Engineered extracellular vesicles

Abstract

The present disclosure provides engineered extracellular vesicles (e.g., exosomes) comprising a fusion polypeptide that includes a Tumor Necrosis Factor-Stimulated Gene-6 (TSG-6) protein or a truncated form thereof displayed on the vesicle surface in a stabilized and biologically active configuration. The engineered extracellular vesicles retain or enhance the anti-inflammatory and cytoprotective activities of TSG-6 and may be used in the prophylaxis or treatment of inflammatory, autoimmune, or injury-related diseases or disorders. Also provided are nucleic acid constructs encoding the fusion polypeptide, compositions comprising the engineered extracellular vesicles, methods for producing the vesicles, and methods for using the vesicles in therapeutic applications.

Description

ENGINEERED EXTRACELLULAR VESICLESREFERENCE TO SEQUENCE LISTINGThis application contains a sequence listing submitted as an electronic xml file named, 63718498_WOPT_ST26” created on November 10, 2025 and having a size in bytes of 89,425 bytes. The information contained in this electronic file is hereby incorporated by reference in its entirety.FIELD OF THE INVENTIONThis invention relates to the generation of extracellular vesicles, including features of extracellular vesicles engineered to deliver signaling, for therapeutic use, including treatment of inflammatory diseases and injury.BACKGROUND

[0001] Extracellular vesicles (EVs) are membrane-bound lipid particles secreted by most types. These vesicles play a central role in intercellular communication by transporting bioactive molecules such as proteins, nucleic acids, lipids, and metabolites to recipient cells. Through these cargo molecules, EVs influence numerous biological processes, including immune modulation, tissue repair, angiogenesis, and neuronal communication. EVs encompass a variety of vesicle types, including micro vesicles, apoptotic bodies, and exosomes, which differ primarily by their size, biogenesis, and molecular composition. Exosomes are a well-characterized subclass of EVs, typically ranging from 30 to 150 nanometers in diameter, originating from the endosomal compartment via a multivesicular body pathway. Upon fusion of multivesicular bodies with the plasma membrane, exosomes are released into the extracellular milieu and can subsequently be taken up by target cells through receptor-mediated endocytosis, phagocytosis, or membrane fusion.

[0002] The biological importance of exosomes arises from their ability to deliver complex molecular signals in a spatially and temporally regulated manner. The lipid bilayer of exosomes provides structural stability and protection to encapsulated molecules, enabling them to circulate systemically and deliver cargo to distal sites without rapid degradation. Moreover, exosomal surface proteins, including tetraspanins (e.g., CD9, CD63, and CD81), integrins, and other membrane-bound molecules, govern tropism and selective uptake by specific recipient cell populations. Consequently, exosomes represent a promising platform for therapeutic delivery because of their inherent biocompatibility, low immunogenicity, and capacity for selective targeting.

[0003] Mesenchymal stem cells (MSCs) have been shown to secrete exosomes with potent antiinflammatory and tissue-protective effects. Numerous studies have demonstrated that MSC-derived exosomes can recapitulate many of the therapeutic benefits associated with MSC therapy, includingimmunomodulation, attenuation of oxidative stress, and enhancement of tissue regeneration. The therapeutic efficacy of MSC-derived exosomes has been attributed, in large part, to the presence of bioactive proteins and enzymes on or within these vesicles. One of these molecules is Tumor Necrosis Factor-Stimulated Gene-6 (TSG-6), a multifunctional glycoprotein with well-documented antiinflammatory and cytoprotective activities.

[0004] TSG-6 (also known as TNFAIP6) is a secreted protein that is rapidly induced in response to inflammatory stimuli, including tumor necrosis factor-alpha (TNFa), interleukin- 1 beta (IL-ip), interferon-gamma (IFNy), and lipopolysaccharide (LPS). It interacts with a variety of extracellular matrix (ECM) components, including hyaluronan, chondroitin sulfate, and other glycosaminoglycans, to modulate inflammatory signaling and tissue remodeling. Mechanistically, TSG-6 exerts anti-inflammatory effects by inhibiting neutrophil migration, reducing pro-inflammatory cytokine expression, and promoting macrophage polarization from a pro-inflammatory (Ml) phenotype toward an anti-inflammatory (M2) phenotype. TSG-6 has been shown to play a key role in models of tissue injury, including myocardial infarction, stroke, arthritis, and traumatic brain injury, where its expression correlates with protection against excessive inflammation and secondary tissue damage.

[0005] In MSCs, TSG-6 is not only secreted as a soluble factor but is also associated with isolated exosomes, where it is believed to contribute significantly to the therapeutic effects of MSC-derived exosomes. Studies have shown that silencing TSG-6 expression in MSCs abolishes the anti-inflammatory and neuroprotective activities of their secretome and exosomes, underscoring TSG-6 as a principal effector molecule. Despite these findings, the use of primary MSCs or their native exosomes for clinical purposes presents substantial limitations. MSC cultures exhibit batch-to-batch variability, finite expansion capacity, and donor-dependent heterogeneity. Additionally, prolonged culture can lead to loss of potency, chromosomal instability, and reduced therapeutic consistency, posing challenges for scalable and reproducible manufacturing. Moreover, mesenchymal stem cells (MSCs) typically require activation by inflammatory stimuli, such as exposure to certain exogenous cytokines, to achieve maximal expression of TSG-6. The requirement for ancillary cytokines during manufacturing may hinder the ability to produce exosomes safely and reproducibly with consistently high expression of this therapeutic molecule.

[0006] Recombinant TSG-6 protein has been developed as a therapeutic agent; however, its clinical translation has been constrained by its physicochemical properties. TSG-6 binds to ECM components and other ligands, leading to aggregation, cell clumping, and rapid clearance from circulation. Moreover, the protein is susceptible to proteolytic cleavage, resulting in degradation and loss of activity during manufacturing and storage. The serum half-life of recombinant human TSG-6 is reported to be less than 10 minutes, which limits its efficacy for systemic administration. These challenges have created a need for more stable and bioavailable forms of TSG-6 that retain biological function but resist degradation and aggregation to achieve effective, stable, and targeted delivery of TSG-6 to inflamed or injured tissues.

[0007] Exosomes represent an advantageous vehicle for TSG-6 delivery because of their ability toprotect and stabilize proteins, extend their circulation half-life, and enable cell-specific targeting. By engineering exosomes to express TSG-6 on their surface, it is possible to recapitulate and enhance the therapeutic mechanisms of MSC-derived exosomes in a defined, manufacturable, and scalable format. Surface expression of TSG-6 on exosomes may improve its bioavailability and biological activity by positioning the protein in an orientation that facilitates interaction with cell-surface receptors and ECM components at sites of injury. Additionally, a high density or valency of TSG-6 on the exosome surface may enhance its ability to modulate target cell signaling by promoting multivalent juxtacrine interactions that drive receptor agonism or antagonism. Moreover, the exosomal membrane environment can stabilize membrane-associated or fusion-linked forms of TSG-6 that resist proteolytic degradation and shedding, further enhancing therapeutic durability.

[0008] The need remains in the art for recombinant, engineered exosomes that display a stabilized form of TSG-6 with enhanced structural integrity, reduced susceptibility to cleavage, and improved manufacturability. Such exosomes could serve as a versatile therapeutic platform for treating a broad range of inflammatory, neurodegenerative, and traumatic conditions, including but not limited to traumatic brain injury, spinal cord injury, ischemic injury, and autoimmune diseases. The development of a TSG-6-expressing exosome provides a means of mimicking the beneficial paracrine effects of MSCs while enabling standardized, large-scale manufacturing using well-characterized cell lines.

[0009] Accordingly, there is a strong rationale for the invention of engineered exosomes that express a stabilized form of TSG-6 on their surface. These exosomes combine the molecular functionality of TSG-6 with the inherent multivalency, targeting, delivery, and pharmacokinetic advantages of exosomes, providing a new class of biologic therapeutic agents for the treatment of inflammation-associated diseases and tissue injury.SUMMARY

[0010] The compositions and methods provided herein are based, in part, on the discovery that extracellular vesicles can be used to express engineered fusion polypeptides that can modulate biological signal generation. These engineered vesicles, also termed engineered extracellular vesicles, adopt the hallmark biophysical and biochemical features of extracellular vesicles, but are further engineered to express one or more fusion polypeptides comprising vesicle targeting domains and signaling domains, optionally joined by a linker with specific functions. The fusion polypeptides provided herein are designed and produced as nucleic acid constructs (e.g., vectors) and expressed in cells, for example, mammalian cells. In particular, the vesicle targeting domain of each fusion polypeptide anchors the fusion polypeptide to the extracellular vesicle lipid membrane, thereby presenting the signaling domain of the fusion polypeptide. The signaling domain is external to the vesicle membrane and can contact recipient cells via target polypeptides (e.g., proteins or receptors on the extracellular surface of a recipient cell) or polysaccharides (e.g., glycosaminoglycan (CAG) or hyaluronan, also known as hyaluronic acid (HA)). Importantly, this strategy can allow for kinetically favorable signal generation and signal propagation.

[0011] This strategy was applied to improve the stability and expression of a fusion polypeptide or protein expressed on the surface of an engineered extracellular vesicle, wherein the extracellular vesicle is an exosome.

[0012] In various embodiments, the present invention provides an engineered extracellular vesicle comprising at least one fusion polypeptide. In some cases, the fusion polypeptide comprises a signaling domain and at least one vesicle targeting domain. In various embodiments, the fusion polypeptide comprises: a signaling domain in an exterior position relative to a phospholipid bilayer of the extracellular vesicle; a polypeptide linker; and at least one vesicle targeting domain spanning at least partly through the phospholipid bilayer of the extracellular vesicle. The polypeptide linker is positioned between the signaling domain and the at least one vesicle targeting domain. The signaling domain comprises at least one fragment from one or more protein of interest (POI).

[0013] In certain cases, the signaling domain comprises a LINK domain. In certain cases, the signaling domain comprises a CUB domain.

[0014] In certain cases, the signaling domain comprises a TNF- Stimulated Gene-6 protein (TSG-6). In certain cases, the signaling domain comprises a truncated TSG-6 protein (tTSG-6), e.g., a truncated TSG- 6 protein comprising a LINK domain and a CUB domain, wherein the C-terminal tail of the TSG-6 is truncated; e.g., a truncated TSG-6 protein wherein amino acid residues 248-277 of the full-length TSG-6 protein are truncated.

[0015] Provided herein, in some aspects, are compositions comprising engineered extracellular vesicles, wherein the extracellular vesicles express a truncated TSG-6 on their surface, constructs encoding said truncated TSG-6 protein, and their methods of manufacture. Provided herein, in some aspects, are compositions comprising engineered extracellular vesicles, wherein the extracellular vesicles are exosomes expressing a truncated TSG-6 on their surface, constructs encoding said truncated TSG-6 protein, and their methods of manufacture.

[0016] Thus, in one aspect, provided herein is an engineered extracellular vesicle comprising: at least one fusion polypeptide comprising: at least one signaling domain comprising a tTSG-6 protein; and at least one vesicle targeting domain. In some embodiments of any of the aspects, the engineered extracellular vesicle is an exosome. In some embodiments, of any of the aspects, the fusion polypeptide further comprises at least one linker. In some embodiments of any of the aspects, the signaling domain comprising tTSG-6 can substantially bind to a target polypeptide. In some embodiments of any of the aspects, the signaling domain comprising tTSG-6 can substantially bind to a target protein. In some embodiments of any of the aspects, the signaling domain comprising tTSG-6 can substantially bind to a target polysaccharide. In some embodiments of any of the aspects, the signaling domain comprising tTSG-6 can substantially bind to a target glycoprotein.

[0017] In one aspect, provided herein is a fusion polypeptide comprising: a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C terminal tail, wherein atleast a part of the C-terminal of the TSG-6 is truncated; and a linker; and at least one vesicle targeting domain, wherein the fusion polypeptide is capable of expressing on a surface of an engineered extracellular vesicle.

[0018] In some embodiments, the fusion polypeptide is displayed on a surface of an engineered extracellular vesicle.

[0019] In some embodiments, the entire C-terminal tail of the TSG-6 protein is truncated. In some embodiments, a fragment of the C-terminal tail of the TSG-6 protein is truncated. In some embodiments, the CUB domain of the TSG-6 protein is truncated or deleted.

[0020] In some embodiments, the truncated TSG-6 protein lacks a fragment comprising an amino acid sequence comprising a sequence as set forth in SEQ ID NO: 19.

[0021] In some embodiments, the vesicle targeting domain is selected from a Glycosylphosphatidylinositol (GPI) anchor, a fatty acetylation site, a prenylation site, and a transmembrane domain.

[0022] In some embodiments, the GPI anchor of the fusion polypeptide comprises a GPI sequence from CD55 or a GPI sequence from CD59 (e.g., sequence SEQ ID NO: 31, or functional fragment thereof). In some embodiments, the GPI anchor is a GPI sequence from CD55 or a GPI sequence from CD59 (e.g., SEQ ID NO: 31, or functional fragment thereof).

[0023] In some embodiments, the vesicle targeting domain comprises a Type I transmembrane protein or fragment thereof. In some embodiments, the vesicle targeting domain comprises a multi-pass transmembrane protein or fragment thereof.

[0024] In some embodiments, the vesicle targeting domain is a Type I transmembrane protein or fragment thereof, or a multi-pass transmembrane protein or fragment thereof.

[0025] In some embodiments, the multi-pass transmembrane protein is a tetraspanin or fragment thereof.

[0026] In some embodiments, the tetraspanin comprises CD9 (SEQ ID NO: 20), or fragment thereof.

[0027] In some embodiments, the tetraspanin is CD9 (SEQ ID NO: 20), or fragment thereof.

[0028] In some embodiments, the fragment of CD9 comprises CD9tm2 (SEQ ID NO: 23), or a functional fragment thereof.

[0029] In some embodiments, the fragment of CD9 is CD9tm2 (SEQ ID NO: 23), or a functional fragment thereof.

[0030] In some embodiments, the vesicle targeting domain comprises an S-Palmitoylation site.

[0031] In some embodiments, the vesicle targeting domain further comprises an S-Famesylation site.

