Pten、Prom1、Psca、Ptch1、Ptf1a、Ptger2、Ptpn13、Ptprj、Ra ra、Rad51、Rassf1、Rb、Rb1、Rb1cc1、Rb12、Recgl4、Ret、Rgs5、R hoc、Rint1、Robo1、Rpl38、S100a4、SCGB1A1、Skp2、Smad2、Smad3、Smad4、Smarcb1、Smo、Snx25、Spata13、Srpx、Ssic1、Sstr2、S str5、Stat3、St5、St7、St14、Stk11、Suds3、Tap1、Tbx21、Terc、 Tnf、Tp53、Tp73、Trpm5、Tsc2、Tsc1、Vhl、Wrn、Wt1、Wt2、Xrcc1、Xrcc5, Xrcc6, and Zac1.

[0079] One particular type of protein that may be desirable to introduce into the intracellular space of diseased cells is an antibody (e.g., a monoclonal antibody). Such an antibody may disrupt the function of intracellular proteins and / or disrupt intracellular protein-protein interactions. Exemplary targets of such monoclonal antibodies include, but are not limited to, proteins involved in the RNAi pathway, telomerase, transcription factors that control disease processes, kinases, phosphatases, proteins required for DNA synthesis, and proteins required for protein translation. Specific examples of targets of exemplary therapeutic antibodies include proteins encoded by the following genes: Dicer, Ago1, Ago2, Trbp, Ras, raf, wnt, btk, Bcl-2, Akt, Sis, src, Notch, Stathmin, mdm2, abl, hTERT, c-fos, c-jun, c-myc, erbB, HER2 / Neu, HER3, VEGFR, PDGFR, c-kit, c-met, c-ret, flt3, API, AML1, axl, alk, fins, fps, gip, lck, Stat, Hox, MLM, PRAD-I, and trk. In addition to monoclonal antibodies, any antigen-binding fragment thereof, such as scFv, Fab fragment, Fab', F(ab')2, Fv, peptibody, diabody, triabody, or minibody, are also contemplated. Any such antibody or antibody fragment may be glycosylated or non-glycosylated.

[0080] Since exosomes are known to contain DICER and active RNA processing RISC complexes (PCT Publication No. WO2014 / 152622, incorporated herein by reference in its entirety), shRNAs transfected into exosomes may be matured into siRNAs bound to the RISC-complex with the exosomes themselves. Alternatively, mature siRNAs themselves can be transfected into exosomes or liposomes. Thus, by way of example, the following are classes of potential target genes that can be used to modulate or attenuate target gene expression in the methods of the invention: wild-type or mutant versions of developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, winged helix family members, Hox family members, cytokines / lymphokines and their receptors, growth or differentiation factors and their receptors, neurotransmitters and their receptors, and siRNAs that are capable of modulating or attenuating target gene expression in the methods of the invention. receptors), tumor suppressor genes (e.g., APC, CYLD, HIN-1, KRAS2b, p16, p19, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2 / JV18, MEN1, MEN2, 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 SEM A3 polypeptide), proapoptotic 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, I L-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, EBRB2, ETS1, ETS1, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, and YES), and enzymes (e.g., ACP desaturase and hycroxylase, ADP-glucose pyrofolylase, ... ATPase, alcohol dehydrogenase, amylase, Myroglycosidase, catalase, cellulase, cyclooxygenase, decarboxylase, dextrinase, esterase, DNA and RNA polymerase, galactosidase, glucanase, glucose oxidase, GTPase, helicase, hemicellulase, integrase, invertase, hemicellulase, kinase, lactase, lipase, lipoxygenase, lysozyme, nuclease, pectinesterase, peroxidase, phosphatase, phospholipase, phosphorylase, polygalacturonase, proteinase and peptidease, planase, recombinase, reverse transcriptase, topoisomerase, xylanase). In some cases, sh / siRNA can be designed to specifically target mutant versions of genes expressed in cancer cells while leaving expression of the corresponding wild-type version unaffected. Indeed, any inhibitory nucleic acid may be applied to the compositions and methods of the present invention, provided that such an inhibitory nucleic acid has been found by any source to be a verified downregulator of a protein of interest.

[0081] In designing RNAi, several factors must be considered, such as the nature of siRNA, the duration of silencing effect, and the choice of delivery system. To produce RNAi effect, the siRNA introduced into organisms generally contains exon sequence. Furthermore, the RNAi process is homology-dependent, and therefore the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. siRNAs are preferably those that show more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences with less than about 80% identity to the target gene are substantially less effective. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes is affected.

[0082] In addition to protein-based and nucleic acid-based therapeutics, exosomes can also be used to deliver small molecule drugs, either alone or in combination with any protein-based or nucleic acid-based therapeutic. Exemplary small molecule drugs contemplated for use in the present embodiment include, but are not limited to, toxins, chemotherapeutic agents, agents that inhibit the activity of intracellular proteins, agents that activate the activity of intracellular proteins, agents for preventing restenosis, agents for treating renal disease, agents used for intermittent claudication, agents used for treating hypotension and shock, angiotensin converting enzyme inhibitors, antianginal agents, antiarrhythmic agents, antihypertensive agents, angiotensin II receptor antagonists, antiplatelet agents, b-blockers b1 selective, beta blockers, botanical products for cardiovascular indications, calcium channel blockers, cardiovascular / diagnostic agents, central alpha-2 agonists, coronary vasodilators, diuretics and tubular inhibitors, neutral endopeptidase / angiotensin converting enzyme inhibitors, peripheral vasodilators, potassium channel openers, potassium salts, anticonvulsants, antiemetics, antiemetics, antiparkinsonian agents, anticonvulsants, cerebral stimulants, agents that can be applied to the treatment of trauma, agents that can be applied to the treatment of Alzheimer's disease or dementia, agents that can be applied to the treatment of migraine, agents that can be applied to the treatment of neurodegenerative diseases, agents that can be applied to the treatment of Kaposi's sarcoma, agents that can be applied to the treatment of AIDS, cancer chemotherapy agents, agents that can be applied to the treatment of immune disorders, agents that can be applied to the treatment of psychiatric disorders, analgesics, epidural and intrathecal anesthetics, systemic, local, regional neuromuscular blocking sedatives, pre-anesthetic adrenal / acth, anabolic steroids, agents that can be applied to the treatment of diabetes drugs for inflammatory bowel disease, dopamine, growth hormone and analogues, hyperglycemic agents, hypoglycemic agents, oral insulin, bulk parenteral drugs (LVPS), lipid altering agents, metabolic testing and inborn errors of metabolism, nutrients / amino acids, nutritional LVPS, obesity drugs (appetite suppressants), somatostatin, thyroid drugs, vasopressin, vitamins, corticosteroids, mucolytics, pulmonary anti-inflammatory agents, pulmonary surfactants, antacids, anticholinergics, antidiarrheals, antiemetics, gallstone dissolving agents, drugs for inflammatory bowel disease, drugs for irritable bowel syndrome, drugs for the liver,Metal chelators, drugs against miscellaneous gastric secretions, drugs against pancreatitis, pancreatic enzymes, prostaglandins, prostaglandins, proton pump inhibitors, sclerosing agents, sucralfate, antiprogestins, contraceptives, oral contraceptives, parenteral dopamine, estrogens, gonadotropins, GNRH, GHRH antagonists, catabolic agents, progestins, uterine agents, antianemic agents, anticoagulants, antifibrinolytic agents, antiplatelet agents, antithrombin agents, coagulants, fibrinolytic agents, hematological agents, heparin inhibitors, metal chelators, prostaglandins, vitamin K, antiandrogens, aminoglycosides, antibacterial agents, sulfonamides, cephalosporins, clindamycin, dermatological agents, surfactants, erythromycin, anthelmintics, antifungals, antimalarials, antimycobacteria, antiparasitics, antiprotozoal agents, antitrichomonas, antituberculous agents, immunomodulators, immunostimulants, macrolides, antiparasitics, corticosteroids, cyclooxygenase inhibitors, enzyme blockers, immunomodulators for rheumatic diseases, metalloproteinase inhibitors, nonsteroidal anti-inflammatory agents), analgesics, antipyretics, alpha adrenergic agonists / blockers, antibiotics, antivirals, beta adrenergic blockers, carbonic anhydrase inhibitors, corticosteroids, immune system regulators, mast cell inhibitors, nonsteroidal anti-inflammatory agents, and prostaglandins.

