Methods and compositions for inducing immune system tolerance

Abstract

This disclosure relates to methods and compositions for the delivery of antigens to host cells for the induction of immune system tolerance. Methods and compositions described herein are useful in the treatment of diseases caused by or relating to immune system dysfunction, including but not limited to autoimmune disorders.

Description

METHODS AND COMPOSITIONS FOR INDUCING IMMUNE SYSTEM TOEERANCEREEATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application U.S.S.N. 63 / 612,539, filed December 20, 2023, the contents of which are incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (H082470453WO00-SEQ-AZW.xml; Size: 52,828 bytes; and Date of Creation: December 12, 2024) are herein incorporated by reference in its entirety.BACKGROUND

[0003] Extracellular vesicle (EV)-based technology has previously been shown to allow membrane proteins to be selectively recruited onto the surface of novel WW domain- activated extracellular vesicles (WAEVs). Methods and compositions comprising WAEVs are described in international patent applications, PCT / US2021 / 055203, filed October 15, 2021; PCT / US2021 / 055158, filed October 15, 2021; and PCT / US2021 / 055154, filed October 15, 2021; which are incorporated herein by reference in their entireties. These previous studies have demonstrated that immunization with viral membrane proteins displayed on WAEVs elicits production of virus-specific neutralizing antibodies and, in the case of influenza antigens, protects mice from the lethal flu infection. See, e.g., PCT / US2021 / 055203, PCT / US2021 / 055158, and PCT / US2021 / 055154

[0004] Autoimmune diseases, such as the non-limiting examples of Myasthenia Gravis, multiple sclerosis, and Type 1 Diabetes are characterized by immune-mediated destruction of the target tissues associated with the respective disease. Common therapies to treat these diseases cause broad immunosuppression with unacceptable safety risks. There remains a need for therapies which selectively suppress autoimmunity.SUMMARY

[0005] Provided is the effective presentation and delivery of non-viral membrane antigens, such as cellular antigens that cause certain autoimmune diseases. Membrane proteins loaded on WAEVs maintain their native-conformations in lipid membranes, thus preserving specificepitope structures that can be recognized by B-cells of the immune system. The present disclosure relates to methods and compositions for the delivery of antigens, such as autoantigens or self-antigens, to target cells for the induction of immune system tolerance, and the methods and compositions described herein are useful in the treatment of diseases caused by or relating to immune system dysfunction, including, but not limited to, autoimmune disorders.

[0006] In one aspect, the present disclosure provides a fusion protein comprising: (a) a WW-containing domain; (b) a transmembrane domain; and (c) an extracellular domain, wherein the extracellular domain is a self-antigen or autoantigen domain. In some embodiments, one or more amino acids from the transmembrane domain may also make up part of the self-antigen domain. In some embodiments, the self-antigen domain is associated with an autoimmune disease. In some embodiments, the autoimmune disease is myasthenia gravis. In some embodiments, the fusion protein does not comprise an arrestin domain containing protein 1 (ARRDC1). In some embodiments, the WW-containing domain comprises at least one WW domain. In some embodiments, the WW-containing domain comprises at least two WW domain. In some embodiments, the WW-containing domain comprises at least three WW domain. In some embodiments, the WW-containing domain comprises at least four WW domain. In some embodiments, the WW-containing domain is contained in the sequence of NEDD4 E3 ligase. In some embodiments, the NEDD4 E3 ligase is selected from the group consisting of ITCH, NEDD4, NEDD4 L, WWP1, WWP2, Smurfl, Smurf2, BUL1, and NEDL2. In some embodiments, the NEDD4 E3 ligase is the ITCH protein. In some embodiments, the WW-containing domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 1. In some embodiments, the WW-containing domain comprises the sequence of SEQ ID NO: 1.

[0007] In one aspect, the present disclosure provides an isolated nucleic acid encoding a fusion protein of any one of the embodiments described herein. In some embodiments, the isolated nucleic acid is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter, an inducible promoter, or a tissue specific promoter. In some embodiments, the isolated nucleic acid comprises at least one additional regulatory sequence. In some embodiments, the present disclosure provides a vector comprising the isolated nucleic acid of any one of the embodiments described herein.

[0008] In one aspect, the present disclosure provides a WW protein domain activated extracellular vesicle (WAEV) comprising: (a) a lipid bilayer; and (b) the fusion protein of any one of the embodiments described herein. In some embodiments, the WAEV further comprises SCAMP3. In some embodiments, the WAEV does not comprise at least one of thefollowing exosomal markers: CD63; CD81, CD9, and PTGFRN. In some embodiments, the WAEV does not comprise any of the following exosomal markers: CD63; CD81, CD9, and PTGFRN. In some embodiments, the WAEV further comprises a tolerogenic substance. In some embodiments, the tolerogenic substance is selected from the listing of Table 1.

[0009] In one aspect, the present disclosure provides a WAEV -producing cell comprising a recombinant expression construct encoding the fusion protein of any one of the embodiments described herein, under the control of a heterologous promoter. In one aspect, the present disclosure provides a WAEV -producing cell comprising the isolated nucleic acid of any one of the embodiments described herein.

[0010] In one aspect, the present disclosure provides a method of delivering WAEVs displaying a self-antigen peptide, the method comprising: delivering the fusion protein of the embodiments described herein, the isolated nucleic acid of any one of the embodiments described herein, the WAEV of any one of the embodiments described herein, or the WAEV- producing cell of any one of the embodiments described herein to a subject, wherein the extracellular protein of the fusion protein comprises a self-antigen. In some embodiments, one or more amino acids from the transmembrane domain may also make up part of the selfantigen. In some embodiments, the subject is mammalian. In some embodiments, the subject is human.[Oil] In some embodiments, the method further comprises simultaneous or subsequent delivery of a tolerogenic substance to the subject. In some embodiments, the WAEV further comprises a tolerogenic substance. In some embodiments, the tolerogenic substance is selected from the listing of Table 1.

[0012] In one aspect, the present disclosure provides a kit comprising one or more of the fusion protein of any one of the embodiments described herein, the isolated nucleic acid of any one of the embodiments described herein, the vector of any one of the embodiments described herein; the WAEV of any one of the embodiments described herein, or the WAEV- producing cell of any one of the embodiments described herein.

[0013] In one aspect, the present disclosure provides the fusion protein of any one of the embodiments described herein, the isolated nucleic acid of any one of the embodiments described herein, the WAEV of any one of the embodiments described herein, the method of any one of the embodiments described herein, or the kit of the embodiments described herein, wherein the self-antigen is selected from (a) a self-antigen associated with the autoantibody- mediated diseases of Table 3.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows a diagram of antigen- specific immune therapy (ASI) for the treatment of autoimmune diseases.

[0015] FIGs. 2A-2E show WAEV structure and function. FIG. 2A shows the WAEV structural components comprised of the extracellular domain (ECD) and transmembrane domain (TM) of the influenza M2 protein fused to the four WW-domains from the ITCH protein. FIG. 2B shows the WAEV budding process from the parental cells transfected with the WAEV construct. FIG. 2C shows extravesicular vesicle (z.e., WAEV) production from HEK293T cells transfected DNA plasmid vectors containing the M2 ECD and TM sequence fused to either the ARRDC1 sequence or to the WW-domain-containing ITCH sequence in which only M2-ITCH (M2-WW) sequence resulted in production of Extracellular vesicles (EVs) termed WAEVs. FIG. 2D shows the quantitation of vesicle numbers and size. FIG. 2E shows the optiprep-based density gradient fractionation and Western Blot analyses of M2- WAEVs.

[0016] FIGs. 3A-3D show M2-WAEV immunization elicits antibody production and protects mice against H1N1 viral infection. FIG. 3A shows the three-shot immunization protocol. CD-I mice (Charles River) were immunized via intraperitoneal (IP) injection with one of the following: PBS with aluminum hydroxide (Alum) as an adjuvant, control EVs (no M2) with Alum, M2-WAEVs with alum, or M2-WAEVs without alum. Sera was collected from mice 3 days after final immunization. One week after the final immunization, all mice were subjected to H1N1 influenza viral infection (strain A / Puerto Rico / 8 / 1934 / HlNl at 800 PFU; given intranasally) and followed by morbidity and mortality measurement for two weeks. FIG. 3B shows the levels of H IN 1 -reactive IgG in serum. Inactivated whole influenza virus (A / Puerto Rico / 8 / 1934 / HlNl) was used to coat 96-well plates. Antibody response was measured using indirect- ELISA using serially diluted serum (1:6250, 1:5000, 1:250, 1:20). FIG. 3C shows the survival rate of immunize mice after influenza infection. Mortality was monitored every day for two weeks after influenza virus infection. FIG. 3D shows HINl-reactive IgG in serum after immunization with or without different adjuvants that were lipid soluble (MPLA and saponin) and aqueous soluble (CpG). * p<0.05, ** p<0.01, *** p<0.001.

[0017] FIG. 4 shows a diagram of autoantibody attachment at the neuromuscular junction which drives myasthenia gravis.

[0018] FIG. 5 shows a chart of the time course of AChR antibody levels in B6 mice. Ten B6 were immunized with AcHR in CFA (50 pg / mouse) and boosted at 4-week intervals (20 pgin IFA [arrows]). Serum antibodies to AChR were measured by kinetic ELISA, and mean values ± SEM at each time point are expressed as units.

[0019] FIG. 6 shows a chart illustrating induction and tol-WAEV-AChR treatment of EAMG in B6 mice. Ten (10) B6 mice per group are immunized with AChR in CFA (50 pg / mouse) and boosted twice at 4-week intervals (20 pg in IFA) and monitored for disease symptoms and bled weekly as indicated (via retro-orbital venipuncture) for serum antibody analyses out to 12 weeks of study.

[0020] FIGs. 7A-7C show characterization of particles. FIG. 7A shows a schematic of nAChR-WAEV. WW domains were fused to the C-terminus of nAChR a-subunit. FIG. 7B shows a western blotting displaying budding of nAChR-WW fusion protein into EVs in HEK293T cells. EVs were isolated by ultracentrifugation from HEK293T cells transfected with pcDNA3.1, M2-WAEV, or nAChR-WAEV construct. Western blotting was done on the EVs along with whole-cell lysates with indicated antibodies. MW, molecular weight. FIG. 7C Shows size distribution and concentration of particles using NanoSight NS300 instrument. Data in the NanoSight particle analysis were obtained from five measurement data. ***p<0.001.DEFINITIONS

[0021] The term “antigen,” as may be used herein refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand and readily appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic nucleic acid. A skilled artisan will understand that any nucleic acid, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated and / or synthesized or can be derived from a biological sample. Such a biological sample can include, but is not necessarily limited to a tissue sample, a tumor sample, a cell, or abiological fluid. In some embodiments, the antigen is a protein, or fragment thereof. In some embodiments, the antigen is a nucleic acid, or fragment thereof.

[0022] The terms “auto-antigen” or “self-antigen” are used herein refers to an antigen derived from the body of an individual. With respect to autoimmune diseases, self-antigens are those cellular proteins and peptides, including those that are post-translationally modified (such as lipoprotein, glycoprotein, and citrullinated proteins or peptides), protein complexes, enzyme complexes, ribonucleoprotein complexes (e.g., proteins complexes with either DNA, and RNA), etc., against which autoantibodies are directed.

[0023] Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated and / or synthesized or can be derived from a biological sample. Such a biological sample can include, but is not necessarily limited to, a tissue sample, a tumor sample, a cell, or a biological fluid. In some embodiments, the antigen is a protein, or fragment thereof. In some embodiments, the antigen is a nucleic acid, or fragment thereof. In some embodiments, the WAEVs of the present disclosure comprise an antigen as an extracellular domain or part of an extracellular domain. In some embodiments, the WAEVs of the present disclosure present an antigen on the membrane of the WAEV. In some embodiments, the fusion proteins of the present disclosure comprise an antigen as an extracellular domain or part of an extracellular domain. In some embodiments, one or more amino acids from the transmembrane domain may also be part of the antigen.

[0024] The term “associated with,” as may be used herein, refers to a property of two or more entities, for example, chemical moieties, molecules (e.g., domains, nucleic acids, peptides), and / or WAEVs, and means that the entities are physically in contact or connected with one another, either directly or via one or more additional moieties that serves as a linker, to form a structure that is sufficiently stable so that the entities remain physically in contact under the conditions in which the structure is used, e.g., physiological conditions. A WAEV can be associated with an agent, for example, a nucleic acid, protein, or small molecule, by a mechanism that involves a covalent or non-covalent association. For example, a WW-domain containing a fusion protein of the present invention can be associated with a protein containing PPXY (SEQ ID NO: 22) motifs, such as a NEDD4 E3 ligase proteins, including, but not limited to, SCAMP3. In certain embodiments, the agent to be delivered (e.g., anextracellular domain cargo protein, which can be or can include an antigen) is covalently bound (e.g. fused) to a transmembrane domain and a WW-containing domain, and this fusion protein can be non-covalently bound to a protein containing a PPXY (SEQ ID NO: 22) motif, including, but not limited to, a SCAMP3 protein or variant thereof. In some embodiments, an association is via a linker, which can be, but is not limited to, a nucleic acid or amino acid linker, for example, a cleavable linker.

[0025] The term “cargo,” as may be used herein, refers to an antigen, protein, or peptide that may be incorporated in a WAEV, for example, as an extracellular domain of the WAEV. The term “delivered” as it relates to cargo refers to any antigen, protein, or peptide that can be delivered via its association with or inclusion in a WAEV to a subject, organ, tissue, or cell. In some embodiments, the cargo is to be delivered to a target cell in vitro, in vivo, or ex vivo. In some embodiments, the cargo to be delivered is an antigen e.g., a self-antigen) that is presented on the surface of a WAEV.