[0032] In some embodiments, the fusion polypeptide comprises a linker between the signaling domain and the vesicle targeting domain, wherein the N terminus of the linker is linked to the C terminus of the signaling domain and the C terminus of the linker is linked to the N terminus of the vesicle targeting domain.

[0033] In some embodiments, the polypeptide linker is positioned N-terminus relative to the at least one vesicle targeting domain.

[0034] In some embodiments, the polypeptide linker is positioned C-terminus relative to the signaling domain.

[0035] In some embodiments, the at least one vesicle targeting domain comprises a secretion signal recognition sequence (S).

[0036] In some embodiments, the secretion signal recognition sequence is positioned N-terminus relative to the signaling domain.

[0037] In some embodiments, the secretion signal recognition sequence is proteolytically cleaved from the signaling domain.

[0038] In some embodiments, a CVIM motif (SEQ ID NO: 27) from KRAS4B (CAAX box) is at the C-terminus of the fusion polypeptide.

[0039] In some embodiments, the CVIM motif (SEQ ID NO: 27) is famesylated by a post translational modification, an isoprenyl group is added to the cysteine residue, and the VIM is cleaved via proteolysis.

[0040] In some embodiments, the vesicle targeting domain comprises CD9tm2-CVIM (SEQ ID NO: 29). In some embodiments, the vesicle targeting domain is CD9tm2-CVIM (SEQ ID NO: 29).

[0041] In some embodiments, the fusion polypeptide further comprises a linker between the signaling domain and the vesicle targeting domain.

[0042] In some embodiments, the linker is selected from the group consisting of Fc domains, Fc from IgGl, Fc from IgG2, Fc from IgG3, Fc from IgG4 (4Fc), and sequences with at least 70%, 80%, or 90% homology with any of the foregoing.

[0043] In some embodiments, the linker of the fusion polypeptide comprises a sequence from SEQ ID NO: 33, or fragment thereof. In some embodiments, the linker of the fusion polypeptide comprises a Fc domain from SEQ ID NO: 33, or fragment thereof. In some embodiments, the linker of the fusion polypeptide comprises a sequence from SEQ ID NO: 35, or fragment thereof. In some embodiments, the linker of the fusion polypeptide comprises a Fc domain from SEQ ID NO: 35, or fragment thereof.

[0044] In some embodiments, the linker of the fusion polypeptide comprises a sequence from IgG2 SEQ ID NO: 39, or fragment thereof. In some embodiments, the linker of the fusion polypeptide comprises a Fc domain from SEQ ID NO: 39, or fragment thereof.

[0045] In some embodiments, the linker of the fusion polypeptide comprises a sequence from IgG3 (SEQ ID NO: 41), or fragment thereof. In some embodiments, the linker of the fusion polypeptide comprises a Fc domain from SEQ ID NO: 41, or fragment thereof.

[0046] In some embodiments, the linker of the fusion polypeptide comprises a sequence from IgG4 (SEQ ID NO: 43), or fragment thereof. In some embodiments, the linker of the fusion polypeptide comprises a Fc domain from SEQ ID NO: 43, or fragment thereof.

[0047] In some embodiments, the nucleic acid sequence of the fusion polypeptide comprises arestriction site. In some embodiments, the nucleic acid sequence of the fusion polypeptide comprises a Clal restriction site (SEQ ID NO: 44). In some embodiments, the fusion polypeptide comprises one or more linkers. In some embodiments, the one or more linker of the fusion polypeptide is SEQ ID NO: 45.

[0048] In some embodiments, the Fc domain comprises one or more mutations (Fc mutein). In some embodiments, the fusion polypeptide comprises a Fc mutein. In some embodiments, the linker of the fusion polypeptide is a Fc mutein. In some embodiments, the Fc mutein comprises SEQ ID NO: 37. In some embodiments the Fc mutein is SEQ ID NO: 37.

[0049] In some embodiments, the TSG-6 protein comprises a sequence as set forth in SEQ ID NOS: 3, 5, 7, 9, 11, 13. In some embodiments, the TSG-6 protein comprises a sequence as set forth in SEQ ID NO: 15

[0050] In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 3, 5, 7, 9, 11, 13, or fragments thereof. In some embodiments, the polypeptide comprises a sequence as set forth in SEQ ID NO: 15.

[0051] In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 47, 49, 51, 53, 55, 57, or a fragment thereof.

[0052] In some embodiments, the nucleic acid encoding the fusion polypeptide is SEQ ID NO: 46. In some embodiments, the fusion polypeptide is SEQ ID NO: 47. In some embodiments, the nucleic acid encoding the fusion polypeptide is SEQ ID NO: 48. In some embodiments, the fusion polypeptide is SEQ ID NO: 49. In some embodiments, the nucleic acid encoding the fusion polypeptide is SEQ ID NO: 50. In some embodiments, the fusion polypeptide is SEQ ID NO: 51. In some embodiments, the nucleic acid encoding the fusion polypeptide is SEQ ID NO: 52. In some embodiments, the fusion polypeptide is SEQ ID NO: 53. In some embodiments, the nucleic acid encoding the fusion polypeptide is SEQ ID NO: 54. In some embodiments, the fusion polypeptide is SEQ ID NO: 55. In some embodiments, the nucleic acid encoding the fusion polypeptide is SEQ ID NO: 56. In some embodiments, the fusion polypeptide is SEQ ID NO: 57

[0053] In some embodiments, provided herein is a nucleic acid encoding a fusion polypeptide disclosed herein. In some embodiments, the nucleic acid sequence of the fusion polypeptide comprises nucleic acid sequence as set forth in SEQ ID NOS: 1, 2, 4, 6, 8, 10, 12, 14, or a fragment thereof. In some embodiments, the nucleic acid sequence of the fusion polypeptide comprises nucleic acid sequence as set forth in SEQ ID NOS: 20, 22, 23, 26, 28, 30, or a fragment thereof. In some embodiments, the nucleic acid sequence of the fusion polypeptide comprises nucleic acid sequence as set forth in SEQ ID NOS: 32, 34, 36, 38, 36, 40, 44, or a fragment thereof.

[0054] In some embodiments, provided herein is an engineered extracellular vesicle comprising a fusion polypeptide disclosed herein.

[0055] In some embodiments, the engineered extracellular vesicle is an exosome.

[0056] In some embodiments, at least a portion of the vesicle targeting domain of the fusionpolypeptide is embedded in a phospholipid bilayer of the engineered extracellular vesicle, and the signaling domain of the fusion polypeptide is in an extracellular position relative to a lipid membrane of the engineered extracellular vesicle.

[0057] In some embodiments, provided herein is a composition comprising a fusion polypeptide, nucleotide, or an engineered extracellular vesicle disclosed herein.

[0058] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0059] In one aspect, provided herein is a method of producing an engineered extracellular vesicle, the method comprising: (a) contacting a population of cells with a nucleic acid construct encoding a fusion polypeptide comprising a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C-terminal tail wherein the C-terminal tail comprises a cleavage site, and wherein at least a part of the C-terminal tail of the TSG-6 is truncated following translation; and at least one vesicle targeting domain; (b) isolating a plurality of the engineered extracellular vesicles from the population of cells; and (c) purifying the plurality of the engineered extracellular vesicles from the population of cells.

[0060] In some embodiments, the fusion polypeptide further comprises at least one linker.A BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0062] FIG. 1A is a schematic illustration of the separation of extracellular vesicles (EVs) from other biomolecules in concentrated conditioned medium (CCM) using a qEV size-exclusion chromatography (SEC) column.

[0063] FIG. IB shows dot blot immunoblot analysis of SEC fractions 7-18 from CCM of TNFa (TNF) and IFNy (IFN)-stimulated mesenchymal stem cells (MSCs).

[0064] FIG. 1C shows Western immunoblots of SEC fractions 7-18 from CCM of Flp-In™-293 cells expressing TSG-6.

[0065] FIG. 2A is a schematic representation of the domain organization of an extracellular vesicle- targeted fusion polypeptide construct.

[0066] FIG. 2B illustrates fusion polypeptide constructs used in various embodiments for EV surface display or affinity purification. Stars indicate putative proteolytic cleavage sites.

[0067] FIG 2C illustrates the predicted organization of native TSG-6 Construct 0 (SEQ ID NO: 3) and fusion polypeptides Constructs 1 (SEQ ID NO: 47) and 3(SEQ ID NO: 51) relative to the EV lipid bilayer.

[0068] FIG. 3A shows dot blot immunoblots of SEC fractions 7-18 from concentrated conditioned medium of Flp-In™-293 cells (Control) or Flp-In™-293 cells expressing Construct 0 (SEQ ID NO: 3) orConstruct 1 (SEQ ID NO: 47), probed with antibodies against ALIX (upper panel) or TSG-6 (lower panel).

[0069] FIG. 3B shows nanoparticle tracking analysis (NTA) of SEC fraction 8 in FIG. 3A, illustrating size distribution of control EVs from unmodified Flp-In™-293 cells.

[0070] FIG. 3C shows NTA of SEC fraction 8 in FIG. 3 A, illustrating size distribution of EVs from Flp-In™-293 cells expressing Construct 1 (SEQ ID NO: 47).

[0071] FIG. 3D shows EV surface marker analysis of SEC fraction 8 from FIG. 3A using the MACSPlex EV Kit.

[0072] FIG. 4A shows results of ExoView R100 single -particle multiplex immunofluorescence analysis of Control EVs bound to antibody-functionalized capture probes and stained with fluorescent antibodies against CD63, CD81, CD9.

[0073] FIG. 4B shows ExoView R100 single-particle multiplex immunofluorescence analysis of Construct 1 (SEQ ID NO: 47) EVs bound to antibody-functionalized capture probes and stained with fluorescent antibodies against CD63, CD81, and CD9. Fluorescence is shown in arbitrary units (A.U.).

[0074] FIG. 5A is an exemplary line graph showing cell growth (cells / mL) over approximately seven days. Plots compare 293H cells producing control EVs, 293H cells expressing Construct 1 (SEQ ID NO: 47) TSG-6 fusion polypeptide, and 293H cells expressing Construct 3 (SEQ ID NO: 51) truncated TSG-6 (tTSG-6) fusion polypeptide.

[0075] FIG. 5B is an exemplary line graph showing glucose concentration (mg / dL) over approximately seven days. Plots compare 293H cells producing control EVs, 293H cells expressing Construct 1 (SEQ ID NO: 47) TSG-6 fusion polypeptide, and 293H cells expressing Construct 3 (SEQ ID NO: 51) tTSG-6 fusion polypeptide.

[0076] FIG. 5C is an exemplary line graph showing both cell density (cells / mL) and glucose concentration (mg / dL) over approximately seven days. Plots compare 293H cells producing control EVs and 293H cells expressing Construct 2 (SEQ ID NO: 49) TSG-6 fusion polypeptide.

[0077] FIG. 6A shows total protein concentration (Qubit assay) and TSG-6 dot blot integrated intensity (LI.) across SEC fractions from concentrated conditioned medium of unmodified 293H cells producing control EVs.

[0078] FIG. 6B shows total protein (Qubit assay) and TSG-6 dot blot LI. across SEC fractions from 293H cells expressing Construct 0 (SEQ ID NO: 3).

[0079] FIG. 6C shows total protein (Qubit assay) and TSG-6 dot blot LI. across SEC fractions from 293H cells expressing Construct 1 (SEQ ID NO: 47).

[0080] FIG. 6D shows total protein (Qubit assay) and dot blot LI. for TSG-6 and ALIX across SEC fractions from unmodified 293H cells producing control EVs.

[0081] FIG. 6E shows total protein (Qubit assay) and dot blot LI. for TSG-6 and ALIX across SEC fractions from 293H cells expressing Construct 2 (SEQ ID NO: 49).

[0082] FIG. 6F shows total protein concentration (Qubit assay) across SEC fractions from 293H cells producing control EVs or EVs expressing Construct 1 (SEQ ID NO: 47) or Construct 3 (SEQ ID NO: 51).

[0083] FIG. 6G shows a TSG-6 dot blot from duplicate SEC fractionations of concentrated conditioned medium from 293H cells producing control EVs or EVs expressing Construct 1 (SEQ ID NO: 47) or Construct 3 (SEQ ID NO: 51), as illustrated in FIG. 3F.

[0084] FIG. 7A shows Western immunoblots of SEC-purified EVs from 293H cells, probed with antibodies against ALIX (left panel) or TSG-6 (right panel). EVs are from unmodified cells or cells expressing fusion polypeptides comprising TSG-6 or fragments thereof.

[0085] FIG. 7B shows Western immunoblots of SEC-purified EVs, probed with antibodies against ALIX (top panel), TSG-6 (middle panel), and Fc (bottom panel). EVs are from unmodified cells or cells expressing TSG-6 fusion constructs. Construct 3 (SEQ ID NO: 51) exhibited increased resistance to proteolytic cleavage.

[0086] FIG. 7C shows Western immunoblots of SEC-purified EVs, probed with anti-ALIX (left panel) or anti-TSG-6 (right panel), from unmodified cells or cells expressing TSG-6 fusion constructs.

[0087] FIG. 8A shows particle size versus concentration distributions for Control EVs, Construct 1 (SEQ ID NO: 47) EVs and Construct 3 (SEQ ID NO: 51) EVs from 293H cells measured by nanoparticle tracking analysis (NTA).

[0088] FIG. 8B shows particle size versus concentration distributions for Control EVs and Construct 3 (SEQ ID NO: 51) EVs from 293H cells measured by tunable resistive pulse sensing (TRPS).

[0089] FIG. 9A shows Gene Ontology (GO) cellular component enrichment analysis of proteins identified by LC-MS shotgun proteomics from 293H Control EVs. Enriched GO terms confirm sample origin and purity.

[0090] FIG. 9B shows GO cellular component enrichment analysis of proteins identified by LC-MS from 293H Construct 1 (SEQ ID NO: 47) EVs.

[0091] FIG. 9C shows GO cellular component enrichment analysis of proteins identified by LC-MS from 293H Construct 3 (SEQ ID NO: 51) EVs.

[0092] FIG. 9D shows a Venn diagram of total proteins identified by LC-MS in 293H Control EVs, Construct 1 (SEQ ID NO: 47) EVs, and Construct 3 (SEQ ID NO: 51) EVs.