[0083] Exosomes can be used to deliver diagnostic agents. Exemplary diagnostic agents include, but are not limited to, magnetic resonance imaging enhancing agents, positron emission tomography products, radioactive diagnostic agents, radioactive therapeutic agents, radiopaque contrast agents, radiopharmaceuticals, ultrasound imaging agents, and angiographic diagnostic agents.

[0084] The term "subject" as used herein refers to any individual or patient on whom the subject method is carried out. Generally, the subject is a human, but as will be understood by those skilled in the art, the subject may be an animal. Thus, other animals are included within the definition of subject, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas).

[0085] "Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject or the performance of a procedure or modality on a subject to obtain a therapeutic benefit with respect to a disease or health-related condition. For example, treatment can include administering chemotherapy, immunotherapy, or radiation therapy, performing surgery, or any combination thereof.

[0086] The term "therapeutic benefit" or "therapeutically effective" as used herein refers to anything that promotes or enhances the well-being of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer can involve, for example, reducing the invasiveness of a tumor, reducing the rate of growth of the cancer, or preventing metastasis. Treating cancer can also refer to prolonging the survival of a subject with cancer.

[0087] The term "cancer" as used herein can be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, the cancer can occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testes, tongue, or uterus.

[0088] The cancer may specifically be of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma adenocarcinoma); papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; adenocarcinoma);mucinous epidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma w / squamous metaplasia;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipocytoma, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomangiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma; Malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymal tumor, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonal carcinoma;teratoma, malignant;ovarian goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;chondroblastoma, malignant;mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Ameloblastic tumor, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Neurilemoma, malignant;Granular cell tumor, Malignant; malignant lymphoma; Hodgkin's disease; Hodgkin; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. Nevertheless, it will be understood that the present invention can also be used to treat non-cancerous diseases (e.g., fungal infections, bacterial infections, viral infections, neurodegenerative diseases, and / or genetic disorders).

[0089] The terms "contacted" and "exposed," as applied to a cell, are used herein to describe the process of delivering a therapeutic agent to or placing it in direct proximity to a target cell. To achieve cell death, for example, one or more agents are delivered to the cell in an amount effective to kill the cell or prevent the cell from dividing.

[0090] Effective response of a patient to a treatment or "responsiveness" of a patient refers to a clinical or therapeutic benefit provided to a patient at risk of or suffering from a disease or disorder. Such benefit can include a cellular or biological response, a complete response, a partial response, stable disease (no progression or recurrence), or a response with subsequent recurrence. For example, an effective response can be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer.

[0091] The methods described herein can predict and monitor treatment outcome and / or identify or select patients who will benefit from such treatment.

[0092] Regarding the treatment of neoplastic conditions, depending on the stage of the neoplastic condition, the treatment of neoplastic conditions involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiation therapy, and chemotherapy.Other therapeutic regimens can be combined with the administration of anticancer agents, such as therapeutic compositions and chemotherapeutic agents.For example, the patient who is treated with such anticancer agents can also undergo radiation therapy and / or surgery.

[0093] For the treatment of disease, the appropriate dosage of the therapeutic composition will depend on the type, severity and course of the disease being treated, as defined above, the patient's clinical history and response to the drug, and the discretion of the attending physician. The drug is suitably administered to the patient at one time or over a series of treatments.

[0094] The therapeutic and preventive methods and compositions can be provided in effective combined amounts to achieve desired effects.Tissue, tumor or cell can be contacted with one or more compositions or pharmacological preparations (several) that contain one or more drugs, or tissue, tumor and / or cell can be contacted with two or more separate compositions or preparations.It is also intended that such combination therapy can be used in conjunction with chemotherapy, radiation therapy, surgery or immunotherapy.

[0095] The administration of combination includes simultaneous administration of two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration.That is, the therapeutic composition of interest and another therapeutic agent can be formulated together in the same dosage form and administered simultaneously.Alternatively, the therapeutic composition of interest and another therapeutic agent can be administered simultaneously, in which case both agents are in separate formulations.In another alternative, a therapeutic agent can be administered and the other therapeutic agent can be administered immediately after, or vice versa.In separate administration protocols, the therapeutic composition of interest and another therapeutic agent can be administered several days apart, or hours apart, or days apart.

[0096] The first anti-cancer treatment (e.g., exosomes expressing a recombinant protein or exosomes with a recombinant protein isolated from exosomes) can be administered prior to, during, after, or in various combinations with the second anti-cancer treatment. The administrations can be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments where the first treatment is provided to the patient separately from the second treatment, generally no significant period of time passes between the time of each delivery, thus ensuring that the two compounds can still exert their advantageous combined effect on the patient. In such instances, it is contemplated to provide the first and second therapies to the patient within about 12 to 24 hours or 72 hours of each other, more particularly within about 6 to 12 hours of each other. In some circumstances, it is desirable to extend the duration of the treatments considerably, where several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between the respective administrations.

[0097] In certain embodiments, the course of treatment lasts for 1-90 days or more (including the ranges in between). It is contemplated that one agent can be administered on any of days 1-90 (including the ranges in between) or any combination thereof, and another agent can be administered on any of days 1-90 (including the ranges in between) or any combination thereof. The agent(s) can be administered to the patient once or multiple times within a day (24 hour period). It is further contemplated that after the course of treatment, there will be a period during which no anti-cancer treatment is administered. This period can be 1-7 days, and / or 1-5 weeks, and / or 1-12 months or more (including the ranges in between), depending on the patient's condition, e.g., prognosis, endurance, health, etc. It is anticipated that the treatment cycle will be repeated as necessary.

[0098] Various combinations can be used. For the following examples, the first anti-cancer therapy is "A" and the second anti-cancer 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

[0099] Administration of any compound or therapy of the invention to a patient follows typical protocols for administering such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0100] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to indicate a compound or composition administered in the treatment of cancer. These agents or drugs are categorized according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink with DNA, to intercalate into DNA, or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0101] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins. (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride oxide hydrochloride, such as melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II and calicheamicin omega II); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicins; and neocarzinostatin chromophore and related chromoprotein enediyne antibacterial chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carubicin, abicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane;Folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate acetate); epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin, etc. oridin A and anguidine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel-gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as Cisplatin, oxaliplatin, and carboplatin, etc.; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above;

[0102] 2. Radiation therapy Other agents that cause DNA damage and are widely used include what is commonly known as gamma radiation, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation are also contemplated. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0103] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy can be used in combination or in conjunction with the method of the present invention. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (Rituxan®) is such an example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody alone can act as an effector of therapy or can recruit other cells to actually affect cell killing. The antibody can also be conjugated with a drug or toxin (such as a chemotherapeutic drug, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply function as a targeting agent. Alternatively, the effector can be a lymphocyte with a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0104] In one aspect of immunotherapy, tumor cells must have some markers that are suitable for targeting, i.e., not present on the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0105] Examples of immunotherapies currently being investigated or used include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1999). 8 years); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2013; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0106] 4.Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, which physically removes, cuts out, and / or destroys all or part of cancerous tissue, and can be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy. Tumor resection refers to physically removing at least a part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0107] When cancerous cells, tissues, or tumors are partially or completely removed, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment can be repeated, for example, every day, every 2, 3, 4, 5, 6, or 7 days, or every week, every 2, 3, 4, and 5 weeks, or every month, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments are similarly subject to dosage variations.

[0108] 5. Other drugs It is contemplated that other agents may be used in combination with certain aspects of the invention to improve the efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the invention to improve the anti-hyperproliferative efficacy of the treatment. It is contemplated that cell adhesion inhibitors improve the efficacy of the invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Additionally, it is contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the invention to improve the efficacy of the treatment.

[0109] III. Pharmaceutical Compositions It is contemplated that exosomes expressing or containing recombinant proteins, inhibitory RNA, and / or small molecule drugs can be administered systemically or locally to inhibit tumor cell growth and, most preferably, to kill cancer cells in cancer patients with locally advanced or metastatic cancer. The exosomes can be administered intravenously, intrathecally, and / or intraperitoneally. The exosomes can be administered alone or in combination with anti-proliferative drugs. In one embodiment, the exosomes can be administered before surgery or other procedures to reduce the cancer burden in patients. Alternatively, the exosomes can be administered after surgery to ensure that any remaining cancer (e.g., cancer that could not be eliminated by surgery) does not survive.

