WO2024064748A2

WO2024064748A2 - Compositions and methods related to exosomal delivery of therapeutic agents

Info

Publication number: WO2024064748A2
Application number: PCT/US2023/074685
Authority: WO
Inventors: Ke CHENG; Shuo LIU
Original assignee: North Carolina State University
Priority date: 2022-09-20
Filing date: 2023-09-20
Publication date: 2024-03-28
Also published as: WO2024064748A3

Abstract

The present disclosure provides compositions and methods related to engineered extracellular vesicles (EVs). In particular, the present disclosure provides a novel exosome-specific delivery scaffold for administering therapeutic agents to a subject. The exosome-specific scaffold can be used to generate and deliver EVs containing biologically active cargo (e.g., mRNA, tumor antigens, small molecule drugs) to a subject to treat and/or prevent disease (e.g., viral infection, cancer, etc.).

Description

COMPOSITIONS AND METHODS RELATED TO EXOSOMAL DELIVERY OF THERAPEUTIC AGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/376,346 filed September 20, 2022, which is incorporated herein by reference in its entirety and for all purposes.

FIELD

[0002] The present disclosure provides compositions and methods related to engineered extracellular vesicles (EVs). In particular, the present disclosure provides a novel exosome- specific delivery scaffold for administering therapeutic agents to a subject. The exosome-specific scaffold can be used to generate and deliver EVs containing biologically active cargo (e.g., mRNA, tumor antigens, small molecule drugs) to a subject to treat and/or prevent disease (e.g., viral infection, cancer, etc.).

BACKGROUND

[0003] Exosomes have emerged with promising applications in nanotechnology and nanomedicine. Exosomes are nanosized extracellular vesicles secreted by numerous cell types and found in almost all biological fluids. Initially regarded as cellular debris, exosomes are now understood to have potent roles in autocrine and paracrine signaling. Originating from the endosomal system and shedding from the plasma membrane, exosomes contain unique cocktails of RNA, protein, and lipid cargo with unique parent-cell signatures. Furthermore, exosomes can be derived from various cell types and can be engineered to deliver therapeutic cargo to a particular target tissue or tissue microenvironment. Additionally, exosomes can be synthetically engineered to enhance cellular targeting and therapeutic efficacy. The combination of vesicle derivation and bioengineering allows for a customizable exosomal delivery platform that can be utilized across many diseases.

SUMMARY

[0004] Embodiments of the present disclosure include an engineered extracellular vesicle (EVs) comprising an exosomal delivery scaffold. In accordance with these embodiments, the exosomal delivery scaffold includes an extracellular immune checkpoint inhibitor domain; a transmembrane domain; and at least one intracellular exosomal sorting domain.

[0005] In some embodiments, the EV is derived from a cell.

[0006] In some embodiments, the EV is derived from a cell selected from the group consisting of: mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs).

[0007] In some embodiments, the EV is derived from a cell selected from the group consisting of: HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO-K1 cells, CHO- derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

[0008] In some embodiments, the EV is an exosome. In some embodiments, the EV is from about 1 nm to about 1000 nm in diameter. In some embodiments, the EV comprises an average size from about 100 nm to about 200 nm in diameter.

[0009] In some embodiments, the extracellular immune checkpoint inhibitor domain comprises PD-1, B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H7, CTLA-4, B7-H5, BTLA, LAG-3, LILRB2, TIGIT, TIM-3, CD70, CD40, OX40L, GITRL, and 4-1BBL.

[0010] In some embodiments, the transmembrane domain is derived from an immune checkpoint inhibitor protein selected from the group consisting of PD-1, B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H7, CTLA-4, B7-H5, BTLA, LAG-3, LILRB2, TIGIT, TIM-3, CD70, CD40, OX40L, GITRL, and 4-1 BBL.

[0011] In some embodiments, the at least one intracellular exosomal sorting domain comprises ALIX, CD9, CD81, and Syntenin, or any combination or derivative thereof.

[0012] In some embodiments, the at least one intracellular exosomal sorting domain comprises Alix, or a derivative thereof.

[0013] In some embodiments, the at least one intracellular exosomal sorting domain further comprises a peptide tag selected from the group consisting of a Flag tag, an HA tag, a Myc tag, a V5 tag, a His tag, an EGFP tag, a GFP tag, an mCherry tag, and a DsRed tag.

[0014] In some embodiments, the exosomal delivery scaffold further comprises at least one tumor therapy modulator. In some embodiments, the at least one tumor therapy modulator comprises a Wnt signaling protein or polypeptide. In some embodiments, the at least one tumor therapy modulator comprises Frizzled-8 (FZD8), or a derivative or variant thereof.

[0015] In some embodiments, the extracellular immune checkpoint inhibitor domain, the transmembrane domain, the at least one intracellular exosomal sorting domain, and/or the at least one tumor therapy modulator are expressed as a single polypeptide.

[0016] In some embodiments, the extracellular immune checkpoint inhibitor domain, the transmembrane domain, the at least one intracellular exosomal sorting domain, and/or the at least one tumor therapy modulator are expressed as multiple polypeptides that are functionally coupled. [0017] Embodiments of the present disclosure also include one or more polynucleotides encoding any of the exosomal delivery scaffolds described herein.

[0018] In some embodiments, the cell is selected from the group consisting of: mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs). In some embodiments, the cell is selected from the group consisting of: HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

[0019] Embodiments of the present disclosure also include a composition comprising a plurality of the EVs described herein, and at least one adjuvant, excipient, and/or carrier.

[0020] Embodiments of the present disclosure also include a method of treating a subject having or suspected of having cancer. In accordance with these embodiments, the method includes administering any of the compositions described herein to the subject.

[0021] Embodiments of the present disclosure also include a chimeric biomolecule. In accordance with these embodiments, the chimeric biomolecule includes an ALIX domain, or a derivative or variant thereof, linked to at least one therapeutically active agent. In some embodiments, the presence of the ALIX domain confers exosome-specific localization of the therapeutic agent.

[0022] In some embodiments, the therapeutic agent comprises one or more of a peptide, a polypeptide, a protein, a nucleic acid aptamer, an anti-sense oligonucleotide, an RNA molecule, and/or an RNA binding protein. In some embodiments, the therapeutic agent is localized to the intracellular portion of the chimeric biomolecule. In some embodiments, the therapeutic agent is localized to the extracellular portion of the chimeric biomolecule. In some embodiments, the chimeric biomolecule further comprises a peptide tag.

[0023] Embodiments of the present disclosure also include an exosome comprising any of the chimeric biomolecules described herein. In some embodiments, the exosome is derived from a cell selected from the group consisting of: mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs). In some embodiments, the exosome is derived from a cell selected from the group consisting of: HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

[0024] Embodiments of the present disclosure also include a composition comprising a plurality of the exosomes described herein, and at least one adjuvant, excipient, and/or carrier.

[0025] Embodiments of the present disclosure also include a method of treating a subject in need thereof by administering any of the compositions described herein to the subject.