[0093] FIG. 9E shows LC-MS protein intensities comparing 293H Control EVs and Construct 3 (SEQ ID NO: 51) EVs, highlighting presence of TSG-6 in Construct 3 (SEQ ID NO: 51).

[0094] FIG. 9F shows a heatmap of selected LC-MS protein intensities comparing 293H Control EVs and Construct 3 (SEQ ID NO: 51) EVs, demonstrating TSG-6 is present in Construct 3 (SEQ ID NO: 51).DETAILED DESCRIPTION

[0095] The details of one or more inventive embodiments are set forth in the accompanying drawings, the claims, and the description herein. Other features, objects, and advantages of the inventive embodiments disclosed and contemplated herein can be combined with any other embodiment unless explicitly excluded. It is to be understood that the embodiments of the disclosures herein are illustrative of the principles of the present disclosure. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto. Any combination of the above - described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure may be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

[0096] The compositions and methods provided herein are based, in part, on the discovery that engineered extracellular vesicles (e.g., exosomes) expressing an engineered fusion polypeptide or protein, comprising (1) a signaling domain of a protein of interest or a fragment thereof (e.g., TSG-6) and (2) a vesicle targeting domain, reduces inflammation in an in vitro model of neuroinflammation. The compositions and methods provided herein are further based, in part, on the discovery that the engineered extracellular vesicles have a more stable and enhanced expression of the engineered fusion polypeptide or protein when the POI domain comprising a TSG-6 signaling domain is truncated at a C-terminal region (e.g., tTSG-6). In some embodiments, the C-terminal region comprises D248-L277.

[0097] In one aspect, provided herein is a fusion polypeptide comprising: a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C-terminal tail, wherein at least a part of the C-terminal tail of the TSG-6 is truncated (e.g., a tTSG-6 protein); and at least one vesicle targeting domain, wherein the fusion polypeptide is capable of expressing on a surface of an engineered extracellular vesicle. In some embodiments, the fusion polypeptide is displayed on a surface of an engineered extracellular vesicle. In some embodiments, at least a part of the C-terminal tail of the TSG-6 protein is truncated. In some embodiments, the entire C-terminal tail of the TSG-6 protein is truncated. In some embodiments, amino acid residues 248 to 277 of the TSG-6 protein are truncated. In some embodiments, the C-terminal tail comprises amino acid residues 248 to 277 of the TSG-6 protein. In some embodiments, the signaling domain comprises a matured tTSG-6 protein; e.g., a tTSG-6 protein wherein the signal peptide is cleaved off. In some embodiments, the vesicle targeting domain is selected from a Glycosylphosphatidylinositol (GPI) anchor, a fatty acetylation site, a prenylation site, and a transmembrane domain. In some embodiments, the GPI anchor is a GPI sequence from CD55 or a GPI sequence from CD59. In some embodiments, the vesicle targeting domain is a transmembrane domain. In some embodiments, the transmembrane domain is a CD9 transmembrane 2 domain. In some embodiments, the vesicle targeting domain comprises CD9 transmembrane 2 domain (CD9tm2). In some embodiments, the vesicle targeting domain comprises CD9 transmembrane 2 domain (CD9tm2) and aCAAX box at the C-terminus. In some embodiments, the CAAX box is the CVIM motif from KRAS4B. In some embodiments, the CAAX box (e.g., CVIM motif from KRAS4B) is added at the C-terminus of the vesicle targeting domain. In some embodiments, the CAAX box is famesylated (a type of prenylation) which is a post translational modification, wherein an isoprenyl group is added to the cysteine residue, and the VIM is cleaved via proteolysis.

[0098] In some embodiments, the vesicle targeting domain further comprises an S-Famesylation site. In some embodiments, of any of the aspects, the fusion polypeptide or protein further comprises at least one linker. In some embodiments, the fusion polypeptide comprises a linker between the signaling domain and the vesicle domain, wherein the N terminus of the linker is linked to the C terminus the signaling domain and the C terminus of the linker is linked to the N terminus of the vesicle targeting domain. In some embodiments, the linker is an Fc domain. In some embodiments, the linker is selected from the group consisting of Fc domain from IgGl, Fc from IgG2, Fc from IgG3, Fc from IgG4, and sequences with at least 70%, 80%, or 90% homology with any of the foregoing. In a preferred embodiment, the linker comprises an Fc from IgGl. In some embodiments, the Fc domain comprises one or more mutations. In some instances, the tTSG-6 or a fragment thereof comprises a domain that can interact with the extracellular matrix (ECM) of a cell (e.g., a domain comprising Clr / Cls, Uegf, and Bmpl, also known as a CUB domain). In other instances, the tTSG-6 or a fragment thereof comprises a structural domain (e.g., a LINK domain) that can bind to the ECM of a cell. Nonlimiting examples of ECM comprise glycosaminoglycans (GAG), e.g., hyaluronan (HA). In some embodiments, of any of the aspects, the signaling domain comprising tTSG-6 or a fragment thereof comprises at least one CUB domain and / or at least one LINK domain. In some embodiments, of any of the aspects, the signaling domain comprising tTSG-6 or a fragment thereof can substantially bind to a target polypeptide. In some embodiments, the TSG-6 protein comprises a sequence as set forth in SEQ ID NOS: 3, 5, 7, 9, 11, 13, or a fragment thereof. In some embodiments, the TSG-6 protein comprises a sequence as set forth in SEQ ID NO: 15.

[0099] In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 3, 5, 7, 9, 11, 13, or a fragment thereof. In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 15. In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 21, 27, 31 or a fragment thereof. In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 22, or a fragment thereof. In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 29, or a fragment thereof. In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 33, 35, 39, 41, 43 or a fragment thereof. In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 37, or a fragment thereof. In some embodiments, the fusion polypeptide comprises a sequence as set forth in SEQ ID NO: 45, or a fragment thereof.

[0100] In some embodiments, the nucleic acid encoding the fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 46, 48, 50, 52, 54, 56, or a fragment thereof. In some embodiments, the TSG-6fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 47, 49, 53, 55, 57, or a fragment thereof. In a preferred embodiment, the fusion polypeptide comprises a sequence as ser forth in SEQ ID NO: 51

[0101] Provided herein, in some aspects, is a nucleic acid encoding a fusion polypeptide as described herein. In one aspect, provided herein is a nucleic acid encoding a fusion polypeptide comprising: a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C- terminal tail, wherein at least a part of the C-terminal tail of the TSG-6 is truncated (e.g., a tTSG-6 protein); and at least one vesicle targeting domain, wherein the fusion polypeptide is capable of expressing on a surface of an engineered extracellular vesicle. In some embodiments, the nucleic acid encodes a fusion polypeptide that is displayed on a surface of an engineered extracellular vesicle. In some embodiments, then nucleic acid encodes a fusion polypeptide of which at least a part of the C-terminal tail of the TSG-6 protein is truncated. In some embodiments, the entire C-terminal tail of the TSG-6 protein is truncated. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising amino acid residues 248 to 277 of the TSG-6 protein that are truncated. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a C-terminal tail comprising amino acid residues 248 to 277 of the TSG-6 protein. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a signaling domain that comprises a matured tTSG-6 protein; e.g., a tTSG-6 protein wherein the signal peptide is cleaved off. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a vesicle targeting domain is selected from a Glycosylphosphatidylinositol (GPI) anchor, a fatty acetylation site, a prenylation site, and a transmembrane domain. In some embodiments, the GPI anchor is a GPI sequence from CD55 or a GPI sequence from CD59. In some embodiments, the vesicle targeting domain is a transmembrane domain. In some embodiments, the transmembrane domain is a ADAM10 transmembrane domain or a CD9 transmembrane domain. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a transmembrane domain that is a CD9 transmembrane 2 domain or a CD9 transmembrane 3 domain. In some embodiments, the vesicle targeting domain comprises CD9 transmembrane 2 domain (CD9tm2).

[0102] In some embodiments, the at least one vesicle targeting domain of the fusion polypeptide is a transmembrane domain from a Type I transmembrane protein or fragment thereof, or a multi-pass transmembrane protein or fragment thereof. In some embodiments of any of the aspects, the multi-pass transmembrane protein is a tetraspanin or fragment thereof. In various embodiments, a fragment of a transmembrane domain or a fragment of a multi-pass transmembrane domain can be at least 75%, 80%, 85%, 90%, or 95% of a transmembrane domain or at least 75%, 80%, 85%, 90%, or 95% of a multi-pass transmembrane domain. In some embodiments of any of the aspects, the linker is positioned N-terminus relative to the at least one vesicle targeting domain. In some embodiments of any of the aspects, a secretion signal recognition sequence is positioned N-terminus relative to the signaling domain. In some embodiments of any of the aspects, the secretion signal recognition sequence is proteolytically cleavedfrom the engineered fusion polypeptide. The secretion signal recognition sequence is cleaved off by cellular proteases after translation, and thus, the final fusion polypeptide on the surface of the extracellular vesicle does not have the secretion signal recognition sequence because the secretion signal recognition sequence is there only for membrane localization during protein translation.