[0110] The present invention is not intended to be limited by the specific nature of the therapeutic preparation.For example, such compositions can be formulated with physiologically acceptable liquid, gel, solid carrier, diluent or excipient.These therapeutic preparations can be administered to mammals for veterinary use, such as with livestock animals, and for clinical use in humans, similar to other therapeutic agents.In general, the dosage required for therapeutic effectiveness varies according to the type of use and the mode of administration, and the specific requirements of each individual subject.

[0111] When clinical applications are intended, it may be necessary to prepare pharmaceutical compositions containing recombinant proteins and / or exosomes in a form suitable for the intended application. In general, pharmaceutical compositions, which may be parenteral formulations, may contain an effective amount of one or more recombinant proteins and / or exosomes and / or additional agents dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human, as appropriate. Preparations of pharmaceutical compositions containing recombinant proteins and / or exosomes, or additional active ingredients, as disclosed herein, are exemplified in Remington's Pharmaceutical Sciences, 18th Edition, 1990, which is incorporated herein by reference in its entirety for all purposes. Furthermore, it will be understood that for administration to animals (e.g., humans), preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0112] Further, according to certain aspects of the present invention, the composition suitable for administration can be provided in a pharma- ceutically acceptable carrier with or without an inert diluent. As used herein, a "pharma- ceutically acceptable carrier" refers to any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, ethanol, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), vegetable oils, and injectable organic esters such as ethyl oleate, etc.), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof), isotonicity agents (isotonic acid agents), tertiary amines, ... The carriers include, for example, absorbent, sorbent, sorbitol, sorbitol-containing ...

[0113] Pharmaceutically acceptable carriers are particularly formulated for administration to humans, although in certain embodiments, it is desirable to use pharma- ceutically acceptable carriers that are formulated for administration to non-human animals and are not acceptable for administration to humans (e.g., by government regulations). Use of carriers in therapeutic or pharmaceutical compositions is contemplated unless any conventional carrier is incompatible with the active ingredient (e.g., harmful to the recipient or to the therapeutic effect of the composition contained in the carrier). According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such procedures are routine for those skilled in the art.

[0114] Certain embodiments of the present invention may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form and whether they need to be sterile for the route of administration, such as injection. The composition may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, through a mucous membrane, orally, locally, topically, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing the target cells directly, via a catheter, via lavage, in lipid compositions (e.g., liposomes), or by other methods known to those skilled in the art, for example, as described in Remington's Pharmaceutical Sciences, 18th Edition, 1990, which is incorporated herein by reference, or any combination of the above.

[0115] The active compound can be formulated for parenteral administration, for example, for injection by intravenous, intramuscular, subcutaneous, or even intraperitoneal route.Thus, the embodiment includes parenteral formulations.Generally, such compositions can be prepared as solution or suspension, and can also be prepared in solid form suitable for use in preparing solution or suspension by adding liquid before injection, and the preparation can also be emulsified.

[0116] Depending on the embodiment of interest, parenteral formulations may include the exosomes disclosed herein together with one or more solutes and / or solvents, one or more buffers and / or one or more antimicrobial agents, or any combination thereof. In some aspects, the solvents may include water, water-miscible solvents, such as ethyl alcohol, liquid polyethylene glycol, and / or propylene glycol, and / or water-immiscible solvents, such as non-volatile oils, including, for example, corn oil, cottonseed oil, peanut oil, and / or sesame oil. In certain versions, the solutes may include one or more antimicrobial agents, buffers, antioxidants, tonicity agents, cryoprotectants, and / or lyoprotectants.

[0117] Antimicrobial agents according to the present disclosure may include those provided elsewhere in the present disclosure, as well as benzyl alcohol, phenol, mercury, and / or parabens. Antimicrobial agents may include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal, or any combination thereof. Antimicrobial agents may be present in various embodiments at concentrations necessary to ensure the sterility required for pharmaceutical products. For example, agents may be present in preparations, such as preparations contained in multi-dose containers, at bacteriostatic or fungistatic concentrations. In various embodiments, the agent may be a preservative and / or may be present at a suitable concentration to prevent the growth of microorganisms during use, such as those accidentally introduced into the preparation during withdrawal of a portion of the contents with a hypodermic needle and syringe. In various aspects, the agent has a maximum volume and / or concentration limit (e.g., phenylmercuric nitrate and thimerosal 0.01%, benzethonium chloride and benzalkonium chloride 0.01%, phenol or cresol 0.5%, and chlorobutanol 0.5%). In various examples, agents such as phenylmercuric nitrate are used at a concentration of 0.002%. A combination of methyl p-hydroxybenzoate 0.18% and propyl p-hydroxybenzoate 0.02%, and benzyl alcohol 2% can also be applied according to embodiments. The antimicrobial agent may include hexylresorcinol 0.5%, phenylmercuric benzoate 0.1%, and / or a therapeutic compound.

[0118] Antioxidants according to the present disclosure may include ascorbic acid and / or its salts, and / or sodium salt of ethylenediaminetetraacetic acid (EDTA). Tonicity agents as described herein may include electrolytes and / or mono- or disaccharides. Cryoprotectants and / or lyoprotectants are additives that protect the biologic from harmful effects resulting from freezing and / or drying of the product during the freeze-drying process. Cryoprotectants and / or lyoprotectants may include sugars (non-reducing), such as sucrose or trehalose, amino acids, such as glycine or lysine, polymers, such as liquid polyethylene glycol or dextran, and polyols, such as mannitol or sorbitol, which are all potential cryoprotectants or lyoprotectants. Targeted embodiments may also include antifungal agents, such as butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid, or any combination thereof. Additional solutes and antimicrobial agents, buffers, antioxidants, tonicity agents, cryoprotectants or lyoprotectants and their properties that can be used in embodiments of the present disclosure, as well as in methods of making the subject parenteral formulations, are described, for example, in Remington's Pharmaceutical Sciences, 21st Edition, 2005, e.g., Chapter 41, which is incorporated herein by reference in its entirety for all purposes.

[0119] Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected.The form must also be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.

[0120] The therapeutic agent can be formulated in the composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of the proteinaceous composition, or those formed with inorganic acids, for example, hydrochloric acid or phosphoric acid, or organic acids, for example, acetic acid, oxalic acid, tartaric acid, or mandelic acid. Similarly, salts formed with free carboxy groups can be derived from inorganic bases, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, or organic bases, for example, isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as those formulated for parenteral administration, such as injection solutions, or those formulated for administration with the diet, such as aerosols for delivery to the lungs, or drug-releasing capsules.

[0121] In certain embodiments of the present invention, the composition is thoroughly combined or mixed with semi-solid or solid carrier.Mixing can be carried out in any convenient manner, such as by grinding.Stabilizers can also be added to the mixing process to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0122] In further embodiments, the present invention may relate to the use of pharmaceutical lipid vehicle compositions comprising one or more lipids and an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide range of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., designed or created by man). However, lipids are usually biological substances. Biological lipids are well known in the art, and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether-linked and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds that are understood to be lipids by those skilled in the art other than those specifically described herein are also encompassed by the compositions and methods.

[0123] Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in lipid vehicles.For example, therapeutic agents can be dispersed in a solution that contains lipids, dissolved with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained in lipids as suspensions, contained in or complexed with micelles or liposomes, or otherwise associated with lipids or lipid structures by any means known to those skilled in the art.Dispersion may or may not result in the formation of liposomes.

[0124] The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of therapeutic composition calculated to produce the desired response discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The amount administered, both as a number of treatments and as a unit dose, depends on the desired effect. The actual dosage of the composition of the present invention administered to a patient or subject can be determined by physical and physiological factors such as the subject's weight, age, health, and sex, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathic diseases of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic agent. For example, the dosage can comprise from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (this range includes doses in between) or more, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include ranges of about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. In any event, the practitioner responsible for administration will determine the concentration of active ingredient(s) in the composition and appropriate dose(s) for the individual subject.

[0125] The actual dosage of the composition administered to an animal patient can be determined by physical and physiological factors such as body weight, the severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the frequency of administration of an effective amount may vary depending on the subject's response. In any event, the practitioner responsible for administration will determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0126] In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise about 2% to about 75%, or about 25% to about 60%, for example, and any range derivable therein, of the weight of the unit. Naturally, the amount of active compound(s) in each therapeutically useful composition can be prepared so that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are taken into account by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0127] In other non-limiting examples, dosages can include from about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight, to about 1000 milligrams / kg / body weight or more per administration, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numbers recited herein include ranges based on the above numbers of about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc.