[0026] In general, a “small molecule” refers to a substantially non-peptide, non-oligomeric organic compound either prepared in the laboratory or found in nature. Small molecules, as used herein, can refer to compounds that are “natural product-like,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 2000 g / mol, less than 1500 g / mol, less than 1250 g / mol, less than 1000 g / mol, less than 750 g / mol, less than 500 g / mol, or less than 250 g / mol, although this characterization is not intended to be limiting for the purposes of the present invention. In certain other embodiments, natural-product-like small molecules are utilized.

[0027] The term “effective amount” refers to an amount of a composition or agent (e.g., a WAEV or fusion protein as described herein) sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a WAEV as described herein may refer to the amount of the WAEV as described herein sufficient to elicit an immune reaction to the extracellular domain contained (e.g., presented) therein or thereon (e.g., antigen, or fragment thereof). As will be appreciated by the skilled artisan, the effective amount of a composition (e.g., a WAEV) as described herein may vary depending on various factors as, for example, the desired biological response, the cell or tissue being targeted, and / or the agent being used.

[0028] The terms “extracellular domain” and “exterior domain,” as may be used interchangeably herein, refer to the domain of an antigen, protein, or peptide which is present on the exterior of a membrane of a membrane-containing molecule (e.g., cell, vesicle, EV, and / or WAEV). In some embodiments, the extracellular domain is a domain of a fusionprotein. The extracellular domain may be the terminal domain of a protein. In some embodiments, the extracellular domain is fused to the transmembrane domain at one terminus. In some embodiments, the extracellular domain is fused to the transmembrane domain through its N-terminus (e.g., directly or indirectly). In some embodiments, the extracellular domain is associated with the transmembrane domain through its C terminus (e.g., directly or indirectly). In some embodiments, extracellular domain is linked or fused directly to the transmembrane domain. In some embodiments, the extracellular domain is linked indirectly to the transmembrane domain, for example through a linker. In some embodiments, the extracellular domain is indirectly linked to the transmembrane domain through another protein domain. In some embodiments, the extracellular domain is indirectly linked to the transmembrane domain through a linker.

[0029] In some embodiments, the extracellular domain is positioned such that all of the extracellular domain is exterior of a membrane to which it is associated. It should be noted that while the term “extracellular” can be used in the context of the membrane of a cell, as used herein, the term shall not solely refer to such context, and shall also refer to domains which are associated with a membrane as described herein which may not be a cell, for example, without limitation, an extracellular vesicle such as a WAEV. In some embodiments, only a portion of the extracellular domain is exterior to a membrane to which it is associated. In some embodiments, the membrane is a lipid-based layer. In some embodiments, the lipid- based layer is a lipid bilayer. In some embodiments, the lipid membrane is a cellular membrane. In some embodiments, the lipid membrane is a lipid layer of an extracellular vesicle. In some embodiments, the extracellular vesicle is a WAEV.

[0030] Any extracellular domain is contemplated for use herein. In some embodiments, the extracellular domain is or comprises an extracellular domain of a known protein. In some embodiments, the extracellular domain is or comprises a fragment of a known protein. In some embodiments, the extracellular domain is or comprises an antigen domain, or fragment thereof. In some embodiments, the extracellular domain is or comprises a protein, or fragment thereof. In some embodiments, the extracellular domain is or comprises a selfantigen protein or domain, or fragment thereof. In some embodiments, the antigen domain is a self-antigen, including but not limited to self-antigens associated with disease conditions recited in Table 1.AHR ligand 2-(l’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) [ligand of the aryl hydrocarbon receptor]TABLE 2 | Selected drugs targeting autoantibody-induced pathology.Drug / intervention Phase Target / mode of action Indications(s) ReferenceAnti-CD20 (Rituximab) Clinical use CD20 (B cells) - Grave’s disease (108)- SSc (109)- TTP- Vasculitis- PemphigusImmunadsorption / Clinical use Temporary removal of all antibodies - MG (110) plasmapheresis - Pemphigus- Pemphigoid- TTP- Autoantibody-induced carditisIVIG Clinical use Several, inhibition of the FcRn reduced - MG (111) circulating autoantibodies - Autoantibody-induced carditis- Pemphigus- NMOSDCaplacizumab Clinical use Anti -von Willebrand nanobody - TTP (112)Eculizumab Off label use C5 - Licensed for paroxysmal (113) nocturnal hemoglobinuria -Proof-of-concept study in NMOSDSHP652 (SM101) Phase II Fc / FcyR interactions - Systemic lupus erythematosus (H4)- Idiopathic thrombocytopenic purpuraR935788 Phase II SYK - Rheumatoid arthritis NCT00665626- Pemphigoid (preclinical) (115)KI -70 Phase I Thyroid-stimulating hormone receptor antagonist Grave’s disease (116)BAX930 Phase I BAX is recombinant AD AMTS 13, given to - TTP NCT02216084 reconstitute ADAMTS13CAAR T cells Preclinical Autoantigen-specific B / plasma cells - Pemphigus (117)TABLE 2 Continued | Selected drugs targeting autoantibody-induced pathology.EGFR inhibition Preclinical EGFR - Pemphigus (118)Tandem peptide Preclinical Tandem peptide consisting of 2 connected - Pemphigus (119) peptide sequences targeting the desmoglein-3Apremilast Preclinical PDE4 Inhibition - Licensed for psoriasis (120, 121)- Preclinical evidence for pemphigoidSSc, systemic sclerosis; MG, myasthenia gravis; IVIG, intravenous immunoglobulin; PDE4, phosphodiesterase 4; FcRn, neonatal Fc-receptors; TTP, thrombotic thrombocytopenic purpura; NMOSD, neuromyelitis optica spectrum disorder; SYK, spleen tyrosine kinase; CAAR, chimeric autoantigen receptor T cells; EGFR, epidermal growth factor receptor.References to websites were all accessed on April 28, 2017.Table 3Autoantibody-Associated Autoimmune DiseasesTable 3Autoantibody-Associated Autoimmune DiseasesTable 3Autoantibody-Associated Autoimmune DiseasesTable 3Autoantibody-Associated Autoimmune Diseases

[0031] The term “fusion protein,” as may be used herein, refers to a hybrid (e.g., chimeric, recombinant) polypeptide which comprises protein domains from at least two different proteins. One protein domain may be located at the amino-terminal (N-terminal) portion of the fusion protein and will contain the free N terminus (e.g., amino (-NH2) group) of the fusion protein, and this protein domain of the fusion protein may be referred to as the “amino-terminal fusion protein” or “amino-terminal fusion protein domain.” Similarly, one protein domain may be located at the carboxy-terminal (C-terminal) portion of the fusion protein and will contain the free C terminus (e.g., carboxyl (-COOH) group) of the fusion protein, this protein domain of the fusion protein may be referred to as the “carboxy-terminal fusion protein” or “carboxy-terminal fusion protein domain.” In some embodiments, fusion proteins may comprise additional protein domains. In some embodiments, the additional protein domains may be similar or distinct from the amino-terminal fusion protein domain and / or carboxy-terminal fusion protein domain. These additional domains are positioned between the amino-terminal fusion protein domain and the carboxy-terminal fusion protein domain. In some embodiments, a protein domain of a fusion protein may comprise a WW- containing domain. In some embodiments, a protein domain of a fusion protein may comprise a transmembrane domain. In some embodiments, a protein domain of a fusion protein may comprise an extracellular domain. Any of the fusion proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for fusion protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. A fusion protein can be encoded by a recombinant nucleic acid (e.g., DNA, RNA).

[0032] The term “isolated,” as may be used herein, refers to a characteristic of a material as provided herein (e.g., nucleic acid (e.g., RNA, DNA, polynucleotide), amino acid, peptide (e.g., polypeptide, protein), vector (e.g., viral vector (e.g., adeno-associated viral vector))), as being altered or removed from its natural state (z.e., native or original environment if it is naturally occurring) where such material would otherwise be found. Therefore, a naturally occurring nucleic acid or peptide present in a living animal is not isolated, but the samenucleic acid or peptide, separated by human intervention from some or all of the coexisting materials in the natural system, is “isolated.” For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state or host is “isolated.” An artificial, recombinant, or engineered material, for example, a non-naturally occurring nucleic acid construct or peptide construct, are, accordingly, also referred to as isolated. An isolated material can exist in substantially purified form, or can exist in a nonnative environment such as, for example, a vector or host cell, however, a material does not have to be purified in order to be isolated. Accordingly, a material may be part of a vector and / or part of a composition, and still be isolated in that such vector or composition is not part of the environment in which the material is found in its natural state.

[0033] The term “linker,” as may be used herein, refers to a chemical moiety linking two molecules or moieties, e.g., a WW-containing domain, transmembrane domain, extracellular domain, and / or any other molecule (e.g., peptide, tag, nucleic acid). Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker comprises an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker comprises a nucleotide (e.g., DNA or RNA) or a plurality of nucleotides (e.g., a nucleic acid). In some embodiments, the linker is an organic molecule, functional group, polymer, or other chemical moiety. In some embodiments, the linker is a cleavable linker, e.g., the linker comprises a bond that can be cleaved upon exposure to, for example, UV light or a hydrolytic enzyme, such as a protease or esterase. In some embodiments, the linker is any stretch of amino acids having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids). In other embodiments, the linker is a chemical bond (e.g., a covalent bond, amide bond, disulfide bond, ester bond, carbon-carbon bond, carbon heteroatom bond).

[0034] The terms “nucleic acid,” “nucleotide sequence,” “polynucleotide,” “oligonucleotide,” and “polymer of nucleotides” as may be used interchangeably herein, refer to a string of at least two, base-sugar-phosphate combinations and includes, among others, single- stranded and double- stranded DNA, DNA that is a mixture of single- stranded and double-stranded regions, single- stranded and double- stranded RNA, and RNA that is mixture of single- stranded and double-stranded regions, hybrid molecules comprising DNA and RNAthat may be single stranded or, more typically, double stranded or a mixture of singlestranded and double- stranded regions. In addition, the terms (e.g., nucleic acid, et al.) as used herein can refer to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often referred to as an oligonucleotide.

[0035] The terms (e.g., nucleic acid, et al.) also encompass such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells. For instance, the terms (e.g., nucleic acid, et al.) as used herein can include DNA or RNA as described herein that contain one or more modified bases. The nucleic acids may also include natural nucleosides (z.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine), nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, 0(6) methylguanine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1 -methyl adenosine, 1 -methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2 '- deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5' N phosphoramidite linkages). Thus, DNA or RNA including unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are nucleic acids as the term is used herein. The terms (e.g., nucleic acid, et al.) also includes peptide nucleic acids (PNAs), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids can contain other types of backbones, but contain the same bases. Thus, DNA or RNA with backbones modified for stability or for other reasons are nucleic acids as that term is intended herein.

[0036] The term “operably linked,” as may be used herein, refers to an arrangement of sequences or regions wherein the components are configured so as to perform their usual or intended function. Thus, a regulatory or control sequence operably linked to a coding sequence is capable of affecting the expression of the coding sequence. The regulatory or control sequences need not be contiguous with the coding sequence, so long as they functionto direct the proper expression or polypeptide production. Thus, as a non-limiting example, intervening untranslated but transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered operably linked to the coding sequence. A promoter sequence, as described herein, is a DNA regulatory region a short distance from the 5' end of a gene that acts as the binding site for RNA polymerase. The promoter sequence may bind RNA polymerase in a cell and / or initiate transcription of a downstream (3' direction) coding sequence. The promoter sequence may be a promoter capable of initiating transcription in prokaryotes or eukaryotes. Some non-limiting examples of eukaryotic promoters include the cytomegalovirus (CMV) promoter, the chicken beta-actin (P-actin) (CBA) promoter, and a hybrid form of the CBA promoter (CBh).

[0037] The terms “percent identity,” “sequence identity,” “% identity,” “% sequence identity,” and % identical,” as they may be interchangeably used herein, refer to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid). The percent identity of genomic DNA sequence, intron and exon sequence, and amino acid sequence between humans and other species varies by species type, with chimpanzee having the highest percent identity with humans of all species in each category.

[0038] Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0039] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and. Genome Projects, Smith, D. W ., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0040] When a percent identity is stated, or a range thereof (e.g., at least, more than, etc.), unless otherwise specified, the endpoints shall be inclusive and the range (e.g., at least 70% identity) shall include all ranges within the cited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity) and all increments thereof (e.g., tenths of a percent (i.e.., 0.1%), hundredths of a percent (i.e., 0.01%), etc.).

[0041] The terms “regulatory sequence,” “regulatory signal,” “control sequence,” and “control signal,” as may be used interchangeably herein, refer to sequences that are responsible for expressing a particular nucleic acid or may include other sequences, such as heterologous, synthetic, or partially synthetic sequences. The sequences can be of eukaryotic, prokaryotic, or viral origin that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory or control regions may include origins of replication, RNA splice sites, introns, chimeric or hybrid introns, promoters, enhancers, transcriptional termination sequences, poly A sites, locus control regions, signal sequences that direct the polypeptide into the secretory pathways ofthe target cell, and introns. A heterologous regulatory region is not naturally associated with the expressed nucleic acid to which it is linked. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences that do not occur in nature, but which are designed by one of ordinary skill in the art.

[0042] The terms “reporter,” “reporter tag,” “signal,” and “signal tag,” as such terms may be used interchangeably herein, refer a molecule (e.g., peptide, nucleic acid, other moiety) which is associated with a subject molecule to identify the subject molecule during use (e.g., in vivo, in vitro, ex vivo). Any suitable reporter is contemplated for use herein. Reporter and signals are well known in the art and the selection and use of such reporters will be readily appreciated by the skilled artisan. For example, without limitation, green fluorescent protein is a protein isolated from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light (e.g., an enhanced or wavelength- shifted version of the protein). In some embodiments, a reporter or signal is green fluorescent protein (GFP).