[0103] In some embodiments of any of the aspects, the vesicle targeting domain comprises amino acid sequences from a type I membrane protein. Exemplary type I membrane proteins include but are not limited to CDla, CDlb, CDlc, CDld, CDle, LEU1 (CD5), CD6, CD7, CD10, ITGB2 (CD18), CD19, CR2 (CD21), CD27, CD28, CD34, integrin alpha-IIb (ITA2B, CD41), platelet glycoprotein IX (CD42a), platelet glycoprotein lb alpha chain (CD42b), platelet glycoprotein lb beta chain (CD42c), platelet glycoprotein V (CD42d), B7-1 (CD80), B7-2 (CD86), 0X40 (CD 134), glucocorticoid-induced TNFR- related protein (GITR, CD357), inducible T-cell costimulatory (ICOS, CD278), ICOS ligand (ICOSL, CD275), Herpes virus entry mediator A (HVEM, CD270), B7-H3 (CD276), B and T lymphocyte attenuator (BTLA, D272), CTLA-4 (CD 152), killer cell immunoglobulin-like receptor family (KIR family, CD158 family: CD158a-k; KIR2DL1, KIR2DL2, KIR2DL3, KIR3DP1, KIR2DL4, KIR3DL1, KIRDsl, KIR2DL5A, KIR2D15B, KIR2DS5, KIR2DS1, KIR2DS4, KIR2DS2, KIR3DL2), PD-1 (CD279), PD-L1 (CD274), PD-L2 (CD273), T-cell immunoglobulin mucin receptor 1 (TIM-1, CD365), T- cell immunoglobulin mucin receptor 3 (TIM-3, CD366), T-cell immunoglobulin and mucin domaincontaining protein 4 (TIM-4), VISTA, sialic acid-binding Ig-like lectin (SIGLEC) 1 (SIGLEC1, CD 169), SIGLEC2 (CD22), SIGLEC3 (CD33), SIGLEC5 (CD170), SIGLEC6 (CD328), SIGLEC7 (CD328), SIGLEC8, SIGLEC9 (CD329), SIGLEC10, TIGIT, PVR (CD155), lysosome associated membrane glycoprotein 1 (LAMP1, CD 107a), lysosome associated membrane glycoprotein 2 (LAMP2, CD 107b), lysosome associated membrane glycoprotein 3 (LAMP3, CD208), PECAM-1 (CD31), STAB-1, NRP2, CEACAM-1 (CD66a), TCR, VTCN1, NCR3LG1, B7-H7 (CD28H), IFNy receptor 1, IFNy receptor 2, CD2, CD4, lymphocyte function-associated antigen 3 (LFA-3, CD58), CD8, CD44, CEACAM3 (CD66d), CD96, IGSF2 (CD101), NECTIN1 (HVEC, CD111), NECTIN2 (CD112), NECTIN3 (CD113), DNAX accessory molecule 1 (DNAM-1, CD226), IL2RB (CD122), tyrosine-protein phosphatase no-receptor type substate 1 (SIRPa, CD 172a), signal -regulatory protein beta-1 (SIRPB1, CD 172b), signal-regulatory protein gamma (SIRPG, CD 172g), OX-2 (CD200), 0X-2R (CD200R), LAG3 (CD223), LAIR-1 (CD305), NKp30 (CD337), TWEAKR (CD266), CD3d, CD3e, CD3g, ITGAL (CDl la), ITGAM (CD1 lb), ITGAX (CD11c), ITGAD (CD1 Id), FCGR3A (CD16a), IL-4 receptor subunit alpha (IL4RA, CD 124), IL-2 receptor subunit alpha (IL2RA, CD25), ITGB1 (CD29), CD30, low affinity immunoglobulin gamma Fc region receptor Il-a (CD32a), low affinity immunoglobulin gamma Fc region receptor Il-b (CD32b), complement receptor type I (CD35), leukosialin (CD43), CD44, receptor-type tyrosine-protein phosphatase C (CD45), membrane cofactor protein (CD46), integrin alpha- 1 (CD49a), integrin alpha-2 (CD49b), integrin alpha-3 (CD49c), integrin alpha-4 (CD49d), integrin alpha-5 (CD49e), integrin alpha-6 (CD49f), intercellular adhesion molecule 3 (ICAM-3, CD50), intercellular adhesionmolecule 1 (ICAM-1, CD54), ICAM-4 (CD242), integrin alpha V (ITGAV, CD51), integrin beta 3 (ITGB3, CD61), complement decay accelerating factor (CD55), neural adhesion molecule 1 (NCAM-1, CD56), CD62E, CD62L, CD62P, High affinity immunoglobulin gamma Fc receptor I (CD64), macrosialin (CD68), B-cell antigen receptor complex-associated protein alpha chain (CD79a), B-cell antigen receptor complex-associated protein beta chain (CD79b), CD83, leukocyte immunoglobulin-like receptor subfamily A members (CD85G, CD85H, CD85I), leukocyte immunoglobulin-like receptor subfamily B members (CD85A, CD85B, CD85C, CD85D, CD85F, CD85J, CD85K), Immunoglobulin alpha Fc receptor (CD89), CD91, CD93, FAS (CD95), T-cell surface protein tactile (CD96), CD99, semaphoring-D (CD100), immunoglobulin superfamily member 2 (CD101), intercellular adhesion molecule 2 (ICAM-2, CD102), integrin alpha-E (CD103), integrin beta-4 (ITGB4, CD104), endoglin (CD105), vascular cell adhesion protein 1 (VCAM1, CD106), thrombopoietin receptor (CD110), CD114, macrophage colony-stimulating factor 1 receptor (CSF1R, CD115), Granulocyte -macrophage colonystimulating factor receptor subunit alpha (CSF2RA, CD116), mast / stem cell growth factor receptor Kit (CD117), leukemia inhibitory factor receptor (LIFR, CD118), interferon gamma receptor 1 (CD119), Tumor necrosis factor receptor superfamily member 1A (TNF-R1, CD 120a), Tumor necrosis factor receptor superfamily member IB (TNF-R2, CD120b), Interleukin-1 receptor type 1 (CD121a), Interleukin-1 receptor type 2 (CD121b), Interleukin-2 receptor subunit beta (CD122), Interleukin-3 receptor subunit alpha (IL3RA, CD 123), Interleukin-4 receptor subunit alpha (IL4RA, CD 124), Interleukin-5 receptor subunit alpha (IL5RA, CD 125), Interleukin-6 receptor subunit alpha (IL6RA, CD126), Interleukin-6 receptor subunit beta (IL6ST, CD130), Interleukin-7 receptor subunit alpha (IL7RA, CD127), Interleukin-9 receptor (CD129), Cytokine receptor common subunit beta (CD131), Cytokine receptor common subunit gamma (CD132), CD135, macrophage stimulating protein receptor (CD136), syndecan-1 (CD138), Platelet-derived growth factor receptor alpha (PDGFRA, CD140a), Platelet-derived growth factor receptor beta (PDGFRB, CD 140b), thrombomodulin (CD 141), CD 142, angiotensin converting enzyme (ACE, CD 143), cadherin-5 (CD 144), melanoma and adhesion molecule (MCAM, CD 146), basigin (BSG, CD 147), CD 148, Signaling lymphocytic activation molecule (SLAM, CD150), SLAM family member 4 (SLAMF4, CD244), signaling lymphocytic activation molecule (SLAM) family member 5 (SLAM5, CD84), SLAM family member 6 (SLAMF6, CD352), SLAM family member 7 (SLAMF7, CD319), SLAM family member 8 (SLAMF8, CD353), SLAM family member 9 (SLAM9), Disintegrin and metalloproteinase domain-containing protein 8 (ADAM8, CD156a), Disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17, CD 156b), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10, CD156c), P-selectin glycoprotein 1 (SELPLG, CD162), CD163, CD164, activated leukocyte cell adhesion molecule (ALCAM, CD166), epithelial discoidin domain containing receptor 1 (CD 167a), discoidin domain containing receptor 2 (CD 167b), neural cell adhesion molecule LI (LI CAM, CD 171), CD 180, endothelial protein C receptor (EPCR, CD201), angiopoietin-1 receptor (CD202b), lymphocyte antigen 75 (CD205), macrophage mannose receptor 1 (CD206), IL- 10 receptor subunit alpha (IL 1 ORA, CD210), IL- 10 receptor subunit beta(IL10RB, CDw210b), IL-12 receptor subunit beta-1 (IL12RB1, CD212), IL-13 receptor subunit alpha-1 (CD213al), IL-13 receptor subunit alpha-2 (CD213a2), IL-15 receptor subunit alpha (CD215), IL-17 receptor A (CD217), IL-18 receptor 1 (CD218a), IL-18 receptor accessory protein (CD218b), insulin receptor (CD220), insulin-like growth factor 1 receptor (CD221), cation-independent mannos-6phosphate receptor (CD222), mucin- 1 (CD227), T-lymphocyte surface antigen Ly-9 (CD229), plexin-Cl (VESPR, CD232), glycophorin-A (CD235a), glycophorin-B (CD235b), basal cell adhesion molecule (CD239), CD246, T-cell surface glycoprotein CD3 zeta chain (CD247), endosialin (CD248), death receptor 3 (DR3, TNFRS25), death receptor 4 (DR4, CD261), death receptor 5 (DR5, CD262), decoy receptor 2 (DcR2, CD264), receptor activator of nuclear factor kappa-B (RANK, CD265), CD271, C-type mannose receptor 2 (CD280), Toll like receptor 1 (CD281), Toll like receptor 2 (CD282), Toll like receptor 3 (CD283), Toll like receptor 4 (CD284), Toll like receptor 6 (CD286), Toll like receptor 8 (CD288), Toll like receptor 9 (CD289), Toll like receptor 10 (CD290), bone morphogenic protein receptor type 1A (CD292), bone morphogenic protein receptor type ID (CwD293), leptin receptor (CD295), CD300a, CD300c, CD302, Neuropilin-1 (CD304), leukocyte-associated immunoglobulin-like receptor 1 (LIAR1, CD305), Fc receptor-like protein 1 (FcRLl, CD307a), Fc receptor-like protein 2 (FcRL2, CD307b), Fc receptor-like protein 3 (FcRL3, CD307c), Fc receptor-like protein 4 (FcRL4, CD307d), Fc receptor-like protein 5 (FcRL5, CD307e), vascular endothelial growth factor receptor 2 (VEGFR2, CD309), prostaglandin F2 receptor negative regulator (PTGFRN, CD315), immunoglobulin superfamily member 8 (IGSF8, CD316), CD320, platelet Fl 1 receptor (Fl 1R, CD321), junctional adhesion molecule B (JAM-B, CD322), cadherin-1 (CD324), cadherin-2 (CD325), epithelial cell adhesion molecule (CD326), fibroblast growth factor 1 (FGFR1, CD331), fibroblast growth factor 2 (FGFR2, CD332), fibroblast growth factor 3 (FGFR3, CD333), fibroblast growth factor 4 (FGFR4, CD334), natural cytotoxicity triggering receptor 1 (NCR1, CD335), natural cytotoxicity triggering receptor 2 (NCR2, CD336), natural cytotoxicity triggering receptor 3 (NCR3, CD337), triggering receptor expressing on myeloid cells 1 (TREM1, CD354), cytotoxic and regulatory T- cell molecule (CRTAM, CD355), tumor necrosis factor receptor superfamily member 21 (CD358), interleukin-21 receptor (IL21R, CD360), protein EVI2B (CD361), syndecan-2 (CD362), V-set and immunoglobulin domain-containing protein 1 (VSIG1), V-set and immunoglobulin domain-containing protein 3 (VSIG3), V-set and immunoglobulin domain-containing protein 4 (VSIG4), V-set and immunoglobulin domain-containing protein 8 (VSIG8), V-set and immunoglobulin domain-containing protein 1 (VSIGl), V-set and immunoglobulin domain-containing protein 3 (VSIG3), V-set and immunoglobulin domain-containing protein 4 (VSIG4), V-set and immunoglobulin domain-containing protein 8 (VSIG8), butyrophilin subfamily 3 member Al (BTN3A1, CD277), butyrophilin subfamily 3 member A2 (BTN3A2), butyrophilin subfamily 2 member Al (BTN2A1), butyrophilin like protein 8 (BTNL8), butyrophilin subfamily 1 member Al (BTN1A1), lymphotoxin beta receptor (LTBR), DNAX- activation protein 10 (DAP 10), TYRO protein tyrosine kinase-binding protein (DAP 12), high affinity immunoglobulin epsilon receptor subunit gamma (FceRIy). Isoforms or fragments thereof, or the like known by one of ordinary skill in the art are also encompassed by the present invention.

[0104] In some embodiments of any of the aspects, the vesicle targeting domain comprises amino acid sequences from a type III membrane protein. Exemplary type III membrane proteins include but are not limited to B cell activating factor (BAFFR, CD268), glycophorin-C (CD236), transmembrane activator and CAML interactor (TACI, CD267), B-cell maturation protein (BCM, CD269) Isoforms or fragments thereof, or the like known by one of ordinary skill in the art are also included in the invention.

[0105] In some embodiments of any of the aspects, the vesicle targeting domain comprises amino acid sequences from a multi-pass membrane protein. In various embodiments, the multi-pass membrane protein vesicle targeting polypeptide or fragment thereof comprises LAT1 (CD98 light subunit encoded by the SLC7A5 gene). Exemplary multi-pass membrane proteins include but are not limited to Alpha-2A adrenergic receptor (A2AR, ADRA2A), adenosine receptor A2b (A2BR, ADORA2B), N0X2, LAT1 (SLC7A5 (CD98 light chain) and SLC7A2 (CD98 heavy chain)), CD39, CD47, PVRIG (CD112R), CD9, CD20, CD36, CD37, CD47, CD53, CD63, CD81, CD82, C5a receptor (CD88), CD92, CD97, prominin-1 (CD133), CD151, high affinity interleukin-8 receptor A (IL8RA, CXCR1, CD181), high affinity interleukin-8 receptor B (IL8RB, CXCR2, CD 182), C-X-C chemokine receptor (CXCR) type 3 (CXCR3, CD 183), CXCR4 (CD 184), CXCR5 (CD 185), CXCR6 (CD 186), C-C chemokine (CCR) type 1 (CCR1, CD191), CCR2 (CD192), CCR3 (CD193), CCR4 (CD194), CCR5 (CD195), CCR6 (CD196), CCR7 (CD 197), CCR8 (CDwl89), CCR9 (CDwl99), CD231, solute carrier family 4 member 1 (SLC4A1, CD233), Duffy antigen / chemokine receptor (DARC, CD234), blood group Rh(CE) polypeptide (CD240CE), blood group Rh(D) polypeptide (CD240D), ammonium transporter Rh type A (CD241), CD243, calcium signal-modulating cyclophilin ligand (CAMLG), prostaglandin D2 receptor 2 (PTGDR2, CD294), EGF-like module receptor 2 (CD312), CD338, frizzled-4 (CD344), frizzled-9 (CD349), frizzled-IO (CD350), sphingosine 1-phosphate receptor 1 (CD363), BAT1 encoded by the SLC7A9 gene, linker for activation of T-cell family member 2 (LAT2), or a fragment thereof.

[0106] In certain embodiments, the at least one multi-pass membrane protein is a tetraspanin selected from the group consisting of TSPAN1 (TSP-1), TSPAN2 (TSP-2), TSPAN3 (TSP-3), TSPAN4 (TSP-4, NAG-2), TSPAN5 (TSP-5), TSPAN6 (TSP-6), TSPAN7 (CD231, TALLA-1, A15), TSPAN8 (CO-029), TSPAN9 (NET-5), TSPAN10 (OCULOSPANIN), TSPAN11 (CD151-like), TSPAN12 (NET-2), TSPAN13 (NET-6), TSPAN14, TSPAN15 (NET-7), TSPAN16 (TM4-B), TSPAN17, TSPAN18, TSPAN19, TSPAN20 (UPlb, UPK1B), TSPAN21 (UPla, UPK1A), TSPAN22 (RDS, PRPH2), TSPAN23 (R0M1), TSPAN24 (CD151), TSPAN25 (CD53), TSPAN26 (CD37), TSPAN27 (CD82), TSPAN28 (CD81), TSPAN29 (CD9), TSPAN30 (CD63), TSPAN31 (SAS), TSPAN32 (TSSC6), TSPAN33, a fragment thereof, and combinations thereof.

[0107] In various embodiments, the vesicle targeting domain is a GPI domain (i.e., GPI, GPI anchor), fatty acetylation site, or prenylation moiety. In some embodiments of any of the aspects, the vesicle targeting domain is a membrane anchoring domain from a GPI anchored membrane protein or fragment thereof. In various embodiments, a fragment of a GPI anchored membrane protein can be at least 75%,80%, 85%, 90%, or 95% of a GPI anchored membrane protein. In certain embodiments, the GPI anchored membrane protein is selected from the group consisting of CD 160, RGMB, CEACAM8 (CD66b, CD67), CEACAM6 (CD66c), CEACAM5 (CD66e), CD73, CD14, FCGR3B (CD16b), CD24, BLAST-1 (CD48), CAMPATH-1 (CD52), CD59, CD87, CD90, semaphorin-7A (CD108), CD109, bone marrow stromal cell antigen 1 (BST1, CD157), CD177, melanotransferrin (CD228), CD230, decoy receptor 1 (DcRl, CD263), CD296, CD297 isoforms thereof, fragments thereof, and combinations thereof. One of skill in the art can appreciate that the aforementioned refer to peptide or protein sites, wherein covalent lipid attachment supports embedding of the lipid in a cell membrane (z.e., phospholipid bilayer). Biochemical forces that anchor EV targeting domains to the EV phospholipid bilayer may include, but are not limited to, electrostatic forces, affinity for EVs through protein-protein interactions with natively resident proteins (e.g., CD81, CD63, CD9, ALIX, TSG101. CD98, CD298, MARCKS, PTGFRN, Lactadherin (MFGe8), ITGB 1, EpCAM, MCAM, CD44, NCAM, ICAM), association or affinity for negatively or positively curved phospholipids, association or affinity for negatively or positively charged domains of resident membrane associated proteins, etc., or the like.

[0108] In some embodiments, the vesicle targeting domain further comprises an S-Famesylation site. In some embodiments, of any of the aspects, the nucleic acid encodes a fusion polypeptide further comprising at least one linker. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a linker between the signaling domain and the vesicle targeting domain, wherein the N terminus of the linker is linked to the C terminus the signaling domain and the C terminus of the linker is linked to the N terminus of the vesicle targeting domain. In some embodiments, the linker is an Fc domain. In some embodiments, the linker is selected from the group consisting of Fc domains, Fc, Fc from IgGl, Fc from IgG2, Fc from IgG3, Fc from IgG4 (4Fc), and sequences with at least 70%, 80%, or 90% homology with any of the foregoing. In some embodiments, the linker comprises an Fc from IgGl. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an Fc domain that comprises one or more mutations. In some instances, the nucleic acid encodes a fusion polypeptide comprising the tTSG-6 or a fragment thereof that comprises a domain that can interact with the extracellular matrix (ECM) of a cell (e.g., a domain comprising Clr / Cls, Uegf, and BMP, also known as a CUB domain). In other instances, the tTSG-6 or a fragment thereof comprises a structural domain (e.g., a LINK domain) that can bind to the ECM of a cell. Nonlimiting examples of a LINK domain binding to the ECM of a cell comprise LINK binding to glycosaminoglycans (GAG), e.g., hyaluronan (HA). In some embodiments, of any of the aspects, the nucleic acid encodes a fusion polypeptide comprising a tTSG-6 or a fragment thereof that comprises at least one CUB domain and / or at least one LINK domain. In a nonlimiting embodiment, the protein or peptide of interest comprises a TSG-6 wherein the CUB domain and the C-terminal of the TSG-6 is cleaved. In some embodiments, of any of the aspects, the nucleic acid encodes a fusion polypeptide comprising a tTSG-6 or a fragment thereof that can substantially bind to a target polypeptide. In some embodiments, the nucleic acid encoding a fusion polypeptide comprising a tTSG-6 protein or fragment thereof comprises a sequence as set forth in SEQ ID NOS: 46, 48, 50, 52, 54,56, or functional fragment thereof.