[0128] IV. Nucleic Acids and Vectors In certain aspects of the present invention, nucleic acid sequences encoding therapeutic proteins or nucleic acid sequences encoding fusion proteins containing therapeutic proteins can be disclosed. Depending on which expression system is used, nucleic acid sequences can be selected based on conventional methods. For example, each gene or its variant can be codon-optimized for expression in a certain system. Various vectors can also be used to express the protein of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0129] V. Recombinant Proteins and Inhibitory RNA Some embodiments relate to recombinant proteins and polypeptides. Certain embodiments relate to recombinant proteins or polypeptides that exhibit at least one therapeutic activity. In some embodiments, the recombinant protein or polypeptide may be a therapeutic antibody. In some aspects, the therapeutic antibody may be an antibody that specifically or selectively binds to an intracellular protein. In another aspect, the protein or polypeptide may be modified to increase its stability in serum. Thus, when the application refers to a function or activity of a "modified protein" or "modified polypeptide," it will be understood by those skilled in the art that this includes, for example, a protein or polypeptide that has additional advantages over an unmodified protein or polypeptide. It is expressly intended that embodiments relating to a "modified protein" may be implemented with respect to a "modified polypeptide," and vice versa.

[0130] Recombinant proteins may have amino acid deletions and / or substitutions, and thus proteins with deletions, substitutions, and deletions and substitutions are modified proteins. In some embodiments, these proteins may further include amino acid insertions or additions, such as fusion proteins or proteins with linkers. A "modified deleted protein" may lack one or more residues of the native protein, but may have the specificity and / or activity of the native protein. A "modified deleted protein" may also have reduced immunogenicity or antigenicity. An example of a modified deleted protein is one in which amino acid residues have been deleted from at least one antigenic region, i.e., a region of the protein that has been determined to be antigenic in a particular organism, such as the type of organism to which the modified protein can be administered.

[0131] Substitution or replacement variants generally contain the exchange of one amino acid for another at one or more sites within the protein and can be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may or may not be conservative, i.e., one amino acid is replaced with an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, an alanine to serine change; an arginine to lysine change; an asparagine to glutamine or histidine change; an aspartic acid to glutamic acid change; a cysteine ​​to serine change; a glutamine to asparagine change; a glutamic acid to aspartic acid change; a glycine to proline change; a histidine to asparagine or glutamine change; an isoleucine to leucine or valine change; a leucine to valine or isoleucine change; a lysine to arginine change; a methionine to leucine or isoleucine change; a phenylalanine to tyrosine, leucine, or methionine change; a serine to threonine change; a threonine to serine change; a tryptophan to tyrosine change; a tyrosine to tryptophan or phenylalanine change; and a valine to isoleucine or leucine change.

[0132] In addition to deletions or substitutions, the modified protein may have the insertion of residues, which generally involves the addition of at least one residue within the polypeptide. This includes the insertion of a targeting peptide or polypeptide or simply a single residue. Terminal additions, referred to as fusion proteins, are discussed below.

[0133] The term "biologically functionally equivalent" is well understood in the art and is further defined in detail herein. Thus, it includes sequences that have between about 70% and about 80%, or between about 81% and about 90%, or even between about 91% and about 99% of amino acids that are identical or functionally equivalent to those of a reference polypeptide, provided that the biological activity of the protein is maintained. In certain embodiments, a recombinant protein is biologically functionally equivalent to its natural counterpart.

[0134] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and still be essentially similar to that set forth in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including, in the case of protein expression, the maintenance of biological protein activity. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or may include various internal sequences, i.e., introns, that are known to be present within genes.

[0135] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids, up to the full length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of from about 3 amino acids to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0136] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous and the sequence of amino acid residues is not interrupted by any non-amino acid. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0137] Thus, the term "protein or peptide" includes an amino acid sequence that contains at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unconventional amino acid.

[0138] Certain embodiments of the present invention relate to fusion proteins. These molecules may have therapeutic proteins linked to heterologous domains at the N-terminus or C-terminus. For example, leader sequences from other species may be used in fusions to allow recombinant expression of proteins in heterologous hosts. Another useful fusion includes the addition of protein affinity tags, such as serum albumin affinity tags or six histidine residues, or immunologically active domains, such as antibody epitopes, which are preferably cleavable to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0139] In certain embodiments, therapeutic proteins can be linked to peptides that increase in vivo half-life, such as XTEN polypeptides (Schellenberger et al., 2009), IgG Fc domains, albumin, or albumin-binding peptides.

[0140] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be made, for example, by de novo synthesis of an entire fusion protein, or by attaching a DNA sequence encoding a heterologous domain and then expressing the intact fusion protein.

[0141] The creation of fusion proteins that restore the functional activity of the parent protein can be facilitated by connecting the genes with a bridging DNA segment that encodes a peptide linker that is spliced ​​between the tandemly connected polypeptides. The linker may be of sufficient length to allow proper folding of the resulting fusion protein.

[0142] siNA (e.g., siRNA) is well known in the art.For example, siRNA and double-stranded RNA are described in U.S. Patent No. 6,506,559 and U.S. Patent No. 6,573,099, and U.S. Patent Application No. 2003 / 0051263, No. 2003 / 0055020, No. 2004 / 0265839, No. 2002 / 0168707, No. 2003 / 0159161 and No. 2004 / 0064842, all of which are incorporated herein by reference in their entirety.

[0143] Within a siNA, the components of the nucleic acid do not have to be of the same type or species throughout (e.g., a siNA may contain nucleotides and nucleic acids or nucleotide analogs). In general, a siNA forms a double-stranded structure, which may result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the invention, a siNA may contain only a single nucleic acid (polynucleotide) or nucleic acid analog, which forms a double-stranded structure by complementing itself (e.g., forming a hairpin loop). The double-stranded structure of the siNA comprises 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutive nucleobases, including all ranges therein. The siNA may comprise 17-35 consecutive nucleobases, more preferably 18-30 consecutive nucleobases, more preferably 19-25 nucleobases, more preferably 20-23 consecutive nucleobases, or 20-22 consecutive nucleobases, or 21 consecutive nucleobases, that hybridize to a complementary nucleic acid (which may be another portion of the same nucleic acid or another complementary nucleic acid) to form a double-stranded structure.

[0144] The agent of the present invention useful for carrying out the method of the present invention includes, but is not limited to, siRNA.In general, the introduction of double-stranded RNA (dsRNA), which may alternatively be referred to herein as small interfering RNA (siRNA), induces strong and specific gene silencing, a phenomenon called RNA interference or RNAi.RNA interference has been referred to as "cosuppression", "post-transcriptional gene silencing", "sense suppression" and "quelling".RNAi is an attractive biotechnology tool, since it provides a means to knock out the activity of specific genes.

[0145] In designing RNAi, several factors must be considered, such as the nature of siRNA, the duration of silencing effect, and the choice of delivery system. To produce RNAi effect, the siRNA introduced into organisms generally contains exon sequence. Furthermore, the RNAi process is homology-dependent, and therefore the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. siRNAs are preferably those that show more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences with less than about 80% identity to the target gene are substantially less effective. Thus, the greater the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes is affected.

[0146] Furthermore, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules that contain at least about 19-25 nucleotides and are capable of modulating gene expression. In the context of the present invention, the siRNA is preferably less than 500 nucleotides, less than 200 nucleotides, less than 100 nucleotides, less than 50 nucleotides, or less than 25 nucleotides in length. More preferably, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

[0147] Target gene generally refers to a polynucleotide that includes a region that codes for a polypeptide, or a polynucleotide that regulates the replication, transcription, or translation or other processes that are important for the expression of the polypeptide, or a polynucleotide that includes both a region that codes for a polypeptide and a region that regulates its expression that is operably linked thereto. Any gene that is expressed in a cell can be targeted. The target gene is preferably one that is involved in or related to the progression of cellular activities that are important for disease, or one that is of particular interest for research purposes.

[0148] siRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of several techniques well known to those skilled in the art.For example, one commercial source of pre-designed siRNA is Ambion®, Austin, Tex. Another commercial source is Qiagen® (Valencia, Calif.).The inhibitory nucleic acid that can be applied to the compositions and methods of the present invention can be any nucleic acid sequence that has been verified by any source to be a down-regulator of the protein of interest.It is understood that, without undue experimentation, using the disclosure of the present invention, additional siRNA can be designed and used to carry out the method of the present invention.

