Telomerase-containing exosomes for the treatment of diseases associated with aging and age-related organ failure

Abstract

To provide telomerase-containing exosomes for the treatment of diseases associated with aging and age-related organ failure. [Solution] This specification provides compositions of lipid-based nanoparticles (e.g., exosomes) containing therapeutic anti-aging agents. Furthermore, methods of using such compositions for treating patients with age-related disorders are also provided. In particular, exosomes containing RNA encoding telomerase, and methods of using them in the treatment of age-related disorders are provided. In one embodiment, this specification provides a composition comprising lipid-based nanoparticles containing a therapeutic agent cargo that enhances the activity of a telomerase complex. In some embodiments, the lipid-based nanoparticles contain CD47 on their surface. In some embodiments, the lipid-based nanoparticles contain a growth factor on their surface.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 803,023, filed on February 8, 2019, the entire content of which is incorporated herein by reference.

[0002] The present invention generally relates to the fields of pharmaceuticals and oncology. More specifically, the present invention relates to a composition for treating age - related disorders by administering exosomes that carry cargo to improve telomerase activity, and a method for said treatment.

Background Art

[0003] Telomerase deficiency is associated with systemic organ dysfunction and age - dependent disorders. With aging, cells lose telomerase, which contributes to cell dysfunction and aging. Genetic studies have shown that re - expression of telomerase can extend cell lifespan and protect organ functions associated with aging. However, a systemic treatment strategy is needed.

Summary of the Invention

Means for Solving the Problems

[0004] Therefore, the present specification provides a composition comprising exosomes engineered to deliver telomerase mRNA or modified telomerase mRNA to cells to reverse age - related disorders (such as organ disorders, etc.), and a method for administering said exosomes.

[0005] In one embodiment, a composition is provided herein comprising lipid-based nanoparticles containing a therapeutic agent cargo that enhances the activity of a telomerase complex. In some embodiments, the lipid-based nanoparticles contain CD47 on their surface. In some embodiments, the lipid-based nanoparticles contain a growth factor on their surface. In some embodiments, the lipid-based nanoparticles are liposomes or exosomes.

[0006] In some embodiments, the therapeutic cargo is a therapeutic protein, an antibody, an inhibitory RNA, a gene editing system, or a small molecule drug. In some embodiments, the therapeutic protein corresponds to the TERT protein. In some embodiments, the antibody binds to an intracellular antigen. In some embodiments, the antibody is a full-length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody. In some embodiments, the inhibitory RNA is siRNA, shRNA, miRNA, or pre-miRNA. In some embodiments, the siRNA knocks down the expression of a protein that downregulates telomerase activity. In some embodiments, the gene editing system is a CRISPR system. In some embodiments, the CRISPR system comprises an endonuclease and a guide RNA (gRNA). In some embodiments, the endonuclease and the gRNA are encoded on a single nucleic acid molecule within an exosome. In some embodiments, the CRISPR system targets mutations in TERT or TERC.

[0007] In one embodiment, a pharmaceutical composition is provided herein comprising nanoparticles based on any one of the lipids of this embodiment and an excipient. In some embodiments, the composition is formulated for parenteral administration. In some embodiments, the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. In some embodiments, the composition further comprises an antimicrobial agent. In some embodiments, the antimicrobial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, or thimerosal.

[0008] In one embodiment, a method is provided herein for treating a disease or disorder in a patient in need thereof, comprising administering one of the compositions of the embodiments to the patient. In some embodiments, the administration delivers the therapeutic cargo to the patient's cells.

[0009] In some embodiments, the disease or disorder is an age-related disease or disorder. In some embodiments, the disease or disorder is pulmonary fibrosis, congenital keratosis, aplastic anemia, muscular dystrophy, atherosclerosis, hypertension, heart disease, cancer, stroke, diabetes, diabetic ulcer, Alzheimer's disease, osteoporosis, macular degeneration, immunosenescence, myocardial infarction, or vascular dementia.

[0010] In some embodiments, the administration is systemic. In some embodiments, the systemic administration is intravenous. In some embodiments, the composition is administered more than once.

[0011] In some embodiments, the method further includes administering at least a second therapy to the patient. In some embodiments, the second therapy includes surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, or immunotherapy.

[0012] In some embodiments, the patient is a human. In some embodiments, the lipid-based nanoparticles are exosomes, and the exosomes are autologous to the patient. In some embodiments, the exosomes are obtained from a bodily fluid sample obtained from the patient. In some embodiments, the bodily fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum. In some embodiments, the exosomes are obtained from mesenchymal cells. In some embodiments, the method is further defined as a method for delivering a therapeutic agent cargo that enhances the activity of telomerase complexes to the liver, brain, and / or pancreas of a patient.

[0013] In one embodiment, this specification provides a method for delivering a therapeutic agent to the liver tissue, brain tissue, and / or pancreatic tissue of a patient, comprising administering mesenchymal cell-derived exosomes carrying the therapeutic agent to the patient. In some embodiments, the exosomes are autologous to the patient. In some embodiments, the therapeutic agent is a therapeutic protein, antibody, inhibitory RNA, gene editing system, or small molecule drug. In some embodiments, the therapeutic agent enhances the activity of the telomerase complex. In some embodiments, the therapeutic agent is a TERT protein. In some embodiments, the exosomes are administered more than once. In some embodiments, the exosomes are administered systemically. In some embodiments, the exosomes are administered topically.

[0014] As used herein, “essentially not present” is used to mean that, with respect to a particular component, that particular component is not intentionally included in the composition at all, and / or is present only as an impurity or only in trace amounts. Thus, the total amount of any particular component derived from any unintended compositional impurities is well less than 0.05%, preferably less than 0.01%. Most preferably, the composition is one in which the particular component is undetectable by standard analytical methods.

[0015] As used herein, "a" or "an" may mean one or more. As used in the claims, as used in combination with the phrase "comprising," the phrase "a" or "an" may mean one or more.

[0016] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly stated that it refers only to substitutes or that the substitutes are not mutually exclusive; however, this disclosure supports the definitions of substitutes only, as well as “and / or.” As used herein, “another” may mean at least a second or more.

[0017] Throughout this application, the term “about” is used to mean that a value includes variations inherent in the error of the device or method used to determine that value, variations that exist between the subjects under investigation, or values ​​within 10 percent of the indicated value.