[0109] In some aspects, provided herein is an engineered extracellular vesicle comprising any fusion polypeptide or protein as described herein. In some embodiments, the extracellular vesicle is an exosome. In one aspect, provided herein is an engineered extracellular vesicle comprising at least one fusion polypeptide or protein comprising: a signaling domain comprising a TSG-6 protein comprising (i) a LINK domain, a CUB domain, and a C-terminal tail, wherein at least a part of the C-terminal tail of the TSG-6 is truncated; and (ii) at least one vesicle domain, wherein the fusion polypeptide is capable of expressing on a surface of an engineered extracellular vesicle. In another aspect, provided herein is an engineered extracellular vesicle comprising at least one fusion polypeptide or protein comprising: at least one signaling domain of a protein of interest (POI), wherein the POI is a tTSG-6 protein; e.g., a truncated TSG-6 protein wherein amino acid residues 248-277 of the full-length TSG-6 protein are truncated; and at least one vesicle-targeting domain, wherein the vesicle targeting domain comprises a transmembrane domain (e.g., CD9tm2), and wherein the fusion polypeptide is capable of expressing on a surface of an engineered extracellular vesicle. In some embodiments, at least a portion of the vesicle targeting domain of the fusion polypeptide is embedded in a phospholipid bilayer of the engineered extracellular vesicle and the signaling domain of the fusion polypeptide is in an extracellular position relative to a lipid membrane of the engineered extracellular vesicle. In some embodiments, a producer cell of the engineered extracellular vesicle comprises a HEK 293 cell.

[0110] In some aspects, provided herein is an engineered extracellular vesicle comprising a nucleic acid or oligonucleotide encoding any fusion polypeptide or protein as described herein. In some embodiments, the engineered extracellular vesicle comprises an oligonucleotide (e.g., mRNA, DNA) encoding any fusion polypeptide or protein as described herein. In one aspect, provided herein is an oligonucleotide encoding a fusion polypeptide or protein comprising at least one signaling domain of a POI, wherein the POI is a tTSG-6 protein (e.g., a truncated TSG-6 protein wherein amino acid residues 248-277 of the full- length TSG-6 protein are truncated); and at least one vesicle targeting domain, wherein the vesicle targeting domain comprises a Glycosylphosphatidylinositol (GPI) anchor, a fatty acetylation site, a prenylation site, or a transmembrane domain (e.g., CD9tm2). In some embodiments, the oligonucleotide is a DNA. In some embodiments, the oligonucleotide is an RNA; e.g., an mRNA. In some embodiments, a producer cell (e.g., HEK 293 cell) of the engineered extracellular vesicle comprises an oligonucleotide encoding said fusion polypeptide or protein. In some embodiments, of any of the aspects, the engineered extracellular vesicle is an exosome. In some embodiments, at least a portion of the vesicle targeting domain of the fusion polypeptide is embedded in a phospholipid bilayer of the engineered extracellular vesicle and the signaling domain of the fusion polypeptide is in an extracellular position relative to a lipid membrane of the engineered extracellular vesicle.

[0111] Provided herein, in some aspects, is a composition comprising any fusion polypeptide as described herein, a nucleic acid encoding any fusion polypeptide as described herein, and / or anyengineered extracellular vesicle as described herein. In one aspect, provided herein is a composition comprising any fusion polypeptide as described herein and any engineered extracellular vesicle as described herein. In another aspect, provided herein is a composition comprising an engineered extracellular vesicle comprising a fusion polypeptide comprising: (i) a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C-terminal tail, wherein at least a part of the C-terminal tail of the TSG-6 is truncated (e.g, a tTSG-6 protein); and (ii) at least one vesicle targeting domain, wherein the fusion polypeptide is capable of expressing on a surface of an engineered extracellular vesicle. In yet another aspect, the composition comprising the engineered extracellular vesicle further comprises a nucleic acid encoding the fusion polypeptide. In some embodiments, the nucleic acid is an mRNA. In other embodiments, the nucleic acid is a DNA.

[0112] Provided herein, in some aspects, is a method of producing an engineered extracellular vesicle, the method comprising:(a) contacting a population of cells with a nucleic acid construct encoding a fusion polypeptide comprising (i) a signaling domain comprising a TSG-6 protein comprising a LINK domain and a CUB domain, wherein the C-terminal tail of the TSG-6 is truncated (e.g., tTSG-6 or a fragment thereof); and (ii) at least one vesicle targeting domain;(b) isolating a plurality of the engineered extracellular vesicles from the population of cells; and(c) purifying the plurality of the engineered extracellular vesicles from the population of cells. In some embodiments, the fusion polypeptide further comprises a linker.

[0113] Generally, the extracellular vesicles (e.g., exosomes) provided herein are produced by contacting a population of cells with a nucleic acid construct encoding the fusion polypeptide provided herein and isolating a plurality of extracellular vesicles. In some nonlimiting embodiments, the cells comprise Human Embryonic Kidney 293 (HEK 293) cells. The extracellular vesicles can then be purified by methods provided herein and are formulated for therapeutic use, including but not limited to, for the treatment or modulation of inflammation in a subject.

[0114] The compositions and methods provided herein are specifically designed to exploit the membrane trafficking mechanisms of extracellular vesicles and rely on the hallmark biophysical and biochemical properties of extracellular vesicles, such as exosomes. The engineered extracellular vesicles provided herein are specifically engineered to propagate biological signaling via a target polypeptide, polysaccharide, or glycoprotein. In some embodiments, the engineered extracellular vesicles provided herein activate pathways to regulate cell migration, extracellular matrix (ECM) organization, and / or reduce inflammation.

[0115] Engineering of the extracellular vesicles provided herein extends these capabilities significantly by incorporating vesicle targeting domain attaching to extracellular vesicles such as exosomes, further coupled with signaling domains of choice. In some embodiments, the signaling domain comprises tTSG-6 or a fragment thereof. For example, attachment of vesicle targeting domain to exosomes, along with theirlinked signaling domains (e.g., tTSG-6 or a fragment thereof), allows for target interaction and biological signal induction and propagation. In this aspect, the aforementioned design achieves the aim of an engineered extracellular vesicle by inducing the desired biological signaling in a target recipient cell. In some instances, a vesicle targeting domain comprises a transmembrane domain of a tetraspanin protein. In one nonlimiting embodiment, a vesicle targeting domain comprises a CD9 tetraspanin transmembrane domain (CD9tm2). Various aspects and embodiments of the compositions and methods are provided herein in detail below.Certain Definitions

[0116] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The abbreviation, “e.g.,” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.,” is synonymous with the term “for example.” The abbreviation, “etc ” is derived from the Latin et cetera, and is used herein to indicate a non-limiting list. Thus, the abbreviation "etc." is synonymous with the term “and other similar things,” or “and so forth.”

[0117] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0118] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0119] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0120] As used herein, ranges and amounts can be expressed as “about” a particular value or range, e.g., ± 15% of a referenced numeral value. About also includes the exact amount. Hence “about 5 pL” means “about 5 pL” and also “5 pL.” Generally, the term “about” includes an amount that would be expected to be within experimental error. The term “about” when used in connection with percentages can mean ± about 1%, about 2%, about 3%, about 4%, or about 5%. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.”

[0121] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two-standard deviation (2SD) difference, above or below a reference value.

[0122] Administration (e.g., transplantation) of cells or exosomes disclosed herein to an area in need of treatment is achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.

[0123] “Transplanting” a composition into a mammal refers to introducing the composition into the body of the mammal by any method established in the art. The composition being introduced is the “transplant,” and the mammal is the “recipient.” The transplant and the recipient can be syngeneic, allogeneic, or xenogeneic. Further, the transplantation can be an autologous transplantation.

[0124] As used herein, the terms “isolate,” “isolated,” and “isolating,” or “purify,” “purified,” and “purifying,” as well as “extracted” and “extracting,” when used in relation to an extracellular vesicle, exosome or a plurality of extracellular vesicle or exosomes, are used interchangeably and refer to the state of a preparation (e.g., a plurality of known or unknown amount and / or concentration) of desired secreted exosomes, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired exosome preparation. A process of purification may refer to the process of removing, partially removing (e.g., a fraction) of the exosomes from a sample containing producer cells (e.g., HEK 293 cells). In some embodiments, an isolated extracellular vesicle or exosome composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other embodiments, an isolated extracellular vesicle or exosome composition has an amount and / or concentration of desired extracellular vesicle or exosomes at or above an acceptable amount and / or concentration. In other embodiments, the isolated extracellular vesicle or exosome composition is enriched as compared to the starting material (e.g., producer cell preparations) from which the composition is obtained. This enrichment can be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material. In some embodiments, isolated extracellular vesicle or exosome preparations are substantially free of residual biological products. In some embodiments, the isolated exosome preparations are 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, or 90% free of any contaminating biological matter (e.g., a contaminant protein). Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products can also mean that the exosome composition contains no detectable producer cells and that only extracellular vesicle or exosomes are detectable.

[0125] As used herein the term “contaminant protein” refers to a protein that is not associated with an exosome. For example, a contaminant protein includes a protein, not enclosed in the exosome, and not attached to or incorporated into the membrane of the exosome. 1

[0126] An “effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose. An effective amount of a composition to treat or ameliorate a disorder is an amount of the composition sufficient to reduce or remove the symptoms of the disorder.

[0127] As used herein, the term “modulates” refers to an effect including increasing or decreasing a given parameter as those terms are defined herein. For example, the terms “modulate,” “modulates,” “modulating,” and “modulation” can refer to upregulation or downregulation of a gene.

[0128] As used herein, a “reference level” can refer to one or more parameters or markers as measured for a normal, otherwise unaffected cell population or tissue (e.g., a biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, or a biological sample that has not yet been contacted with a pathogen as described herein). For measuring or monitoring therapeutic efficacy, a level determined prior to treatment or earlier in treatment can also provide a reference level for a given parameter or value.

[0129] The terms “increased,” “increase,” “increases,” or “enhance” or “activate” are all used herein to generally mean an increase of a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms “increased,” “increase,” “enhance,” or “activate” mean an increase of at least about 10% as compared to a reference level, for example, an increase of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more, or any increase between about 10 and about 100% as compared to a reference level, or at least about 1-fold, about 2-fold, about 3 -fold, about 4-fold, about 5- fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95 -fold, about 99-fold, about 100-fold, about 1000-fold increase or more as compared to a reference level. For example, increasing activity can refer to activating a receptor or a signaling pathway (e.g., antibody production or inflammation).

[0130] The terms “decrease,” “reduced,” “reduction,” or “inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter by a statistically significant amount. In some embodiments of any of the aspects, “reduce,” “reduction,” “decrease,” or “inhibit” can mean a decrease by at least about 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more, or at least about 1-fold, about 2-fold, about 3 -fold, about 4-fold, about 5 -fold, about 6-fold, about 7-fold,about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 99-fold, about 100-fold, about 1000-fold decrease or more as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0131] As used herein, the term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. “Expression products” can include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.

[0132] As used herein, the term “engineered extracellular vesicle”, “extracellular vesicle”, and “vesicle” are used interchangeably and refer to a particle, wherein the particle comprises a phospholipid bilayer that encloses an internal space and an exterior surface and may or may not be derived from a cell. The size of extracellular vesicles can range between 20 nm to 3 pm in diameter but may be smaller than 20 nm or larger than 3 pm. Examples of extracellular vesicles include, but is not limited to, exosomes (for example, small exosomes and large exosomes), ectosomes, macrovesicles, microparticles, apoptotic bodies, vesicular organelles, oncosomes (for examples large oncosomes), exospheres, exomeres, cell- derived nanovesicles (CDN) (e.g., by genesis via grating or shearing cells), liposomes or the like known by one of ordinary skill in the art. Extracellular vesicles may originate naturally via known or unknown biosynthetic pathways. Extracellular vesicles may be promoted to originate by using mechanical methods such as cell grating or cell shearing wherein a cell is grated or sheared causing portions or parts of the cell membrane to from vesicles. For example, CDNs may be formed by using mechanical methods such as cell grating or cell shearing wherein a cell is grated or sheared causing portions or parts of the cell membrane to from vesicles.

[0133] As used herein, the term “control” refers to a biological sample (such as a “control cell”, “control exosome”, or “control extracellular vesicle”) that serves as a reference against which one or more experimental samples are compared. In some embodiments, a control exosome or control extracellular vesicle is derived from the same cell type or production process as the experimental exosome or the experimental extracellular vesicle but does not include the introduced modification, treatment, or expressed engineered fusion polypeptide. The control thereby represents a baseline condition for evaluating changes in composition, biological activity, or function attributable to the experimental manipulation. As used herein, unless otherwise indicated, the term “control” encompasses control cells, control exosomes, and control extracellular vesicles interchangeably.