[0149] The siRNA may contain modifications of one or more nucleotides. Such modifications may include the addition of non-nucleotide material, such as to the end(s) or internal (one or more nucleotides of the RNA) of the 19-25 nucleotides. In certain embodiments, the RNA molecule contains a 3'-hydroxyl group. The nucleotides in the RNA molecules of the invention may also contain non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotides may contain modified backbones, such as phosphorothioates, phosphorodithioates, or other modified backbones known in the art, and may contain non-natural internucleoside linkages. Additional modifications of siRNA (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in US Patent Application Publication No. 2004 / 0019001 and US Patent No. 6,673,611 (each of which is incorporated by reference in its entirety). Collectively, all of the above modified nucleic acids or RNAs are referred to as modified siRNAs.

[0150] VI. Kits and Diagnostics In various aspects of the present invention, kits are envisioned that contain the components necessary to purify exosomes from body fluids or tissue culture media. In other aspects, kits are envisioned that contain the components necessary to isolate exosomes and transfect them with a therapeutic nucleic acid, a therapeutic protein, or a nucleic acid encoding a therapeutic protein. In yet other aspects, kits are envisioned that contain the components necessary to isolate exosomes and determine the presence of markers specific for cancer cell-derived exosomes within the isolated exosomes.

[0151] The kit may include one or more sealed vials containing any of such components. In some embodiments, the kit may also include suitable container means, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes, which are containers that do not react with the components of the kit. The containers may be made of sterilizable materials, such as plastic or glass.

[0152] The kit may further comprise instructions outlining the procedural steps of the methods described herein and may follow substantially the same procedures as described herein or known to one of skill in the art. The instructional information may be in a computer readable medium containing machine readable instructions that, when executed using a computer, display actual or hypothetical procedures for purifying exosomes from a sample, transfecting exosomes with a therapeutic nucleic acid, expressing a recombinant protein in the exosomes, electroporating exosomes with a recombinant protein, or identifying cancer cell derived markers in or on the exosomes. EXAMPLES

[0153] VII. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples are techniques that the inventors have discovered to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for practicing it. However, those skilled in the art should, in light of this disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0154] material and method Exosome isolation and purification. Exosomes were purified by a differential centrifugation process as previously described (Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). (2012). Supernatants were collected from cells cultured for 48 h in medium containing exosome-depleted FBS and then subjected to sequential centrifugation steps at 800 g for 5 min and 2000 g for 10 min. The resulting supernatant was then filtered using a 0.2 μm filter in a culture bottle and the pellet was collected after ultracentrifugation at 28,000 g for 2 h in a SW 32 Ti rotor (Beckman). The supernatant was aspirated and the pellet was resuspended in PBS and then ultracentrifuged for another 2 h. The purified exosomes were then analyzed and used for experimental procedures.

[0155] Electroporation of exosomes and liposomes. 1×10 8Exosomes (measured by nanosight analysis) and 1 μg of siRNA (Qiagen) or shRNA were mixed in 400 μl of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep™). Exosomes were electroporated using a Gene Pulser Xcell™ Electroporation System (BioRad) using a 4 mm cuvette as previously described (Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). A similar procedure was performed using liposomes (100 nm, purchased from Encapsula Nano Sciences). After electroporation, protease-free RNAse A (Sigma Aldrich) was used to treat the exosomes, followed by the addition of 10× concentrated RNase inhibitor (Ambion) and washing with PBS under ultracentrifugation as above.

[0156] Immunogold labeling and electron microscopy. Specimens fixed at optimal concentrations were placed on 300 mesh carbon / formvar-coated grids and absorbed in formvar for a minimum of 1 min. For immunogold staining, grids were placed in blocking buffer for a blocking / permeabilization step for 1 h. Grids were placed immediately, without rinsing, in the appropriate dilution of primary antibody overnight at 4°C (monoclonal anti-CD9, 1:10, Abcam). As a control, some grids were not exposed to primary antibody. The following day, all of the grids were rinsed in PBS and then suspended in a drop of the appropriate secondary antibody with attached 10 nm gold particles (AURION, Hatfield, PA) for 2 h at room temperature. Grids were rinsed in PBS and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 min. After rinsing with PBS and distilled water, grids were dried and stained for counterstaining using uranyl acetate. Samples were examined using a Tecnai™ Bio Twin transmission electron microscope (FEI, Hillsboro, OR) and images were acquired using an AMT CCD Camera (Advanced Microscopy Techniques, Danvers, MA).

[0157] Quantification of Alexa Fluor 647 in cells treated with exosomes or liposomes. Exosomes isolated from BJ fibroblasts were electroporated with Alexa Fluor® 647-tagged siRNA (Qiagen, SEQ ID NO: 1), treated with PBS, proteinase K (Qiagen, 1×, 15 min at room temperature, and ultracentrifuged with PBS at 4° C. for 1 h), or trypsinized (Life Technologies, 10×, 15 min at room temperature, and ultracentrifuged with PBS at 4° C. for 1 h), washed with PBS for 2 h, and added to Panc-1 cell cultures on glass coverslips for 3 h. The cells were then washed with cold PBS and fixed by incubating with 4% PFA for 20 min at room temperature. The cells were then washed with PBS, incubated with 0.05% Triton-X for 10 min, washed with PBS, and stained with Sytox® green nuclear stain (Invitrogen). The coverslips were then mounted on glass slides with fluorescent mounting medium. Focal retention of Alexa Fluor® 647 was visualized using a Zeiss Observer Z1 inverted microscope. The number of cells with Alexa Fluor® 647 label was counted per field (400×) and the results were expressed as the percentage of cells with positive label out of the total number of cells counted per field.

[0158] Real-time PCR analysis. After total RNA purification using TRIzol® (Invitrogen) according to the manufacturer's instructions, RNA was retrotranscribed using MultiScribe reverse transcriptase (Applied Biosystems) and oligo-d(T) primers. Real-time PCR analysis was performed using SYBR® Green Master Mix (Applied Biosystems) on an ABI PRISM® 7300HT Sequence Detection System Instrument. Transcripts of interest were normalized to 18S transcript levels. Kras G12DPrimers for K-ras were designed as described (Rachagani et al., 2011) and K-ras wild-type primers were designed as described (Poliseno et al., 2010). Each measurement was performed in triplicate. Threshold cycle number, amplified target The fractional cycle number at which the amount of target protein reaches a fixed threshold is determined, and expression is measured by 2 -ΔCt The primer sequences are listed in Table 1. [Table 1]

[0159] Cell culture. Human foreskin fibroblast (BJ) cells were cultured in DMEM supplemented with 20% exosome-depleted FBS and 1% penicillin-streptomycin. Panc-1 and BxPC-3 cells (obtained from American Type Culture Collection [ATCC]) were cultured in RPMI 10% FBS. Panc-1 and BxPC3 cells (transfected with luciferase promoter) were a kind gift from Dr. Thiru Arumugam, UT MDACC. Ptf1acre / +;LSL-KRas G12D / + ;Tgfbr2 flox / flox Mouse (PKT) fibroblasts were isolated from the pancreas of PKT mice by mincing and incubating the isolated tumors overnight in unsupplemented DMEM and collagenase 4 (400 units / ml). The following day, the medium was then aspirated and the cells were then cultured in DMEM supplemented with 20% exosome-depleted FBS and 1% penicillin-streptomycin-ampicillin.

[0160] RNAi strategy. [ka] Both are Kras G12DThe mutation specifically targets the glycine to aspartic acid amino acid substitution and reflects a G to A nucleotide deviation from the wild-type Kras gene sequence, as well as including a TT nucleotide overhang to enhance silencing efficiency. G12D The central position of the deviated nucleotide in siRNA enhances its specificity to wild-type mRNA sequence.It was also labeled with Alexa Fluor® 647 fluorophore at the 3' end of the sense strand to track its delivery.siRNA was obtained from Qiagen (Cat. No. 1027424).All Stars Negative siRNA was obtained from Qiagen (Cat. No. 1027287) for use as scrambled siRNA.GFP-targeting shRNA was used as scrambled shRNA.

[0161] Exosome transfection. For in vitro transfection using exosomes and liposomes, both electroporated as above and washed with PBS, 200,000 cells in 6-well plates were treated with exosomes and liposomes for the required time periods described for each assay, then washed with PBS and used for further analysis.