[0018] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while this detailed description and specific examples illustrate preferred embodiments of the present invention, 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 and are therefore provided solely for illustrative purposes. The present invention provides, for example, the following items. (Item 1) A composition comprising lipid-based nanoparticles containing a therapeutic agent cargo that enhances the activity of the telomerase complex. (Item 2) The composition according to item 1, wherein the lipid-based nanoparticles contain CD47 on their surface. (Item 3) The composition according to item 1, wherein the lipid-based nanoparticles contain a growth factor on their surface. (Item 4) The composition according to item 1, wherein the lipid-based nanoparticles are liposomes or exosomes. (Item 5) The composition according to item 1, wherein the therapeutic agent cargo is a therapeutic protein, an antibody, inhibitory RNA, a gene editing system, or a small molecule drug. (Item 6) The composition according to item 5, wherein the therapeutic protein corresponds to the TERT protein. (Item 7) The composition according to item 5, wherein the antibody binds to an intracellular antigen. (Item 8) The composition according to item 5, wherein the antibody is a full-length antibody, scFv, Fab fragment, (Fab)2, diabody, triabody, or minibody. (Item 9) The composition according to item 5, wherein the inhibitory RNA is siRNA, shRNA, miRNA, or pre-miRNA. (Item 10) The composition according to item 9, wherein the siRNA knocks down the expression of a protein that downregulates telomerase activity. (Item 11) The composition according to item 9, wherein the gene editing system is a CRISPR system. (Item 12) The composition according to item 11, wherein the CRISPR system contains an endonuclease and a guide RNA (gRNA). (Item 13) The composition according to item 12, wherein the endonuclease and the gRNA are encoded on a single nucleic acid molecule within the exosome. (Item 14) The composition according to item 11, wherein the CRISPR system targets a TERT or TERC mutation. (Item 15) A pharmaceutical composition comprising the lipid-based nanoparticle according to any one of items 1 to 14 and an excipient. (Item 16) The composition according to item 15, wherein the composition is formulated for parenteral administration. (Item 17) The composition according to item 16, wherein the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. (Item 18) The composition of item 16, further comprising an antimicrobial agent. (Item 19) The composition according to item 18, wherein the antimicrobial 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 20) A method of treating a disease or disorder in a patient needing the same, the method comprising administering to the patient a composition according to any one of items 15 to 19. (Item 21) The method according to item 20, wherein the administration results in delivery of the therapeutic cargo to the cells of the patient. (Item 22) The method according to item 20, wherein the disease or disorder is an age-related disease or disorder. (Item 23) The method according to item 20, wherein the disease or disorder is pulmonary fibrosis, congenital keratosis, aplastic anemia, muscular dystrophy, atherosclerosis, hypertension, heart disease, cancer, stroke, diabetes, diabetic ulcer, Alzheimer's disease, osteoporosis, macular degeneration, immunosenescence, myocardial infarction, or vascular dementia. (Item 24) The method according to item 20, wherein the administration is systemic administration. (Item 25) The method according to item 24, wherein the systemic administration is intravenous administration. (Item 26) The method according to item 20, further comprising administering at least the second therapy to the patient. (Item 27) The method according to item 26, wherein the second therapy described above includes surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, or immunotherapy. (Item 28) The method according to item 20, wherein the patient is a human. (Item 29) The method according to item 28, wherein the lipid-based nanoparticles are exosomes, and the exosomes are autologous to the patient. (Item 30) The method according to item 29, wherein the exosome is obtained from a bodily fluid sample obtained from the patient. (Item 31) The method according to item 30, wherein the bodily fluid sample is blood, lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirate, ocular exudate / tears, or serum. (Item 32) The method described in item 29, wherein the exosome is obtained from mesenchymal cells. (Item 33) The method according to item 32, further defined as a method for delivering a therapeutic agent cargo that enhances the activity of telomerase complexes to the liver, brain, and / or pancreas of the patient. (Item 34) The method according to item 20, wherein the composition is administered more than once. (Item 35) A method for delivering a therapeutic agent to the liver tissue, brain tissue, and / or pancreatic tissue of a patient, comprising administering mesenchymal cell-derived exosomes that carry the therapeutic agent to the patient. (Item 36) The method according to item 35, wherein the exosome is autologous to the patient. (Item 37) The method according to item 35, wherein the therapeutic agent is a therapeutic protein, antibody, inhibitory RNA, gene editing system, or small molecule drug. (Item 38) The method according to item 35, wherein the therapeutic agent enhances the activity of the telomerase complex. (Item 39) The method according to item 38, wherein the therapeutic agent is the TERT protein. (Item 40) The method according to item 35, wherein the exosome is administered more than once. (Item 41) The method according to item 35, wherein the exosome is administered systemically. (Item 42) The method according to item 35, wherein the exosome is administered locally.

[0019] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments shown herein. [Brief explanation of the drawing]

[0020] [Figure 1-1] Figures 1A-E show the in vivo distribution of mesenchymal stem cell-derived exosomes in monkeys. Figures 1A-B show the localization to the pancreas. Figures 1C-D show the localization to the liver. Figure 1E shows the localization to the brain. [Figure 1-2] Same as above.

[0021] [Figure 2-1]Figure 2A shows the structure of mRNA or modRNA transcribed in vitro. Figure 2B shows hTERT mRNA expression in BJ cells treated with hTERT mRNA and lipofectamine in vitro, as measured by qPCR. Figures 2C-D show the telomerase activity of BJ cells treated with hTERT mRNA and lipofectamine in vitro. Figure 2E shows hTERT modRNA expression in BJ cells treated with hTERT modRNA and lipofectamine in vitro, as measured by qPCR. Figure 2F shows the telomerase activity of BJ cells treated with hTERT modRNA and lipofectamine in vitro. Figure 2G shows that transfection with dominant-negative hTERT modRNA does not increase telomerase activity. Figure 2H shows that hTERT mRNA induces cell death, but modRNA does not. Figure 2I shows the effect of prolonged treatment of BJ cells with hTERT modRNA using lipofectamine on cellular senescence. Figure 2J shows that transfection with dominant-negative hTERT modRNA does not improve cellular senescence. Figure 2K shows the effect of prolonged treatment of BJ cells with lipofectamine and hTERT modRNA on telomere signaling. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above. [Figure 2-7] Same as above. [Figure 2-8] Same as above.

[0022] [Figure 3-1] Figures 3A and 3B show the electroporation of hTERT mRNA into exosomes. Figure 3A shows modRNA expression in exosomes after electroporation. Figure 3B shows primer designs for exogenous and endogenous hTERT. [Figure 3-2]Same as above.

[0023] [Figure 4-1] Figures 4A-E show the treatment of BJ cells with exosomes electroporated with hTERT mRNA. Figure 4A shows mRNA expression in BJ cells after treatment with hTERT exosomes. Figure 4B shows the effect of hTERT exosome treatment on telomerase activity. Figure 4C shows the effect of hTERT exosome treatment on aging. Figure 4D shows that hTERT overexpressing exosomes increase C12FDG signaling. BJ cells were treated twice with hTERT overexpressing exosomes, then harvested and evaluated for β-galactosidase signaling using a fluorescent substrate (C12FDG). A decrease in C12FDG MFI indicates a lower degree of aging. Figure 4E shows that BJ hTERT cells emit a weaker aging signal. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above.

[0024] [Figure 5] Figures 5A and 5B show the treatment of U2OS cells with hTERT exosomes (Exofect transfection). Figure 5A shows hTERT mRNA expression in U2OS cells after treatment with hTERT exosomes. Figure 5B shows telomerase activity in U2OS cells after treatment with hTERT exosomes.

[0025] [Figure 6-1]Figures 6A-E show hTERT overexpressing cell lines. Figure 6A shows that 293T hTERT overexpressing cells express more hTERT protein. Figure 6B shows that 293T hTERT cells exhibit high telomerase activity. Figure 6C shows that 293T hTERT exosomes express more hTERT mRNA. Figure 6D shows that hTERT overexpressing cells have higher hTERT mRNA levels. Figure 6E shows that BJ and U2OS hTERT overexpressing cells express more hTERT protein. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0026] [Figure 7] Figure 7 shows cell treatment with 293T hTERT exosomes. The data indicate that U2OS hTERT cells exhibit higher telomerase signaling.

[0027] [Figure 8-1]Figures 8A-J show the delivery of tdTomato mRNA into exosomes and the transfection of tdTomato mRNA with Exofect. Figure 8A shows the transfection of tdTomato plasmid and mRNA into 293T cells by lipofectamine, as measured by FACS. Figure 8B shows the transfection of tdTomato plasmid and mRNA into 293T cells by lipofectamine, as measured by immunofluorescence. Figure 8C shows the structure of tdTomato mRNA or modRNA transcribed in vitro. Figure 8D shows the delivery of tdTomato mRNA to 293T cells by exosomes, as measured by FACS. Figure 8E shows the treatment of 293T cells by exosomes treated with Exofect and tdTomato mRNA / plasmid, as measured by FACS. Figure 8F shows the treatment of 293T cells by exosomes treated with Exofect and tdTomato mRNA, as measured by FACS. Figure 8G shows immunofluorescence of treatment of 293T cells with exosomes treated with Exofect and tdTomato mRNA. Figures 8H-I show the effects of Exofect and tdTomato mRNA delivery on cell viability. Figure 8J shows visualization of mRNA delivery to U2OS cells by exosomes using Exofect. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above. [Figure 8-6] Same as above. [Figure 8-7] Same as above. [Modes for carrying out the invention]

[0028] Extracellular vesicles (EVs), including exosomes and microvesicles, are nano-sized intracellular delivery vehicles involved in several physiological processes. Due to their biological properties and ability to invade other cells when injected into mice and monkeys, extracellular vesicles (EVs) can be used for systemic delivery of therapeutic compounds (e.g., mRNA, microRNA, siRNA, shRNA, CRISPR-Cas9 gene-editing constructs, therapeutic proteins, cytokines, chemotherapeutic drugs, nucleic acids, and bacterial and viral vectors). Intraperitoneal or intravenous injection of mesenchymal cell or 293T cell-derived exosomes into healthy mice and monkeys results in the localization / accumulation of therapeutic exosomes in several organs (including the brain, liver, lungs, and pancreas).

[0029] This specification provides a method for delivering a functional mRNA molecule to a cell using exosomes. Exosomes are superior in their ability to deliver mRNA into cells, along with their functional benefits. For this reason, exosomes may be used to deliver telomerase-encoding mRNA to cells in order to reverse age-related organ dysfunction.