[0134] Extracellular vesicles comprise cargo, wherein the term “cargo” refers to peptides, proteins, nucleic acids (e.g, miRNA, mRNA, DNA, dsDNA, ssDNA, IncRNA, siRNA), lipids, metabolites,carbohydrates, biomolecules, small molecules, large molecules, vesicles, organelles, or fragments thereof. In some embodiments, cargo may refer to existing drugs or therapeutics. Extracellular vesicle cargo may be located within the internal space of the extracellular vesicle. Extracellular vesicle cargo may be membrane-bound and span one or both layers of the extracellular vesicle phospholipid bilayer (for example, a transmembrane protein). Extracellular vesicle cargo may be in contact with the external or internal surface of the extracellular vesicle, for example, through a covalent bond or a non-covalent bond. The phospholipid bilayer of the extracellular vesicle may comprise one or more transmembrane proteins, wherein a portion of the one or more transmembrane membrane proteins is located within the internal space of the extracellular vesicle. The phospholipid bilayer of the extracellular vesicle may comprise one or more transmembrane proteins, wherein the one or more transmembrane membrane proteins comprises a domain on the exterior of the extracellular vesicle. The phospholipid bilayer of the extracellular vesicle may comprise one or more transmembrane proteins, wherein the one or more transmembrane membrane proteins comprises a domain on the interior of the extracellular vesicle. Cargo may refer to a protein on the luminal side (e.g., in the internal space) of the extracellular vesicle wherein said protein encodes a vesicle targeting domain that may be in contact with the interior phospholipid layer of the extracellular vesicle. Cargo may refer to a protein on the luminal side (e.g., in the internal space) of the extracellular vesicle wherein said protein encodes a vesicle -targeting domain that may be in contact with the interior phospholipid layer of the extracellular vesicle and wherein said protein may be presented into the internal space of the extracellular vesicle.

[0135] As used herein, the terms “vesicle targeting domain” and “anchor protein” are used interchangeably and refer to a protein that is covalently or non -covalently attached to at least one lipid, wherein the one or more lipids is embedded within a membrane (e.g., a cell membrane), and the lipid serves to anchor the protein to the membrane. The terms “vesicle targeting domain” and “anchor protein” can also mean a protein sequence that encodes for one or more transmembrane domains, wherein the one or more transmembrane domains spans at least partly through a phospholipid bilayer, for example, the phospholipid bilayer of an extracellular vesicle. The transmembrane domain can be of a Type I or Type II membrane protein. Transmembrane domains can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains.

[0136] A vesicle targeting domain may include, but is not limited to, one or more prenylation sites, fatty acylation sites, and / or glycosylphosphatidylinositol (GPI) linked proteins. In some embodiments, a vesicle targeting domain comprises a transmembrane domain from a tetraspanin. In some embodiments, sequences for vesicle-targeting domains include transmembrane regions of CD9 (for example, transmembrane 2 or 3 of CD9, also known as CD9tm2 or CD9tm3, respectively). In some embodiments, a vesicle targeting domain is the transmembrane 2 domain from CD9, or CD9tm2. In some embodiments, a vesicle targeting domain comprises the transmembrane 3 domain from CD9, or CD9tm3. In some embodiments, sequences for vesicle-targeting domains include glycosylphosphatidylinositol (GPI) linkedproteins (for example, the GPI sequence from CD55, or the GPI sequence from CD59), a domain from milk fat globule epidermal growth factor 8 (for example, C1C2 domain from MFGE8), KRAS (for example, KRAS4A and KRAS4B), transmembrane domain from A Disintegrin and Metalloproteinase Domain-containing protein 10 (ADAM10, also known as CDwl56 or CD156c) or other ADAM proteins. Vesicle targeting domains may include one or more sequences from 4F2 (for example, 4F2 encoded by the solute carrier family 3 member 2 (SLC3A2) gene, which makes up the heavy subunit of CD98). Vesicle targeting domains may include transmembrane sequences from Homo sapiens transferrin receptor 2 (TFR2), transcript variant 1 (transferrin receptor protein 2 isoform 1) or versions thereof. In some embodiments, the vesicle targeting domain may be a transmembrane domain from CD298. All variants, isoforms, or fragments or the like known by one of ordinary skill in the art are encompassed by the present invention.

[0137] As used herein, the terms “signal peptide,” “secretion signal,” and “secretion signal recognition sequence” are used interchangeably and refer to an N-terminal amino acid sequence of a polypeptide that directs the nascent polypeptide to the endoplasmic reticulum (ER) for translocation and secretion or for localization to membrane -bound compartments. In some embodiments, the signal peptide facilitates co- translational translocation of the polypeptide into the ER lumen or integration into the ER membrane, where it may subsequently be processed, folded, and trafficked to its final cellular or extracellular destination.

[0138] Signal peptides are typically 15-30 amino acids in length and comprise a tripartite structure including: (i) a positively charged N-terminal region (n-region), (ii) a central hydrophobic core (h-region), and (iii) a polar C-terminal region (c-region) containing a recognition site for signal peptidases. The hydrophobic core mediates insertion into the ER membrane, while the c-region includes the cleavage site at which the signal peptide is removed by cellular proteases following translocation. Signal peptides are found in a wide variety of secreted or membrane -bound proteins and are highly conserved in their general function but vary in sequence among proteins and species. See, e.g., von Heijne G. Signal sequences: the limits of variation. J Mol Biol. 1985;184:99-105; Rapoport TA. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature. 2007;450:663-669; which are incorporated herein by reference in their entirety.

[0139] As used herein, a “signal peptide” or “secretion signal” may be naturally occurring or heterologous and can be derived from the N-terminal sequence of any protein known in the art that directs polypeptides to the secretory pathway. In some embodiments, the signal peptide is proteolytically cleaved from the mature polypeptide following translocation, such that the final, mature polypeptide (e.g., displayed on an extracellular vesicle) does not contain the signal peptide sequence. In certain embodiments, the signal peptide is selected or engineered to optimize expression, secretion, or membrane localization of the polypeptide of interest.

[0140] In the context of the engineered exosomes described herein, the signal peptide (also referred toherein as a secretion signal or secretion signal recognition sequence) functions to direct TSG-6, truncated TSG-6 (tTSG-6), or other fusion polypeptides to the secretory pathway, ensuring proper folding and translocation, and facilitating display on the surface of extracellular vesicles. In some embodiments, the signal peptide comprises the amino acid sequence as set forth in SEQ ID NO: 17 or is encoded by the nucleic acid sequence as set forth in SEQ ID NO: 16. In other embodiments, the polypeptide lacks a signal peptide.

[0141] As used herein the term “famesylation” is used herein to mean one of the important steps in the posttranslational modification of proteins associated with intracellular signal transduction. The process is catalyzed by a heterodimeric zinc protein known as famesyltransferase, and involves the transfer of a famesyl group from famesylpyrophosphate to the C-terminal cysteine sulfur of the target proteins such as Ras. These protein substrates, in general, have characteristic C-terminal consensus sequences of the type CAAX (C, cysteine; A, aliphatic amino acid; X, serine or methionine) and prenylation is important for their translocation to the membrane where they form part of the signaling network. Since constitutive activation of Ras is a major contributory factor in a number of malignant human tumors, its inactivation by interfering with the famesylation step has been extensively studied as a strategy to develop new anticancer agents. Famesylation facilitates their membrane association and also promotes protein-protein interaction.

[0142] As used herein, the terms “proteins,” “peptides,” and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “peptide” as used herein refers to peptides, polypeptides, proteins, and fragments of proteins, unless otherwise noted. The terms “protein” and “peptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary peptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0143] As used herein, the terms “linker” and “linker polypeptide” and “polypeptide linker” in the context of peptides, polypeptides and proteins refer to a protein sequence of amino acids that is used to connect two polypeptides. The term linker in the context of nucleic acids refer to the nucleic acid sequence which encodes for a linker polypeptide. The linker may be flexible, rigid, or cleavable. Further, the linker can be linked directly or via another linker (e.g., a peptide of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids) to the fusion polypeptides described herein. Linkers can be configured according to a specific need, e.g., based on at least one of the following characteristics. In some embodiments of any of the aspects, linkers can be configured to have a sufficient length and flexibility such that it can allow for a proteolysis at a target cleavage site, for example proteolytic cleavage at a proteolytic site. In some embodiments of any of the aspects, linkers can be configured toallow multimerization of the fusion polypeptides provided herein, for example two fusion polypeptides multimerizing to form a dimer. In some embodiments of any of the aspects, linkers can be configured to allow multimerization of one or more fusion polypeptides provided herein, for example two or more fusion polypeptides multimerize to form a multimer, wherein the multimerization of the two or more fusion polypeptides is mediated by covalent or noncovalent intermolecular forces between linkers of the said two or more fusion polypeptides. In some embodiments of any of the aspects, linkers can be configured to facilitate expression and purification of the fusion polypeptides or engineered extracellular vesicles provided herein. In some embodiments of any of the aspects, a linker can be configured to have any length in a form of a peptide, peptidomimetic, an aptamer, a protein, a nucleic acid (e.g., DNA or RNA), or any combinations thereof. In some embodiments, the linker can be an Fc domain or Fc mutein. In some embodiments the linker is a synthetic protein sequence.

[0144] As used herein, the term “fusion polypeptide” and “fusion protein” are used interchangeably and refers to a single chimeric protein comprising a protein of interest joined to an exogenous protein or protein fragment (e.g., an anchor protein), wherein the components of the fusion polypeptide are linked to each other by peptide bonds, either directly or through a peptide linker. The anchor protein of the fusion polypeptide may enhance incorporation of the fusion polypeptide onto and / or into the membrane of a vesicle, for example, the internal and / or external leaflet of the phospholipid bilayer of an exosome membrane. The fusion polypeptide may have at least a part of an amino acid sequence of an HA binding protein. The fusion polypeptide may have at least a part of an amino acid sequence for a hyaluronan (HA)-binding motif, such as a LINK domain or a functional fragment thereof. The fusion polypeptide may have a polypeptide linker sequence (e.g., an Fc domain and / or a GSSG linker), followed by an amino acid sequence coding for an anchor protein sequence (e.g., a prenylation site, fatty acylation site, a GPI sequence, or a transmembrane domain) or any isoform, fragment, variation thereof, or a ligand to the aforementioned proteins thereof, or the like known by one of ordinary skill in the art. All variants are encompassed by the present invention.

[0145] As used herein, the term “truncated” or “fragment” or “active fragment” refers to a portion of a nucleic acid or polypeptide provided herein that retains the ability to be expressed by the engineered EVs provided herein. In some embodiments, the active fragment retains the ability to activate a target polypeptide, thereby increasing the activity of said target polypeptide (e.g., suppressing an immune response). In some embodiments, a fusion polypeptide construct comprising truncated protein is more stable than a fusion polypeptide construct comprising the full-length protein. As used herein, the term “fragment” also refers to any portion of a polypeptide, protein, nucleic acid, or extracellular vesicle (EV) associated molecule that comprises a subset of a sequence or structure described herein and retains at least one biological, structural, or functional property of the corresponding sequence. A protein or polypeptide fragment may include domains, subunits, or regions that mediate a specific function or interaction, such as receptor binding, enzymatic activity, signal transduction, or effector function. Exemplary fragments ofimmunoglobulins include Fc, Fab, Fab', and F(ab')2 regions. In some embodiments, fragments of EV or exosome-associated proteins, such as tetraspanins (e.g., CD9, CD63, CD81), targeting peptides, or fusion domains, may retain membrane localization or cargo-loading activity. A nucleic acid fragment may comprise a subsequence of a larger nucleic acid molecule, such as a portion encoding a functional domain, regulatory element, or epitope. Fragments may be naturally occurring, recombinantly expressed, or synthetically generated, and may include truncations, deletions, or variants with conservative substitutions or minor modifications that preserve the desired function or property of the parent molecule. Unless otherwise indicated, the term “fragment” encompasses both naturally occurring and engineered fragments.

[0146] As used herein, the terms “specifically bind” and / or “specifically recognize” or “substantially binds” refers to the affinity of a binding molecule for a target molecule compared to the binding molecule's affinity for non-target molecules. A binding molecule (e.g., a signaling domain or a POI domain) that specifically binds a target molecule (e.g., a target polypeptide or polysaccharide provided herein) does not substantially recognize or bind non-target molecules, e.g., an antibody “specifically binds” and / or “specifically recognize” another molecule, meaning that this interaction is dependent on the presence of the binding specificity of the molecule structure, e.g., an antigenic epitope. As used herein, “non-specific binding” and “background binding” refers to the interaction that does not depend on the presence of specific structure (e.g., a specific antigenic epitopes). Methods of measuring binding of a polypeptide to a target are known in the art (e.g., differential scanning calorimetry, isothermal titration calorimetry, spectroscopy, crystallography, surface plasmon resonance, co-immunoprecipitation, pulldown assays, crosslinking, yeast two-hybrid system, tandem affinity purification-mass spectroscopy, protein microarrays, bio-layer interferometry, far-Westem blots, computational prediction, analytical ultracentrifugation, light scattering, fluorescence spectroscopy, resonance energy transfer, ELISA or ELISPOT assays, or the like known by one of ordinary skill in the art).

[0147] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to obtain a desired pharmacologic and / or physiologic effect, such as to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with inflammation. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In someinstances, an individual (e.g., an individual suspected to be suffering from and / or genetically pre-disposed to an inflammation-associated disease or disorder) is treated prophylactically with a preparation of EVs described herein and such prophylactic treatment completely or partially prevents an inflammation- associated disease or disorder or sign or symptom thereof. In some instances, an individual is treated therapeutically (e.g., when an individual is suffering from an inflammation-associated disease or disorder), such therapeutic treatment causes a partial or complete cure for the disease or disorder and / or reverses an adverse effect attributable to the disease or disorder and / or stabilizes the disease or disorder and / or delays progression of the disease or disorder and / or causes regression of the disease or disorder.

[0148] As used herein, “preventing” or “prevention” refers to any methodology where the disease state does not occur due to the actions of the methodology (such as, for example, administration of a composition or construct as described herein). In one aspect, it is understood that prevention can also mean that the disease is not established to the extent that occurs in untreated controls. Accordingly, prevention of a disease encompasses a reduction in the likelihood that a subject can develop the disease, relative to an untreated subject (e.g., a subject who is not treated with the methods or compositions described herein).

[0149] As used herein, the terms “inflammatory condition,” “inflammatory disorder,” and “inflammation” interchangeably and refer to any disease characterized by elevated inflammatory markers, such as, for example, inflammation due to injury, inflammation due to trauma (e.g., physical trauma), and / or neuroinflammation. Inflammation can comprise acute inflammation, chronic inflammation, and / or subacute inflammation. Acute inflammation is a sudden and temporary response to injury, infection, or illness. Symptoms include redness, swelling, pain, and heat. Chronic inflammation is a long-term inflammation that can last for a long period of time, such as, for example, weeks, months, or years. Chronic inflammation can be a factor in many diseases, such as, for example, heart disease, diabetes, arthritis, and some forms of cancer. Subacute inflammation comprises the period between acute and chronic inflammation that can last between about 2 and about 6 weeks.