[0043] The term “subject,” as used herein, refers to any organism in need of the use of the subject matter herein. In some embodiments, the use includes treatment using, or diagnosis using, the subject matter herein. For example, without limitation, subjects may include mammals and non-mammals. As used herein, a “mammal,” refers to any animal constituting the class Mammalia (e.g., a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Marmoset, Macaque)). In some embodiments, the mammal is a human.

[0044] The term “target cell” as used herein, refers to a cell which is the intended or desired target of the intervention, action, or effect which is intended or desired by the intervention of a method or composition. In some embodiments, the target cell is a cell that can host, replicate, and express an isolated nucleic acid, fusion protein, microvesicle, or WAEV as described herein. In some embodiments, the target cell is the cell to which the delivery of a therapeutic molecule is directed, for example, such as when a WAEV displays a homing molecule for such a target cell. In some embodiments, a host cell is taken from a subject. In some embodiments, the host cell is derived from cells not taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa- S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL- 2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4,COS, COS-1, COS-6, C0S-M6A, BS-C-1 monkey kidney epithelial, BALB / 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H- 10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr - / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV- 434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HEK293T, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCL H69 / CPR, NCI- H69 / LX10, NCI-H69 / LX20, NCLH69 / LX4, NIH-3T3, N ALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (e.g., the American Type Culture Collection (ATCC) (Manassus, Va.)).

[0045] The term “transmembrane domain,” as may be used herein, refers to the domain of a protein or polypeptide which spans the membrane of a membrane-contained molecule (e.g., cell, vesicle, EV, or WAEV), potentially associating multiple domains of a larger protein structure (e.g., WW-containing domain, extracellular domain). In some embodiments, the transmembrane domain comprises a domain of a fusion protein. In some embodiments, the transmembrane domain is positioned centrally to a domain located interior of a membrane and a domain exterior to a membrane. In some embodiments, the membrane is a lipid-based layer. In some embodiments, the lipid-based layer is a lipid bilayer. In some embodiments, the lipid layer is a lipid monolayer. In some embodiments, the lipid membrane is a cellular membrane. In some embodiments, the lipid membrane is a lipid layer of an extracellular vesicle. In some embodiments, the extracellular vesicle is a WAEV. The transmembrane domain may span the membrane one time or multiple times and can be responsible for connecting the domains of the fusion protein across the membrane. Any transmembrane domain is contemplated for use herein. Transmembrane domains can be identified using any method known in the art or described herein, e.g., by using the UniProt Database.

[0046] The terms “treatment,” “treat,” and “treating,” as may be used interchangeably herein, refer to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or moresymptoms or features of a particular indication, disease, disorder, condition, and / or symptom thereof. In some embodiments, the treatment refers to a clinical intervention. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms (e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease). For example, treatment may be administered to a susceptible individual (e.g., subject) prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). In some embodiments, the treatment is used and / or administered as a prophylaxis. Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0047] The terms “WW-containing domain” and “WW domain” as may be used interchangeably herein, refer to a protein domain having two basic residues at the C terminus that mediates protein-protein interactions with short proline-rich or proline-containing motifs. It should be appreciated that the two basic residues (e.g., any two of: histidine (H), arginine (R), and / or lysine (K)) of the WW-containing domain are not required to be at the absolute C terminus of the WW-containing protein domain (e.g., the final residues of the C terminus). Rather, the two basic residues may be at a C-terminal portion of the WW-containing protein domain (e.g., the C-terminal half of the WW-containing protein domain). In some embodiments, the WW-containing domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tryptophan (W) residues. In some embodiments, the WW-containing domain contains at least two W residues. In some embodiments, the at least two W residues are spaced apart by from 15-25 amino acids. In some embodiments, the at least two W residues are spaced apart by from 19-23 amino acids. In some embodiments, the at least two W residues are spaced apart by from 20-22 amino acids. The WW-containing domain possessing the two basic C- terminal amino acid residues may have the ability to associate with short proline-rich or proline-containing motifs (e.g., a PPXY (SEQ ID NO: 22) motif). WW-containing domains bind a variety of distinct peptide ligands including motifs with core proline-rich sequences, such as PPXY (SEQ ID NO: 22), such as is found in SCAMP3 (among others). A WW- containing domain may be a 30-40 amino acid protein interaction domain with two signature tryptophan residues spaced by 20-22 amino acids. The three-dimensional structure of WW- containing domains shows that they generally fold into a three-stranded, antiparallel P sheet with two ligand-binding grooves.

[0048] WW-containing domains are found in many eukaryotes and are present in approximately 50 human proteins (Bork, P. & Sudol, M. The WW domain: a signaling site in dystrophin? Trends Biochem Sci 19, 531-533 (1994)). WW-containing domains may bepresent together with several other interaction domains, including membrane targeting domains, such as C2 in the NEDD4 family proteins, the phosphotyrosine-binding (PTB) domain in FE65 protein, FF domains in CA150 and FBPI1, and pleckstrin homology (PH) domains in PEEKHA5. The NEDD4 E3 ligase proteins include, but are not necessarily limited to, ITCH, NEDD4, NEDD4 E, WWP1, WWP2, Smurfl, Smurf2, BUE1, and NEDE2. WW-containing domains are also linked to a variety of catalytic domains, including HECT E3 protein-ubiquitin ligase domains in NEDD4 family proteins, rotomerase or peptidyl prolyisomerase domains in Pinl, and Rho GAP domains in ArhGAP9 and ArhGAP12.

[0049] In the instant disclosure, the WW-containing domain may be a WW-containing domain that naturally possesses two basic amino acids at the C terminus. In some embodiments, a WW-containing domain or WW-containing domain variant may be from the human ubiquitin ligase WWP1, WWP2, Nedd4-1, Nedd4-2, Smurfl, Smurf2, ITCH, NEDL1, or NEDL2. Exemplary amino acid sequences of WW-containing domain containing proteins (WW-containing domains underlined) are listed below. It should be appreciated that any of the WW-containing domains or WW-containing domain variants of the exemplary proteins may be used in the invention, described herein, and are not meant to be limiting.DETAILED DESCRIPTION

[0050] This disclosure demonstrates that autoantigen-loaded WAEVs induce tolerance that protects against underlying autoimmune diseases, such as in Myasthenia Gravis disease, which is caused by the autoantibodies against the membrane-expressed nicotinic acetylcholine receptor (nAChR). We demonstrate that WAEVs are a versatile platform generally for presenting and delivering autoantigens, including membrane-expressed autoantigens, as “tolerance vaccines” against autoimmune diseases whose etiology is linked to B-cell responses to membrane autoantigens.

[0051] Autoimmune diseases refers to organ- specific or systemic, primary or secondary autoimmune and / or inflammatory diseases, including those associated with pathogenic autoantibodies. Autoimmune diseases such as myasthenia gravis, multiple sclerosis, and type 1 diabetes are characterized by immune-mediated destruction of the target tissues associated with the respective disease. Other autoimmune diseases are shown in Table 3. Such autoimmune mechanisms include both autoantibodies derived from autoreactive B cells and inflammation / cytotoxicity by autoantigen-specific T cells. In contrast to therapies that cause broad immunosuppression with unacceptable safety risks, antigen- specific immunotherapies (ASIs) are a therapeutic approach designed to target only the specific autoimmune components contributing to disease. This autoantigen-targeted immunoregulatory approachmay likely require repetitive administration of ASI to maintain the selective suppression of autoimmunity, a process termed peripheral immune tolerance, throughout the life of the individual who would be expected to start such therapy at the earliest clinical signs of autoimmunity (e.g., via biomarker tests). Therefore, ASIs must meet a high safety standard in contrast to that of general immunosuppressive therapies known to have significant side effects that prohibit chronic use. ASI’s are “tolerance vaccines” that are different from the typical “infectious disease vaccines” in that the general type of immune cell responses involved in protection from infectious disease are also of the type that mediate cell destruction in autoimmune diseases, in which ASIs induce the counteracting regulatory response [1]. The two main immune tolerance mechanisms of action of ASIs are i) repression of pathogenic T effector cells (Teffs), and ii) induction of suppressor T regulatory cells (Tregs) in which the disease-associated autoantigen (“signal 1”) must be delivered to antigen-presenting cells (APCs) in such a way as to avoid inflammatory stimulation of the APC (“signal 2” co-stimulatory molecules) (FIG. ID). (Note that such signal 2 molecules are induced by adjuvants required for vaccination against infectious disease, but would be counteractive to tolerance induction by potentially enhancing Teffs [2].)

[0052] There are a significant number of tolerance-delivery systems (TDS) of ASI’s that have different features and compositions, many of which have demonstrated positive results in preclinical studies, but most have not demonstrated clinical efficacy in autoimmune disease [3]. Such current TDSs include nanoparticles containing tolerogenic substances, anti-inflammatory-modified DNA plasmid, apoptotic cells, tolerized dendritic cells, and mucosal delivery of autoantigen [4-7]. These ASIs are predominantly designed to target pathogenic T cell “peptide” antigens because of the default use of soluble autoantigens that do not have the native conformation of “membrane target antigens” recognized by B cell autoantibody-mediated autoimmune diseases [8]. To solve this hurdle, we have created a technology that enables the genetic engineered-expression of membrane protein antigens in an extracellular vesicle (EV) [9, 10] via a proprietary delivery system called, WW-domain- Activated Extracellular Vesicles (WAEVs) (FIG. 1A). WAEVs, like other extracellular vesicles e.g., exosomes), are inherently immunologically inert tolerogenic vehicles [5, 11, 12] and are designed to allow native conformational expression of membrane autoantigen(s) that can be recognized by antigen receptors on pathogenic B cells (FIG. IB;

[0010] ) in addition to being loaded with appropriate tolerogenic substances such as mTOR inhibitors (rapamycin, everolimus), calcineurin inhibitors (tacrolimus), ITE [ligand of the aryl hydrocarbon receptor], Vitamin D3, Ret Acid, and RORyt inhibitors [13, 14] (FIGs. IB, 1C; Table 1). Because such B cells produce pathogenic autoantibodies (FIG. ID), delivery of tolerogenicsubstances via the WAEV vehicle is expected to suppress such B cells from producing autoantibodies in addition to converting its antigen-presenting function to tolerance induction that can stimulate Tregs and repress pathogenic Teff cells (FIG. IB). Likewise, tol-WAEVs are predicted also convert other APCs, such as dendritic cells, to tolerogenic state (z.e., tol- DC) that stimulate de novo Tregs (FIG. 1C) [1].

[0053] In some embodiments, the WW-containing domain consists essentially of a WW- containing domain or WW-containing domain variant. Consists essentially of means that a domain, peptide, or polypeptide consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example, from about 1 to about 10 or so additional residues, typically from 1 to about 5 additional residues in the domain, peptide, or polypeptide.

[0054] Alternatively, the WW-containing domain may be a WW-containing domain that has been modified to include two basic amino acids at the C-terminus of the domain. Techniques are known in the art and are described in the art, for example, in Sambrook et al. ((2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press). Thus, a skilled person could readily modify an existing WW-containing domain that does not normally have two C-terminal basic residues so as to include two basic residues at the C- terminus.

[0055] Basic amino acids are amino acids that possess a side-chain functional group that has a pKa of greater than 7 and includes lysine, arginine, and histidine, as well as basic amino acids that are not included in the twenty a- amino acids commonly included in proteins. The two basic amino acids at the C terminus of the WW-containing domain may be the same basic amino acid or may be different basic amino acids. In one embodiment, the two basic amino acids are two arginine residues.

[0056] The term WW-containing domain also includes variants of a WW-containing domain provided that any such variant possesses two basic amino acids at its C-terminus and maintains the ability of the WW-containing domain to associate with the PPXY (SEQ ID NO: 22) motif. A variant of such a WW-containing domain refers to a WW-containing domain which retains the ability of the variant to associate with the PPXY (SEQ ID NO: 22) motif, (i.e. the PPXY (SEQ ID NO: 22) motif of SCAMP3) and that has been mutated at one or more amino acids, including point, insertion, and / or deletion mutations, but still retains the ability to associate with the PPXY (SEQ ID NO: 22) motif. A variant or derivative therefore includes deletions, including truncations and fragments; insertions and additions, for example conservative substitutions, site-directed mutants and allelic variants; and modifications, including one or more non- amino acyl groups (e.g., sugar, lipid, etc.) covalently linked to thepeptide and post-translational modifications. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.

[0057] The WW-containing domain may be part of a longer protein. Thus, the protein, in various different embodiments, comprises the WW-containing domain, consists of the WW- containing domain or consists essentially of the WW-containing domain, as defined herein. The polypeptide may be a protein that includes a WW domain as a functional domain within the protein sequence.

[0058] The present disclosure relates, at least in part, to novel extracellular vesicles (EVs) which contain WW-domain containing proteins that comprise an extracellular domain (WW- domain- Activated Extracellular Vesicles, or WAEVs). Such extracellular domains can be presented on the surface of the WAEV through the introduction of WW-domain containing proteins that are fused to a transmembrane domain and the extracellular domain. Direct fusions of transmembrane-containing proteins to arrestin domain containing protein 1 (ARDDC1) result in decreased or abolished budding activity of ARRCC1. WAEVs are able to bud independent of ARRDC1, and do not appear to be enhanced by ARDDC1 overexpression. In addition, WAEVs do not appear to be like classical exosomes because they do not contain one or more of the typical exosomal markers (e.g., CD63; CD81, CD9, and PTGFRN). Instead, other proteins may be responsible for mediating WAEV budding, including the secretory carrier-associated membrane protein 3 (SCAMP3). WAEVs can be used to deliver and present antigens useful for vaccine development; to display homing molecules for targeted delivery of therapeutic molecules to specific cells or tissues; and for packaging and delivery of therapeutic molecules via interactions with the WW domains. WW- Domain- Activated Extracellular Vesicles (WAEVs)

[0059] In some aspects, the disclosure relates to a WW-domain-activated extracellular vesicle (WAEV), comprising: (a) a lipid bilayer; and (b) a fusion protein as described herein.