[0026] Embodiments of the present disclosure also include compositions, methods, and systems for delivering RNA-based therapeutics to exosomes. In some embodiments, the RNA comprises siRNA, microRNA, shRNA, and/or mRNA. In some embodiments, the composition and systems include at least one intracellular exosomal sorting domain coupled to an RNA binding protein. In some embodiments, the at least one intracellular exosomal sorting domain coupled to an RNA binding protein facilitates the delivery of a corresponding RNA molecule to an exosome. In some embodiments, the system comprises at least four distinct vectors: a vector for expressing a modified shRNA, a second vector for expressing an RNA-binding protein, a third for expressing a Vsvg protein, and a fourth for expressing a Dicer-knockout. In some embodiments, this system facilitates the delivery and enrichment of RNA molecules in exosomes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGS 1A-1G: Efficient PD1 protein sorting into exosomes by Alix motif. (A) The schematic illustration of the construction of plasmids for expression of exosome-marker-infused PD1 protein. (B) Detection of PD1 expression in HEK293T cells by western blot. (C) NanoSight characterization of exosomes acquired from indicated cells. (D) Detection of PD1 in exosomes acquired from indicated cells. (E) ELISA detection of PD1 in indicated exosomes.

[0028] FIGS. 2A-2C: External display of PD1 protein on the surface of exosomes. (A) His- tag-based PD-L1 pulldown assay to detect the distribution of PD1 in exosomes. (B) PD1 exosomes block PD-L1 binding to PD1 recombinant protein. (C) Confocal imaging detection of PD1 exosomes binding with PD-L1 in tumor cells.

[0029] FIGS. 3A-3G: PD1 exosomes for tumor immunotherapy. (A) The study design of PD1 exosomes for tumor immunotherapy. (B) Tumor growth curve at indicated time points. (C) Tumors of indicated groups at the therapeutic endpoint. (D) Tumor weight at the endpoint. (E) Flow cytometric analysis of CD8⁺/CD4⁺ T cells in the tumor-draining lymph node. (F) Flow cytometric analysis of CD8⁺/Tregs in the tumor-draining lymph node. (G) Detection of IFNy⁺CD8⁺ cytotoxic T cells in the tumor-draining lymph node.

[0030] FIGS. 4A-4D: Characterization of exosomes. (A) Nanosight analysis of the size distribution of exosomes. (B-D) Detection of the expression levels of various commonly used exosome markers (CD63, CD81, CD9, Alix, TsglOl and Syn) in exosomes secreted by WT HEK293T cells by flow cytometry (B, C), and quantitative data (D). These results showed that the size of exosomes is around 180nm, and Alix is the most abundant marker expressed in the HEK293T exosomes, followed by CD9 and CD81.

[0031] FIG. 5: Sanger sequencing results confirmed success in the plasmid constructs.

[0032] FIGS. 6A-6C: Immunofluorescent staining of stable cells to show the expression of PD 1. (A) Immunofluorescence staining of Flag tag confirmed the establishment of stable cell lines. (B and C) Immunostaining confirmed the colocalization of PD 1 with Alix and CD63 in a vesiclelike aggregation pattern. These results confirmed the success in establishing stable cell lines, and fusing PD1 with Alix sorting domain promoted PD1 enrichment in the exosomes.

[0033] FIGS. 7A-7H: Displaying PD1, FZD8 and FZD8+PD1 to exosome surface. (A) Schematic illustration of protein display by using Alix scaffold. (B) Engineered plasmid showing the assembly of target protein gene sequence. (C) Western blot analysis of PD1, FZD8 and FZD8+PD1 expression in HEK293 cells. (D) Western blot analysis of PD1 , FZD8 and FZD8+PD1 expression in engineered exosomes. (E-F) ELISA assay to detect PD1 and FZD8 in exosomes. (G- H) His-tag pulldown assays to demonstrate the binding ability of exosome displayed PD1 and FZD8 to their corresponding ligands. [0034] FIG. 8: Display of PD1 to the surface of engineered exosomes. Representative cryoelectron microscopy images of the WT-exo and PDl-exo. The arrow indicated the protein corona formed surrounding the exosomes membrane after surface display (right panel). Scale bar, 50 nm. [0035] FIGS. 9A-9C: Tandem FZD8+PD1 displaying exosomes counteract tumor immunotherapy resistance. (A) Experimental design outlining the tumor modeling and administration of exosomes for immunotherapy. (B) Ex vivo IVIS imaging of tumor-afflicted lungs of the indicated groups at the therapeutic endpoint, and accordingly, the luminescent signals were acquired for quantitative analysis of the tumor size (C).

[0036] FIGS. 10A-10F: Enrich RNA therapeutics to Exosomes by using Alix motif. To augment siRNA delivery to exosomes, a specific RNA binding protein was introduced and fused with Alix (A). Post-transfection with this system, HEK293 cells will release exosomes that are rich with targeted siRNA (B). Four distinct loop modifications of the shRNA were tested: CDbox, Bbox, MS2, and PP7 (C). While the knockdown ability of shRNA remained unaffected by CDbox, Bbox, and MS2 modifications (D), cells transfected with PP7-modified shRNA did not match the efficiency of the standard control. The corresponding RNA binding proteins (L7Ae for CDbox, N22 for Bbox, and MCP for MS2), when fused with Alix (E), were co-transfected to assess any potential interference in the knockdown ability due to shRNA-protein binding. These results showed that both L7Ae+CDbox and MS2+MCP pairings markedly down-regulated GAPDH expression, matching the efficacy of shRNA alone (F).

DETAILED DESCRIPTION

[0037] The present disclosure provides compositions and methods related to engineered extracellular vesicles (EVs). In particular, the present disclosure provides a novel exosome- specific delivery scaffold for delivering therapeutic agents to exosomes and their use in treating disease. The exosome-specific scaffold can be used to generate therapeutic EVs with biologically active cargo (e.g., mRNA, tumor antigens, small molecule drugs), and deliver them to a subject to treat and/or prevent disease (e.g., viral infection, cancer, etc.).

[0038] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. 1. Definitions

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0040] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. [0041] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0042] “Correlated to” as used herein refers to compared to.

[0043] The terms “administration of’ and “administering” a composition as used herein refers to providing a composition of the present disclosure to a subject in need of treatment (e.g., antiviral treatment). The compositions of the present disclosure may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, nebulization, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.

[0044] The term “composition” as used herein refers to a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such a term in relation to a pharmaceutical composition is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure and a pharmaceutically acceptable carrier and/or excipient. When a compound of the present disclosure is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of the present disclosure is contemplated. Accordingly, the pharmaceutical compositions of the present disclosure include those that also contain one or more other active ingredients, in addition to a compound of the present disclosure. The weight ratio of the compound of the present disclosure to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Combinations of a compound of the present disclosure and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used. In such combinations the compound of the present disclosure and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).

[0045] The term “pharmaceutical composition” as used herein refers to a composition that can be administered to a subject to treat or prevent a disease or pathological condition in the patient (e.g., viral infection). The compositions can be formulated according to known methods for preparing pharmaceutically useful compositions. Furthermore, as used herein, the phrase “pharmaceutically acceptable carrier” means any of the standard pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations containing pharmaceutically acceptable carriers are described in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W, Remington's Pharmaceutical Sciences, Easton Pa., Mack Publishing Company, 19.sup.th ed., 1995) describes formulations that can be used in connection with the subject invention.

[0046] Formulations suitable for nebulizing administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question.

[0047] The term “pharmaceutically acceptable carrier, excipient, or vehicle” as used herein refers to a medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. A carrier, excipient, or vehicle includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials such as absorbents that may be needed in order to prepare a particular composition. Examples of carriers etc. include but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The use of such media and agents for an active substance is well known in the art.