[0162] Growth kinetics and apoptosis assays. Panc-1 and BxPC-3 cells were seeded (2.5 × 10 5 ) and grown for 12 hours, after which they were treated with si / shRNA-electroporated exosomes. Every 24 hours thereafter, the number of viable cells was counted by trypsinizing the cells, mixing with trypan blue, and then counting the cells using a hemocytometer. This process was repeated every 24 hours until 84 hours after seeding. Apoptosis by TUNEL was assessed using the In Situ Cell Death Kit, TMR red (Roche) was used to assess according to the manufacturer's instructions. Cells were fixed as above and nuclei were stained using Sytox® green (Invitrogen, 1:10,000 in PBS, 10 min at room temperature). Images were acquired using a Zeiss LSM 510 confocal microscope and quantified by counting the number of TUNEL-positive cells per field (400×), and results were expressed as the percentage of cells with positive labeling out of the total number of cells counted per field.

[0163] Western blot. To estimate cellular protein expression 24 hours after treatment with exosomes, Panc-1 cells were harvested in RIPA buffer and protein lysates were normalized using quantification by the Bradford method. 40 μg of lysate was loaded onto an acrylamide gel for electrophoretic separation of proteins under denaturing conditions and transferred to a PVDF membrane (ImmobilonP) by wet electrophoretic transfer. The membrane was then blocked with 5% nonfat dry milk / 0.05% Tween-20 in PBS for 1 hour at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-rabbit p-Erk-p44 / p42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (Cell Signaling, 4376, 1:1000), anti-rabbit p-AKT-anti-AKT1 (phospho S473) (Abcam, ab81283, 1:5000), anti-rabbit β-actin (Cell Signaling, 4967, 1:1000). The secondary antibody was incubated for 1 hour at room temperature. After antibody incubation, washing with 1×PBS, 0.05% Tween®-20 was performed three times at 15 minute intervals on an orbital shaker. The membrane was developed using chemiluminescence reagent from Pierce according to the manufacturer's instructions, and chemiluminescence was captured on film.

[0164] Northern blot. 20 μg of sucrose gradient exosomal RNA was loaded with 1× RNA loading dye for 2 min at 95° C., followed by 2 min on ice using a urea / acrylamide 15% gel. MicroRNA markers were used according to the manufacturer's instructions (N2102, New England BioLabs). Electrophoresis was performed for 3 h at 4° C. using 1× TBE. Transfer was performed for 2 h at 4° C. using Whatman blotting paper and BrightStar®-Plus Positively Charged Nylon Membrane (Ambion) using 0.5× TBE. RNA was crosslinked to the membrane for 20 min using a UV transilluminator. Membranes were prehybridized by rotating in Ambion's ULTRAhyb®-Oligo hybridization solution (Ambion) for 1 h at 42° C. The probe was then thawed on ice and incubated at 95°C for 5 minutes before adding 150ng per mL of hybridization buffer, after which the membrane was rotated overnight at 42°C. The following washing steps were performed: 2xSSPE / 0.5%SDS-15 minutes, twice; 0.2xSSPE / 0.5%SDS-30 minutes, twice, and 2xSSPE-5 minutes. These initial washing steps were followed by additional washes, and then the blots were developed using a BrightStar® BioDetect™ Kit according to the manufacturer's instructions (Ambion). The blots were exposed to a 4-stack of film overnight. The Alexa Fluor® 647 fluorophore was directly detected using a LI-COR Biosciences Odyssey Infrared Imaging System.

[0165] Immunocytochemistry. Cells were plated on glass coverslips and immunofluorescence stained for Kras G12DsiRNA was treated with either electroporated exosomes or liposomes for 3 hours. Coverslips were then washed with cold 1x PBS, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.5% Triton™ X-100 in PBS for 10 minutes at room temperature, and nuclei were stained with Sytox green resuspended in 2% BSA. Images were obtained using a Zeiss LSM510 Upright Confocal System, using the recycle tool to maintain identical settings. Aggregated exosomes containing Alexa Fluor® 647-tagged siRNA allowed the visualization of focal retention of the label detectable by confocal microscopy. For data analysis, images were selected from a pool drawn from at least two independent experiments. The number of cells with Alexa Fluor 647 label per field (x400) was counted, and results were expressed as the percentage of cells with positive label out of the total number of cells counted per field.

[0166] Mice and imaging. Four- to six-week-old female athymic nu / nu mice (Charles Rivers) were housed in individually ventilated cages with a 12-h light / dark cycle, 21–23 °C, and 40%–60% humidity. Mice were fed irradiated diet and sterile water ad libitum. Under general anesthesia, Panc-1 or BXPC-3 cells (10 6 100 mg / kg luciferin (200 μl of 10 mg / ml in PBS) was injected i.p. into the tail of the pancreas using a 27-gauge syringe. To detect luciferase expression, mice were injected i.p. with 100 mg / kg luciferin (200 μl of 10 mg / ml in PBS) 12–15 min before imaging, anesthetized with isoflurane, and imaged using an IVIS (Xenogen Spectrum). For orthotopic tumor analysis, Living Image version 4.4 (Caliper All tumor measurements were quantified using a 3D CT scanner (Digital Life Sciences). A circular region of interest (ROI) around the pancreas and tumor was defined and set as a standard for comparing all images within the same experimental group. Furthermore, exposure conditions (time, aperture, stage position, binning) were kept identical for all measurements in all experimental groups. Subsequent tumor measurements (p / sec / cm) were then calculated. 2 / sr) were obtained under the same conditions for all experimental groups. Mice were imaged periodically and randomly assigned to treatment groups. Mice were administered 2 × 10 8 Each mouse received 100 μg of exosomes or liposomes ip once every 2 days. Exosomes or liposomes were electroporated with 2 μg of siRNA or shRNA, washed with PBS, and then injected. When using PKT (Ptf1acre / +; LSL-KrasG12D / +; Tgfbr2flox / flox) (Ozdemir et al., 2014) genetically engineered mice, Treatment with tRNAs was initiated at 33 days of age, when mice showed PaNIN and PDAC lesions. All animal procedures were reviewed and approved by the Institute for Animal Care and Use Committee at UT MDACC.

[0167] Macrophage clearance. Immunocompetent adult mice were injected ip with either exosomes or liposomes containing Alexa Fluor® 647-tagged siRNA. The blood of these mice was collected 12 hours after injection and processed for flow cytometry analysis. RBCs were depleted using ACK lysis buffer (Invitrogen), and peripheral cells were blocked with FC block (1:1000, BD Pharmingen), stained with Sytox green (1:200, Invitrogen) and CD11b (1:200, BD Pharmingen, PerCP / Cye 5.5) antibodies for 30 minutes, washed with PBS, and analyzed using an LSR Fortessa™ X-20 cell analyzer. Preincubating mouse cell suspensions with FC Block for a few minutes prior to staining with specific antibodies ensures that any staining observed is due to the interaction of the antigen-binding portion of the antibody with the antigen on the cell surface.

[0168] Histology, histopathology, and immunohistochemistry. Tissues were fixed in formalin and paraffin embedded. Five-μm-thick tissue sections were cut and stained for hematoxylin and eosin (H&E) and Masson's trichrome (MTS) (Leica). For histopathology scoring, H&E stained slides were scored based on the morphological stage of pancreatic cancer: normal, pancreatic intraepithelial neoplasia (PaNIN), and pancreatic ductal adenocarcinoma (PDAC). For each tissue section, a percentage score for each of the three stages (normal, PaNIN, PDAC) was obtained manually in a blinded manner by an expert in pancreatic histology, which were then averaged to obtain an overall score out of 100 for each cohort. These percentage scores were then averaged for each mouse within each cohort.

[0169] For the analysis of fibrosis in mice, eight 200× fields were randomly selected for each MTS-stained pancreatic section, and fibrosis was manually assessed by grid intersection analysis using Adobe Photoshop. For the evaluation of each image, a grid of 100 squares was overlapped on each image, and each intersection was counted for blue (fibrotic areas) and purple / red (non-fibrotic areas). A percentage score was then obtained for each tissue section. Tissue sections were also subjected to antigen retrieval (15 min in 10 nM citrate buffer, pH 6 and 98° C.) before immunostaining. Tissue sections were incubated with 4% CWFS gelatin (Aurion) in TBS or PBS for 1 h, followed by overnight incubation with primary antibodies. The following primary antibodies were used for staining: anti-rabbit p-Erk-p44 / p42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (Cell Signaling, 4376, 1:400), anti-rabbit p-AKT-anti-AKT1 (phospho S473) (Abcam, ab81283, 1:100), anti-rabbit Ki-67 (Thermo Scientific, RM-9106-S, 1:400). For all staining, sections were incubated with biotinylated goat anti-rabbit and streptavidin-HRP (Biocare Medical) for 10 min each and counterstained with hematoxylin. DAB positivity was analyzed. Ki-67 staining was quantified by counting the number of positively stained nuclei per field (400×), while p-Erk and p-AKT staining were quantified using ImageJ by designing a macro to include only the darkly stained parts of the image for each antibody and then consider it as the positive staining area. This was performed for eight 200× images per tissue section, and an average of the positive scores was obtained for each tissue section. TUNEL assay was performed using the in situ cell death detection kit, TMR Red (Roche), according to the manufacturer's instructions. Alexa 647 on frozen tissue sections was detected by staining of tissue nuclei with Sytox® green (1:10,000 in PBS, 10 min). Images were acquired using a Zeiss LSM 510 confocal microscope and quantified by counting the number of TUNEL-positive cells per field (×400), and results were expressed as the percentage of positively labeled cells out of the total number of cells counted per field.