[0060] In some embodiments, a WAEV as described herein, further comprises WAEV- mediating protein. WAEV-mediating proteins can contain either the PPXY (SEQ ID NO: 22) motif or the PSAP (SEQ ID NO: 17) motif, and preferably contain both. The PPXY (SEQ ID NO: 22) and PSAP (SEQ ID NO: 17) motifs are critical elements in the ARDDC1 protein that are required for ARMMs budding. The WAEV-mediating protein can interact with fusion proteins WW-containing domain through the PPXY (SEQ ID NO: 22) motif, and the WAEV-mediating protein can recruit TSG101 via the PSAP (SEQ ID NO: 17) motif to the cell membrane to drive the budding of WAEVs.

[0061] A non-limiting example of a WAEV-mediating protein is SCAMP3. Secretory carrier-associated membrane protein 3 (SCAMP3) is a protein that in humans is encoded by the SCAMP3 gene, which is a member of the SCAMP family of proteins that are secretory carrier membrane proteins. These proteins are known to function as carriers of proteins to the cell surface in post-golgi recycling pathways. SCAMP3 is an integral membrane protein that has four transmembrane domains and contains a PPXY (SEQ ID NO: 22) motif at its N- terminal cytosolic segment. In addition, SCAMP3 has a PSAP (SEQ ID NO: 17) motif that is known to interact with TSG101, the ESCRT I complex protein required for budding of ARMMs (see United States Patent Serial Number 9,737,480) as well as other multivesicular bodies. Thus, SCAMP3 shares both PPXY (SEQ ID NO: 22) and PSAP (SEQ ID NO: 17) motif with ARRDC1 but differs from ARRDC1 in that SCAMP3 is integrated in the plasma membrane via its transmembrane domain whereas ARRDC1 transiently associates with plasma membrane via its arrestin domain. It is believed that the that fusion protein WW- containing domain (e.g., WW-containing domain protein fused to a transmembrane domain and extracellular domain) interacts with the PPXY (SEQ ID NO: 22) motif of SCAMP3, which subsequently recruits TSG101 via the PSAP (SEQ ID NO: 17) motif to the cell membrane to drive the budding of WAEVs. The extracellular domain can include a cargo domain.

[0062] Tumor susceptibility gene 101 (TSG101), refers to a group of seemingly inactive homologs of ubiquitin-conjugating enzymes. The protein contains a coiled-coil domain that interacts with stathmin, a cytosolic phosphoprotein implicated in tumorigenesis. TSG101 can interact with proteins that comprises a PSAP (SEQ ID NO: 17) motif. TSG101, in budding viruses, drives budding through direct plasma membrane budding (DPMB). TSG101 is a protein that comprises a UEV domain and can interact with SCAMP3. As referred to herein, UEV refers to the Ubiquitin E2 variant domain of approximately 145 amino acids. The structure of the domain contains a a / p fold similar to the canonical E2 enzyme but has an additional N-terminal helix and further lacks the two C-terminal helices. Often found in TSG101 / Vps23 proteins, the UEV interacts with a ubiquitin molecule and is essential for the trafficking of a number of ubiquitylated pay loads to multivesicular bodies (MVBs). Furthermore, the UEV domain can bind to Pro-Thr / Ser- Ala-Pro peptide ligands, a fact exploited by viruses such as HIV. Thus, the TSG101 UEV domain binds to the PTAP tetrapeptide motif in the viral Gag protein that is involved in viral budding. The disclosure also contemplates variants of TSG101, such as fragments of TSG101 and / or TSG101 proteins that have a degree of identity (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity) to a TSG101 protein and are capable of interacting with PSAP-containing proteins likeSCAMP3. Accordingly, an TSG101 protein may be a protein that comprises a UEV domain and interacts with SCAMP3. In some embodiments, the TSG101 protein is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 58, comprises a UEV domain, and interacts with PSAP-containing proteins like SCAMP3. In some embodiments, the TSG101 protein has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, or at least 390, identical contiguous amino acids of any one of SEQ ID NO: 58, comprises a UEV domain, and interacts PSAP-containing proteins like SCAMP3. In some embodiments, the TSG101 protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NO: 58 and comprises a UEV domain. Exemplary, non-limiting TSG101 protein sequences are provided herein, and additional, suitable TSG101 protein sequences, isoforms, and variants are known in the art. It will be appreciated by those of skill in the art that this invention is not limited in this respect.

[0063] The structure of UEV domains is known to those of skill in the art (see, e.g., Owen Pomillos et al., Structure and functional interactions of the TsglOl UEV domain, EMBO J. 2002 May 15; 21(10): 2397-2406, the entire contents of which are incorporated herein by reference).

[0064] In some embodiments, the fusion proteins of the disclosure do not comprise an arrestin domain containing protein 1 (ARRDC1). ARRDC1, as described elsewhere herein, is a protein that comprises a PSAP (SEQ ID NO: 17) motif and a PPXY (SEQ ID NO: 22) motif in its C-terminus and interacts with TSG101. However, as can be shown herein, the present WAEVs do not require the presence or action of ARRDC1 to form and / or bud. Accordingly, in some embodiments, the WAEVs of the present disclosure lack an ARRDC1 protein.

[0065] The WAEVs of the present disclosure further are distinguishable from various other exosomes and / or extracellular vesicles in markers they carry. Typical EVs carry a variety of proteins used as markers to identify exosomes, as well as imbue qualities to the exosome for use in experiments and diagnostics. Exosomal markers are known in the art, and are known, for example, to belong to various functional groups, such as tetraspanins (CD9, CD63 and CD81), heat shock proteins (HSC70 and HSC90), membrane transporters (GTPases) andlipid-bound proteins. Some of the most prevalent exosomal markers include: heat shock protein 8 (HSPA8), CD63 antigen (CD63), beta actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase 1 alpha (EN01), cytosolic heat shock protein 90 alpha (HSP90AA1), CD9, CD81, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), muscle pyruvate kinase (PKM2). However, the WAEVs of the present disclosure can lack one or more (or all) of the anticipated markers found in EVs, for example: CD9; CD63; CD81; and / or PTGFRN. Accordingly, in some embodiments, the WAEVs of the present disclosure are enriched for a number of proteins.

[0066] In some embodiments, the WAEVs as described herein do not comprise at least one of the following exosomal markers: CD9; CD63; CD81; and / or PTGFRN. In some embodiments, a WAEV as described herein does not comprise at least two of the following exosomal markers: CD9; CD63; CD81; and / or PTGFRN. In some embodiments, a WAEV as described herein does not comprise at least three of the following exosomal markers: CD9; CD63; CD81; and / or PTGFRN. In some embodiments, a WAEV as described herein does not comprise any of the following exosomal markers: CD9; CD63; CD81; and / or PTGFRN.

[0067] In some aspects, the disclosure relates to a fusion protein comprising: (a) a WW- containing domain; (b) a transmembrane domain; and (c) an extracellular domain. In some embodiments, the WW-containing domain is positioned at the N-terminus of the fusion protein. The fusion proteins of the present disclosure, may facilitate (e.g., increase the likelihood of, influence the production of) the production of WAEVs. In some embodiments, the fusion proteins, by containing extracellular domains as described in further detail herein, may facilitate the expression of, or presentation, of domains on the surface, or protruding from the surface of WAEVs.

[0068] Accordingly, in some embodiments, the WW-containing domain of any of the fusion proteins of the disclosure comprise at least one WW domain. In some embodiments, the WW-containing domain is positioned at the C-terminus of the fusion protein. In some embodiments, the WW-containing domain is positioned between the N-terminus and C- terminus of the fusion protein (e.g., between two other domains). In some embodiments, the WW-containing domain is positioned at the N-terminus of the fusion protein.

[0069] In some embodiments, the WW-containing domain of any of the fusion proteins of the disclosure comprise at least two WW domains. In some embodiments, the WW-containing domain of any of the fusion proteins of the disclosure comprise at least three WW domains. In some embodiments, the WW-containing domain of any of the fusion proteins of the disclosure comprise at least four WW domains. In some embodiments, the WW-containing domain of any of the fusion proteins of the disclosure comprise more than four WW domains.In some embodiments, the fusion protein comprises at least one WW domain which is an ITCH protein WW domain. In some embodiments, the WW-containing domain of any of the fusion proteins of the disclosure comprise a sequence having at least 95% identity to the sequence of SEQ ID NO: 1. In some embodiments, the WW-containing domain of any of the fusion proteins of the disclosure comprise the sequence of SEQ ID NO: 1. In some embodiments, where the WW-containing domain contains more than one WW domain, the WW domains may be oriented in the fusion protein such that they are adjacent to one another without another domain in between. In some embodiments, where the WW-containing domain contains more than one WW domain, the WW domains may be oriented in the fusion protein such that they are not adjacent to one another (e.g., with an intervening domain). In some embodiments, the intervening domain may be a linker domain. In some embodiments, the intervening domain may be another domain (e.g., peptide, molecule, nucleic acid). In some embodiments at least one of the WW domains of the fusion protein is positioned such that it has a free N-terminus. In some embodiments at least one of the WW domains of the fusion protein is positioned such that it has a free C-terminus.

[0070] In some embodiments, the fusion proteins of the disclosure comprise an extracellular domain. The extracellular domain is a portion (e.g., domain) of the fusion protein, which will be oriented (e.g., located, positioned), such that at least a portion of the extracellular domain is physically located outside of the membrane of the molecule (e.g., cell, EV) to which it is associated. In some embodiments, the entirety of the extracellular domain is located exterior to the membrane. In some embodiments, the extracellular domain comprises a known protein. In some embodiments, the extracellular domain comprises a portion of a known protein (e.g., fragment). In some embodiments, an extracellular domain of the fusion protein is the extracellular domain or a known protein, or fragment thereof. In some embodiments, the extracellular domain may be a recombinant protein, or fragment thereof (e.g., recombinant or engineered protein, fusion protein, or fragment thereof). In some embodiments, the extracellular domain may comprise a protein, or fragment thereof, which is known to provoke an immune response in an organism. In some embodiments, the extracellular domain may comprise a protein, or fragment thereof, which is believed to provoke an immune response in an organism. In some embodiments, the extracellular domain may comprise a protein, or fragment thereof, which is anticipated to provoke an immune response in an organism. In some embodiments, the extracellular domain may comprise a protein, or fragment thereof, which is desired to provoke an immune response in an organism. In some embodiments, the extracellular domain may comprise one or more carbohydrate unit that may or may not be responsible for, or involved in, provoking an immune response in an organism. In someembodiments, the extracellular domain may comprise one or more lipid unit that may or may not be responsible for, or involved in, provoking an immune response in an organism. As used herein, the term “provoke” is intended to describe a cause or impetus, the introduction of which into an organism influences or affects, at least in part, an immune reaction therein. Any action, beneficial or harmful (e.g., deleterious) is encompassed by the term. A direct reaction is not required (e.g., the reaction may be only partial caused by, or driven by, the introduction of the cause (e.g., domain, extracellular domain, protein, fusion protein), and may further be a component of, or step in, a larger cascade or reaction), nor must the reaction be substantial or complete. The immune reaction may further require the addition of other components.

[0071] In some embodiments, any of the isolated nucleic acids of the disclosure are operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter, an inducible promoter, or a tissue specific promoter. In some embodiments, the promoter is a chicken beat-actin (CBA) promoter. In other embodiments, that promoter is EF-l-alpha. In some embodiments, the promoter is a viral promoter such as CMV, SV40. In some embodiments, the promoter is a prokaryotic promoter.

[0072] In some embodiments, any of the isolated nucleic acids of the disclosure comprise at least one additional regulatory sequence. In some embodiments, the regulatory sequence is an enhancer. In some embodiments, the regulatory sequence is a self-amplifying RNA.

[0073] In some aspects, the disclosure relates to a WAEV-producing cell, comprising: (a) at least one of any of the isolated nucleic acids of the disclosure. In some embodiments, the WAEV-producing cell further comprises a heterologous promoter operably linked to a heterologous promoter. The WAEV-producing cell may be any type of suitable cell. For example, without limitation, the cell may be a target cell as described elsewhere herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0074] In some aspects, the disclosure relates to a method of delivering WAEVs displaying an antigenic peptide, comprising: delivering at least one of any of the fusion proteins of the disclosure, at least one of any of the isolated nucleic acids of the disclosure, at least one of any of the WAEVs of the disclosure, and / or at least one of any of the WAEV-producing cells of the disclosure, wherein the extracellular protein of the fusion protein comprises an antigenic peptide. In some embodiments, one or more amino acids from the transmembrane domain may also be part of the antigenic peptide.