[0030] Telomeres are repeating DNA sequences at the ends of chromosomes. Sufficiently long telomeres form loops that prevent the chromosome ends from being used as substrates in DNA repair processes. However, telomeres shorten over time, resulting in exposed chromosome ends, which destabilize the chromosomes and can lead to cellular aging, apoptosis, or cancer. I. Lipid-based nanoparticles

[0031] Lipid-based nanoparticles may be liposomes, exosomes, lipid preparations, or other lipid-based nanoparticles, such as lipid-based vesicles (e.g., DOTAP: cholesterol vesicles). Lipid-based nanoparticles may be positively charged, negatively charged, or neutral. Lipid-based nanoparticles may contain components necessary to enable transcription and translation, signal transduction, chemotaxis, or other cellular functions.

[0032] Lipid-based nanoparticles may contain CD47 on their surface. CD47 (integrin-related protein) is a transmembrane protein expressed on most tissues and cells. CD47 is a ligand for signal regulatory protein alpha (SIRP-α), which is expressed on phagocytic cells such as macrophages and dendritic cells. Activated CD47-SIRP-α initiates a signaling cascade that suppresses phagocytosis. Therefore, although not theoretically bound, expression of CD47 on the surface of exosomes may inhibit macrophage phagocytosis (see WO2016 / 201323, which is incorporated herein by reference in its entirety). A. Liposomes

[0033] "Liposomes" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes are generally characterized as having a vesicle-like structure comprising a phospholipid-containing bilayer membrane and an internal medium generally comprising an aqueous composition. Liposomes provided herein include monolayer liposomes, multilayer liposomes, and multivesicular liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged.

[0034] Multilayer liposomes have multiple lipid layers separated by an aqueous medium. Such liposomes spontaneously form when lipids, including phospholipids, are suspended in an excess amount of aqueous solution. The lipid components undergo self-reorganization before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions can similarly dissolve within the lipid bilayers or associate with them.

[0035] In certain embodiments, polypeptides, nucleic acids, or small molecule drugs may, for example, be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, bound to liposomes by linking molecules that bind to both liposomes and polypeptides / nucleic acids, captured within liposomes, or form complexes with liposomes.

[0036] Liposomes used according to this embodiment can be prepared by various methods, as is known to those skilled in the art. For example, a phospholipid (e.g., dioleoylphosphatidylcholine (DOPC), which is a neutral phospholipid) is dissolved in TERT-butanol. This lipid(s) is then mixed with polypeptides, nucleic acids, and / or other components(s). Tween® 20 is added to this lipid mixture so that it accounts for about 5% of the composition weight. An excess amount of TERT-butanol is added to this mixture so that the volume of TERT-butanol accounts for at least 95%. This mixture is vortexed and frozen in a dry ice / acetone bath and freeze-dried overnight. The freeze-dried preparation is stored at -20°C and is usable for up to 3 months. If necessary, the freeze-dried liposomes are reconstituted in 0.9% saline.

[0037] Alternatively, liposomes can be prepared by mixing lipids in a solvent in a container, such as a glass pear-shaped flask. The container should have a capacity 10 times that of the expected volume of the liposome suspension. The solvent is removed at approximately 40°C under negative pressure using a rotary evaporator. Depending on the desired volume of liposomes, the solvent is usually removed within about 5 minutes to 2 hours. The composition may be further dried in a desiccator under vacuum. Since the dried lipids tend to degrade over time, they are usually discarded after about a week.

[0038] The dried lipids can be hydrated with approximately 25–50 mM phospholipids by shaking in sterile pyrogen-free water until all lipid films are resuspended. The aqueous liposomes may then be divided into aliquots, each aliquot placed in a vial, and freeze-dried and sealed under vacuum.

[0039] The dried lipids or lyophilized liposomes prepared as described above may be dehydrated, reconstituted in a protein or peptide solution, and diluted to an appropriate concentration in a suitable solvent, such as DPBS. The mixture is then vigorously shaken in a vortex mixer. Excess unencapsulated material, such as but not limited to hormones, drugs, nucleic acid constructs, etc., is removed by centrifugation at 29,000 × g, and the resulting liposome pellet is washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, for example, about 50–200 mM. The amount of excess material or encapsulated active agent can be determined according to standard methods. After determining the amount of excess material or encapsulated active agent in the liposome preparation, the liposomes may be diluted to an appropriate concentration and stored at 4°C until use. Pharmaceutical compositions containing liposomes typically contain a sterile, pharmaceutically acceptable carrier or diluent, such as water or a saline solution.

[0040] Further liposomes that may be useful in this embodiment include cationic liposomes, such as those described in WO02 / 100435A1, U.S. Patent No. 5,962,016, U.S. Patent Application No. 2004 / 0208921, WO03 / 015757A1, WO04 / 029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068 (all of which are incorporated herein by reference in their entirety without disclaimer).

[0041] In the preparation of such liposomes, any protocol described herein or any protocol known to those skilled in the art may be used. Further non-limiting examples of liposome preparations are described in U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; International Applications PCT / US85 / 01161 and PCT / US89 / 05040 (which are incorporated herein by reference, respectively).

[0042] In certain embodiments, the lipid-based nanoparticles are neutral liposomes (e.g., DOPC liposomes). “Neutral liposome” or “uncharged liposome” is defined, as used herein, 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, if any, only a small number of lipid components in a given population (e.g., a population of liposomes) have a charge that is not canceled out 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 have an uncharged charge). In certain embodiments, the neutral liposome may primarily consist of lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., at about pH 7).

[0043] The liposomes and / or lipid-based nanoparticles of this embodiment may contain phospholipids. In certain embodiments, a single type of phospholipid may be used to produce liposomes (for example, a neutral phospholipid such as DOPC may be used to produce neutral liposomes). In other embodiments, more than one type of phospholipid may be used to produce liposomes. The phospholipids may be of natural origin or of synthetic origin. Examples of phospholipids include phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine; since phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at about pH 7), these compounds may be particularly useful for the production of neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce uncharged liposomes. In certain embodiments, lipids other than phospholipids (e.g., cholesterol) may be used.

[0044] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauroylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), and distearoyl phosph Phosphatidylcholine ("DSPC"), 1-Myristoyl-2-Palmitoylphosphatidylcholine ("MPPC"), 1-Palmitoyl-2-Myristoylphosphatidylcholine ("PMPC"), 1-Palmitoyl-2-Stearoylphosphatidylcholine ("PSPC"), 1-Stearoyl-2-Palmitoylphosphatidylcholine ("SPPC"), Dilauroylphosphatidylglycerol (" DLPG, dimyristoyl phosphatidylglycerol ("DMPG"), dipalmitoyl phosphatidylglycerol ("DPPG"), distearoyl phosphatidylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoyl phosphatidylethanolamine ("DSPE"), dioleoyl phosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphati Dimethyl phosphate ("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,Examples include 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoylphosphatidylcholine ("POPC"), palmitoyloeoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine. B. Exosome

[0045] The terms “microvesicle” and “exosome,” as used herein, refer to membrane-bound particles with a diameter (or, if the particle is not spherical, its maximum dimension) of approximately 10 nm to 5000 nm, more typically 30 nm to 1000 nm, and most typically 50 nm to 750 nm, where at least a portion of the exosome membrane is obtained directly from the cell. Most commonly, exosomes have a size (average particle size) of up to 5% of the size of the donor cell. Therefore, exosomes particularly intended are those expelled from cells.

[0046] Exosomes can be detected in any suitable sample type (e.g., body fluids) or isolated from such sample types. As used herein, the term “isolated” means separated from its natural environment and is intended to include at least partial purification, and may include substantial purification. As used herein, the term “sample” means any sample suitable for the methods provided by the present invention. A sample may be any sample containing exosomes suitable for detection or isolation. Sources of samples include blood, bone marrow, pleural fluid, ascites, 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 embodiment, a sample is a blood sample, and a blood sample includes, for example, whole blood or any fraction or component thereof. A blood sample suitable for use with the present invention can be extracted from any source known to contain blood cells or components thereof, such as a venous source, arterial source, peripheral source, tissue source, umbilical cord source, etc. For example, samples can be obtained and processed by well-known and routine clinical methods (e.g., procedures for collecting and processing whole blood). In one embodiment, an exemplary sample may be peripheral blood collected from a subject with cancer.

[0047] Exosomes can also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells. When isolating exosomes from a tissue source, it may be necessary to homogenize the tissue to obtain a single cell suspension, then lyse the cells, and release the exosomes. When isolating exosomes from tissue samples, it is important to select homogenization and lysis procedures that do not destroy the exosomes. The exosomes intended herein are preferably isolated from body fluids in a physiologically acceptable solution (e.g., buffered saline, growth medium, various aqueous media).