[0150] As used herein, the terms “autoimmune condition” and “autoimmune disease” are used interchangeably and refer to any disease characterized by abnormal functioning of the immune system and may include, but is not limited to, achalasia, Addison’s disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / Anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), osteoarthritis, Churg-Strauss syndrome (CSS), eosinophilic granulomatosis(EGPA), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn’s disease, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), Paget' s disease, discoid lupus, Dressier’s syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammalglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, type 1 diabetes juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, Lyme disease chronic, Meniere’s disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal Lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDA, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes type I, II, III, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud’s phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia (SO), takayasu’s arteritis, temporal arteritis / Giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease. An autoimmune condition or autoimmune diseases may be caused by, but not limited to, a natural predisposition, an infection (e.g., bacteria or virus), drugs, vaccination, environmental triggers (e.g., toxins or chemicals such as dust, silica, oil, benzene, tri- or perchloroethylene etc.), stress, cancer, blood or tissue or organ transplantation, or unknown etiology. Autoimmune disorders may result in, but are not limited to, the destruction of body tissue, abnormal growth of an organ or tissue, changes in organ or tissue function (e.g., changes in blood vessels, connective tissue, function of endocrine glands, joints, muscles, blood cells, skin, etc.).

[0151] As used herein, the terms “subject,” “individual,” “host,” and “patient” are used interchangeablyand may refer to any animal, mammal, bird, fish, reptile, and amphibian, for example, human, monkey, dog, cat, horse, pig, cattle, ox, donkey, rabbit, sheep, goat, mouse, rat, guinea pig, llama, chicken, goose, duck, turkey, or the like receiving or registered to receive a therapeutic amount of a composition of the present invention for medical care or treatment.

[0152] As used herein, the term “injection” refers to any process or method which allows the person skilled in the art to administer any therapeutic to a target site by penetration. Examples of injection are, but not limited to, subcutaneous, subcuticular, subcapsular, subarachnoid, intradermal, intramuscular, intravenous, intra-arterial, intraventricular, intracapsular, intraorbital, intraocular, intrathoracic, intraperitoneal, intravitreal, retro-orbital, intranasal, intracerebral, intrathymic, intraspinal, intrastemal, intra-articular, intracavemous, intracardiac, intraosseous, intrathecal, transtracheal, epidural, or the like as known in the art. A therapeutic does of the present invention may be delivered to a patient by means of controlled release, for example, but not limited to, implantable pump and implantable cannulas to provide continuous access to the venous or arterial system.

[0153] As used herein, the term “topical application” refers to applying or spreading a composition of the present invention onto surfaces on or in the body, both internally and / or externally, in a therapeutically effective amount for local and / or systemic treatment. Topical application may be epicutaneous, wherein a composition of the present invention may be directly applied onto a localized surface of the skin or mucous membranes. Topical application may include transdermal application wherein a composition of the present invention may be absorbed into the body to obtain systemic delivery and systemic distribution. For example, a transdermal patch may be applied onto the body to deliver a therapeutic dose of a composition of the invention presented herein. Topical application formulations may include, but are not limited to, creams, foams, gels, lotions, solutions, ointments, dermal patch, transdermal patches, powder, solid, sponge, tape, vapor, paste, film, liposomes, balm, shampoo, spray, or tincture. A therapeutic dose of a composition of the present invention may be delivered vaginally (for example, a vaginal suppository, vaginal ring, douche, intrauterine device, intravesical infusion, and the like) or urethra.

[0154] As used herein, the term “enteral administration” refers to a composition of the present invention administered via the gastrointestinal tract in a therapeutically effective amount for local or systemic treatment. Enteral administration may include, but is not limited to, delivery of a composition of the present invention via the mouth, sublingual, esophagus, gastric (for example, the stomach), small intestines, large intestines, or rectum. Oral delivery of the present invention may include, but is not limited to, the use of a capsule, pastille, pill, tablet, solution, gel, suspension, emulsion, syrup, elixir, tincture, mouthwash, lozenges, chewing gum, lollipop, osmotic-controlled release oral delivery system, or the like. Gastric delivery may involve the use of a tube or nasal passage that leads directly to the stomach, for example, a percutaneous endoscopic gastrostomy tube. Gastric delivery may involve direct injection made through the abdominal wall. Rectal delivery may involve, but is not limited to, the use of a suppository, ointment, enema, murphy drip, or the like. A therapeutic does of the present invention maybe delivered to a patient by means of controlled release, for example, but not limited to, controlled release drug delivery pellet or pill.

[0155] As used herein, the term “mucosal administration” refers to a composition of the present invention administered via any mucous membrane in a therapeutically effective amount for local or systemic treatment. Mucosal administration may include, but is not limited to, delivery of a composition of the present invention via the nose (intranasal) and / or mouth (inhalation).

[0156] As used herein, the term “inhalation” (e.g., pulmonary delivery, pulmonary administration) refers to delivery to the respiratory system through the respiratory route, including but not limited to, intranasal administration, oral administration, and oral inhalative administration (e.g., intratracheal instillation and intratracheal inhalation) of a therapeutically effective amount for local or systemic treatment. Pulmonary delivery of a therapeutically effective amount of a composition of the present invention may be achieved by dispersion, for example by using a syringe. Pulmonary delivery of a composition of the present invention may be achieved by aerosol administration, wherein aerosol administration may deposit a therapeutically effective amount of the present invention by gravitational sedimentation, inertial impaction, or diffusion.

[0157] As used herein, the term “molecular weight” or “MW” refers to the theoretical or measured mass of a molecule, polymer, polypeptide, or macromolecule expressed either as a numerical value in Daltons (Da) or kilodaltons (kDa). Unless otherwise indicated, molecular weight may be expressed as the molecular mass of a single molecule calculated from its chemical composition, or as an average molecular weight (e.g., number-average or weight-average) determined by an appropriate analytical method. Molecular weight values are typically determined or estimated by methods known in the art, such as mass spectrometry, analytical ultracentrifugation, light scattering, electrophoretic mobility (e.g., SDS-PAGE), size exclusion chromatography, or other accepted analytical technique.Engineered extracellular vesicle (EV) compositions

[0158] The compositions provided herein comprises at least one extracellular vesicle, wherein the extracellular vesicle comprises at least one fusion polypeptide or a plurality of fusion polypeptides comprising at least one vesicle targeting domain and at least one protein of interest domain, also termed “signaling domain”.

[0159] Extracellular vesicles (EVs) are lipid particles that are released from various cell types that function to transfer “cargo” such as nucleic acids and proteins to other cells. EVs are not able to replicate but serve as cell messengers. EV-mediated signals can be transmitted by all the different biomolecule categories - e.g., protein, lipids, nucleic acids, and sugars - and the unique package of this information provides both protection and the option of simultaneous delivery of multiple different messengers even to sites remote to the vesicular origin. EVs may also be able to modulate a milieu of cellular signaling processes.

[0160] There are various types of extracellular vesicles that are named for their site of origin in a cell,size, and structural and / or functional properties. In some embodiments of any of the aspects provided herein, the extracellular vesicle is an exosome, ectosome, macrovesicle, microparticle, apoptotic body, vesicular organelle, oncosome, exosphere, exomeres, or cell-derived nanovesicles (CDN) (e.g., by genesis via grating or shearing cells), liposomes or the like, known by one of ordinary skill in the art. In various embodiments, the extracellular vesicle comprises a phospholipid bilayer with an exterior phospholipid layer and an interior phospholipid layer, wherein the exterior phospholipid layer has an external surface and an internal surface, wherein the interior phospholipid layer has an internal surface and an external surface, and the internal surface of the exterior phospholipid layer faces the internal surface of the interior phospholipid layer, and the phospholipid bilayer encloses an internal space, wherein the external surface of the interior phospholipid layer faces the internal space and wherein the external surface of the exterior phospholipid layer faces an extracellular environment, and the external surface of the inner phospholipid layer is the internal surface of the extracellular vesicle.

[0161] In various embodiments, the extracellular vesicles range in size from about 30 nanometers (nm) to about 300 nm. In various embodiments, the plurality of EVs range in size from about 30 nm to about 150 nm. In various embodiments, the plurality of engineered extracellular vesicles includes one or more engineered extracellular vesicles that are about 10 nm to about 250 nm in diameter, including those about 10 nm to about 15 nm, about 15 nm to about 20 nm, about 20 nm to about 25 nm, about 25 nm to about 30 nm, about 30 nm to about 35 nm, about 35 nm to about 40 nm, about 40 nm to about 50 nm, about 50 nm to about 60 nm3 about 60 nm to about 70 nm, about 70 nm to about 80 nm, about 80 nm to about 90 nm, about 90 nm to about 95 nm, about 95 nm to about 100 nm, about 100 nm to about 105 nm, about 105 nm to about 110 nm, about 110 nm to about 115 nm, about 115 nm to about 120 nm, about 120 nm to about 125 nm, about 125 nm to about 130 nm, about 130 nm to about 135 nm, about 135 nm to about 140 nm, about 140 nm to about 145 nm, about 145 nm to about 150 nm, about 150 to about 200 nm, about 200 nm to about 250 nm, or about 250 nm or more in diameter.

[0162] In some embodiments of any of the aspects provided herein, the extracellular vesicle is an exosome. Exosomes are membrane -bound EVs that are produced in the endosomal compartment of most eukaryotic cells. As used herein, the term “exosome” refers to a species of extracellular vesicle from about 20 nm to about 400 pm in diameter, e.g., about 30 nm - 200 nm in diameter by inward invagination of a portion of a membrane of an endosome (for example, an early or late endosome), wherein the endosome is within a cell comprising a plasma membrane, and the exosome is released from the cell upon fusion of another portion of the endosome membrane with the plasma membrane. An exosome can have a specific density (flotation at from about 1.13 to about 1.21 g / ml on a sucrose gradient) and an endocytic origin. An exosome may refer to a species of extracellular vesicle between 20 nm - 400 pm in diameter, more preferably, 30 nm - 200 nm in diameter, that originates by budding of a portion of a plasma membrane from a cell wherein the budded portion of the plasma membrane is released to the extracellular environment. An exosome can comprise lipid or fatty acid and polypeptide, a payload (e.g., a therapeuticagent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid such as RNA or DNA), a sugar (e.g., a simple sugar, polysaccharide, or glycan), and / or other molecules. The exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. Exosomes generally contain a lipid bilayer with a center containing fluid, DNA, RNA, and / or proteins. The bilayer comprises lipids including, but not limited to, cholesterol, a diglyceride, a sphingolipid (e.g., sphingomyelin, ceramide, etc.), a phospholipid, a glycerophospholipid (such as, for example, phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and a polyglycerophospholipid (e.g., bisphosphate)).

[0163] The EVs (e.g., exosomes or cell derived vesicles) provided herein may comprise cargo, for example, peptides, proteins, nucleic acids, lipids, metabolites, carbohydrates, biomolecules, small molecules, large molecules, vesicles, organelles, or fragments thereof. Exosome cargo may be located within the internal space of the exosome. EV cargo may be membrane-bound, spanning one or both layers of the exosome phospholipid bilayer (for example, a transmembrane protein). EV cargo may be in contact with the exterior or interior surface of the exosome, for example, through a covalent bond or a non- covalent bond. The phospholipid bilayer of the EV or exosome provided herein may comprise one or more transmembrane proteins, wherein a portion of the one or more transmembrane membrane proteins is located within the internal space of the exosome. The phospholipid bilayer of the EV or exosome provided herein may comprise one or more transmembrane proteins, wherein a portion of the one or more transmembrane membrane proteins traverses the EV phospholipid bilayer. The phospholipid bilayer of the EV may comprise one or more transmembrane proteins, wherein the one or more transmembrane membrane proteins comprises a domain on the exterior of the exosome. Cargo proteins or peptide may include therapeutic peptides or proteins that act on a target (e.g., a target cell) that is contacted with the exosome. Cargo proteins may comprise a fusion polypeptide comprising a targeting protein or peptide or fragment or modification thereof, such that the cargo fusion polypeptide can be targeted to an exosome lumen.

[0164] In some embodiments of any of the aspects, the extracellular vesicles or exosomes provided herein have positive expression of CD81, CD82, CD37, CD63, CD9, CD151, CD105, or any combination thereof. In one nonlimiting embodiment, the extracellular vesicles or exosomes provided herein have positive expression of CD81, CD82, CD37, CD63, CD9, CD151, and CD105. In various embodiments, the plurality of engineered extracellular vesicles (EVs) includes one or more EV expressing one or more engineered biomarkers. In certain embodiments, the biomarkers tetraspanins. In other embodiments, the tetraspanins are one or more selected from the group comprising CD63, CD81, CD82, CD53, CD 151, and CD37. In one nonlimiting embodiment, an EV has positive expression of CD63, CD81, CD82, CD53, CD151, and CD37. In an embodiment, an EV has positive expression of CD63, CD81, CD82, CD53, CD 151, and CD37 and at least one engineered tetraspanin. In another nonlimiting embodiment, the extracellular vesicles or exosomes provided herein have positive expression of CD81, CD82, CD37,CD53, CD63, CD9, CD151, and CD105. In other embodiments, the EV express one or more lipid raft associated proteins (e.g., glycosylphosphatidylinositol-anchored proteins and flotillin), cholesterol, sphingolipids such as sphingomyelin, and / or hexosylceramides.

[0165] In other embodiments, EVs express one or more proteins related to exosome formation and packaging of cytosolic proteins, e.g., Hsp70, Hsp90, 14-3-3 epsilon, PKM2, GW 182 and AGO2. In certain embodiments, the EV express CD63, HSP70, CD105 or combinations thereof. In one nonlimiting embodiment, the EV express CD63, HSP70, and CD 105. In other embodiments, the EV do not express CD9 or CD81, or express neither. In another nonlimiting embodiment, the EV do not express CD9. In yet another nonlimiting embodiment, the EV do not express CD81. In a nonlimiting example, a plurality of EV can include one or more EV that are CD63+, HSP70+, CD105+, CD9 , and CD81 . In another nonlimiting example, a plurality of EV can include one or more EV that are CD63+, HSP90+, CD 105+, CD9’, and CD81’.