[0075] Some aspects of this invention provide a method of delivering an extracellular domain (e.g., antigen), for example, by delivering a WAEV comprising a fusion protein comprising aWW-containing domain, transmembrane domain, and extracellular domain to a target cell. The target cell can be contacted with the WAEV in different ways. For example, a target cell may be contacted directly with a WAEV, including but not necessarily limited to, an isolated WAEV from a microvesicle-producing cell. The contacting can be done in vitro by administering the WAEV, fusion protein, and / or isolated nucleic acid, to the target cell in a culture dish, or in vivo by administering the WAEV, fusion protein, isolated nucleic acid, and / or a microvesicle-producing cell comprising a fusion protein and / or isolated nucleic acid to a subject. Alternatively, the target cell can be contacted with a microvesicle producing cell as described herein, either directly or indirectly, for example, in vitro by co-culturing the target cell and the microvesicle producing cell, or in vivo by administering a microvesicle producing cell to a subject harboring the target cell. Accordingly, the method may include contacting the target cell with a microvesicle, for example, a WAEV, as described herein. The target cell may be contacted with a microvesicle-producing cell, either directly or indirectly as described herein, or with an isolated microvesicle, wherein the produced or isolated microvesicle has a lipid bilayer, a SCAMP3 protein or variant thereof, and an extracellular domain.

[0076] It should be appreciated that the target cell may be of any origin. For example, the target cell may be a human cell. The target cell may be a mammalian cell. Some non- limiting examples of a mammalian cell include a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell. It should also be appreciated that the target cell may be of any cell type. For example the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells). In other cases, the target cell may be any differentiated cell type found in a subject. In some embodiments, the target cell is a cell in vitro, and the method includes administering the microvesicle to the cell in vitro, or coculturing the target cell with the microvesicle-producing cell in vitro. In some embodiments, the target cell is a cell in a subject, and the method comprises administering the microvesicle or the microvesicle -producing cell to the subject. In some embodiments, the subject is a mammalian subject, for example, a rodent, a mouse, a rat, a hamster, or a non-human primate. In some embodiments, the subject is a human subject.EXAMPLESExample 1: Production of WAEV Particles

[0077] This example demonstrates the production of WAEV particles for use in methods for delivering peptides to host cells, such as in methods for inducing immune system tolerance.

[0078] HEK293T cells were engineered to express viral membrane antigens, including those of influenza (Matrix 2 protein (M2) and hemagglutinin 2, (HA2)) and HIV (the membrane- proximal external region (MPER) of the envelope antigen), for recruitment to lipid membrane EV’s -100 nm in diameter, i.e., WAEVs, as infectious disease vaccines formulated with strong inflammatory adjuvants

[0010] . This was accomplished by using genetic sequences to peptides containing specific WW domains (double tryptophan residues) as the intracellular moiety fused to a transmembrane-extracellular protein antigen sequence that allows such expressed transmembrane proteins to be recruited into EVs that are robustly budded from the cellular membrane as WAEVs (FIG. 2A). Note that WAEVs are distinct from known EVs such as exosomes and other microvesicles that cannot be engineered to express and display “extracellular membrane-bound antigens” [9, 15]. The WAEV budding is likely dependent on secretory carrier-associated membrane protein 3 (SCAMP3), which through its PPXY motif can specifically interact with the WW domains to recruit the fused membrane proteins such as the influenza antigen M2 onto the surface of WAEVs (FIG. 2B). We have also demonstrated WAEV budding of influenza HA2 and HIV MPER antigens

[0010] .

[0079] Results are shown in FIGs. 2C-2E. The ability of WW-domain sequences from the signaling protein, ITCH, to present M2 antigen on the surface of from WAEVs were compared to the known EV recruitment protein, ARRDC1 (that gives rise to the EVs termed, ARMMs)

[0016] . M2 fusions to the WW domain-containing ITCH protein (M2-WW fusion proteins), similar to M2 fusions to ARRDC1, resulted in secretion of the M2-WW fusion proteins into EVs as detected by the Western blotting (FIG. 2C). Moreover, M2-WW fusion proteins led to a robust increase in the number of EVs produced (FIG. 2D). We further characterized M2-WW fusion protein EVs using Optiprep-based density gradient fractionation. M2-WW fusion protein EVs were fractionated into ten fractions on the density gradient. The peak of M2-WW fusion protein EVs as indicated by M2 (FLAG-tagged) Western blotting occurred in the same fraction as that of ARMMs, as indicated by ARRDC1 staining, and that of exosomes (indicated by the exosomal marker CD9) (FIG. 2E), suggesting that M2-WW fusion protein EVs, M2-WAEVs, are of similar size as ARMMs and exosomes. To confirm that M2 protein is presented on the surface of the WAEVs, we performed immuno-gold staining of un-permeabilized M2-WW fusion protein EVs using an M2-specific antibody that recognizes the extracellular domain of the protein. Electron microscopy of immuno-gold- stained EVs showed the presence of gold particles on the surface of M2-WW fusion protein EVs but not on control EVs (not shown; see

[0010] ).Together, these results indicate that fusion to WW-domains allow M2 protein to be recruited and displayed onto the surface of WAEVs.

[0080] Such M2-WAEVs formulated with the inflammatory adjuvants, alum (FIG. 3A-3C) and the lipid soluble saponin and MPLA (FIG. 3D), induced specific viral-neutralizing and in vivo protective antibodies titers to influenza virus in mice demonstrating that WAEV expression of native conformational membrane antigen can productively present such antigens to B cells to elicit the expected response. Note that, as expected, the non-lipophilic substance, CpG, did not show an adjuvant effect when complexed with WAEV, demonstrating the lipophilic nature of substances that can be considered for loading WAEVs (FIG. 3D). While induction of anti-M2 antibodies may appear counterintuitive to an “immune tolerance” vaccine, such infectious disease antibodies were induced because these WAEV vaccines 1) contained foreign antigens, not self-antigens; 2) were derived from xenogeneic immunostimulatory cells (human HEK293 cells); and 3) contained a “vaccine adjuvant” that induces inflammation and signal 2 on APCs, which would not be used for a tolerance-WAEV (tol-WAEV). Rather, tol-WAEVs can be derived from autologous cells (z.e., mouse) and formulated with different tolerogenic lipophilic substances mentioned above to ensure tolerogenic function by APCs such as B cells and DCs.Example 2: tol-WAEV induction of immune tolerance in the treatment of autoimmune disease

[0081] This example demonstrates the production and delivery of tol-WAEV particles comprising self-antigens for inducing immune system tolerance, such as in the treatment of autoimmune disease. The example uses Myasthenia Gravis (MG) as illustrative.

[0082] MG is caused by autoantibodies against the membrane-expressed nicotinic acetylcholine receptor (nAChR). MG is a chronic autoimmune disease caused by the immune attack of the neuromuscular junction (FIG. 4). Antibodies directed against nAChR induce receptor degradation, complement cascade activation, and postsynaptic membrane destruction, resulting in a functional reduction of AChR availability. While AChR appears to be the dominant autoantigen in human MG, autoantibodies to other self-antigen targets, in a minority of MG patients, include muscle specific kinase (MuSK) and low-density lipoprotein related protein 4 (LRP4) that are known to play pathogenic roles in MG (FIG. 4) [17, 18]. Note that the majority of AChR antibodies recognize an extracellular domain of the receptor, defined as the main immunogenic region, localized between residues 67 and 76 of the a- subunit of the receptor

[0017] . Experimental autoimmune MG (EAMG) models have been used to successfully uncover the pathophysiological role of specific autoantibodies and autoreactive T helper lymphocytes, in addition enabling new therapies for prevention and modulation of the disease. EAMG is induced in susceptible strains of mice and rats uponimmunization with adjuvanted purified AChR or immunodominant peptides of the AChR a- chain that result clinical symptoms that reflect those of the human disease [19-22].

[0083] Tol-WAEV particles comprising MG self-antigens are produced and administered to MG model animals as outlined below. Results can demonstrate that tol-WAEVs expressing the full membrane-associated mouse AChR can prevent disease onset when administered prior to disease induction, thereby demonstrating immune tolerance induction.

[0084] Production of autoantigen-specific WAEV material. A fusion construct, AChR- 4WW, is produced comprising the full-length mouse AChR gene and the 4 WW domains of the ITCH protein. AChR-4WW is transfected into autologous mouse cells to produce AChR- WAEVs. AChR-WAEVs are isolated and purified via density gradient fractionation and ultracentrifugation, as described in

[0010] . Budding of AChR-associated WAEVs are confirmed via Western blotting, and presentation of AChR on the surface of WAEVs are confirmed via immune-gold labeling (with an n AChR- specific antibody) followed by electron-microscopy. Nanosight Analysis measures the size and number of AChR-WAEVs.

[0085] The production is anticipated to yield ~2-5 x 109WAEVs per 1 million transfected cultured cells. Based on the WAEV-M2 project, each mouse should receive a maximum of 109WAEV particles. When performing 4 mouse studies of ~60 mice / study, in which each mouse receives up to 5 doses, 4 x 60 x 5 x 109WAEV-AChR particles should be produced totaling 1.2 x 1012particles from ~ 109cells during the first 9 months. Such WAEV-AChR particles are mixed with the different tolerogenic agents.

[0086] Induction of antigen-specific tolerance with tol-WAEV treatment in the C57BL / 6 EAMG mouse model. Different tolerance agents (z.e., rapamycin, tacrolimus, Vitamin D3, Ret Acid) are formulated with AChR- WAEV and injected up to 14 days before (to prevent) or up to 14 days after (to treat) induction of autoimmunity in the C57BL / 6 mouse model, experimental autoimmune MG (EAMG) [23, 24].

[0087] EAMG is induced by immunizing 8- to 10-week-old female B6 mice (The Jackson Laboratory) with 100 pL s.c. injections of 25 pg of tAChR (purified from the electric organ of Torpedo califomica stingray; cat. # 28601, Cube Biotech, Wayne, PA) emulsified in equal volumes of PBS and Complete Freund’s Adjuvant containing 2.5 mg / ml M. tuberculosis H37 Ra (CFA, Difco). After the initial injection, 2 additional booster injections are given at 4 and 8 weeks later that contain the same amount of tAChR but emulsified in Incomplete Freund’ s Adjuvant (IFA, Difco). This procedure triggers the production of autoantibodies to tAChR and to murine AChR along with AChR-specific Thl cells, in which myasthenic symptoms typically appear 7-14 days after the second injection [20, 23, 24] (see time course example in FIG. 5;

[0025] ). Anti-tAChR and anti-mouse AChR IgG in sera are measured viaradioimmunoprecipitation assay via a method entailing AChR labelled with125I-labeled a- bungarotoxin (125I-aBTX) that binds serum anti-tAChR or anti-mouse AChR IgG, and IgG isotype subclasses (to evaluate Thl- and Th2-like responses) are measured via ELISA as described elsewhere [25, 26]. Clinical manifestations are monitored daily and graded as follows: grade 0, no definite muscular weakness after exercise; grade 1, moderate muscular weakness after exercise; grade 2, weakness at rest; and grade 3, severe muscle weakness, paralysis, dehydration, moribund. In FIG. 5, clinical weakness developed 1 to 2 weeks after the first boost, paralleling the increased levels of AChR antibodies, in which 88% of the B6 mice developed EAMG (15 of 17 mice; not shown,

[0025] ). This model is used to determine if tol-WAEV-AChR administration with or without loading of tolerogenic substances can prevent and / or treat disease and modulate anti- AChR IgG (FIG. 6). Negative controls are tolerogenic substances with WAEVs containing no antigen or irrelevant antigen (e.g., influenza M2) to rule out the possibility that WAEV-delivered tolerogenic substance alone could be responsible for suppression of disease. Once each tolerogenic substance has been evaluated, those that demonstrate significant tolerance induction (z.e., suppression of disease signs and anti-AChT IgG titers) are evaluated in combinations to optimize the tol-WAEV activity.

[0088] The following studies are performed:

[0089] A first study tests 5 formulations of WAEV-AChR containing no adjuvant, rapamycin, tacrolimus, Vitamin D3, and Ret Acid, respectively, administered at an expected high dose level of 109WAEV-AChR given every 3 days for 12 days in which the first disease immunization dose is given on 14thday (N=12 mice / formulation group = 60 mice total). Because each tolerogenic substance has an established history of demonstrating tolerogenic activity in the presence of antigen- specific immunotherapy in several animal models of autoimmunity, each of these “lipophilic” substances should effectively load into WAEVs and promote tolerance in vivo. Also, the relative potency among the 4 tolerogenic substances is determined such that selection of the leading 2 substances advances to the next study.

[0090] A second study evaluates the 2 lead substances individually and in combination using the prevention study design described in study 1. A final optimal formulation advances to the next study that addresses whether such a lead formulation can impact disease. (Up to 60 mice are expected.)

[0091] A third study addresses whether 5 doses given every 3 days immediately after the initial disease immunization suppresses the disease (FIG. 6). (Up to 60 mice are expected.)

[0092] A 4thstudy refines and confirms features of the first 3 studies.

[0093] Demonstration of induction of antigen-specific T cell immunorcgulatory responses in protected, WAEV-treated EAMG animals. In addition to reductions in clinical symptoms and anti-AChR IgG titers evaluated, it is critical to evaluate how tol-WAEV treatment modulated critical T cell compartments as illustrated in FIG. 1. AChR- specific Tregs (CD25+ / FOXP3+), suppressor Th2 (IL-4) and Th3 (TGF- ) cells, and pathogenic CD4+ Thl (IFN-y) / Thl7 (IL-17) effector derived from spleen and lymph nodes are evaluated during and after tol-WAEV treatment in parallel “terminal” groups of mice via ELISPOT (D Systems, Minneapolis, MN) and flow cytometry assays in which an enhancement of Tregs and Th2 / 3 cells and reduction of Th 1 / 17 cells is associated with tolerance induction. Each splenocyte and lymph node lymphocyte sample is also evaluated for proliferative responses to AChR a- peptide antigen T cell epitopes. For such proliferation assays, splenocytes and lymph node cells from each mouse are depleted of CD8+ T cells using rat anti-mouse CD8 Ab (BD Biosciences, https: / / www.bdbiosciences.com / en-us) and paramagnetic beads coated with goat anti-rat IgG (as described previously

[0026] ) resulting in a CD4+ T cell enriched population. Cells are cultured for 4 days in the presence or absence 20-mer peptide antigens spanning the tAChR a subunit sequence (10 pg / ml), or the whole tAChR (2.5 pg / ml), or phytohemagglutinin (PHA; 5 pg / ml) as a positive control. Cell proliferation is determined using the BRDU incorporation method (kit #6813, Cell Signaling Technology, www.cellsignal.com). Because these T cell studies are contingent on the efficacy outcomes of tol-WAEV-AChR , the expected T cells profiles described above should be associated with suppression of disease and anti-AChR IgG titers.