[0048] Exosomes may be isolated from freshly collected samples or from frozen or refrigerated samples. In some embodiments, exosomes may be isolated from cell culture media. Although not essential, higher purity exosomes may be obtained if the liquid sample is clarified and any debris is removed from the sample before precipitation with volume exclusion polymer. Clarification methods include centrifugation, ultracentrifugation, filtration, or ultrafiltration. Most typically, exosomes can be isolated by a number of methods known in the art. One preferred method is fractional centrifugation from body fluids or cell culture supernatants. Exemplary methods for exosome isolation are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Alternatively, exosomes may also be isolated by flow cytometry, as described in (Combes et al., 1997).

[0049] One established protocol for isolating exosomes is ultracentrifugation, often combined with a sucrose density gradient or sucrose cushion to suspend relatively low-density exosomes. Isolation of exosomes by sequential fractionation centrifugation is complex because their size distribution may overlap with other microvesicles or macromolecular complexes. Furthermore, the size-based vesicle separation methods provided by centrifugation may be insufficient. However, when combined with sucrose gradient ultracentrifugation, sequential centrifugation can achieve high exosome enrichment.

[0050] Size-based exosome isolation using alternatives to ultracentrifugation routes is another option. Successful purification of exosomes using ultrafiltration procedures, which are less time-consuming and do not require specialized equipment than ultracentrifugation, has been reported. Similarly, a commercially available kit (EXOMIR®, Bioo Scientific) is available that utilizes positive pressure to push the fluid, removing cells, platelets, and cellular debris on one microfilter and capturing vesicles larger than 30 nm on a second microfilter. However, in this process, exosomes are not recovered; their RNA contents are extracted directly from the material captured on the second microfilter and can then be used for PCR analysis. HPLC-based protocols may potentially allow for the acquisition of very pure exosomes, but these processes require specialized equipment and are difficult to scale up. A significant problem is that both blood and cell culture media contain a large number of nanoparticles (somewhat non-vesicular) within the same size range as exosomes. For example, some miRNAs may be contained within extracellular protein complexes rather than in exosomes, but treatment with proteases (e.g., proteinase K) can eliminate any possible contamination by "extra-exosome" proteins.

[0051] In another embodiment, cancer cell-derived exosomes may be captured by techniques commonly used to enrich a sample for exosomes, such as techniques that utilize immunospecific interactions (e.g., immunomagnetic capture). Immunomagnetic capture (also known as immunomagnetic cell separation) typically involves attaching antibodies against proteins found on a particular cell type to small paramagnetic beads. When these antibody-coated beads are mixed with a sample (e.g., blood), the beads adhere to and surround the specific cells. The sample is then placed in a strong magnetic field, thereby pelleting the beads to one side. After the blood is removed, the captured cells are retained by the beads. Many variations of this common method are well known in the art and are suitable for use in isolating exosomes. In one example, exosomes may be attached to magnetic beads (e.g., aldehyde / sulfate beads), and then antibodies may be added to the mixture to recognize epitopes on the surface of the exosomes attached to the beads. Exemplary proteins known to be found on cancer cell-derived exosomes include ATP-binding cassette subfamily A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), and glypican 1 (GPC1). Cancer cell-derived exosomes may be isolated, for example, using antibodies or aptamers against one or more of these proteins.

[0052] It should be noted that not all proteins expressed in a cell are found in the exosomes secreted by that cell. For example, calnexin, GM130, and LAMP-2 are all proteins expressed in MCF-7 cells, but are not found in the exosomes secreted by MCF-7 cells (Baietti et al., 2012). As another example, one study found that 190 out of 190 patients with pancreatic ductal adenocarcinoma had higher levels of GPC1+ exosomes than healthy controls (Melo et al., 2015 (the entire report is incorporated herein by reference)). Notably, only 2.3% of healthy controls, on average, had GPC1+ exosomes. 1. Exemplary protocol for extracting exosomes from cell cultures

[0053] On day 1, seed a sufficient number of cells (e.g., about 5 million cells) in a T225 flask in a medium containing 10% FBS so that the cells reach approximately 70% confluence the following day. On day 2, aspirate the medium from the cells, wash the cells twice with PBS, and then add 25-30 mL of basic medium (i.e., without PenStrep or FBS) to the cells. Incubate the cells for 24-48 hours. While 48 hours of incubation is preferable, some cell lines are more sensitive to serum-free medium and therefore the incubation time should be shortened to 24 hours. Note that FBS contains exosomes, which can significantly distort NanoSight results.

[0054] On days 3 and 4, collect the culture medium and centrifuge at 800 × g for 5 minutes at room temperature to pellet dead cells and large debris. Transfer the supernatant to a new conical tube and centrifuge the medium for a further 10 minutes at 2000 × g to remove other large debris and large vesicles. Pass the medium through a 0.2 μm filter and then dispense 35 mL per tube into ultracentrifuge tubes (e.g., 25 × 89 mm Beckman Ultra-Clear). If the amount of medium per tube is less than 35 mL, fill the remaining part of the tube with PBS to make 35 mL. Ultracentrifuge the medium at 28,000 rpm for 2–4 hours at 4°C using an SW32Ti rotor (k-factor 266.7, maximum RCF 133,907). Carefully aspirate the supernatant until the liquid volume is approximately 1 inch. Tilt the centrifuge tube to allow the remaining medium to slowly enter the aspirator pipette. If desired, the exosome pellet may be resuspended in PBS and ultracentrifugation at 28,000 rpm may be repeated for 1-2 hours to further purify the exosome population.

[0055] Finally, resuspend the exosome pellet in 210 μL of PBS. If multiple ultracentrifuge tubes are available for each sample, sequentially resuspend each exosome pellet using the same 210 μL of PBS. Take 10 μL of each sample and add it to 990 μL of H2O for use in nanoparticle tracking analysis. Use the remaining 200 μL of exosome-containing suspension for downstream processes or store immediately at -80°C. 2. Exemplary protocol for extracting exosomes from serum samples

[0056] First, thaw the serum sample on ice. Next, dilute 250 μL of cell-free serum sample in 11 mL of PBS and filter through a 0.2 μm pore filter. Ultracentrifuge the diluted sample overnight at 150,000 × g at 4°C. The following day, carefully remove the supernatant and wash the exosome pellet in 11 mL of PBS. Perform a second ultracentrifuge at 150,000 × g for 2 hours at 4°C. Finally, carefully remove the supernatant and resuspend the exosome pellet in 100 μL of PBS for analysis. Exemplary protocol for electroporation of C. exosomes and liposomes

[0057] 1 x 10 8 Mix exosomes (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula NanoSciences) with 1 μg of siRNA (Qiagen) or shRNA in 400 μL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). Electroporate the exosomes or liposomes using a 4 mm cuvette (see Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After electroporation, treat the exosomes or liposomes with protease-free RNAse, then add a 10-fold enrichment of RNAse inhibitor. Finally, wash the exosomes or liposomes with PBS under ultracentrifugation as described above. II. Treatment of Diseases

[0058] Certain aspects of the present invention provide treatment for a patient by expressing or containing an exosome a therapeutic agent that enhances intracellular telomerase activity. “Therapeutic agent,” as used herein, is an atom, molecule, or compound useful in the treatment of age-related disorders or other conditions. Examples of therapeutic agents include, but are not limited to, drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes, nucleic acids, nucleases, hormones, immunomodulators, antisense oligonucleotides, gene editing systems, chelating agents, boron compounds, photoactivators, and dyes.

[0059] Examples of genetic disorders associated with shortened telomeres include idiopathic pulmonary fibrosis, congenital keratosis, and aplastic anemia. Other diseases known to be associated with shortened telomeres include muscular dystrophy, atherosclerosis, hypertension, heart disease, cancer, stroke, diabetes, diabetic ulcers, Alzheimer's disease, osteoporosis, macular degeneration, immunosenescence, myocardial infarction, and vascular dementia.

[0060] As exosomes are known to contain DICER and an active RNA-processing RISC complex (see International Patent Application Publication WO2014 / 152622, which is incorporated herein by reference in its entirety), shRNA transfected into an exosome can mature into a RISC complex-binding siRNA within that exosome. Alternatively, the mature siRNA itself may be transfected into an exosome or liposome. Thus, for example, inhibitory RNA may be used in the methods of the present invention to modulate telomerase activity (e.g., Menin, SIP1, pRB, p38, p53, p73, MKRN1, CHIP, HSP70, androgen, TGF-beta, Arc1, cAbl, Pinx1, CRM1, POT1, p19 / Arf). Any inhibitory nucleic acid can be applied to the compositions and methods of the present invention, provided that the inhibitory nucleic acid is found by any source to be a reliable down-regulator of the protein of interest.