[0170] Statistical analysis. Statistical analyses used are detailed in the figure legends. Statistical significance was established using one-way ANOVA or unpaired two-tailed Student's t-tests using GraphPad Prism (GraphPad Software). For survival analysis, Kaplan-Meier plots were generated and statistical differences were assessed using the log-rank Mantel-Cox test. A p-value <0.05 was considered statistically significant.

[0171] Example 1 Antitumor properties of exosomes containing inhibitory RNA siRNA and shRNA constructs were administered to the Kras G12D was designed to specifically target [ka] Kras found in cell lines and animal models G12D The mutation reflects a G to A nucleotide deviation from the wild-type Kras gene sequence (underlined and bold) to specifically target the glycine to aspartic acid amino acid substitution, and a TT nucleotide overhang (underlined) to promote silencing efficiency (Rejiba et al., 2007; Ma et al., 2004; Du et al., 2005). G12D The central location of the nucleotide deviation in the siRNA enhances its specificity for the wild-type mRNA sequence (Du et al., 2005). The shRNA sequence (SEQ ID NO:2) was G12D To facilitate specific targeting of mRNA, the seed sequence was designed to contain a specific G to A nucleotide deviation. G12D siRNA oligonucleotides against were labeled with the Alexa Fluor® 647 fluorophore to track their delivery ( FIG. 4A ).

[0172] To insert shRNA and siRNA constructs into exosomes without functionally impairing the exosomes (siKras G12D / shKras G12D To obtain exosomes (Fig. 4A-C), a new electroporation method was developed and optimized (Fig. 4A-C). To this end, exosomes were electroporated from human foreskin glands using an established ultracentrifugation method (Kahlert et al., 2014). Exosomes were isolated from fibroblasts (BJ fibroblasts). The purity and homogeneity of exosomes (particles with diameters of 80-150 nm) were verified by Nanosight™ measurements (Figure 4B), transmission electron microscopy (Figure 4C), and CD9 immunogold labeling (Figure 4D). The purity of the exosome extraction and the presence of Alexa Fluor 647 in the exosomes were also verified by sucrose gradient ultracentrifugation and Northern blot (Figure 4E). Exosomes containing scrambled siRNA and scrambled shRNA (siScrbl / shScrbl exos), liposomes containing scrambled siRNA and scrambled shRNA (siScrbl / shScrbl), and Kras G12D Liposomes containing siRNA / shRNA (siKras G12D / shKras G12D lipos were also produced. asp12 (Rejiba et al., 2007; Sun et al., 2001)) cells were stained with Alexa Fluor® )647-labeled siRNA-containing exosomes and liposomes were incubated for 3 hours, and immunofluorescence imaging revealed significant focal retention of label in exosome-treated cells compared to liposome-treated cells (Figure 1A). Treatment of exosomes with proteinase K or trypsin attenuated cellular staining, whereas treatment of liposomes with proteinase K or trypsin maintained low cellular staining, supporting the idea that exosome surface proteins enhance delivery of labeled siRNA to cells (Figure 1A). G12D Processing with exos and shKras G12D Treatment with exos significantly reduced Kras expression in Panc-1 cells compared to siScrbl / shScrbl exos or non-electroporated (control, no RNAi payload) exos. G12D The mRNA levels were reduced (approximately 70% and 50%, respectively) (Figure 1B). G12D and shKras G12DTreatment with lipos also significantly reduced Kras expression in Panc-1 cells compared with siScrbl / shScrbl lipos. G12D The mRNA levels of mutant Kras were decreased (approximately 20% decrease, respectively) (Fig. 1B). G12D Specific knockdown of the transcript was performed using the G12D The siKras expression level was measured using quantitative real-time PCR (qPCR) with primers that specifically amplify siKras but not wild-type Kras (Table 1). G12D exos and shKras G12D Treatment with exos did not reduce wild-type Kras mRNA levels, demonstrating the feasibility of Kras expression using this methodology. G12D This supports mRNA-specific targeting (Figure 1C). G12D The efficacy of transcript knockdown was greater when exosomes were used instead of liposomes (Figure 1B), likely reflecting impaired delivery of liposomes compared to exosomes (Figure 1A). G12D lipos and shKras G12D Increasing the concentration of lipos or the time liposomes were incubated with Panc-1 cells increased the expression of Kras G12D The efficiency of mRNA targeting was not improved (Figure 4F), confirming that the endogenous nature of exosomes is superior to liposomes in delivering RNAi cargo for effective mRNA targeting. Further experimental optimization demonstrated that Kras G12D A ratio of approximately 400 exosomes per Panc-1 cell was found to be superior to a ratio of 700 exosomes per Panc-1 cell for suppressing transcript levels (Figure 4G). G12D BxPC3 pancreatic cancer cells without Kras mutation wt,Gly (Sun et al., 2001)) was used as a control. siKras G12D and shKras G12D Treatment with exos did not suppress wild-type Kras expression in these cells (Figure 4H), thus demonstrating that oncogenic Kras Kras mRNA levels suppressed G12D siRNA constructs and Kras G12D Further supporting the specificity of the shRNA construct. G12D exos or shKras G12D Oncogenic Kras suppression in Panc-1 cells treated with exos was associated with decreased levels of phosphorylated ERK and phosphorylated AKT protein, supporting reduced downstream signaling of oncogenic Kras (Figure 1D). In contrast to Panc-1 cells treated with shScrbl exos or non-electroporated control exos, siKras suppression was associated with decreased levels of phosphorylated ERK and AKT protein, supporting reduced downstream signaling of oncogenic Kras (Figure 1D). G12D exos or shKras G12D The proliferation of Panc-1 cells incubated with exos was significantly reduced (Fig. 1E). In contrast, the proliferation of BxPC3 cells was significantly reduced by siKras. G12D exos or shKras G12D Treatment with exos was not affected by the siKras gene (Figure 4I). G12D exos or shKras G12D The reduced proliferation of Panc-1 cells treated with exos was associated with enhanced apoptosis as measured by the TUNEL assay, which suggested that siKras G12D exos or shKras G12D The reduced proliferation of these cells when treated with exos is confirmed (Figure 1E).