[0094] Results. Animals receiving the tol-WAEV particles should show a specific immune response and an induction of immune tolerance as compared to control animals. The induction of immune tolerance should also ameliorate MG symptoms and disease progression.Example 3: nAChR WAEV construct

[0095] To assess whether nAChR fusion with the four WW domains from the ITCH protein could be used to present nAChR a subunit on the surface of WW domain-activated extracellular vesicles (WAEV), a fusion construct, nAChR-WAEV, having WW domain fused to the modified nAChR a subunit was designed (FIG. 7A). HEK 293T cells were transfected with pcDNA3.1, M2-WAEV or nAChR-WAEV constructs and whole cell lysates and EVs were collected for Western blotting. As shown in FIG. 7B, nAChR WW fusion protein budded out into WAEVs as efficiently as compared to M2 WW fusion protein. NanoSight analysis showed that the size distribution and the number of EVs harvested fromnAChR-WAEV transfected HEK293T cells were similar to EVs harvested from M2-WAEV transfected HEK 293T cells (FIG. 7C).References1. Moorman, C.D., S.J. Sohn, and H. Phee, Emerging Therapeutics for Immune Tolerance: Tolerogenic Vaccines, T cell Therapy, and. IL-2 Therapy. Front Immunol, 2021. 12: p. 657768.2. Guimaraes, L.E., et al., Vaccines, adjuvants and autoimmunity. Pharmacol Res, 2015. 100: p. 190-209.3. Richardson, N. and D.C. Wraith, Advancement of antigen- specific immunotherapy: knowledge transfer between allergy and autoimmunity. Immunother Adv, 2021. 1(1): p. ltab009.4. Mosanya, C.H. and J.D. Isaacs, Tolerising cellular therapies: what is their promise for autoimmune disease? Ann Rheum Dis, 2019. 78(3): p. 297-310.5. Nazimek, K. and K. Bryniarski, Approaches to inducing antigen- specific immune tolerance in allergy and autoimmunity: Focus on antigen-presenting cells and extracellular vesicles. Scand J Immunol, 2020. 91(6): p. el2881.6. Steinman, L., et al., Antigen- specific tolerance to self-antigens in protein replacement therapy, gene therapy and autoimmunity. Curr Opin Immunol, 2019. 61: p. 46-53.7. Streeter, H.B. and D.C. Wraith, Manipulating antigen presentation for antigenspecific immunotherapy of autoimmune diseases. Curr Opin Immunol, 2021. 70: p. 75-81.8. Ludwig, R.J., et al., Mechanisms of Autoantibody-Induced Pathology. Front Immunol, 2017. 8: p. 603.9. Murphy, D.E., et al., Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking. Exp Mol Med, 2019. 51(3): p. 1-12.10. Choi, S., et al., Displaying and Delivering Viral Membrane Antigens via IVIV domain- Activated Extracellular Vesicles (WAEVs) Sci. Adv., 2023. 9(4): eade2708..11. Fu, Y., et al., Umbilical cord mesenchymal stem cell-derived exosomes alleviate collagen-induced arthritis by balancing the population ofTh!7 and regulatory T cells. FEBS Lett, 2022.12. Ou, Q., et al., Small extracellular vesicles derived from PD-L1 -modified mesenchymal stem cell promote Tregs differentiation and prolong allograft survival. Cell Tissue Res, 2022. 389(3): p. 465-481.13. Carballido, J.M. and P. Santamaria, Taming autoimmunity: Translating antigenspecific approaches to induce immune tolerance. J Exp Med, 2019. 216(2): p. 247-250.14. 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[0096] Human WWP1 amino acid sequence (uniprot.org / uniprot / Q9H0M0). The four underlined WW domains correspond to amino acids 349 - 382 (WW1), 381 - 414 (WW2), 456 - 489 (WW3), and 496 - 529 (WW4).MATASPRSDTSNNHSGRLQLQVTVSSAKLKRKKNWFGTAIYTEVVVDGEI 50 TKTAKSSSSSNPKWDEQLTVNVTPQTTLEFQVWSHRTLKADALLGKATID 100 LKQALLIHNRKLERVKEQLKLSLENKNGIAQTGELTVVLDGLVIEQENIT 150NCSSSPTIEIQENGDALHENGEPSARTTARLAVEGTNGIDNHVPTSTLVQ 200NSCCSYVVNGDNTPSSPSQVAARPKNTPAPKPLASEPADDTVNGESSSFA 250PTDNASVTGTPVVSEENALSPNCTSTTVEDPPVQEILTSSENNECIPSTS 300AELESEARSILEPDTSNSRSSSAFEAAKSRQPDGCMDPVRQQSGNANTET 350LPSGWEQRKDPHGRTYYVDHNTRTTTWERPQPLPPGWERRVDDRRRVYYV 400DHNTRTTTWQRPTMESVRNFEOWOSORNOLOGAMQQFNORYLYSASMLAA 450ENDPYGPLPPGWEKRVDSTDRVYFVNHNTKTTQWEDPRTQGLQNEEPLPE 500GWEIRYTREGVRYFVDHNTRTTTFKDPRNGKSSVTKGGPOIAYERGFRWK 550LAHFRYLCQSNALPSHVKINVSRQTLFEDSFQQIMALKPYDLRRRLYVIF 600RGEEGLDYGGLAREWFFLLSHEVLNPMYCLFEYAGKNNYCLQINPASTIN 650 PDHLSYFCFIGRFIAMALFHGKFIDTGFSLPFYKRMLSKKLTIKDLESID 700TEFYNSLIWIRDNNIEECGLEMYFSVDMEILGKVTSHDLKLGGSNILVTE 750ENKDEYIGLMTEWRFSRGVQEQTKAFLDGFNEVVPLQWLQYFDEKELEVM 800 LCGMQEVDLADWQRNTVYRHYTRNSKQIIWFWQFVKETDNEVRMRLLQFV 850 TGTCRLPLGGFAELMGSNGPQKFCIEKVGKDTWLPRSHTCFNRLDLPPYK 900SYEQLKEKLLFAIEETEGFGQE (SEQ ID NO: 25) 922

[0097] WWI (349-382): ETLPSGWEQRKDPHGRTYYVDHNTRTTTWERPQP (SEQ ID NO: 26).

[0098] WW2 (381-414):QPLPPGWERRVDDRRRVYYVDHNTRTTTWQRPTM (SEQ ID NO: 27).

[0099] WW3 (456-489):ENDPYGPLPPGWEKRVDSTDRVYFVNHNTKTTQWEDPRT (SEQ ID NO: 28).

[0100] WW4 (496-529):EPLPEGWEIRYTREGVRYFVDHNTRTTTFKDPRN (SEQ ID NO: 29).

[0101] Human WWP2 amino acid sequence (uniprot.org / uniprot / 000308). The four underlined WW domains correspond to amino acids 300 - 333 (WW1), 330 - 363 (WW2), 405 - 437 (WW3), and 444 - 547 (WW4).MASASSSRAG VALPFEKSQL TLKVVSAKPK VHNRQPRINS YVEVAVDGLP 50SETKKTGKRI GSSELLWNEI IILNVTAQSH LDLKVWSCHT LRNELLGTAS 100VNLSNVLKNN GGKMENMQLT LNLQTENKGS VVSGGELTIF LDGPTVDLGN 150VPNGSALTDG SQLPSRDSSG TAVAPENRHQ PPSTNCFGGR SRTHRHSGAS 200ARTTPATGEQ SPGARSRHRQ PVKNSGHSGL ANGTVNDEPT TATDPEEPSV 250VGVTSPPAAP LSVTPNPNTT SLPAPATPAE GEEPSTSGTQ QLPAAAQAPD 300ALPAGWEORE LPNGRVYYVD HNTKTTTWER PLPPGWEKRT DPRGRFYYVD 350HNTRTTTWQR PTAEYVRNYE QWQSQRNQLQ GAMQHFSQRF LYQSSSASTD 400HDPLGPLPPG WEKRQDNGRV YYVNHNTRTT QWEDPRTQGM IQEPALPPGW 450EMKYTSEGVR YFVDHNTRTT TFKDPRPGFE SGTKQGSPGA YDRSFRWKYH 500QFRFECHSNA EPSHVKISVS RQTEFEDSFQ QIMNMKPYDE RRREYIIMRG 550EEGEDYGGIA REWFFEESHE VENPMYCEFE YAGKNNYCEQ INPASSINPD 600HETYFRFIGR FIAMAEYHGK FIDTGFTEPF YKRMENKRPT EKDEESIDPE 650FYNSIVWIKE NNEEECGEEE YFIQDMEIEG KVTTHEEKEG GESIRVTEEN 700KEEYIMEETD WRFTRGVEEQ TKAFEDGFNE VAPEEWERYF DEKEEEEMEC 750GMQEIDMSDW QKSTIYRHYT KNSKQIQWFW QVVKEMDNEK RIREEQFVTG 800TCREPVGGFA EEIGSNGPQK FCIDKVGKET WEPRSHTCFN REDEPPYKSY 850EQLREKLLYA IEETEGFGQE (SEQ ID NO: 30) 870

[0102] WW1 (300-333):DALPAGWEQRELPNGRVYYVDHNTKTTTWERPLP (SEQ ID NO: 31).

[0103] WW2 (330-363):PLPPGWEKRT DPRGRFYYVDHNTRTTTWQRPTA (SEQ ID NO: 32).

[0104] WW3 (405-437):HDPLGPLPPGWEKRQDNGRVYYVNHNTRTTQWEDPRT (SEQ ID NO: 33)

[0105] WW4 (444-477):PALPPGWEMKYTSEGVRYFVDHNTRTTTFKDPRP (SEQ ID NO: 34).

[0106] Human Nedd4-1 amino acid sequence (uniprot.org / uniprot / P46934). The four underlined WW domains correspond to amino acids 610 - 643 (WW1), 767 - 800 (WW2), 840 - 873 (WW3), and 892 - 925 (WW4).MAQSLRLHFAARRSNTYPLSETSGDDLDSHVHMCFKRPTRISTSNVVQMK 50LTPRQTALAPLIKENVQSQERSSVPSSENVNKKSSCLQISLQPTRYSGYL 100QSSNVLADSDDASFTCILKDGIYSSAVVDNELNAVNDGHLVSSPAICSGS 150LSNFSTSDNGSYSSNGSDFGSCASITSGGSYTNSVISDSSSYTFPPSDDT 200FLGGNLPSDSTSNRSVPNRNTTPCEIFSRSTSTDPFVQDDLEHGLEIMKL 250PVSRNTKIPLKRYSSLVIFPRSPSTTRPTSPTSLCTLLSKGSYQTSHQFI 300ISPSEIAHNEDGTSAKGFLSTAVNGLRLSKTICTPGEVRDIRPLHRKGSL 350QKKIVLSNNTPRQTVCEKSSEGYSCVSVHFTQRKAATLDCETTNGDCKPE 400MSEIKLNSDSEYIKLMHRTSACLPSSQNVDCQININGELERPHSQMNKNH 450GILRRSISLGGAYPNISCLSSLKHNCSKGGPSQLLIKFASGNEGKVDNLS 500RDSNRDCTNELSNSCKTRDDFLGQVDVPLYPLPTENPRLERPYTFKDFVL 550HPRSHKSRVKGYLRLKMTYLPKTSGSEDDNAEQAEELEPGWVVLDQPDAA 600CHLQQQQEPSPLPPGWEERQDILGRTYYVNHESRRTQWKRPTPQDNLTDA 650ENGNIQLQAQRAFTTRRQISEETESVDNRESSENWEIIREDEATMYSNQA 700FPSPPPSSNLDVPTHLAEELNARLTIFGNSAVSQPASSSNHSSRRGSLQA 750YTFEEOPTLPVLLPTSSGLPPGWEEKODERGRSYYVDHNSRTTTWTKPTV 800OATVETSOLTSSOSSAGPOSOASTSDSGOOVTOPSEIEQGFLPKGWEVRH 850APNGRPFFIDHNTKTTTWEDPRLKIPAHLRGKTSLDTSNDLGPLPPGWEE 900RTHTDGRIFYINHNIKRTOWEDPRLENVAITGPAVPYSRDYKRKYEFFRR 950KLKKQNDIPNKFEMKLRRATVLEDSYRRIMGVKRADFLKARLWIEFDGEK 1000GLDYGGVAREWFFLISKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDH 1050LSYFKFIGRVAGMAVYHGKLLDGFFIRPFYKMMLHKPITLHDMESVDSEY 1100YNSLRWILENDPTELDLRFIIDEELFGQTHQHELKNGGSEIVVTNKNKKE 1150YIYLVIQWRFVNRIQKQMAAFKEGFFELIPQDLIKIFDENELELLMCGLG 1200DVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIRLLQFVTGTS 1250RVPMNGFAELYGSNGPQSFTVEQWGTPEKLPRAHTCFNRLDLPPYESFEE 1300LWDKLQMAIENTQGFDGVD (SEQ ID NO: 35) 1319

[0107] WW1 (610-643):SPLPPGWEERQDILGRTYYVNHESRRTQWKRPTP (SEQ ID NO: 36)

[0108] WW2 (767-800):SGLPPGWEEKQDERGRSYYVDHNSRTTTWTKPTV (SEQ ID NO: 37).