[0061] In designing RNAi, several factors must be considered, including the properties of the siRNA, the persistence of the silencing effect, and the choice of delivery system. The siRNA introduced into an organism to produce an RNAi effect typically contains an exon sequence. Furthermore, because the RNAi process is homology-dependent, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA exhibits greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or even 100% identity between its sequence and the gene it inhibits. Sequences with less than approximately 80% identity to the target gene are substantially ineffective. Therefore, the greater the homology between the siRNA and the gene it inhibits, the less likely it is that the expression of unrelated genes will be affected.

[0062] Since exosomes are known to contain the mechanisms necessary to complete mRNA transcription and protein translation (see PCT / US2014 / 068630, which is incorporated herein by reference), mRNA or DNA nucleic acids encoding therapeutic proteins may be transfected into exosomes. Alternatively, the therapeutic protein itself may be electroporated into exosomes or directly incorporated into liposomes. An example of therapeutic RNA is the telomerase RNA component (TERC). Examples of therapeutic proteins include, but are not limited to, telomerase reverse transcriptase (TERT (NP_937983.2 or NP_001180305.1)), TCAB1, diskelin, Gar1, Nhp2, Nop10, RHAU, helicase, UPF1, HSP90, PKC, Shp-2, NFκB p65, TPP1, ATM, DAT, TRF1, TRF2, Rap1, Rif1, TIN2, NBS, MRE17, RAD50, EGF, IGF-1, FGF-2, VEGF, IL-2, IL-4, IL-6, IL-7, IL-13, IL-15, and Akt.

[0063] Certain types of proteins that may be desirable to introduce into the intracellular space of diseased cells are antibodies (e.g., monoclonal antibodies) that can specifically or selectively bind to intracellular antigens. Such antibodies can 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, Menin, SIP1, pRB, p38, p53, p73, MKRN1, CHIP, HSP70, androgens, TGF-beta, Arc1, cAbl, Pinx1, CRM1, POT1, and p19 / Arf. In addition to monoclonal antibodies, any antigen-binding fragments thereof, e.g., scFv, Fab fragment, Fab', F(Ab')2, Fv, pepti-bodies, dia-bodies, tria-bodies, or mini-bodies are also intended. Either such antibody or antibody fragment may or may not be glycosylated.

[0064] The exosome may also be engineered to include a gene editing system (e.g., a CRISPR / Cas system) that corrects a genetic defect in the telomerase complex (e.g., a mutation in TERT or TERC). Generally, the “CRISPR system” refers collectively to transcripts and other elements that are involved in or direct the activity of CRISPR-related (“Cas”) genes, and CRISPR-related (“Cas”) genes include the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr mate sequence (in the context of the endogenous CRISPR system, including “direct repeats” and tracrRNA-processed partial direct repeats), the guide sequence (also called a “spacer” in the context of the endogenous CRISPR system), and / or other sequences and transcripts derived from the CRISPR site. In some embodiments, the Cas nuclease and gRNA (including a fusion of a target sequence-specific crRNA and a sequence-defined tracrRNA) are introduced into the cell. Generally, the target site at the 5' end of a gRNA directs the Cas nuclease to its target site (e.g., a gene) using complementary base pairing. The target site may be selected based on its position, being immediately 5' to the protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, the gRNA is directed to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of its guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, the “target sequence” refers to a sequence to which the guide sequence is designed to be complementary, where hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and facilitate the formation of the CRISPR complex.A CRISPR system within an exosome, engineered to contain such a system, can function to edit genomic DNA within a target cell, or such a system can edit DNA within the exosome itself. For further embodiments relating to the use of exosomes as a means of delivering gene editing systems, see U.S. Patent Application No. 62 / 599,340, which is incorporated herein by reference in its entirety.

[0065] In addition to protein-based and nucleic acid-based therapeutic agents, exosomes may be used to deliver small molecule drugs, either alone or in combination with protein-based or nucleic acid-based therapeutic agents. Examples of small molecule drugs intended for use in this embodiment include, but are not limited to, TA-65 (Harley et al., Rejuvenation Research, 14:45-56, 2011), estrogen, erythropoietin, resveratrol, cycloastragenol (TAT2), TA-65, TAT153, and okadaic acid.

[0066] The term "subject," as used in this invention, refers to any individual or patient on whom the subject method is performed. Generally, the subject is human, but as will be understood by those skilled in the art, the subject may also be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cattle, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas), are included in the definition of subject.

[0067] "Treatment" and "treating" refer to administering or applying a therapeutic agent to a subject, or performing a procedure or modality on a subject, with the aim of obtaining a therapeutic benefit from a disease or health-related condition. For example, treatment may include administering cargo-carrier exosomes, administering chemotherapy, immunotherapy, or radiation therapy, performing surgery, or a combination thereof.

[0068] When used throughout this application, the terms “therapeutic benefit” or “therapeutic effectiveness” refer to anything that promotes or enhances the well-being of a subject with respect to medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of signs or symptoms of the disease. For example, treatment of cancer may include, for instance, a reduction in the invasiveness of the tumor, a decrease in the rate of cancer growth, or a prevention of metastasis. Treatment of cancer may also refer to an extension of the survival period of a subject with cancer.

[0069] The terms “contact” and “expose” are used herein, when applied to cells, to describe the process by which a therapeutic agent is delivered to target cells, or the process by which a therapeutic agent is directly juxtaposed with respect to target cells. For example, to achieve telomere elongation, one or more amounts of a drug effective to enhance telomerase function are delivered to the cells.

[0070] A patient's effective response to a treatment, or "responsiveness," refers to the clinical or therapeutic benefit derived to a patient who is at risk of or suffering from a disease or disorder. Such benefits include cellular or biological responses, complete responses, partial responses, stable conditions (no exacerbations or relapses), or responses that later relapse. The methods described herein can be used to predict and monitor the outcomes of a treatment and / or to identify or select patients who will benefit from such a treatment.

[0071] Regarding the treatment of a disease, the appropriate dosage of the therapeutic composition is determined by the type of disease being treated, the severity and course of the disease, the patient's clinical history and response to the medication, and the discretion of the attending physician. The medication should be administered to the patient appropriately, either as a single dose or over a series of treatments.

[0072] Methods and compositions for treatment and prevention may be provided in combined amounts effective to achieve the desired effect. Tissues, tumors, or cells may be brought into contact with one or more compositions or pharmacological preparations containing one or more agents, or tissues, tumors, and / or cells may be brought into contact with two or more separate compositions or preparations. Such combination therapies are also intended to be used in combination with chemotherapy, radiotherapy, surgery, or immunotherapy.

[0073] Concomitant administration may include simultaneous administration of two or more drugs in the same dosage form, simultaneous administration of different dosage forms, and separate administration. That is, the therapeutic composition of interest and another therapeutic agent may be formulated together in the same dosage form and administered simultaneously. Alternatively, the therapeutic composition of interest and another therapeutic agent may be administered simultaneously, with both drugs present in separate formulations. In another option, the therapeutic agent of interest may be administered immediately after the other therapeutic agent, or vice versa. In separate administration protocols, the therapeutic composition of interest and the other therapeutic agent may be administered at intervals of several minutes, several hours, or several days. Mai. Pharmaceutical composition

[0074] Exosomes expressing or containing therapeutic agents are intended to be administered systemically or topically to enhance telomerase activity. These exosomes may be administered intravenously, intrathecally, and / or intraperitoneally. They may be administered alone or in combination with a second drug.

[0075] The present invention is not intended to be limited by the specific properties of the therapeutic preparations. For example, such compositions may be provided as formulations in combination with physiologically tolerable liquids, gels, solid carriers, diluents, or excipients. These therapeutic preparations may be administered to mammals in a manner similar to other therapeutic agents, for veterinary use (e.g., to domesticated animals) and for clinical use in humans. Generally, the dose required for therapeutic efficacy will vary depending on the type of use and mode of administration, as well as the specific needs of the individual subject.