[0048] The term “culturing” as used herein refers to growing cells or tissue under controlled conditions suitable for survival, generally outside the body (e.g., ex vivo or in vitro). The term includes “expanding,” “passaging,” “maintaining,” etc. when referring to cell culture of the process of culturing. Culturing cells can result in cell growth, differentiation, and/or division.

[0049] The term “derived from” as used herein refers to cells or a biological sample (e.g., blood, tissue, bodily fluids, etc.) and indicates that the cells or the biological sample were obtained from the stated source at some point in time. For example, a cell derived from an individual can represent a primary cell obtained directly from the individual (e.g., unmodified). In some instances, a cell derived from a given source undergoes one or more rounds of cell division and/or cell differentiation such that the original cell no longer exists, but the continuing cell (e.g., daughter cells from all generations) will be understood to be derived from the same source. The term includes directly obtained from, isolated and cultured, or obtained, frozen, and thawed. The term “derived from” may also refer to a component or fragment of a cell obtained from a tissue or cell, including, but not limited to, a protein, a nucleic acid, a membrane or fragment of a membrane, and the like.

[0050] The term “exosomes” as used herein refers to small secreted vesicles (typically about 30 nm to about 250 nm (or largest dimension where the particle is not spheroid)) that may contain, or have present in their membrane or contained within their membrane, nucleic acid(s), protein, small molecule therapeutics, or other biomolecules and may serve as carriers of this cargo between diverse locations in a body or biological system. The term “exosomes” as used herein advantageously refers to extracellular vesicles that can have therapeutic properties, including, but not limited to LSC exosomes.

[0051 ] Exosomes may be isolated from a variety of biological sources including mammals such as mice, rats, guinea pigs, rabbits, dogs, cats, bovine, horses, goats, sheep, primates or humans. Exosomes can be isolated from biological fluids such as serum, plasma, whole blood, urine, saliva, breast milk, tears, sweat, joint fluid, cerebrospinal fluid, semen, vaginal fluid, ascetic fluid and amniotic fluid. Exosomes may also be isolated from experimental samples such as media taken from cultured cells (“conditioned media,” cell media, and cell culture media). Exosomes may also be isolated from tissue samples such as surgical samples, biopsy samples, and cultured cells. When isolating exosomes from tissue sources it may be necessary to homogenize the tissue in order to obtain a single cell suspension followed by lysis of the cells to release the exosomes. When isolating exosomes from tissue samples it is important to select homogenization and lysis procedures that do not result in disruption of the exosomes. Exosomes may be isolated from freshly collected samples or from samples that have been stored frozen or refrigerated. Although not necessary, higher purity exosomes may be obtained if fluid samples are clarified before precipitation with a volume-excluding polymer, to remove any debris from the sample. Methods of clarification include centrifugation, ultracentrifugation, filtration or ultrafiltration. [0052] The genetic information within the extracellular vesicle such as an exosome may easily be transmitted by fusing to the membranes of recipient cells, and releasing the genetic information into the cell intracellularly. Though exosomes as a general class of compounds represent great therapeutic potential, the general population of exosomes are a combination of several class of nucleic acids and proteins which have a constellation of biologic effects both advantageous and deleterious.

[0053] The term “vesicle” or “extracellular vesicle” as used herein can refer to a vesicle secreted by cells or derived from cells (e.g., via extrusion process) that may have a larger diameter than that referred to as an “exosome.” Vesicles and nanovesicles (alternatively named “microvesicle” or “membrane vesicle”) may have a diameter (or largest dimension where the particle is not spheroid) of between about 10 nm to about 5000 nm (e.g., between about 50 nm and 1500 nm, between about 75 nm and 1500 nm, between about 75 nm and 1250 nm, between about 50 nm and 1250 nm, between about 30 nm and 1000 nm, between about 50 nm and 1000 nm, between about 100 nm and 1000 nm, between about 50 nm and 750 nm, etc.). Typically, at least part of the membrane of the extracellular vesicle is directly obtained from a cell (also known as a donor cell).

[0054] The term “isolating” or “isolated” when referring to a cell or a molecule (e.g., nucleic acids or protein) indicates that the cell or molecule is or has been separated from its natural, original or previous environment. For example, an isolated cell can be removed from a tissue derived from its host individual, but can exist in the presence of other cells (e.g., in culture), or be reintroduced into its host individual.

[0055] As used herein, the term “subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (e.g., a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, macaque, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In one embodiment, the subject is a human. The subject or patient may be undergoing various forms of treatment.

[0056] As used herein, the term “treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and/or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease (e.g., viral infection). A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease.

[0057] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

2. Engineered Extracellular Vesicles and Related Compositions

[0058] Embodiments of the present disclosure include an engineered extracellular vesicle (EVs), or a plurality of EVs, comprising an exosomal delivery scaffold. In accordance with these embodiments, the exosomal delivery scaffold includes an extracellular immune checkpoint inhibitor domain, a transmembrane domain, and at least one intracellular exosomal sorting domain. [0059] In some embodiments, the exosomal delivery scaffold comprises an extracellular immune checkpoint inhibitor domain. In some embodiments, the immune checkpoint inhibitor domain includes, but is not limited to, PD-1, B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H7, CTLA-4, B7-H5, BTLA, LAG-3, LILRB2, TIGIT, TIM-3, CD70, CD40, OX40L, GITRL, and 4- 1BBL, and any combinations thereof. As would be appreciated by one of ordinary skill in the art based on the present disclosure, embodiments of the engineered EVs disclosed herein can be developed using any other immune checkpoint inhibitor domain currently available or identified at some point in the future.

[0060] In some embodiments, the exosomal delivery scaffold includes a transmembrane domain. In some embodiments, the transmembrane domain is derived from an immune checkpoint inhibitor protein. In some embodiments, the transmembrane domain is selected from an immune checkpoint inhibitor including, but not limited to, PD-1, B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H7, CTLA-4, B7-H5, BTLA, LAG-3, LILRB2, TIGIT, TIM-3, CD70, CD40, OX40L, GITRL, and 4-1 BBL. In other embodiments, the transmembrane domain is selected from a protein that is not an immune checkpoint inhibitor. As would be appreciated by one of ordinary skill in the art based on the present disclosure, embodiments of the engineered EVs disclosed herein can be developed using any other transmembrane domain currently available or identified at some point in the future.

[0061] In some embodiments, the exosomal delivery scaffold includes at least one exosomal sorting domain. In some embodiments, the exosomal sorting domain is an intracellular exosomal sorting domain. In some embodiments, the at least one intracellular exosomal sorting domain comprises ALIX, CD9, CD81, and Syntenin, or any combination or derivative thereof. In some embodiments, the at least one intracellular exosomal sorting domain is an ALIX domain. In accordance with these embodiments, and the Examples described herein, the presence of an exosomal sorting domain (e.g., an ALIX domain) confers exosome-specific localization of a therapeutic agent. In some embodiments, the at least one intracellular exosomal sorting domain additionally comprises a peptide tag. In some embodiments, the peptide tag facilitates isolation, purification, and/or visualization of the exosomal delivery scaffold or EVs comprising the exosomal delivery scaffold. In some embodiments, the peptide tag includes, but is not limited to a Flag tag, an HA tag, a Myc tag, a V5 tag, a His tag, an EGFP tag, a GFP tag, an mCherry tag, and a DsRed tag.