[0173] In vitro experiments showed that siKras G12D exos or shKras G12D These results suggest that exos specifically targets oncogenic Kras and induces apoptosis by impairing downstream oncogenic Kras signaling. G12D or shKras G12D Exosomes containing Kras in pancreatic tumors G12DWe explored the ability of exosomes to silence expression in mice. Focal retention of Alexa Fluor 647-labeled siRNA derived from exosomes injected intraperitoneally into mice was detected in pancreatic tissue 24 hours after injection. Furthermore, exosomes containing Alexa Fluor 647-labeled siRNA were detected using flow cytometry in the serum of mice 24 hours after ip injection (Figure 5A). These results suggest that exosomes administered ip to mice enter the systemic circulation and reach the pancreas. After a significant number of exosomes were confirmed to approach the pancreatic parenchyma, 1 × 10 6 Luciferase-expressing Panc-1 human pancreatic (Panc-1-luc) cells were orthotopically implanted into nude mice treated with ip injection of exosomes or liposomes. Ten days after injection of the cancer cells, all mice showed tumors detectable by bioluminescence imaging, with a radiance of 1 × 10 5 p / sec / cm 2 / sr to 1×10 6 p / sec / cm 2 Mice were randomized to receive 1 × 10 6 Each mouse was subjected to repeated ip injections of exosomes or liposomes. Note that the liposomes used were 100 nm in size, close to the size range of exosomes, and were injected at a similar concentration and dose as the exosomes. Cohorts of mice were also injected with PBS vehicle and non-electroporated exosomes. Tumors in mice receiving PBS or non-electroporated exosomes grew at an exponential rate and upregulated the siKras G12D exos or shKras G12D Tumors in mice treated with exos were significantly reduced to baseline bioluminescence detection levels 30 days after treatment initiation (Figure 2A). G12D lipos or shKras G12DTumor growth was also slowed in mice treated with lipos, but to a much lesser extent than with exosomes (Figure 2A). We also detected increased macrophage clearance of liposomes compared to exosomes, with a greater number of macrophages containing Alexa Fluor 647-labeled RNAi in the systemic circulation of mice treated with liposomes containing labeled RNAi compared to mice treated with exosomes containing labeled RNAi (Figure 5A). G12D exos or shKras G12D Exos had no effect on orthotopic BxPC3 tumor growth (Figure 2B) or overall survival (Figure 5D), indicating that siKras G12D exos or shKras G12D Exos treatment, Kras G12D Note that the specific antitumor effect against cancer cells harboring the mutation was confirmed. G12D Histopathological findings in mice treated with exos showed that short-term (16-day) siKras treatment improved tumor cell survival as early as 26 days after cancer cell injection. G12D A significant reduction in pancreatic cancer disease following exos treatment was demonstrated. At 77 days after cancer cell injection, control mice treated with PBS showed extensive tumor burden as assayed by bioluminescence imaging, whereas mice treated with shKras showed no significant tumor burden. G12D Tumor burden in mice treated with exos was reduced to nearly undetectable levels (Figure 2C, Figure 6B). G12D All Panc-1 tumor-bearing mice treated chronically with exos, treated with PBS, and treated with control exos required euthanasia based on moribund criteria or excessive tumor burden 130 days after cancer cell injection and were subsequently treated with shKras. G12D All mice treated with exos were healthy and showed minimal tumor burden as detected by bioluminescence imaging 200 days after cancer cell injection (Figure 2D, Figure 6B). Immunolabeling of tumors for p-ERK (Figure 2E) and p-AKT (Figure 5C) revealed that shKras tumors were significantly more aggressive than control (PBS-treated) mice. G12DWe also found that Kras signaling was suppressed in tumors from mice treated with exos. These data support the finding that shKras G12D Exosomal delivery of shKras resulted in a reduction in tumor growth and demonstrated sustained inhibition of tumor growth. Histopathological analysis of the pancreas at these time points demonstrated that shKras G12D Mice treated with PBS showed tumor progression (130 days) involving the entire pancreas, in contrast to the small tumor foci without much involvement of the pancreas in mice treated with exos (Figure 2D). Percent tumor burden (based on pancreatic mass at the experimental endpoint) (Figure 2F) and survival (Figure 2G) were also significantly improved in mice bearing shKras tumors bearing Panc-1 tumors. G12D After exos, pancreatic tumors were much improved, whereas all control mice succumbed to pancreatic tumor burden. In the PBS- and control exos-treated groups, mice were euthanized when they reached a moribund state, 88–130 days after cancer cell implantation, whereas shKras mice were euthanized. G12D In the exos-treated group, nearly all mice were healthy and alive 200 days after cancer cell implantation (one mouse was found to have died on day 59, but autopsy analysis revealed that this mouse had a minimal tumor burden and confirmed that the death was unrelated to cancer).

[0174] The antitumor properties of treatment with siRNA iExosomes (i.e., exosomes containing drug entities such as siRNA) in nude mice bearing Panc-1 tumors warranted further evaluation in genetically engineered mouse models (GEMMs) of PDAC. Rapidly progressive Ptf1acre / +;LSL-KRasG12D / +;Tgfbr2flox / flox mice (PKT mice (Ozdemir et al., 2014)) were treated with siKras G12D ex These mice spontaneously develop pancreatic adenocarcinomas that recapitulate the clinical and histopathological characteristics of human pancreatic cancer (Ozdemir et al., 2014). This model is sufficient PKT mice develop pancreatic intraepithelial neoplasia (PaNIN) stage at approximately 28 days of age, invasive adenocarcinoma at approximately 32 days of age, and death at 45–55 days of age. Mice are treated with non-electroporated control exosomes or siKras G12D exos or shKras G12D Exos was injected ip every 48 hours starting at 33 days of age (mice with PDAC) (Figure 3A). Focal retention of Alexa Fluor® 647 label derived from exos containing tagged siRNA was detected in the pancreatic tumors of mice at the time of sacrifice. siKras G12D exos or shKras G12D Mice treated with exos showed a significant increase in lifespan, with median survival longer than those treated with shKras. G12D For mice treated with exos, it was 50 days, and for mice treated with siKras, G12D Mice treated with control exos showed a median survival of 43 days compared to 60 days for mice treated with exos (Figure 3B). The increased survival was consistent with siKras compared to mice treated with control exos, both at age-matched time points (Figure 3C) and at each experimental end point (Figure 7A). G12D In addition to a significant survival advantage (Figure 3B), siKras was associated with a significant reduction in tumor burden in mice treated with exos. G12D Histopathological characterization of tumors from exos-treated mice (age-matched to control exos-treated mice at 44 days of age) revealed a relative increase in normal parenchymal and PaNIN-stage lesions, in contrast to the near-complete transformation of pancreatic to cancerous tissue with invasive features in control mice at 44 days of age (Figure 3D). G12DPancreata from exos-treated mice still demonstrated improved histopathological features compared to control mice (Figure 7B). Experiments with GEMMs were first performed using exosomes derived from BJ human fibroblasts. To address the potential impact of species differences in the efficacy of siRNA exos in GEMMs, syngeneic fibroblasts were isolated from the pancreas of PKT mice and transfected with siKras. G12D Exos were generated from these primary cell cultures. Similar improvements in survival, tumor burden, and histopathology were observed with mouse fibroblast-derived siRNA exos compared with BJ fibroblast-derived siRNA exos and control exos-treated mice (Figures 3E-F; Figure 7C). G12D Treatment with exos significantly reduced the desmoplastic response associated with pancreatic cancer progression in PKT mice (reduced fibrosis-associated extracellular matrix deposition in PKT tumors, increased cancer cell apoptosis as assayed by TUNEL staining, reduced cancer cell proliferation (reduced Ki67 staining)), and reduced phosphorylated ERK and phosphorylated AKT staining in tumors (Figure 3G, Figure 7D).

[0175] Example 2 CD47 prevents the uptake of exosomes by circulating monocytes Circulating monocytes were found to phagocytose liposomes (100 nm; purchased from Encapsula Nanosciences), but not exosomes (Figures 8A-8B). Exosomes isolated from BJ fibroblasts were found to contain CD47 on their surface (Figures 9A and 9C), whereas liposomes were determined to lack CD47 on their surface (Figure 9B). Treatment of exosomes with anti-CD47 antibodies was found to stimulate exosome uptake by circulating monocytes in vivo (Figure 10).

[0176] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0177] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. [ka] [ka] [ka] [ka] [ka]

Claims

1. A pharmaceutical composition comprising an exosome and an excipient, wherein the exosome comprises a therapeutic agent, and the exosome is isolated from a fibroblast that overexpresses CD47 and comprises CD47 on its surface.

2. The composition described in claim 1, wherein the therapeutic agent is a therapeutic protein, antibody, inhibitory RNA, or a small molecule drug.

3. The composition described in claim 2, wherein the antibody binds to an intracellular antigen.

4. The composition of claim 2, wherein the antibody is a full-length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody.

5. The composition described in claim 2, wherein the inhibitory RNA is siRNA, shRNA, miRNA, or pre-miRNA.

6. The composition described in claim 2, wherein the therapeutic protein is a kinase, phosphatase, or transcription factor.

7. The composition of claim 2, wherein the small molecule drug is an imaging agent.

8. The composition of claim 1, wherein the composition is formulated for parenteral administration.

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

10. The composition of claim 8, further comprising an antibacterial agent.

11. The composition of claim 10, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

12. The composition of claim 1, wherein the exosome comprises an inhibitory RNA targeting Kras G12D .

13. A composition according to any one of claims 1 to 12 for use in treating a disease in a patient.

14. The composition described in claim 13, wherein the disease is cancer.

15. The composition described in claim 14, wherein the therapeutic agent is an inhibitory RNA targeting an oncogene.

16. The composition of claim 15, wherein the inhibitory RNA targets Kras G12D .

17. The composition of claim 14, wherein the therapeutic agent is a tumor suppressor protein.

18. The composition of claim 13, wherein the composition is formulated for parenteral administration.

19. The composition of claim 18, wherein the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

20. The composition of claim 18, further comprising an antibacterial agent.

21. The composition of claim 20, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

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