[0076] When clinical application is intended, it may be necessary to prepare a pharmaceutical composition containing exosomes in a form appropriate for the intended application. Generally, a pharmaceutical composition may contain one or more effective amounts of exosomes and / or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase “pharmaceutically or pharmacologically acceptable” means a molecular entity or composition that does not produce adverse reactions, allergic reactions, or other undesirable reactions when administered to an animal (e.g., a human, where appropriate). The preparation of exosome-containing pharmaceutical compositions or additional active ingredients disclosed herein will be understood by those skilled in the art in consideration of this disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th edition, 1990 (as incorporated herein by reference). Furthermore, for administration to an animal (e.g., a human), it will be understood that the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0077] Furthermore, according to certain embodiments of the present invention, compositions suitable for administration may be provided in a pharmaceutically acceptable carrier, with or without an inert diluent. As used herein, “pharmaceutically acceptable carrier” is known to those skilled in the art, and may include all aqueous solvents (e.g., water, alcoholic / aqueous solutions, ethanol, saline solutions, parenteral vehicles (e.g., sodium chloride, ringer's dextrose), non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and injectable organic esters (e.g., ethyl oleate), 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), isotonic agents (e.g., sugars and sodium chloride), and absorbents. This includes agglutination retarders (e.g., aluminum monostearate and gelatin), salts, drugs, drug stabilizers, gels, resins, fillers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluids and nutrients, similar materials, and combinations thereof. The carrier should be absorbable and may be a liquid, semi-solid (i.e., paste), or solid carrier. In addition, optionally, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffers. The pH and the precise concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0078] pharmaceutically acceptable carriers are formulated particularly for administration to humans, but in certain embodiments, the use of pharmaceutically acceptable carriers formulated for administration to non-human animals but not acceptable for administration to humans (e.g., due to government regulations) may be desirable. Any conventional carrier is intended for use in therapeutic or pharmaceutical compositions unless it is incompatible with the active ingredient (e.g., detrimental to the recipient or detrimental to the therapeutic efficacy of the composition it contains). According to certain embodiments of the present invention, the composition is mixed with the carrier in any convenient and practical manner, namely by dissolution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such procedures are routine operations for those skilled in the art.

[0079] Certain embodiments of the present invention may include different types of carriers, depending on whether the administration is in solid, liquid, or aerosol form and whether it needs to be sterile for the route of administration (e.g., injection). The composition can be administered intravenously, intradermally, percutaneously, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, transmucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion directly immersing target cells, by catheter, by lavage, in a lipid composition (e.g., liposomes), or by any other method or any combination thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990 (materially incorporated herein by reference)).

[0080] Exosomes may be formulated for parenteral administration, for example, for intravenous, intramuscular, or subcutaneous injection, or even for injection via an intraperitoneal route. Typically, such compositions may be prepared as either a liquid solution or a suspension; they may be prepared in a solid form suitable for use in preparing a solution or suspension by adding liquid before injection; or the preparation may be emulsified.

[0081] Suitable pharmaceutical formulations for use by injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the formulations must be sterile and fluid enough to be easily injected. They must also be stable under manufacturing and storage conditions and protected from microbial contamination (e.g., bacteria and fungi).

[0082] Once formulated, the liquid formulation is administered in a manner appropriate to the dosage form and in a therapeutically effective amount. The formulation is readily administered in various dosage forms, such as those formulated for parenteral administration (e.g., injectable liquid formulations or aerosol formulations for delivery to the lungs) or those formulated for gastrointestinal administration (e.g., drug-releasing capsules).

[0083] The term “unit dose” or “dosage” refers to a physically separate unit suitable for use in a subject, each unit comprising a predetermined amount of the therapeutic composition calculated to produce the desired response in relation to its administration (i.e., appropriate route and treatment regimen) as described above. The amount administered (by both the number of treatments and the unit dose) depends on the desired effect. The actual dose 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 status, and sex, the type of disease being treated, the extent of disease penetration, past or parallel therapeutic interventions, the patient’s idiopathic disease, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, doses may also include amounts from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (such a range includes intermediate doses) or greater, and any range that can be derived within that. In non-limiting examples derived from the numbers listed herein, doses can be administered in ranges such as approximately 5 μg / kg / body weight to approximately 100 mg / kg / body weight, approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight, etc. In another example, doses may also include approximately 1 billion to approximately 500 billion exosomes per dose (such ranges include intermediate doses) or more exosomes, and any range that can be derived within that. In non-limiting examples derived from the numbers listed herein, doses can be administered in ranges such as approximately 1 million to approximately 500 billion exosomes, approximately 5 million to approximately 250 billion exosomes, etc. In one example, a dose may contain approximately 150 billion exosomes in a volume of 5 mL, and such a dose may be administered to a human patient weighing 70 kg. In any case, the person responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual subject.

[0084] The actual dosage of a composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of condition, type of disease being treated, past or concurrent therapeutic interventions, the patient's idiopathic disease, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or number of effective doses may be modified depending on the subject's response. In any case, the person responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual subject.

[0085] 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 constitute, for example, about 2% to about 75% or about 25% to about 60% of the unit weight, and any range that can be derived therein. Naturally, the amount of the active compound(s) in each therapeutically useful composition may be prepared so that an appropriate dose is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations are intended by those skilled in the art of preparing such pharmaceutical formulations, and thus various dosages and treatment regimens may be desirable.

[0086] In other non-limiting examples, the dose may also include amounts from approximately 1 microgram / kg / body weight to approximately 5 micrograms / kg / body weight, approximately 10 micrograms / kg / body weight, approximately 50 micrograms / kg / body weight, approximately 100 micrograms / kg / body weight, approximately 200 micrograms / kg / body weight, approximately 350 micrograms / kg / body weight, approximately 500 micrograms / kg / body weight, approximately 1 milligram / kg / body weight, approximately 5 milligrams / kg / body weight, approximately 10 milligrams / kg / body weight, approximately 50 milligrams / kg / body weight, approximately 100 milligrams / kg / body weight, approximately 200 milligrams / kg / body weight, approximately 350 milligrams / kg / body weight, approximately 500 milligrams / kg / body weight, approximately 1000 milligrams / kg / body weight, or more, and any range that can be derived therefrom. In non-limiting examples that can be derived from the numbers listed herein, the dose can be administered in ranges such as approximately 5 milligrams / kg / body weight to approximately 100 milligrams / kg / body weight, or approximately 5 micrograms / kg / body weight to approximately 500 milligrams / kg / body weight, based on the numbers above. IV. Exosome Cargo A. Nucleic acids and vectors

[0087] In certain embodiments of the present invention, nucleic acid sequences encoding therapeutic proteins or antibodies may be disclosed. Depending on the expression system used, the nucleic acid sequences may be selected based on conventional methods. For example, each gene or its variant may be codon-optimized for expression in a particular system. Various vectors can also be used to express the protein of interest. Exemplary vectors, but not limited to them, include plasmid vectors, viral vectors, transposons, or liposome-based vectors. B. Recombinant Protein

[0088] Some embodiments relate to recombinant proteins and polypeptides, such as therapeutic antibodies. In some embodiments, the therapeutic antibody may be an antibody that specifically or selectively binds to intracellular proteins. In further embodiments, the protein or polypeptide may be modified to increase serum stability. Thus, when the function or activity of “modified protein” or “modified polypeptide” is referred to in this application, those skilled in the art will understand that this includes, for example, proteins or polypeptides that have further advantages over unmodified proteins or polypeptides. Embodiments relating to “modified protein” can be carried out with respect to “modified polypeptide” and vice versa, and so on.

[0089] As used herein, protein or peptide generally refers to proteins, but not limited to, that are greater than about 200 amino acids and up to the full-length sequence translated from a gene; polypeptides greater than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein.

[0090] As used herein, “amino acid residue” means any naturally occurring amino acid, any amino acid derivative, or any amino acid mime known in the art. In certain embodiments, the residues of a protein or peptide are continuous and do not contain any non-amino acids that interrupt the sequence of amino acid residues. In other embodiments, the sequence may contain 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.

[0091] Therefore, the term “protein or peptide” encompasses amino acid sequences that include at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid. C. Inhibitory RNA

[0092] siRNA (e.g., siNA) is well known in the art. For example, siRNA and double-stranded RNA are described in U.S. Patent Nos. 6,506,559 and 6,573,099, and U.S. Patent Applications 2003 / 0051263, 2003 / 0055020, 2004 / 0265839, 2002 / 0168707, 2003 / 0159161, and 2004 / 0064842 (all of which are incorporated herein by reference in their entirety).