[0062] In some embodiments, the exosomal delivery scaffold further comprises at least one tumor therapy modulator. In some embodiments, the at least one tumor therapy modulator comprises a Wnt signaling protein or polypeptide. In some embodiments, the at least one tumor therapy modulator comprises Frizzled-8 (FZD8), or a derivative or variant thereof.

[0063] In some embodiments, the extracellular immune checkpoint inhibitor domain, the transmembrane domain, the at least one intracellular exosomal sorting domain, and/or the at least one tumor therapy modulator are expressed as a single polypeptide. In some embodiments, the extracellular immune checkpoint inhibitor domain, the transmembrane domain, the at least one intracellular exosomal sorting domain, and/or the at least one tumor therapy modulator are expressed as multiple polypeptides that are functionally coupled. [0064] In some embodiments, the EV is derived from a cell. In some embodiments, the EV is derived from a cell that includes, but is not limited to, mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs). In some embodiments, the EV is derived from a cell that includes, but it not limited to, HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells. In some embodiments, the plurality of EVs comprise liposomes. In some embodiments, the plurality of EVs comprise exosomes.

[0065] As would be understood by one of ordinary skill in the art based on the present disclosure, the term “vesicle” or “extracellular vesicle” generally refers to a vesicle secreted by cells or derived from cells (e.g., via extrusion process) that may have a larger diameter than that referred to as an “exosome.” Vesicles and nanovesicles (alternatively named “microvesicle” or “membrane vesicle”) may have a diameter (or largest dimension where the particle is not spheroid) of between about 10 nm to about 5000 nm (e.g., between about 50 nm and 1500 nm, between about 75 nm and 1500 nm, between about 75 nm and 1250 nm, between about 50 nm and 1250 nm, between about 30 nm and 1000 nm, between about 50 nm and 1000 nm, between about 100 nm and 1000 nm, between about 50 nm and 750 nm, etc.). Typically, at least part of the membrane of the extracellular vesicle is directly obtained from a cell (also known as a donor cell).

[0066] In some embodiments, the EV is an exosome. In some embodiments, the EV is from about 1 nm to about 1000 nm in diameter. In some embodiments, the EV is from about 10 nm to about 1000 nm in diameter. In some embodiments, the EV is from about 100 nm to about 1000 nm in diameter. In some embodiments, the EV is from about 250 nm to about 1000 nm in diameter. In some embodiments, the EV is from about 500 nm to about 1000 nm in diameter. In some embodiments, the EV is from about 750 nm to about 1000 nm in diameter. In some embodiments, the EV is from about 1 nm to about 750 nm in diameter. In some embodiments, the EV is from about 1 nm to about 500 nm in diameter. In some embodiments, the EV is from about 1 nm to about 250 nm in diameter. In some embodiments, the EV is from about 1 nm to about 100 nm in diameter. In some embodiments, the EV is from about 1 nm to about 10 nm in diameter. In some embodiments, the EV is from about 100 nm to about 750 nm in diameter. In some embodiments, the EV is from about 250 nm to about 500 nm in diameter.

[0067] In some embodiments, the EV comprises an average size from about 100 nm to about 200 nm in diameter. In some embodiments, the EV comprises an average size from about 125 nm to about 200 nm in diameter. In some embodiments, the EV comprises an average size from about 150 nm to about 200 nm in diameter. In some embodiments, the EV comprises an average size from about 175 nm to about 200 nm in diameter. In some embodiments, the EV comprises an average size from about 100 nm to about 175 nm in diameter. In some embodiments, the EV comprises an average size from about 100 nm to about 150 nm in diameter. In some embodiments, the EV comprises an average size from about 100 nm to about 125 nm in diameter. In some embodiments, the EV comprises an average size from about 125 nm to about 175 nm in diameter. [0068] Embodiments of the present disclosure also include one or more polynucleotides encoding any of the exosomal delivery scaffolds described herein. The polynucleotide can include any domains typically included in expression vectors or plasmids that are used to store and/or express a protein or polypeptide of interest. The polynucleotide encoding any of the immune checkpoint inhibitors described herein can be transformed or transfected into any cell type, including but not limited to, mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs). In some embodiments, the cell includes, but is not limited to, HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO- K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

[0069] Embodiments of the present disclosure also include a chimeric biomolecule. In accordance with these embodiments, the chimeric biomolecule includes an ALIX domain, or a derivative or variant thereof, linked to at least one therapeutically active agent. In some embodiments, and as described further herein, the presence of the ALIX domain confers exosome- specific localization of the therapeutic agent.

[0070] In some embodiments, the therapeutic agent comprises one or more of a peptide, a polypeptide, a protein, a nucleic acid aptamer, an anti-sense oligonucleotide, an RNA molecule, and/or an RNA binding protein. In some embodiments, the therapeutic agent is localized to the intracellular portion of the chimeric biomolecule. In some embodiments, the therapeutic agent is localized to the extracellular portion of the chimeric biomolecule. In some embodiments, the chimeric biomolecule further comprises a peptide tag.

[0071] Embodiments of the present disclosure also include an exosome comprising any of the chimeric biomolecules described herein. In some embodiments, the exosome is derived from a cell that includes, but is not limited to, mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs). In some embodiments, the exosome is derived from a cell that includes, but is not limited to, HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

[0072] Embodiments of the present disclosure also include compositions, methods, and systems for delivering RNA-based therapeutics to exosomes. In some embodiments, the RNA comprises siRNA, microRNA, shRNA, and/or mRNA. In some embodiments, the composition and systems include at least one intracellular exosomal sorting domain coupled to an RNA binding protein. In some embodiments, the at least one intracellular exosomal sorting domain coupled to an RNA binding protein facilitates the delivery of a corresponding RNA molecule to an exosome. In some embodiments, the system comprises at least four distinct vectors: a vector for expressing a modified shRNA, a second vector for expressing an RNA-binding protein, a third for expressing a Vsvg protein, and a fourth for expressing a Dicer-knockout. In some embodiments, this system facilitates the delivery and enrichment of RNA molecules in exosomes.

3. Therapeutic Methods

[0073] Embodiments of the present disclosure also include a method of treating a subject by administering any of the compositions comprising the engineered EVs or exosomes described herein to the subject. Embodiments of the present disclosure also include a composition comprising a plurality of the engineered EVs or exosomes described herein, and at least one adjuvant, excipient, and/or carrier. In some embodiments, the composition is administered orally, parenterally, intramuscularly, intraperitoneally, intravenously, intracerebroventricularly, intracistemally, subcutaneously, via injection or infusion, via inhalation, spray, nasal, vaginal, rectal, sublingual, or topical administration. In some embodiments, the composition treats a disease or condition in the subject (e.g., cancer).

[0074] In some embodiments, administration of compositions comprising the plurality of EVs or exosomes described herein treats one or more symptoms in the subject. As would be recognized by one of ordinary skill in the art based on the present disclosure, pharmaceutical compositions comprising a plurality of EVs or exosomes can be administered in an amount effective such that a desired therapeutic result is achieved (e.g., immunogenic response). In some embodiments, the composition is administered at a dose of about IxlO⁷ to about IxlO¹³ particles per kg of body weight. In some embodiments, the composition is administered at a dose of about IxlO⁸ to about IxlO¹² particles per kg of body weight. In some embodiments, the composition is administered at a dose of about IxlO⁹ to about IxlO¹¹ particles per kg of body weight. In some embodiments, the composition is administered at a dose of about IxlO⁷ particles per kg of body weight, about IxlO⁸ particles per kg of body weight, about IxlO⁹ particles per kg of body weight, about IxlO¹⁰ particles per kg of body weight, about IxlO¹¹ particles per kg of body weight, about IxlO¹² particles per kg of body weight, about IxlO¹³ particles per kg of body weight, about IxlO¹⁴ particles per kg of body weight, or about IxlO¹⁵ particles per kg of body weight.