[0109] WW3 (840-873):GFLPKGWEVRHAPNGRPFFIDHNTKTTTWEDPRL (SEQ ID NO: 38).

[0110] WW4 (892-925):GPLPPGWEERTHTDGRIFYINHNIKRTQWEDPRL (SEQ ID NO: 39)

[0111] Human Nedd4-2 amino acid sequence Qgil213614721reflNP_056092.21 E3 ubiquitin- protein ligase NEDD4-like isoform 3 [Homo sapiens]). The four underlined WW domains correspond to amino acids 198 - 224 (WW1), 368 - 396 (WW2), 480 - 510 (WW3), and 531 - 561 (WW4).MATGLGEPVYGLSEDEGESRILRVKVVSGIDLAKKDIFGASDPYVKLSLYVADENRE LALVQTKTIKKTLNPKWNEEFYFRVNPSNHRLLFEVFDENRLTRDDFLGQVDVPLSH LPTEDPTMERPYTFKDFLLRPRSHKSRVKGFLRLKMAYMPKNGGQDEENSDQRDD MEHGWEVVDSNDSASOHOEELPPPPLPPGWEEKVDNLGRTYYVNHNNRTTQWHRP SLMDVSSESDNNIRQINQEAAHRRFRSRRHISEDLEPEPSEGGDVPEPWETISEEVNIAGDSLGLALPPPPASPGSRTSPQELSEELSRRLQITPDSNGEQFSSLIQREPSSRLRSCSVT DAVAEOGHLPPPSVAYVHTTPGLPSGWEERKDAKGRTYYVNHNNRTTTWTRPIMQ LAEDGASGSATNSNNHLIEPQIRRPRSLSSPTVTLSAPLEGAKDSPVRRAVKDTLSNP OSPOPSPYNSPKPOHKVTQSFLPPGWEMRIAPNGRPFFIDHNTKTTTWEDPRLKFPVH MRSKTSENPNDEGPEPPGWEERIHEDGRTFYIDHNSKITOWEDPREQNPAITGPAVPYSREFKQKYDYFRKKEKKPADIPNRFEMKEHRNNIFEESYRRIMSVKRPDVEKAREWI EFESEKGEDYGGVAREWFFEESKEMFNPYYGEFEYSATDNYTEQINPNSGECNEDHE SYFTFIGRVAGEAVFHGKEEDGFFIRPFYKMMEGKQITENDMESVDSEYYNSEKWIE ENDPTEEDEMFCIDEENFGQTYQVDEKPNGSEIMVTNENKREYIDEVIQWRFVNRVQ KQMNAFEEGFTEEEPIDEIKIFDENEEEEEMCGEGDVDVNDWRQHSIYKNGYCPNHP VIQWFWKAVEEMDAEKRIREEQFVTGTSRVPMNGFAEEYGSNGPQEFTIEQWGSPE KEPRAHTCFNREDEPPYETFEDEREKEEMAVENAQGFEGVD ( SEQ ID NO: 40 )

[0112] WW1 (198 - 224):GWEEKVDNEGRTYYVNHNNRTTQWHRP (SEQ ID NO: 41)

[0113] WW2 (368 - 396):PSGWEERKDAKGRTYYVNHNNRTTTWTRP (SEQ ID NO: 42).

[0114] WW3 (480 - 510):PPGWEMRIAPNGRPFFIDHNTKTTTWEDPRL (SEQ ID NO: 43).

[0115] WW4 (531 - 561):PPGWEERIHLDGRTFYIDHNSKITQWEDPRL (SEQ ID NO : 44)

[0116]

[0117] Human Smurf 1 amino acid sequence (uniprot.org / uniprot / Q9HCE7). The two underlined WW domains correspond to amino acids 234 - 267 (WW 1) and 306 - 339 (WW2).MSNPGTRRNG SSIKIRLTVL CAKNLAKKDF FRLPDPFAKI VVDGSGQCHS 50 TDTVKNTLDP KWNQHYDLYV GKTDS ITI SV WNHKKIHKKQ GAGFLGCVRL 100 LSNAI SRLKD TGYQRLDLCK LNPSDTDAVR GQIVVSLQTR DRIGTGGSVV 150 DCRGLLENEG TVYEDSGPGR PLSCFMEEPA PYTDSTGAAA GGGNCRFVES 200 PSQDQRLQAQ RLRNPDVRGS LQTPQNRPHG HQSPELPEGY EQRTTVQGQV 250 YFLHTQTGVS TWHDPRIPSP SGTIPGGDAA FLYEFLLQGH TSEPRDLNSV 300 NCDELGPLPP GWEVRSTVSG RIYFVDHNNR TTQFTDPRLH HIMNHQCQLK 350 EPSQPLPLPS EGSLEDEELP AQRYERDLVQ KLKVLRHELS LQQPQAGHCR 400 IEVSREEIFE ESYRQIMKMR PKDLKKRLMV KFRGEEGLDY GGVAREWLYL 450 LCHEMLNPYY GLFQYSTDNI YMLQINPDSS INPDHLSYFH FVGRIMGLAV 500 FHGHYINGGF TVPFYKQLLG KPIQLSDLES VDPELHKSLV WILENDITPV 550 LDHTFCVEHN AFGRILQHED KPNGRNVPVT EENKKEYVRL YVNWRFMRGI 600 EAQFLALQKG FNELIPQHLL KPFDQKELEL IIGGLDKIDL NDWKSNTRLK 650 HCVADSNIVR WFWQAVETFDEERRARLLQF VTGSTRVPLQ GFKALQGSTG 700 AAGPRLFTIH LIDANTDNLP KAHTCFNRID IPPYESYEKL YEKLLTAVEE 750 TCGFAVE (SEQ ID NO: 45)

[0118] WW1 (234-267):PELPEGYEQRTTVQGQVYFLHTQTGVSTWHDPRI (SEQ ID NO: 46).

[0119] WW2 (306-339):GPLPPGWEVRSTVSGRIYFVDHNNRTTQFTDPRL (SEQ ID NO: 47).

[0120] Human Smurf2 amino acid sequence (uniprot.org / uniprot / Q9HAU4). The three underlined WW domains correspond to amino acids 157 - 190 (WW1), 251 - 284 (WW2), and 297 - 330 (WW3).MSNPGGRRNGPVKLRLTVLCAKNLVKKDFFRLPDPFAKVVVDGSGQCHST 50 DTVKNTLDPKWNQHYDLYIGKSDSVTISVWNHKKIHKKQGAGFLGCVRLL 100 SNAINRLKDTGYQRLDLCKLGPNDNDTVRGQIVVSLQSRDRIGTGGQVVD 150 CSRLFDNDLPDGWEERRTASGRIQYLNHITRTTQWERPTRPASEYSSPGR 200PLSCFVDENTPISGTNGATCGQSSDPRLAERRVRSQRHRNYMSRTHLHTP 250 PDLPEGYEQRTTQQGQVYFLHTQTGVSTWHDPRVPRDLSNINCEELGPLP 300PGWEIRNTATGRVYFVDHNNRTTQFTDPRLSANLHLVLNRQNQLKDQOOQ 350QVVSLCPDDTECLTVPRYKRDLVQKLKILRQELSQQQPQAGHCRIEVSRE 400 EIFEESYRQVMKMRPKDLWKRLMIKFRGEEGLDYGGVAREWLYLLSHEML 450 NPYYGLFQYSRDDIYTLQINPDSAVNPEHLSYFHFVGRIMGMAVFHGHYI 500DGGFTLPFYKQLLGKSITLDDMELVDPDLHNSLVWILENDITGVLDHTFC 550 VEHNAYGEIIQHELKPNGKSIPVNEENKKEYVRLYVNWRFLRGIEAQFLA 600LQKGFNEVIPQHLLKTFDEKELELIICGLGKIDVNDWKVNTRLKHCTPDS 650NIVKWFWKAVEFFDEERRARLLQFVTGSSRVPLQGFKALQGAAGPRLFTI 700HQIDACTNNLPKAHTCFNRIDIPPYESYEKLYEKLLTAIEETCGFAVE 748(SEQ ID NO: 48)

[0121] WW1 (157-190):NDLPDGWEERRTASGRIQYLNHITRTTQWERPTR (SEQ ID NO: 49).

[0122] WW2 (251-284):PDLPEGYEQRTTQQGQVYFLHTQTGVSTWHDPRV (SEQ ID NO: 50).

[0123] WW3 (297-330):GPLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRL ( SEQ ID NO : 51).

[0124] Human ITCH amino acid sequence (uniprot.org / uniprot / Q96J02). The four underlined WW domains correspond to amino acids 326 - 359 (WW1), 358 - 391 (WW2), 438 - 471 (WW3), and 478 - 511 (WW4).MSDSGSQLGSMGSLTMKSQLQITVISAKLKENKKNWFGPSPYVEVTVDGQ 50 SKKTEKCNNTNSPKWKQPLTVIVTPVSKLHFRVWSHQTLKSDVLLGTAAL 100 DIYETLKSNNMKLEEVVVTLQLGGDKEPTETIGDLSICLDGLQLESEVVT 150 NGETTCSENGVSLCLPRLECNSAISAHCNLCLPGLSDSPISASRVAGFTG 200ASQNDDGSRSKDETRVSTNGSDDPEDAGAGENRRVSGNNSPSLSNGGFKP 250SRPPRPSRPPPPTPRRPASVNGSPSATSESDGSSTGSLPPTNTNTNTSEG 300ATSGLIIPLTISGGSGPRPLNPVTQAPLPPGWEQRVDQHGRVYYVDHVEK 350RTTWDRPEPLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLESVRNYEQW 400QLQRSQLQGAMQQFNQRFIYGNQDLFATSQSKEFDPLGPLPPGWEKRTDS 450 NGRVYFVNHNTRITQWEDPRSQGOLNEKPLPEGWEMRFTVDGIPYFVDHN 500 RRTTTYIDPRTGKSALDNGPOIAYVRDFKAKVOYFRFWCOQLAMPOHIKI 550TVTRKTLFEDSFQQIMSFSPQDLRRRLWVIFPGEEGLDYGGVAREWFFLL 600 SHEVLNPMYCLFEYAGKDNYCLQINPASYINPDHLKYFRFIGRFIAMALF 650 HGKFIDTGFSLPFYKRILNKPVGLKDLESIDPEFYNSLIWVKENNIEECD 700LEMYFSVDKEILGEIKSHDLKPNGGNILVTEENKEEYIRMVAEWRLSRGV 750EEQTQAFFEGFNEILPQQYLQYFDAKELEVLLCGMQEIDLNDWQRHAIYR 800 HYARTSKQIMWFWQFVKEIDNEKRMRLLQFVTGTCRLPVGGFADLMGSNG 850 PQKFCIEKVGKENWLPRSHTCFNRLDLPPYKSYEQLKEKLLFAIEETEGF 900GQE 903 (SEQ ID NO: 1)

[0125] ITCH WW1 (326-359):APLPPGWEQRVDQHGRVYYVDHVEKRTTWDRPEP (SEQ ID NO : 13)

[0126]

[0127] ITCH WW2 (358-391):EPLPPGWERRVDNMGRI YYVDHFTRTTTWQRPTL ( SEQ ID NO : 14).

[0128] ITCH WW3 (438-471):GPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRS ( SEQ ID NO : 4).

[0129] ITCH WW4 (478-511):KPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRT ( SEQ ID NO : 6).

[0130] Human NEDL1 amino acid sequence (uniprot.org / uniprot / Q76N89). The two underlined WW domains correspond to amino acids 829 - 862 (WW1), and 1018 - 1051 (WW2).MLLHLCSVKNLYQNRFLGLAAMASPSRNSQSRRRCKEPLRYSYNPDQFHN 50 MDLRGGPHDGVTIPRSTSDTDLVTSDSRSTLMVSSSYYSIGHSQDLVIHW 100 DIKEEVDAGDWIGMYLIDEVLSENFLDYKNRGVNGSHRGQIIWKIDASSY 150 FVEPETKICFKYYHGVSGALRATTPSVTVKNSAAPIFKSIGADETVQGQG 200 SRRLISFSLSDFQAMGLKKGMFFNPDPYLKISIQPGKHSIFPALPHHGQE 250 RRSKIIGNTVNPIWQAEQFSFVSLPTDVLEIEVKDKFAKSRPIIKRFLGK 300 LSMPVQRLLERHAIGDRVVSYTLGRRLPTDHVSGQLQFRFEITSSIHPDD 350EEISLSTEPESAQIQDSPMNNLMESGSGEPRSEAPESSESWKPEQLGEGS 400VPDGPGNQSIELSRPAEEAAVITEAGDQGMVSVGPEGAGELLAQVQKDIQ 450PAPSAEELAEQLDLGEEASALLLEDGEAPASTKEEPLEEEATTQSRAGRE 500EEEKEQEEEGDVSTLEQGEGRLQLRASVKRKSRPCSLPVSELETVIASAC 550GDPETPRTHYIRIHTLLHSMPSAQGGSAAEEEDGAEEESTLKDSSEKDGL 600SEVDTVAADPSALEEDREEPEGATPGTAHPGHSGGHFPSLANGAAQDGDT 650HPSTGSESDSSPRQGGDHSCEGCDASCCSPSCYSSSCYSTSCYSSSCYSA 700SCYSPSCYNGNRFASHTRFSSVDSAKISESTVFSSQDDEEEENSAFESVP 750DSMQSPELDPESTNGAGPWQDELAAPSGHVERSPEGLESPVAGPSNRREG 800ECPILHNSOPVSQLPSLRPEHHHYPTIDEPLPPNWEARIDSHGRVFYVDH 850VNRTTTWORPTAAATPDGMRRSGSIOQMEOLNRRYONIORTIATERSEED 900SGSQSCEQAPAGGGGGGGSDSEAESSQSSLDLRREGSLSPVNSQKITLLL 950QSPAVKFITNPEFFTVLHANYSAYRVFTSSTCLKHMILKVRRDARNFERY 1000OHNRDLVNFINMFADTRLELPRGWEIKTDQOGKSFFVDHNSRATTFIDPR 1050IPLQNGRLPNHLTHRQHLQRLRSYSAGEASEVSRNRGASLLARPGHSLVA 1100AIRSQHQHESLPLAYNDKIVAFLRQPNIFEMLQERQPSLARNHTLREKIH 1150YIRTEGNHGLEKLSCDADLVILLSLFEEEIMSYVPLQAAFHPGYSFSPRC 1200SPCSSPQNSPGLQRASARAPSPYRRDFEAKLRNFYRKLEAKGFGQGPGKI 1250KLIIRRDHLLEGTFNQVMAYSRKELQRNKLYVTFVGEEGLD YSGPSREFF 1300FLLSQELFNPYYGLFEYS ANDTYTVQISPMS AFVENHLEWFRFSGRILGL 1350ALIHQYLLDAFFTRPFYKALLRLPCDLSDLEYLDEEFHQSLQWMKDNNIT 1400DILDLTFTVNEEVFGQVTERELKSGGANTQVTEKNKKEYIERMVKWRVER 1450GVVQQTEALVRGFYEVVDSRLVS VFDARELELVIAGTAEIDLNDWRNNTE 1500YRGGYHDGHLVIRWFWAAVERFNNEQRLRLLQFVTGTSSVPYEGFAALRG 1550SNGLRRFCIEKWGKITSLPRAHTCFNRLDLPPYPS YSMLYEKLLTA VEET 1600STFGLE (SEQ ID NO: 52) 1606