[0093] Within an siRNA, the nucleic acid components do not need to be of the same type or homogenous throughout (for example, an siRNA may contain nucleotides and nucleic acids or nucleotide analogs). Typically, an siRNA forms a double-stranded structure; this double-stranded structure may result from two distinct nucleic acids that are partially or completely complementary. In certain embodiments of the present invention, an siRNA may contain only a single nucleic acid (polynucleotide) or nucleic acid analog, forming a double-stranded structure by complementing itself (for example, forming a hairpin loop). The double-stranded structure of siRNA may contain 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more consecutive nucleic acid bases (including the entire range within that number). siRNA may contain 17 to 35 consecutive nucleic acid bases, more preferably 18 to 30 consecutive nucleic acid bases, more preferably 19 to 25 consecutive nucleic acid bases, more preferably 20 to 23 consecutive nucleic acid bases, or 20 to 22 consecutive nucleic acid bases, or 21 consecutive nucleic acid bases, which hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[0094] Useful agents of the present invention for carrying out the methods of the present invention include, but are not limited to, siRNA. Typically, the introduction of double-stranded RNA (dsRNA) (sometimes referred to herein as small interfering RNA (siRNA)) induces potent and specific gene silencing (a phenomenon called RNA interference or RNAi). RNA interference has been called "co-repression," "post-transcriptional gene silencing," "sense repression," and "quelling." RNAi is an attractive biotechnology tool because it provides a means of knocking out the activity of specific genes.

[0095] In designing RNAi, several factors must be considered, including the properties of the siRNA, the persistence of the silencing effect, and the choice of delivery system. The siRNA introduced into an organism to produce an RNAi effect typically contains an exon sequence. Furthermore, because the RNAi process is homology-dependent, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA exhibits greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or even 100% identity between its sequence and the gene it inhibits. Sequences with less than approximately 80% identity to the target gene are substantially ineffective. Therefore, the greater the homology between the siRNA and the gene it inhibits, the less likely it is that the expression of unrelated genes will be affected.

[0096] Furthermore, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to an siRNA molecule containing at least about 19 to 25 nucleotides and 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 to about 25 nucleotides in length.

[0097] A target gene generally refers to a polynucleotide containing a polypeptide-coding region, or a polynucleotide region that regulates replication, transcription, translation, or other processes important for polypeptide expression, or a polynucleotide containing both a polypeptide-coding region and an expression-regulating region operably linked thereto. Any gene expressed in a cell can be targeted. Preferably, the target gene is one that is involved in or related to the exacerbation of disease-related cellular activity, or one of particular interest for research purposes.

[0098] siRNA can be obtained from commercial sources, from natural sources, or synthesized using any of the many techniques well known to those skilled in the art. For example, one commercial source of pre-designed siRNA is Ambion® (Austin, Tex). Another is Qiagen® (Valencia, Calif.). The inhibitory nucleic acid that can be applied in the compositions and methods of the present invention may be any nucleic acid sequence that has been confirmed by any source to be a reliable down-regulator of the protein of interest. It is understood that, without excessive experimentation, further siRNAs can be designed and used to carry out the methods of the present invention using the disclosures of the present invention.

[0099] siRNA may also include one or more nucleotide modifications. Such modifications may include, for example, the addition of non-nucleotide material to the 19-25 nucleotide terminus (one or more) of the RNA, or internal additions (in one or more nucleotides of the RNA). In certain embodiments, the RNA molecule contains a 3'-hydroxyl group. The nucleotides in the RNA molecule of the present invention may also include non-standard nucleotides (including nucleotides or deoxyribonucleotides that do not exist in nature). The double-stranded oligonucleotide may include a modified backbone, such as a phosphorothioate, phosphorodithioate, or other modified backbone known in the art, or it may include non-natural nucleoside bonds. Further modifications of siRNA (e.g., incorporation of 2'-O-methylribonucleotides, 2'-deoxy-2'-fluororibonucleotides, "universal base" nucleotides, 5-C-methylnucleotides, one or more phosphorothioate nucleotide interbondings, and inverted deoxy-base residues) can be found in U.S. Patent Application Publication No. 2004 / 0019001 and U.S. Patent No. 6,673,611 (both of which are invoked in whole by reference, respectively). All of the above modified nucleic acids or RNAs are collectively referred to as modified siRNA. D. Gene editing systems

[0100] Generally, the “CRISPR system” refers collectively to the transcripts and other elements involved in or instructing the expression of CRISPR-related (“Cas”) genes, which include the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracrmate sequence (in the context of the endogenous CRISPR system, encompassing “direct repeats” and tracrRNA-processed partial direct repeats), the guide sequence (also called “spacers” in the context of the endogenous CRISPR system), and / or other sequences and transcripts derived from CRISPR sites.

[0101] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide) RNA (which binds to DNA in a sequence-specific manner) and a Cas protein (e.g., Cas9) having nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a type I, type II, or type III CRISPR system, or from a specific organism containing an endogenous CRISPR system, such as Streptococcus pyogenes.

[0102] In some embodiments, a Cas nuclease and gRNA (including a fusion of a target sequence-specific crRNA and a sequence-defined tracrRNA) are introduced into cells. Generally, a target site at the 5' end of the gRNA directs the Cas nuclease to its target site (e.g., a gene) using complementary base pairing. The target site may be selected based on its location, being immediately 5' to a protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, the gRNA is directed to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of its guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, a "target sequence" refers to a sequence to which a guide sequence is designed to be complementary, where hybridization between the target and guide sequences promotes the formation of the CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex.

[0103] The CRISPR system can induce double-strand breaks (DSBs) at a target site and subsequently disrupt them, as described herein. In other embodiments, a Cas9 variant (considered a "nickase") is used to introduce a nick into the single strand at the target site. Pairs of nickases can be used, for example, to enhance specificity, with each nickase being oriented by a set of different gRNA targeting sequences such that a 5' overhang is introduced when the nick is introduced simultaneously. In other embodiments, a catalytically inactive Cas9 is fused to a heterogeneous effector domain (e.g., a repressor or activator) to influence gene expression.

[0104] The target sequence may contain any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of a cell, for example, within a cell organelle. Generally, a sequence or template that can be used for recombination into a target site containing the target sequence is called an “editing template,” or “edited polynucleotide,” or “editing sequence.” In some embodiments, an exogenous template polynucleotide is called an editing template. In some embodiments, the recombination is homologous recombination.

[0105] Typically, in the context of endogenous CRISPR systems, the formation of a CRISPR complex (which includes a guide sequence that hybridizes with the target sequence and forms a complex with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). A tracr sequence (which may include the entire wild-type tracr sequence, be composed of it, or include a portion of the wild-type tracr sequence (for example, a wild-type tracr sequence containing about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides, or more than 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides)) may also form part of a CRISPR complex, for example, by hybridization along at least a portion of the tracr sequence to the whole or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to hybridize with the tracr mate sequence and participate in the formation of the CRISPR complex; for example, when optimally aligned, it has sequence complementarity of at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% along the length of the tracr mate sequence.

[0106] One or more vectors that promote the expression of one or more elements of the CRISPR system may be introduced into cells so that the expression of elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Components may also be delivered to cells as proteins and / or RNA. For example, the Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence may each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined within a single vector, and one or more additional vectors may bring any components of the CRISPR system not contained in the first vector. The vector may contain one or more insertion sites (also called "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements in one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to direct CRISPR activity to multiple different corresponding target sequences within the cell.

[0107] The vector may include a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-exclusive examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, their homologs, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0108] The CRISPR enzyme may be Cas9 (e.g., derived from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct the cleavage of one or both strands at a location within the target sequence, e.g., within the target sequence and / or within the complementary sequence of the target sequence. The vector may encode a modified CRISPR enzyme relative to the corresponding wild-type enzyme, such that the modified CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from S. pyogenes (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). In some embodiments, the Cas9 nickase may be used in combination with one or more guide sequences, such as two guide sequences, each targeting the sense and antisense strands of a DNA target. This combination allows for the introduction of nicks into both strands, which can then be used to introduce an NHEJ or HDR.

[0109] In some embodiments, the enzyme coding sequences encoding CRISPR enzymes are codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells may be cells of specific organisms, such as mammals, or cells derived from such organisms. Mammals include, but are not limited to, humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying nucleic acid sequences by replacing at least one codon in a native sequence with a codon that is more frequently or most frequently used in the gene of the target host cell, while maintaining its native amino acid sequence, in order to increase expression in the target host cell. In various forms, a specific bias is observed for certain codons of certain amino acids. Codon bias (differences in codon usage frequency between organisms) is often related to the translation efficiency of messenger RNA (mRNA), which is also thought to depend, among other things, on the properties of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of selected tRNAs in a cell generally reflects the codons that are most frequently used in peptide synthesis. Therefore, based on codon optimization, genes can be tuned for optimal gene expression in a given organism.