[0075] In accordance with these embodiments, the plurality of EVs or exosomes of the present disclosure can persist in the subject’s tissues for at least 72 hours after administration. In some embodiments, the plurality of EVs or exosomes persist in a subject for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, and at least 96 hours. In some embodiments, the plurality of EVs or exosomes are administered every 24 hours, every 48 hours, every 72 hours, or every 96 hours, depending on the dose being administered and the subject’s physiological characteristics.

[0076] In some embodiments, a single dose of the plurality of EVs or exosomes of the present disclosure can exert a beneficial effect (e.g., induce an immunogenic response) on a subject. In some embodiments, two or more doses are required to provide a beneficial effect. In some embodiments, three or more doses are required to provide a beneficial effect. In some embodiments, four or more doses are required to provide a beneficial effect. In some embodiments, five or more doses are required to provide a beneficial effect. In some embodiments, six or more doses are required to provide a beneficial effect. In some embodiments, seven or more doses are required to provide a beneficial effect. In some embodiments, eight or more doses are required to provide a beneficial effect. In some embodiments, nine or more doses are required to provide a beneficial effect. In some embodiments, ten or more doses are required to provide a beneficial effect.

[0077] In some embodiments, the present disclosure encompasses methods of treating a pathological condition of a human subject, wherein the method comprises administering to the human subject a pharmaceutical composition comprising a plurality of EVs in an amount effective in modulating a pathological condition when delivered to the human subject in need thereof. In some embodiments, the pathological condition is a viral infection, an immune disorder, and/or cancer. The various compositions of the present disclosure provide dosage forms, formulations, and methods that confer advantages and/or beneficial pharmacokinetic profiles. A composition of the disclosure can be utilized in dosage forms in pure or substantially pure form, in the form of its pharmaceutically acceptable salts, and also in other forms including anhydrous or hydrated forms. A beneficial pharmacokinetic profile may be obtained by administering a formulation or dosage form suitable for once, twice a day, or three times a day, or more administration comprising one or more composition of the disclosure present in an amount sufficient to provide the required concentration or dose of the composition to an environment of use to treat a disease disclosed herein.

[0078] A subject may be treated with a composition of the present disclosure or composition or unit dosage thereof on substantially any desired schedule. They may be administered one or more times per day, in particular 1 or 2 times per day, once per week, once a month or continuously. However, a subject may be treated less frequently, such as every other day or once a week, or more frequently. A composition or composition may be administered to a subject for about or at least about 24 hours, 2 days, 3 days, 1 week, 2 weeks to 4 weeks, 2 weeks to 6 weeks, 2 weeks to 8 weeks, 2 weeks to 10 weeks, 2 weeks to 12 weeks, 2 weeks to 14 weeks, 2 weeks to 16 weeks, 2 weeks to 6 months, 2 weeks to 12 months, 2 weeks to 18 months, 2 weeks to 24 months, or for more than 24 months, periodically or continuously. A beneficial pharmacokinetic profile can be obtained by the administration of a formulation or dosage form suitable for once, twice, or three times a day administration in an amount sufficient to provide a required dose of the composition. Certain dosage forms and formulations may minimize the variation between peak and trough plasma and/or brain levels of compositions of the disclosure and in particular provide a sustained therapeutically effective amount of the compositions. The present disclosure also contemplates a formulation or dosage form comprising amounts of one or more composition of the disclosure that results in therapeutically effective amounts of the composition over a dosing period, in particular a 24 h dosing period. A medicament or treatment of the disclosure may comprise a unit dosage of at least one composition of the disclosure to provide therapeutic effects. A “unit dosage or “dosage unit” refers to a unitary (e.g., a single dose), which is capable of being administered to a subject, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active agents as such or a mixture with one or more solid or liquid pharmaceutical excipients, carriers, or vehicles.

[0079] In some embodiments, the composition further comprises at least one pharmaceutically acceptable excipient or carrier. A pharmaceutically acceptable excipient and/or carrier or diagnostically acceptable excipient and/or carrier includes but is not limited to, sterile distilled water, saline, phosphate buffered solutions, amino acid-based buffers, or bicarbonate buffered solutions. An excipient selected and the amount of excipient used will depend upon the mode of administration. An effective amount for a particular subject/patient may vary depending on factors such as the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. Guidance for methods of treatment and diagnosis is available (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK). For any compositions described herein comprising the EVs, a therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data which are known to exhibit similar pharmacological activities, such as other adjuvants. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered EVs and any corresponding cargo (e.g., vaccine). Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled person in the art.

[0080] Embodiments of the present disclosure also includes methods of generating a plurality of EVs for the treatment and/or prevention of a disease. In accordance with these embodiments, the methods include culturing a plurality of parental cells from which the EVs are derived. Parental cells can be cultured in 2D or 3D cell culture platforms. In some embodiments, the method includes subjecting the plurality of parental cells to an extrusion process to produce the plurality of EVs having the desired characteristics. In some embodiments, the extrusion process comprises passing the parental cells through an extruder comprising at least one of a 5 pm, a 1 pm, and/or a 400 nm pore-sized membrane filters. As would be recognized by one of ordinary skill in the art, other filter sizes and combinations can be used in the extrusion process, depending on the EV size and characteristics desired. In some embodiments, the method further includes purifying and concentrating the plurality of EVs using ultrafiltration or other filtration means known in the art. In some embodiments, the EVs can be selected, sorted, purified, or concentrated based on the use of one or more cell surface proteins.

[0081] In accordance with the above embodiments, the compositions of the present disclosure can be formulated as a pharmaceutically acceptable composition for administering to a subject in need thereof to treat and/or prevent a disease or condition. In some embodiments, the compositions of the present disclosure are stable at room temperature (e.g., 15-25°C). In some embodiments, the compositions of the present disclosure are stable below room temperature. In some embodiments, the compositions of the present disclosure are stable above room temperature. In some embodiments, the compositions of the present disclosure are stable at room temperature for at least 6 hours. In some embodiments, the compositions of the present disclosure are stable at room temperature for up to an including 6 months. In some embodiments, the compositions of the present disclosure are stable at room temperature from about 1 day to about 6 months, from about 1 day to about 5 months, from about 1 day to about 4 months, from about 1 day to about 3 months, from about 1 day to about 2 months, from about 1 day to about 1 month, from about 1 day to about 4 weeks, from about 1 day to about 3 weeks, from about 1 day to about 2 weeks, and from about 1 day to about 1 week.

[0082] In some embodiments, the compositions of the present disclosure can be formulated as a composition that comprises a pharmaceutically acceptable excipient and/or carrier or diagnostically acceptable excipient and/or carrier, including but not limited to, sterile distilled water, saline, phosphate buffered solutions, amino acid-based buffers, or bicarbonate buffered solutions. An excipient selected and the amount of excipient used will depend upon the mode of administration. An effective amount for a particular subject/patient may vary depending on factors such as the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. Guidance for methods of treatment and diagnosis is available (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch PubL, London, UK). For any compositions described herein comprising the EVs, a therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data which are known to exhibit similar pharmacological activities, such as other adjuvants. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered EVs and any corresponding cargo. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled person in the art.