[0131] WW1 (829-862):PLPPNWEARIDSHGRVFYVDHVNRTTTWQRPTA (SEQ ID NO : 53)

[0132] WW2 (1018-1051):LELPRGWEIKTDQQGKSFFVDHNSRATTFIDPRI (SEQ ID NO: 54)

[0133]

[0134] Human NEDL2 amino acid sequence (uniprot.org / uniprot / Q9P2P5). The two underlined WW domains correspond to amino acids 807 - 840 (WW1) and 985 - 1018 (WW2).MASSAREHLLFVRRRNPQMRYTLSPENLQSLAAQSSMPENMTLQRANSDT 50DLVTSESRSSLTASMYEYTLGQAQNLIIFWDIKEEVDPSDWIGLYHIDEN 100 SPANFWDSKNRGVTGTQKGQIVWRIEPGPYFMEPEIKICFKYYHGISGAL 150 RATTPCITVKNPAVMMGAEGMEGGASGNLHSRKLVSFTLSDLRAVGLKKG 200MFFNPDPYLKMSIQPGKKSSFPTCAHHGQERRSTIISNTTNPIWHREKYS 250 FFALLTDVLEIEIKDKFAKSRPIIKRFLGKLTIPVQRLLERQAIGDQMLS 300 YNLGRRLPADHVSGYLQFKVEVTSSVHEDASPEAVGTILGVNSVNGDLGS 350PSDDEDMPGSHHDSQVCSNGPVSEDSAADGTPKHSFRTSSTLEIDTEELT 400STSSRTSPPRGRQDSLNDYLDAIEHNGHSRPGTATCSERSMGASPKLRSS 450FPTDTRLNAMLHIDSDEEDHEFQQDLGYPSSLEEEGGLIMFSRASRADDG 500 SLTSQTKLEDNPVENEEASTHEAASFEDKPENLPELAESSLPAGPAPEEG 550 EGGPEPQPSADQGSAELCGSQEVDQPTSGADTGTSDASGGSRRAVSETES 600LDQGSEPSQVSSETEPSDPARTESVSEASTRPEGESDLECADSSCNESVT 650 TQLSSVDTRCSSLESARFPETPAFSSQEEEDGACAAEPTSSGPAEGSQES 700 VCTAGSLPVVQVPSGEDEGPGAESATVPDQEELGEVWQRRGSLEGAAAAA 750ESPPQEEGSAGEAQGTCEGATAQEEGATGGSQANGHQPLRSLPSVRQDVS 800RYORVDEALPPNWEARIDSHGRIFYVDHVNRTTTWORPTAPPAPOVLORS 850NSIQQMEQLNRRYQSIRRTMTNERPEENTNAIDGAGEEADFHQASADFRR 900 ENILPHSTSRSRITLLLQSPPVKFLISPEFFTVLHSNPSAYRMFTNNTCL 950 KHMITKVRRDTHHFERYOHNRDLVGFLNMFANKOLELPRGWEMKHDHOGK 1000AFFVDHNSRTTTFIDPRLPLOSSRPTSALVHROHLTRORSHSAGEVGEDS 1050RHAGPPVLPRPSSTFNTVSRPQYQDMVPVAYNDKIVAFLRQPNIFEILQE 1100RQPDLTRNHSLREKIQFIRTEGTPGLVRLSSDADLVMLLSLFEEEIMSYV 1150 PPHALLHPSYCQSPRGSPVSSPQNSPGTQRANARAPAPYKRDFEAKLRNF 1200 YRKLETKGYGQGPGKLKLIIRRDHLLEDAFNQIMGYSRKDLQRNKLYVTF 1250VGEEGLDYSGPSREFFFLVSRELFNPYYGLFEYS ANDTYTVQISPMS AFV 1300DNHHEWFRFSGRILGLALIHQYLLDAFFTRPFYKALLRILCDLSDLEYLD 1350EEFHQSLQWMKDNDIHDILDLTFTVNEEVFGQITERELKPGGANIPVTEK 1400NKKEYIERMVKWRIERGVVQQTESLVRGFYEVVDARLVSVFDARELELVI 1450AGTAEIDLSDWRNNTEYRGGYHDNHIVIRWFWAAVERFNNEQRLRLLQFV 1500TGTSSIPYEGFASLRGSNGPRRFCVEKWGKITALPRAHTCFNRLDLPPYP 1550SFSMLYEKLLTAVEETSTFGLE 1572 (SEQ ID NO: 55)

[0135] WW1 (807-840):EALPPNWEARIDSHGRIFYVDHVNRTTTWQRPTA (SEQ ID NO: 56)

[0136]

[0137] WW2 (985-1018):LELPRGWEMKHDHQGKAFFVDHNSRTTTFIDPRL (SEQ ID NO: 57)

[0138]

[0139] Exemplary TSG101 sequences include the following sequences (the UEV domain in these sequences includes amino acids 1-145 and is underlined in the sequences below):

[0140] )gil54541401reflNP_006283.11 tumor susceptibility gene 101 protein [Homo sapiens]MAVSESQLKKMVSKYKYRDLTVRETVNVITLYKDLKPVLDSYVFNDGSSRELMNLTGTIPVPYRGNTYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKI YLPYLHEWKHPQSDLLGLIQVMIVVFGDEPPVFSRPISASYPPYQATGPPNTSYMPGM PGGISPYPSGYPPNPSGYPGCPYPPGGPYPATTSSQYPSQPPVTTVGPSRDGTISEDTIR ASLISAVSDKLRWRMKEEMDRAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDT IFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY (SEQ ID NO: 58)

[0141] )gil 112307801reflNP_068684.ll tumor susceptibility gene 101 protein [Mus musculus]MAVSESQLKKMMSKYKYRDLTVRQTVNVIAMYKDLKPVLDSYVFNDGSSRELVNLTGTIPVRYRGNIYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHDWKHPRSELLELIQIMIVIFGEEPPVFSRPTVSASYPPYTATGPPNTSYMPGM PSGISAYPSGYPPNPSGYPGCPYPPAGPYPATTSSQYPSQPPVTTVGPSRDGTISEDTIR ASLISAVSDKLRWRMKEEMDGAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVA EVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDT IFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY (SEQ IDNO: 59)

[0142] )gil483740871reflNP_853659.21 tumor susceptibility gene 101 protein [Rattus norvegicus] MAVSESQLKKMMSKYKYRDLTVRQTVNVIAMYKDLKPVLDSYVFNDGSSR ELVNLTGTIPVRYRGNIYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHDWKHPRSELLELIQIMIVIFGEEPPVFSRPTVSASYPPYTAAGPPNTSYLPSM PSGISAYPSGYPPNPSGYPGCPYPPAGPYPATTSSQYPSQPPVTTAGPSRDGTISEDTIR ASLISAVSDKLRWRMKEEMDGAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVA EVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDT IFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY (SEQ ID NO: 60).

Claims

CLAIMSWhat is claimed is:

1. A fusion protein comprising:(a) a WW-containing domain;(b) a transmembrane domain; and(c) an extracellular domain, wherein the extracellular domain is a self-antigen domain.2 The fusion protein of claim 1, wherein the self-antigen domain is associated with an autoimmune disease.3 The fusion protein of any of claims 1-2, wherein the fusion protein does not comprise an arrestin domain containing protein 1 (ARRDC1).4 The fusion protein of any one of claims 1-3, wherein the WW-containing domain comprises at least one WW domain.5 The fusion protein of any one of claims 1-4, wherein the WW-containing domain comprises at least two WW domain.6 The fusion protein of any one of claims 1-5, wherein the WW-containing domain comprises at least three WW domain.7 The fusion protein of any one of claims 1-6, wherein the WW-containing domain comprises at least four WW domain.8 The fusion protein of any of claims 4-7, wherein the WW-containing domain is obtained from a NEDD4 E3 ligase domain.9 The fusion protein of claim 8, wherein the NEDD4 E3 ligase is selected from the group consisting of ITCH, NEDD4, NEDD4 L, WWP1, WWP2, Smurfl, Smurf2, BUL1, and NEDL2.10 The fusion protein of claim 8, wherein the NEDD4 E3 ligase is the ITCH protein.11 The fusion protein of any one of claims 1-10, wherein the WW-containing domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 1.

12. The fusion protein of any one of claims 1-11, wherein the WW-containing domain comprises the sequence of SEQ ID NO: 1.

13. An isolated nucleic acid encoding a fusion protein of any one of claims 1-12.

14. The isolated nucleic acid of claim 13, operably linked to a promoter.

15. The isolated nucleic acid of claim 14, wherein the promoter is a constitutive promoter, an inducible promoter, or a tissue specific promoter.

16. The isolated nucleic acid of any of claims 13-15 comprising at least one additional regulatory sequence.

17. A WW protein domain activated extracellular vesicle (WAEV) comprising:(a) a lipid bilayer; and(b) the fusion protein of any one of claims 1-12.

18. The WAEV of claim 17 further comprising SCAMP3.

19. The WAEV of claims 17 or 18, wherein the fusion protein does not comprise at least one of the following exosomal markers: CD63; CD81, CD9, and PTGFRN.

20. The WAEV of any one of claims 17-19, wherein the fusion protein does not comprise any of the following exosomal markers: CD63; CD81, CD9, and PTGFRN.21 The WAEV of any one of claims 17-20, wherein the WAEV further comprises a tolerogenic substance.

22. The WAEV of any one of claims 17-21, wherein the tolerogenic substance is selected from the listing of Table 1.

23. A WAEV -producing cell comprising a recombinant expression construct encoding the fusion protein of any one of claims 1-12 under the control of a heterologous promoter.

24. A WAEV -producing cell comprising the isolated nucleic acid of any one of claims 13-16.

25. A method of delivering WAEVs displaying a self-antigen peptide, the method comprising: delivering the fusion protein of any one of claims 1-12, the isolated nucleic acid of any one of claims 13-16, the WAEV of any one of claims 17-22, or theWAEV-producing cell of claim 23 or 24 to a subject, wherein the extracellular protein of the fusion protein comprises a self-antigen.

26. The method of claim 25, wherein the subject is mammalian.

27. The method of claims 25 or 26, wherein the subject is human.

28. The method of any one of claims 25-27, wherein the method further comprises simultaneous or subsequent delivery of a tolerogenic substance.

29. The method of any one of claims 25-27, wherein the WAEV further comprises a tolerogenic substance.

30. The method of claim 28 or 29, wherein the tolerogenic substance is selected from the listing of Table 1.

31. A kit comprising one or more of the fusion protein of any one of claims 1-12, the isolated nucleic acid of any one of claims 13-16, the WAEV of any one of claims 17- 22, or the WAEV-producing cell of claim 23 or 24.

32. The fusion protein of any one of claims 1-12, the isolated nucleic acid of any one of claims 13-16, the WAEV of any one of claims 17-22, the WAEV-producing cell of claim 23 or 24, the method of any one of claims 25-30, or the kit of claim 31, wherein the self-antigen is selected from (a) a self-antigen associated with the autoantibody- mediated diseases of Table 3.

33. A pharmaceutical composition comprising the fusion protein of any one of claims 1- 12 or 32, the isolated nucleic acid of any one of claims 13-16, the WAEV of any one of claims 17-22, or the WAEV-producing cell of claim 23 or 24.

34. A method of treating or preventing an autoimmune disease comprising delivering the fusion protein of any one of claims 1-12 or 32, the isolated nucleic acid of any one of claims 13-16, the WAEV of any one of claims 17-22, or the WAEV-producing cell of claim 23 or 24 to a subject.

35. The use of a fusion protein of any one of claims 1-12 or 32, the isolated nucleic acid of any one of claims 13-16, the WAEV of any one of claims 17-22, the WAEV- producing cell of claim 23 or 24, or the kit of claim 31, to treat an autoimmune disease.

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