[0110] Generally, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence, hybridizes with the target sequence, and directs the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99%, or greater than or equal to.

[0111] The optimal alignment can be determined using any suitable algorithm for aligning sequences, and such algorithms include, but are not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0112] A CRISPR enzyme may be part of a fusion protein containing one or more heterogeneous protein domains. A CRISPR enzyme fusion protein may also contain any additional protein sequences and, optionally, linker sequences between any two domains. Examples of protein domains that can fusion to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-exclusive examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, and autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes may be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other molecules (including, but not limited to, maltose-binding protein (MBP), S-tags, Lex A DNA-binding domain (DBD) fusions, GAL4A DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions). Further domains that may form part of a fusion protein containing a CRISPR enzyme are described in US20110059502 (incorporated herein by reference). V. Kits and Diagnostics

[0113] In various embodiments of the present invention, a kit is intended that comprises components necessary for purifying exosomes from body fluids or tissue culture media. In other embodiments, a kit is intended that comprises components necessary for isolating exosomes and transfecting them with therapeutic nucleic acids, therapeutic proteins, or inhibitory RNA. The kit may comprise one or more sealed vials containing any of these components. In some embodiments, the kit may also comprise suitable container means (e.g., Eppendorf tubes, assay plates, syringes, bottles, or tubing) that do not react with the components of the kit. The containers may be made of sterilizable material such as plastic or glass. The kit may further comprise instructions outlining the steps of the procedure of the method described herein, which are substantially the same as those described herein or are known to those skilled in the art. The information in the instructions may be in a computer-readable medium, including machine-readable instructions that, when performed using a computer, display the actual or virtual procedure for purifying exosomes from a sample and transfecting the exosomes with therapeutic cargo. VI. Examples

[0114] The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in carrying out the present invention and may therefore be considered to constitute preferred modes for carrying out the present invention. However, those skilled in the art should understand that, in consideration of this disclosure, many modifications can be made to the particular embodiments disclosed, and similar or comparable results can still be obtained without departing from the spirit and scope of the present invention. [Examples]

[0115] Distribution of mesenchymal stem cell-derived exosomes in monkeys Three adult male rhesus macaques (weighing 6 kg) were used. One received intravenous administration of PHK-67-labeled exosomes; one received intravenous administration of DiR-labeled exosomes; and one received intraperitoneal administration of DiR-labeled exosomes. Prior to exosome administration, 5 mL of whole blood was collected from each monkey. The exosome administration consisted of 2.5 mL containing 68 billion exosomes, as evaluated by NanoSight post-labeling. The rhesus macaques were euthanized 24 hours after exosome administration. Urine was collected. Blood was collected as follows: 2 × 5 mL whole blood, 2 × 5 mL EDTA, 2 × 5 mL heparin. Organs were collected, treated for formalin fixation, paraffin embedded for HE staining, rapidly frozen in liquid nitrogen, OCT embedded, and slowly cooled on dry ice, or organs were collected and kept fresh for IVIS imaging. Bone marrow was collected from the femur. Imaging results showed that exosomes were localized in the pancreas (Figure 1A-B), liver (Figure 1C-D), and brain (Figure 1E). [Examples]

[0116] tdTomato mRNA delivery using exosomes As a principal demonstration, 293T cells were transfected with either plasmid DNA or RNA encoding tdTomato (Figure 8C). Transfected cells were assayed 24 hours after transfection using FACS (Figure 8A) and immunofluorescence (Figure 8B). Next, 293T cells were treated with exosomes electroporated with tdTomato mRNA and assayed 24 hours later using FACS (Figure 8D). Similarly, 293T cells were transfected with Exofect and exosomes treated with tdTomato mRNA or plasmid DNA. Cells were assayed 24 hours later using FACS for tdTomato expression (Figures 8E and F) and cell viability (Figures 8H and I). Cells were similarly assayed for tdTomato expression using immunofluorescence (Figure 8G). Finally, mRNA delivery by exosomes using Exofect was visualized using U2OS cells (Figure 8J). [Examples]

[0117] Telomerase exosomes for anti-aging therapy As a demonstration of the principal, BJ cells were transfected with in vitro transcribed hTERT mRNA (Figure 2A) using lipofectamine over a 96-hour time course. During this time course, mRNA was isolated from the cells and the level of hTERT mRNA was evaluated by qPCR. The hTERT mRNA level remained relatively constant over 24 hours (Figure 2B). Similarly, during this time course, the protein was isolated and tested for telomerase activity. Relative telomerase activity remained elevated for 24 hours after transfection (Figures 2C and D).

[0118] BJ cells were similarly transfected with modified hTERT mRNA (hTERT modRNA) transcribed in vitro using lipofectamine over a 96-hour time course. During this time course, mRNA was isolated from the cells and hTERT mRNA levels were evaluated by qPCR. hTERT mRNA levels remained relatively constant over 24 hours (Figure 2E). Similarly, during this time course, proteins were isolated and tested for telomerase activity. Relative telomerase activity remained elevated for 24 hours after transfection (Figure 2F). Notably, dominant-negative hTERT modRNA did not increase telomerase activity (Figure 2G).

[0119] The effects of hTERT mRNA and hTERT modRNA on cell viability were tested by transfecting cells for either 24 or 48 hours and assaying the cultures for cell death. It was found that hTERT mRNA induced cell death, but hTERT modRNA did not (Figure 2H).

[0120] The effect of hTERT modRNA on cellular senescence was tested by treating cells with hTERT modRNA four times over three weeks using lipofectamine. Lipofectamine alone was used as a control. Finally, cells were harvested and β-galactosidase expression was evaluated. Treatment with hTERT modRNA reduced the level of cellular senescence, but treatment with dominant-negative hTERT modRNA did not (Figure 2I and J).

[0121] The effect of hTERT modRNA on telomere signaling detected by FISH was investigated by treating cells with lipofectamine four times over a three-week period. Lipofectamine alone was used as a control. Finally, cells were harvested and telomere signaling was evaluated using PNA-FISH. Cells were imaged, and telomere signaling was measured by signal integrated density (SHI). Evaluation was performed using software based on density. Treatment with hTERT modRNA increased the relative frequency of cells showing higher telomere signals (Figure 2K).

[0122] Next, BJ cells were examined using exosomes electroporated with hTERT modRNA. ModRNA expression in exosomes after electroporation (using the primers shown in Figure 3B) was more efficient (Figure 3A). BJ cells were treated with exosomes containing hTERT modRNA at 0 and 48 hours. Cells were harvested at 72 hours and tested for hTERT mRNA and modRNA levels (Figure 4A). Cells were similarly tested for telomerase activity (Figure 4B) and senescence (Figures 4C-E).

[0123] U2OS cells were treated with exosomes transfected with hTERT modRNA using Exofect, and after 24 hours, hTERT mRNA expression (Figure 5A) and telomerase activity (Figure 5B) were tested.

[0124] hTERT was overexpressed in 293T cells (Figure 6A). hTERT-overexpressing 293T cells were found to exhibit higher telomerase activity (Figure 6B) and express more hTERT mRNA (Figure 6C). hTERT was also overexpressed in BJ cells and U2OS cells, which similarly showed higher hTERT mRNA levels (Figure 6D) and protein levels (Figure 6E). Exosomes isolated from hTERT-overexpressing 293T cells were used to treat BJ cells and U2OS cells. Treated U2OS cells showed stronger telomere signaling (Figure 7).

[0125] All methods described and claimed herein can be carried out or performed without excessive experimentation in consideration of this disclosure. Although the compositions and methods of the present invention have been described in preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the methods and to the steps or sets of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, certain chemically and physiologically relevant agents may be substituted for the agents described herein, and it will be apparent that the same or similar results will still be achieved. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references are incorporated herein by reference to the extent that they provide exemplary procedural or other details that supplement what is described herein. U.S. Patent No. 4,870,287 U.S. Patent No. 5,739,169 U.S. Patent No. 5,760,395 U.S. Patent No. 5,801,005 U.S. Patent No. 5,824,311 U.S. Patent No. 5,830,880 U.S. Patent No. 5,846,945

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Claims

[Claim 1] The invention as shown in the drawings.

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