[0083] The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

[0084] The pharmaceutically acceptable compositions described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, inhalable, topical, injectable, immediate -release, pulsatile-release, delayed-release, or sustained release). The pharmaceutically acceptable compositions may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

4. Materials and Methods

[0085] Cell culture. Human embryonic kidney 293 cells (HEK293T cells) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Coming Incorporated, Coming, NY, USA), 1% sodium pymvate (ThermoFisher Scientific, Waltham, MA, USA), 0.5% Gentamicin (ThermoFisher Scientific, Waltham, MA, USA). Fresh medium was changed for cell culture every other day. The cells were allowed to reach 70-80% confluence before collecting conditioned medium.

[0086] Generation of stable cells by lentiviral infection. The medium was changed at 24 h after transfection and supernatant collected after an additional 48 h. The centrifuged supernatants were filtered using 0.45 pm filters. Adherent cells (30% confluency) were cultured in lentivims- containing medium supplemented with polybrene (8 pg/ml; EMD Millipore, Burlington, MA, USA) for 24 h, and then the cells subjected to puromycin (2 pg/ml; InvivoGen, San Diego, CA, USA) incubation for 72 h to select stable expressing cells.

[0087] Exosome isolation and characterization. HEK293T conditioned medium was collected 48h after changing to serum-free culture. The conditioned medium was first passing through a 0.22 pm filter to remove cell debris, followed by exosomes isolation using an ultrafiltration method. Filtered conditioned medium was pipetted into a lOOkDa Amicon centrifugal filter unit (MilliporeSigma, Burlington, MA, USA), followed by centrifugation at 4°C, 4000 rpm for 30 min. After all media passed through the centrifugal filter unit, the remaining exosomes were resuspended using 1 xDulbecco’s phosphate -buffered saline (DPBS; ThermoFisher Scientific, Waltham, MA, USA) for further analysis. Exosome concentrations and particle size distribution were characterized by nanoparticle tracking analysis (NanoSight NS3000, Malvern Panalytical, Malvern, UK).

[0088] Immunoblotting. Cells were lysed in RIPA buffer (for H2AX-S139p, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors aprotinin (4 mg/ml) and PMSF (1 mM), and phosphatase inhibitors (ThermoFisher, Cat# 88667). Cell lysates were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA and subjected to SDS-PAGE separation. Protein samples were then transferred onto polyvinylidene fluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA) using the BioRad wet electroblotting transfer system (Bio-Rad, Hercules, CA, USA). Then the membranes were blocked using non-fat milk (5% w/v in PBST) for one hour at room temperature. Primary antibodies, anti-p-Actin (ab6276, Abeam), anti-Flag (#14793, Cell signaling technology), anti- TSG101 (MAI-23296, ThermoFisher) diluted in 5% milk were added to incubate membranes at 4°C overnight. After primary antibody incubation and three washes with PBST, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit (ab6721, Abeam) or goat anti-mouse (ab6789, Abeam) secondary antibodies for 1 hour at room temperature. Then the protein bands can be visualized using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and imaged in a Bio-Rad Imager (Bio-Rad, Hercules, CA, USA).

[0089] Immunofluorescence. Cells were seeded on coverslips and fixed with cold methanol for 15 min at -20°C. Then the cells were washed 3 times with PBST, followed by blocking with 5% normal goat serum diluted by PBS. Then the cells were incubated with anti-Flag (#14793, Cell signaling technology), anti-PD-Ll (#13684, Cell signaling technology), anti-Alix (MAI-83977, ThermoFisher) and anti-CD63 (MAI-83977, ThermoFisher) primary antibodies diluted in 5% normal goat serum at 4°C overnight. The cells were then washed with PBST for 3 times, and subsequently were incubated with diluted fluorescent-labeled secondary antibodies at room temperature for 1 hour. After washing and staining the nuclei with DAPI, the slides were sealed with a quenching-preventive mounting medium. Images were recorded by a confocal microscope (Olympus, FV-3000).

[0090] ELISA assay. PD1 Elisa assay kit was purchased from Abeam (ab210971) and performed according to the manufacturer’s instructions. 5 * 10⁶ exosomes in 50pl PBS were added to the antibody-coated wells. 50 pl antibody cocktails were added to each well followed by 1 hour incubation at room temperature. After 3 washes, 100 pl TMB development solution was added to each well and incubated for 10 minutes in the dark. Then 100 pl stop solution was added and OD450 was recorded.

[0091] Immunoprecipitation assay. PD-L 1 -His protein was incubated with anti-His magnetic beads (10103D, ThermoFisher) overnight at 4 °C. Beads were washed 5 times with lx PBS buffer containing 0.005% NP-40 (PBS-NP-40) and incubated with WT Exo or PD 1 Exo dissolved in PBS for 4 h at 4 °C, followed by washing 5 times with 1 ml PBS buffer. Beads with proteins were eluted in lx SDS sample buffer and subjected to Western blot assay.

[0092] Flow cytometry. Cells were washed in pre-cooling PBS once and suspended in 100 ml cell staining buffer (CSB; BioLegend). The cells were then incubated with fluorescence- conjugated antibodies ice for 30 min followed by washing with CSB. Afterwards, the cells were incubated with 1ml Fixation/Permeabilization working solution (00-5523-00, ThermoFisher) for 30min at 4°C. After 3 times wash with Permeabilization Buffer (00-5523-00, ThermoFisher), cells were incubated with antibodies diluted in Permeabilization Buffer for 30min at room temperature. Cells were washed with Permeabilization Buffer for 3 times and resuspended in CSB. Data were acquired using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA).

[0093] PD-1 -Fc binding assay. Adherent tumor cells (2 x 10⁵) were suspended in 50 pl CSB following trypsin digestion and FBS neutralization. Cells were then cultured with WT Exo or PD1 Exo in 100 pl volume for 1 h at 4 °C. Then, 10 ng recombinant mouse (rm)-PD-l-Fc protein (1021- PD-100, R&D) was added and incubated for 1 h, followed by incubating with anti-human IgG-Fc- Alexa FluroTM 488 antibody for 20 min on ice in the darkness. Cells were washed, and analyzed using a BD LSRII flow cytometer.

[0094] Animal studies. Animals were purchased from Charles River Laboratory (Wilmington, MA, USA), and all experimental procedures were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of North Carolina State University. The experiment design is briefly described in the results and FIG. 3A. Tumor cells (IxlO⁵ cells) were subcutaneously injected into C57BL/6 (6-week-old females) mice (one tumor per mouse). Tumor bearing mice were randomized into treatment groups when tumors reached approximately 100 mm³. Tumor volumes were calculated according to the following formulas: cm³=[(length,cm)x(width,cm)²]/2.

[0095] Exosomes were administered to mice every three days by intratumor injection for a total of 6 injections (2xlO¹⁰ exosomes/mouse/treatment in 20 pl PBS). Equimolar anti-mPD-Ll antibody was given as control. On day 19, mice in each group were euthanized and tumors were collected for immunoblots and flow cytometry analysis. No animals were excluded from the analysis in this study. 5. Examples

[0096] ft will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0097] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

Example 1

[0098] Efficient PD1 sorting into exosomes by infusing Alix. To develop an effective scaffolding protein that can enrich and display PD1 in the exosomes and enable the therapeutic applications of engineered exosomes for cancer, a series of mammalian cell expression plasmids was first constructed, which linked the extracellular and transmembrane domains of PD 1 to the sorting domains of common exosome markers at the C-terminus (FIG. 1A). In this way, the truncated PD1 should be signaling incompetent. The sorting domain used in the present disclosure is from Alix, CD9, CD81, and Syntenin. In addition, Flag tags were added as an alternative indicator for detection and characterization.

[0099] Then the plasmids were used for lentivirus packaging, followed by lentiviral infection of HEK293T cells. After acquiring stable cell lines, the expression of modified PD1 proteins at the cellular level was analyzed by western blot (FIG. IB). Next, the efficacy of different sorting domains to sort PD1 into the exosomes was compared. Exosomes derived from HEK293T-PD1 cells were prepared and isolated by centrifugation and ultrafiltration. The characterization of exosomes by NanoSight was shown (FIG. 1 C). Western blot detection of PD 1 in exosomes showed that Alix is most efficient at enriching PD1 into exosomes (FIG. ID). This is further supported by ELISA analysis (FIG. E). These results indicated that Alix is the most efficient sorting domain for enriching PD1 into exosomes. CD9 has the moderate capability to sort PD1 into the exosomes, followed by Syntenin and CD81.

Example 2

[0100] External display of PD1 on the surface of exosome membrane. Given the interaction between the receptor (PD1) and the ligand (PD-L1), a his-tag -based PD-L1 pulldown assay was performed to detect the exhibition pattern of PD1 protein in engineered exosomes. Immunoprecipitation- western blot result showed that the Alix fused PD1 exosomes have the strongest binding with PD-L1 -beads complex (Fig. 2A), suggesting the external display of PD1 protein on the surface of the exosome membrane. In addition, CD9 fused PD1 exosomes showed the moderate binding capability to the PD-L1 beads and followed by Syntenin and CD81 (Fig. 2A). This data is consistent with the sorting efficacy shown in Figure 1, further revealing an external display pattern of PD1 protein on the engineered exosomes.

Example 3

[0101] PD1 exosomes block PDl-Fc recombinant protein binding to PD-L1. Experiments were then conducted to investigate the effects of PD1 exosomes on PDl-Fc recombinant protein binding to PD-L1. Co-culture of PDl-Fc with PD-Ll-high expressing tumor cells significantly increased PD1 -Fc-positive cells. Control-PDl exosomes have negligible effects on PDl-Fc-PD- L1 binding (FIG. 2B). However, treatment of Alix-PDl and CD9-PD1 exosomes, profoundly reduced the number of PDl-Fc positive cells, suggesting well-preserved bioreactivity of engineered PD1 exosomes to block the interaction between PD-L1 and PD1.

[0102] Furthermore, confocal imaging was performed to study the consequent outcomes of PD 1 exosome treatment in tumor cells. Co-localization of flag tag with PD-L1 suggested the binding of PD1 exosomes with cellular PD-L1 (FIG. 2C). Following binding, endocytosis of exosomes into the cell, as well as reduced distribution of PD-L1 across the tumor cell membrane was observed (FIG. 2C).

Example 4

[0103] PD1 exosomes for tumor immunotherapy. To validate the anti-tumor ability of PD1 exosomes in vivo, a melanoma-bearing mice model was established (FIG. 3A). After the tumors reached a volume of 100 mm³, intratumoral injections of PD1 exosomes were performed at a dose of 2 X 10¹⁰ exosomes per tumor, and a total of 6 injections were performed. As shown, PDl-Alix exosomes significantly inhibited tumor growth (FIG. 3B). The tumor size and weight at the endpoint showed the most reduction in Alix-PDl exosomes treated mice (FIGS. 3C, 3D). Two tumors were completely regressed after Alix-PD 1 exosomes treatment.

[0104] The T cell subpopulations were then detected in the tumor-draining lymph nodes by flow cytometry. The ratios of CD8⁺/CD4⁺ T cells and CD8⁺/Treg cells were significantly increased in Alix-PDl exosomes treated mice (FIGS. 3E, 3F). Moreover, the number of IFNy⁺CD8⁺ T cells was increased in Alix-PDl treated mice (FIG. 3G). Besides to the most potent effect of Alix-PDl exosomes, CD9-PD1 exosomes also exhibit effectiveness in inhibiting tumor growth (FIGS. 3B- 3G). Taken together, these results suggested the immunotherapeutic potential of PD1 exosomes to induce tumor regression.

Example 5

[0105] Displaying functional proteins (PD1, FZD8 and FZD8+PD1) to the surface of exosomes. Experiments were conducted to test the use of Alix as an intracellular exosomal sorting domain for a therapeutic exosome delivery scaffold to display proteins (PD1, FZD8, FZD8+PD1) to the exosome surface (FIG. 7A). Engineered plasmids carrying the open reading frame (ORF) of Alix and the above genes (FIG. 7B) were used to transfect HEK293 cells, followed by isolation of exosomes from the conditioned medium. Expression of PD1, FZD8 and FZD8+PD1 was characterized in HEK293 cells and exosomes, respectively (FIGS. 7C-7D). The results showed significantly increased levels of the proteins (PD1, FZD8 and FZD8+PD1) (FIGS. 7C-7D). Next, enzyme-linked immunoassays (ELISAs) were performed using unlysed exosomes to detect the surface protein levels. The results further confirmed the increase of PD1 and FZD8 protein levels in engineered exosomes (FIGS. 7E-7F). Notably, the data implied the presence of the proteins (PD1, FZD8 and FZD8+PD1 tandem) to the surface of exosomes. Pulldown assays were also performed to determine the binding affinity of engineered PD1 exosomes, FZD8 exosomes and FZD8+PD1 exosomes to their ligands (FIGS. 7G-7H). Taken together, the above data showed the display of functional proteins to exosome surface.

Example 6

[0106] Display of functional proteins to the surface of exosomes. Experiments were conducted using cryo-electron microscopy to visualize the distribution of proteins displayed on exosomes (FIG. 8). Here, the PD1 exosomes (PDl-exo) were exhibited as a representative. Exosomes without protein display (wild type exosome, WT-exo) exhibit the typical structure of phospholipid bilayer with clear boundary (left panel). Engineered protein-displaying exosomes (PDl-exo, for example) also exhibit a typical phospholipid bilayer (right panel), however, a significant protein corona formation surrounding the exosome membrane was observed after engineered protein displaying (PDl-exo, right). Consistent with the expectations as shown in FIG. 7A, the above data demonstrated the enrichment of displayed proteins to the surface of exosomes.

Example 7

[0107] Therapeutic potential of tandem FZD8+PDl-displaying exosomes. Overexpression of Wnt7b, the ligand of FZD8, and PD-L1, ligand of PD1, in cancer cells leads to resistance to PD1 therapy. To investigate the therapeutic potential of FZD8+PD1 exosomes in suppressing cancer progression, PD1 therapy-resistant, Wnt7b-overexpressing melanoma cell line was used to establish a lung metastasis model. About 10 days after tumor cell implantation, engineered exosomes (WT-exo, PDl-exo, FZD8-exo, and FZD8+PDl-exo) or PBS vehicle were administrated via inhalation (FIG. 9A). Inhalation of FZD8+PD1 exosomes remarkably suppressed tumor growth in the lungs (FIGS. 9B-9C), whereas exosomes displaying solely FZD8 or PD1 showed no significant effects on the growth of PD1 therapy -resistant tumors (FIGS. 9B- 9C). These results suggested the superior therapeutic potentials of tandem FZD8+PD1 -displaying exosomes for PD 1 -therapy resistant cancer.

Example 8

[0108] Enrich RNA therapeutics to Exosomes by using Alix motif. RNA therapeutics can be siRNA, microRNA and mRNA of any sequence. To enhance the delivery of these RNA therapeutics to exosomes, an RNA-binding protein that is conjugated to the Alix motif was designed. Here siRNA was used as a representative to test the feasibility.

[0109] To augment siRNA delivery to exosomes, a specific RNA binding protein was introduced and fused with Alix (A). The RNA enriching system comprises four distinct vectors: first for expressing a modified shRNA, second for expressing the RNA-binding protein, third for expressing the Vsvg protein, and fourth for Dicer-knockout. Vsvg was included to facilitate the escape of the engineered exosome from the endosome, allowing for the release of siRNA cargo into the cytoplasm. Post-transfection with this system, HEK293 cells will release exosomes that are rich with targeted siRNA (B). [0110] Initially, four distinct loop modifications of the shRNA were tested: CDbox, Bbox, MS2, and PP7 (C). While the knockdown ability of shRNA remained unaffected by CDbox, Bbox, and MS2 modifications (D) cells transfected with PP7-modified shRNA did not match the efficiency of the standard control. Hence, experiments proceeded with CDbox, Bbox, and MS2.

[0111] Pursuing this further, the corresponding RNA binding proteins (L7Ae for CDbox, N22 for Bbox, and MCP for MS2), when fused with Alix (E), were co-transfected to assess any potential interference in the knockdown ability due to shRNA-protein binding. These results showed that both L7Ae+CDbox and MS2+MCP pairings markedly down-regulated GAPDH expression, matching the efficacy of shRNA alone (F). These findings indicate that modified shRNA, when bound with RNA binding proteins, do not compromise its knockdown capability. This holds significant promise as a strategy to enrich siRNA within exosomes. Alternatively, the other RNA therapeutics can be modified with the above indicated loops for intra-exosomes sorting.

Claims

CLAIMS What is claimed is:

1. An engineered extracellular vesicle (EV) comprising an exosomal delivery scaffold comprising: an extracellular immune checkpoint inhibitor domain; a transmembrane domain; and at least one intracellular exosomal sorting domain.

2. The engineered EV of claim 1 , wherein the EV is derived from a cell.

3. The composition of claim 2, wherein the EV is derived from a cell selected from the group consisting of: mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs).

4. The composition of claim 2, wherein the EV is derived from a cell selected from the group consisting of: HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

5. The EV of any one of claims 1 to 4, wherein the EV is an exosome.

6. The EV of any one of claims 1 to 5, wherein the EV is from about 1 nm to about 1000 nm in diameter.

7. The EV of any one of claims 1 to 5, wherein the EV comprises an average size from about 100 nm to about 200 nm in diameter.

8. The EV of any one of claims 1 to 7, wherein the extracellular immune checkpoint inhibitor domain comprises PD-1, B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H7, CTLA-4, B7-H5, BTLA, LAG-3, LILRB2, TIGIT, TIM-3, CD70, CD40, OX40L, GITRL, and 4-1BBL.

9. The EV of any one of claims 1 to 8, wherein the transmembrane domain is derived from an immune checkpoint inhibitor protein selected from the group consisting of PD-1, B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H7, CTLA-4, B7-H5, BTLA, LAG-3, LILRB2, TIGIT, TIM-3, CD70, CD40, OX40L, GITRL, and 4-1 BBL.

10. The EV of any one of claims 1 to 9, wherein the at least one intracellular exosomal sorting domain comprises ALIX, CD9, CD81, and Syntenin, or any combination or derivative thereof.

11. The EV of any one of claims 1 to 9, wherein the at least one intracellular exosomal sorting domain comprises Alix, or a derivative thereof.

12. The EV of any one of claims 1 to 11, wherein the at least one intracellular exosomal sorting domain further comprises a peptide tag selected from the group consisting of a Flag tag, an HA tag, a Myc tag, a V5 tag, a His tag, an EGFP tag, a GFP tag, an mCherry tag, and a DsRed tag.

13. The EV of any one of claims 1 to 12, wherein the exosomal delivery scaffold further comprises at least one tumor therapy modulator.

14. The EV of claim 13, wherein the at least one tumor therapy modulator comprises a Wnt signaling protein or polypeptide.

15. The EV of claim 13, wherein the at least one tumor therapy modulator comprises Frizzled-8 (FZD8), or a derivative or variant thereof.

16. The EV of any one of claims 1 to 15, wherein the extracellular immune checkpoint inhibitor domain, the transmembrane domain, the at least one intracellular exosomal sorting domain, and/or the at least one tumor therapy modulator are expressed as a single polypeptide.

17. The EV of any one of claims 1 to 15, wherein the extracellular immune checkpoint inhibitor domain, the transmembrane domain, the at least one intracellular exosomal sorting domain, and/or the at least one tumor therapy modulator are expressed as multiple polypeptides that are functionally coupled.

18. A polynucleotide encoding the exosomal delivery scaffold of any one of claims 1 to 17.

19. A cell comprising the polynucleotide of claim 18, wherein the cell is selected from the group consisting of: mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs).

20. A cell comprising the polynucleotide of claim 18, wherein the cell is selected from the group consisting of: HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

21. A composition comprising a plurality of EVs of any one of claims 1 to 17, and at least one adjuvant, excipient, and/or carrier.

22. A method of treating a subject having or suspected of having cancer, the method comprising administering the composition of claim 21 to the subject.

23. A chimeric biomolecule, wherein the chimeric biomolecule comprises an ALIX domain, or a derivative or variant thereof, linked to at least one therapeutically active agent, wherein the presence of the ALIX domain confers exosome-specific localization of the therapeutic agent.

24. The chimeric biomolecule of claim 23 , wherein the therapeutic agent comprises one or more of a peptide, a polypeptide, a protein, a nucleic acid aptamer, an anti-sense oligonucleotide, an RNA molecule, and/or an RNA binding protein.

25. The chimeric biomolecule of claim 23, wherein the therapeutic agent is localized to the intracellular portion of the chimeric biomolecule.

26. The chimeric biomolecule of claim 23, wherein the therapeutic agent is localized to the extracellular portion of the chimeric biomolecule.

27. The chimeric biomolecule of any one of claims 23 to 26, wherein the chimeric biomolecule further comprises a peptide tag.

28. An exosome comprising the chimeric biomolecule of claim 23, wherein the exosome is derived from a cell selected from the group consisting of: mesenchymal stem cells (MSCs), dendritic cells (DCs), amniotic epithelial cells (AECs), and neural stem cells (NSCs).

29. An exosome comprising the chimeric biomolecule of claim 23, wherein the exosome is derived from a cell selected from the group consisting of: HeLa cells, HEK293 cells, HEK293 derived cells, Vero cells, CHO cells, CH0-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, and RK cells.

30. A composition comprising a plurality of the exosomes of claim 29 or claim 29, and at least one adjuvant, excipient, and/or carrier.

31. A method of treating a subject in need thereof, the method comprising administering the composition of claim 30 to the subject.