[0166] The EVs provided herein are specifically engineered to express fusion polypeptides that elicit biological signaling via a target cell. In some embodiments, the fusion polypeptide is overexpressed to elicit a biological response on a target cell or target polypeptide. The engineered EV comprises at least one fusion polypeptide and can comprise a plurality of the same or different fusion polypeptides provided herein. The fusion polypeptides provided herein comprise a protein of interest (POI) domain, also termed the signaling domain. In some embodiments, the POI comprises a tTSG-6 or a fragment thereof. In one embodiment, the POI comprises a truncated TSG-6 (e.g., tTSG-6). In certain embodiments, the fusion polypeptides provided herein comprise a fragment of a truncated TSG-6 (e.g., a truncated tTSG-6). In one embodiment, the truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., a truncated tTSG-6) comprises a CUB domain and a LINK domain.

[0167] The fusion polypeptides provided herein can comprise one or more of a POI domain, wherein the POI is tTSG-6 or a fragment thereof, such that expression of said fusion polypeptide is permitted and that the number of signaling domains, does not impede protein expression or folding. Furthermore, the EVs provided herein can express more than one fusion polypeptide (e.g., encoded by multiple different nucleic acid constructs). One of skill in the art can appreciate that an engineered EV can include one or more combinations of different signaling domains and / or vesicle targeting domains, or that one can use a plurality of engineered EVs, each including one or more vesicle targeting domains and one or more signaling domains.

[0168] In some embodiments, the compositions provided herein comprises at least one extracellular vesicle, wherein the extracellular vesicle comprises at least one fusion polypeptide or a plurality of fusion polypeptides comprising at least one vesicle-targeting domain and at least one POI domain, or termed signaling domain, wherein the POI comprises a comprises a hyaluronan (HA)-binding motif. In some embodiments, the HA-binding motif comprises a LINK module or LINK domain. The LINK domain may mediate specific, non-covalent binding to HA, a linear polysaccharide consisting of repeating disaccharideunits of D-glucuronic acid and N-acetyl-D-glucosamine.

[0169] In some embodiment, the composition provided herein comprises at least one extracellular vesicle, wherein the extracellular vesicle comprises at least one fusion polypeptide or a plurality of fusion polypeptides comprising at least one vesicle targeting domain and at least one POI domain, also termed “signaling domain,” wherein the POI is a TNF-Stimulated Gene-6 protein (TSG-6) protein or a fragment thereof.

[0170] In some embodiments, the EVs provided herein comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more fusion polypeptides. The fusion polypeptides can be encoded by the same vector or separate vectors. In some embodiments of any of the aspects, the engineered extracellular vesicle comprises at least two POI domains, wherein the POI is a truncated TSG-6 or a fragment thereof, and / or at least two vesicle targeting domains. In one embodiment, the truncated TSG-6 or a fragment thereof comprises a CUB domain and a LINK domain.

[0171] In some embodiments, the fusion polypeptide comprises one or more, two or more, three or more, four or more, five or more, or six or more POI domains on the same polypeptide or nucleic acid construct encoding said polypeptide, wherein the POI domain is a truncated TSG-6. For example, the fusion polypeptides provided herein can express a fusion polypeptide encoding one or more, two or more, three or more, four or more, five or more, or six or more signaling domains.

[0172] In some embodiments, the EV comprises one or more, two or more, three or more, four or more, five or more, or six or more fusion polypeptides on the same EV. For example, EVs comprising one or more, two or more, three or more, four or more, five or more, or six or more fusion polypeptides wherein the fusion polypeptides encode a signaling domain. In a nonlimiting embodiment, the signaling domain comprises a tTSG-6 domain. In another example, EVs comprising one or more, two or more, three or more, four or more, five or more, or six or more fusion polypeptides wherein the fusion polypeptides encode for one or proteins.

[0173] In various embodiments, the signaling domain comprises a protein or peptide of interest, or a fragment thereof. In various embodiments, the signaling domain comprises a truncated protein. In various embodiments, the signaling domain comprises a hyaluronan (HA)-binding motif, such as a LINK domain or a functional fragment thereof.

[0174] In some embodiments of any of the aspects provided herein, the signaling domain comprises a protein or a fragment thereof, selected from the group consisting of tumor necrosis factor stimulated gene- 6 (TSG-6), cartilage link protein 1 (HAPLN1), brain link proteins (e.g., HAPLN2, HAPLN3, HAPLN4), and members of the hyalectan family, including aggrecan (ACAN), versican (VCAN), brevican (BCAN), and neurocan (NCAN). Additional HA-binding proteins that contain LINK domains or functionally related HA-binding motifs include the hyaluronan-binding receptors CD44, receptor for hyaluronan- mediated motility (RHAMM / HMMR), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1),stabilin- 1 , stabilin-2 (also known as hyaluronan receptor for endocytosis, HARE), hyaluronan-binding protein 1 (HABPl / p32 / gClqR), hyaluronectin, inter-a-inhibitor (lai) heavy chains (HC1, HC2, HC3), pentraxin 3 (PTX3), intercellular adhesion molecule-1 (ICAM-1), and layilin.

[0175] In some embodiments of any of the aspects provided herein, the protein of interest domain (also referred to as the signaling domain) comprises a polypeptide or a fragment thereof, or a nucleic acid encoding said polypeptide or fragment thereof, selected from the group consisting of: Table 1 (below). In some embodiments, the POI domain comprises a polypeptide or a fragment thereof, or a nucleic acid encoding said polypeptide or fragment thereof, of TSG-6. In one embodiment, the POI domain comprises a fragment of TSG-6 (e.g., tTSG-6). In some embodiment, the POI domain comprises a truncated TSG-6 (e.g., tTSG-6), e.g., a tTSG-6 comprising an amino acid sequence as set forth in SEQ ID NO: 7 or is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 6. In yet another embodiment, the POI domain comprises a fragment of a tTSG-6 (e.g., a truncated tTSG-6); a truncated tTSG-6 comprising an amino acid sequence as set forth in SEQ ID No. 9 or is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 8 In yet another embodiment, the POI domain comprises a fragment of a tTSG-6 (e.g., a truncated tTSG-6); a truncated tTSG-6 comprising an amino acid sequence as set forth in SEQ ID No. 11 or is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 10. In yet another embodiment, the POI domain comprises a fragment of a tTSG-6 (e.g., a truncated tTSG-6); a truncated tTSG-6 comprising an amino acid sequence as set forth in SEQ ID No. 13 or is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TSG-6, tTSG-6, or the truncated tTSG-6 described herein comprises a signal peptide comprising an amino acid sequence as set forth in SEQ ID NO: 17 or is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 16. In some embodiments, the TSG-6, tTSG-6, or the truncated tTSG-6 described herein lacks a signal peptide. Non-limiting examples of nucleic acid sequences and amino acid sequences that encode the POI domains, TSG-6, tTSG-6, truncated tTSG-6, or T-SG6 LINK domain provided herein are also provided in Table 1.Table 1. Sequences for Proteins of Interest

[0176] The polypeptides (e.g., TSG-6, tTSG-6) provided in Table 1 above are involved in a range of biological processes, including but not limited to, suppressing the adaptive arm of the immune. The POI domains comprising a tTSG-6 or a fragment thereof can also be used to track, purify, or identify the engineered EVs from native EVs. The genes, transcripts, polypeptides, and fragments and variants thereof can be used in any combination from Table 1 to be expressed by an engineered EV provided herein. In some embodiments, the POI domain is a human polypeptide. In some embodiments, the POI domain is a homologue of the human polypeptide (e.g., mouse).

[0177] In some embodiments of any of the aspects, the engineered cell or EVs provided herein comprises an exogenous nucleic acid encoding one or more exogenous polypeptide (s) selected from the group consisting of the polypeptides listed in Table 1.

[0178] In some embodiments, the composition comprises a fusion polypeptide comprising: a signaling domain comprising a LINK domain, a CUB domain, and a C terminal tail, wherein the at least a part of the C-terminal of the signaling domain is truncated; and at least one vesicle targeting domain. In some embodiments, the composition comprises a plurality of fusion polypeptides wherein each fusion polypeptide comprises a signaling domain comprising a LINK domain, and a CUB domain, and a C terminal tail, wherein the at least a part of the C-terminal of the signaling domain is truncated; and at least one vesicle targeting domain. In some embodiments, the composition comprises an engineered extracellular vesicle further comprising at least one fusion polypeptide, wherein at least one fusion polypeptide described herein is displayed on the surface of the engineered extracellular vesicle, e.g., an engineered extracellular vesicle described herein. In some embodiments, the composition comprises a plurality of engineered extracellular vesicles wherein at least one fusion polypeptide described herein is displayed on the surface of the each engineered extracellular vesicle.

[0179] In some embodiments, the fusion polypeptide described herein comprises a hyaluronan (HA)- binding motif. In some embodiments, the HA-binding motif comprises a LINK module or LINK domain, which is a conserved structural domain found in various HA-binding proteins. The LINK domain may mediate specific, non-covalent binding to HA, a linear polysaccharide consisting of repeating disaccharideunits of D-glucuronic acid and N-acetyl-D-glucosamine.

[0180] In certain embodiments, the fusion polypeptide described herein comprises a hyaluronan (HA)- binding motif, such as a LINK domain or a functional fragment thereof. The LINK domain is a conserved structural module of approximately 100 amino acids that mediates specific binding to HA and related glycosaminoglycans. The LINK domain or other HA-binding motif may be derived from any HA-binding protein known in the art. Exemplary LINK domain containing or HA-binding proteins include, but are not limited to, tumor necrosis factor stimulated gene-6 (TSG-6), cartilage link protein 1 (HAPLN1), brain link proteins (e.g., HAPLN2, HAPLN3, HAPLN4), and members of the hyalectan family, including aggrecan (ACAN), versican (VCAN), brevican (BCAN), and neurocan (NCAN). Additional HA-binding proteins that contain LINK domains or functionally related HA-binding motifs include the hyaluronan-binding receptors CD44, receptor for hyaluronan-mediated motility (RHAMM / HMMR), lymphatic vessel endothelial hyaluronan receptor- 1 (LYVE-1), stabilin-1, stabilin-2 (also known as hyaluronan receptor for endocytosis, HARE), hyaluronan-binding protein 1 (HABPl / p32 / gClqR), hyaluronectin, inter-a-inhibitor (lai) heavy chains (HC1, HC2, HC3), pentraxin 3 (PTX3), intercellular adhesion molecule-1 (ICAM-1), and layilin. The LINK domain may be used in its full-length form or as a fragment, variant, or derivative retaining HA-binding activity. In some embodiments, the LINK domain is engineered or modified to enhance HA affinity, structural stability, or tissue retention when incorporated into a fusion polypeptide.

[0181] In some embodiments, the HA-binding motif may comprise a sequence derived from one or more of the above-listed proteins, or a fragment, variant, or derivative thereof that retains HA-binding activity. For example, the LINK module from TSG-6 (nucleic acid SEQ ID NO: 14 and amino acid sequence SEQ ID NO: 15) may be used as an HA-binding domain. In other embodiments, the HA- binding motif may be engineered or synthetic, provided that it retains the ability to specifically bind HA.

[0182] In certain embodiments, inclusion of an HA-binding motif in the fusion polypeptide facilitates binding to extracellular HA and may enhance tissue localization, retention, or bioactivity of the fusion construct within HA-rich environments such as the extracellular matrix, synovial fluid, or inflamed tissues. In some embodiments, the HA-binding motif promotes association of the fusion polypeptide with CD44-expressing cells through HA-mediated interactions, thereby modulating cell adhesion, migration, signaling, or immune responses.

[0183] Accordingly, in various embodiments, the fusion polypeptide may comprise one or more HA- binding motifs derived from any HA-binding protein or functional equivalent thereof, enabling targeted engagement with HA and HA-containing structures in vivo or in vitro.

[0184] In some embodiments of any of the aspects, the fusion polypeptide comprises a signaling domain, also referred to as the protein of interest (POI) domain. In some embodiments of any of the aspects, the POI domain is TSG-6 or tTSG-6 or a fragment thereof. In some embodiments of any of the aspects, the POI domain substantially binds to one or more of a target. In some embodiments of any of the aspects, the target is a polypeptide. In some embodiments of any of the aspects, the target polypeptide is acellular receptor. In some embodiments of any of the aspects, the target polypeptide is an immunosuppressive polypeptide. In some embodiments of any of the aspects, the target polypeptide is an immunostimulatory polypeptide. In some embodiments of any of the aspects, the target is a polysaccharide.

[0185] In some embodiments of any of the aspects, the target is hyaluronan (HA). In some embodiments, the fusion polypeptide binds to HA via a LINK domain or an HA binding motif of the signaling domain, modifying and promoting the formation of stable HA-rich matrices. In some embodiments of any of the aspects, the target is inter-a-inhibitor (lai) / heavy chains (HC1, HC2, HC3). In some embodiments, the POI catalyzes the transfer of heavy chains from lai onto HA, forming HC-HA complexes that stabilize cellular extracellular matrix during inflammation. In some embodiments of any of the aspects, the POI interacts with Versican (a large chondroitin sulfate proteoglycan). In some embodiments of any of the aspects, the POI interacts with aggrecan. In some embodiments of any of the aspects, the POI binds thrombospondin- 1 (TSP-1). In some embodiments of any of the aspects, the POI interacts with TNFa. In some embodiments of any of the aspects, the POI binds and inhibits TNFa activity. In some embodiments of any of the aspects, the POI binds to CXCL8 (IL-8). In some embodiments of any of the aspects, the POI reduces neutrophil chemotaxis by sequestering IL-8 receptor interaction. In some embodiments of any of the aspects, the POI binds to CXCL12 (SDF-1). In some embodiments of any of the aspects, the POI modulates stem / progenitor cell migration. In some embodiments of any of the aspects, the POI modulates HA-CD44 interaction. In some embodiments of any of the aspects, the POI modulates HA-CD44 interaction through HC-HA formation. In some embodiments of any of the aspects, the POI enhances HA-CD44 interaction. In some embodiments of any of the aspects, the POI inhibits H-CD44 interaction. In some embodiments of any of the aspects, the POI influences TLR2 / TLR4 mediated macrophage activation. In some embodiments of any of the aspects, the POI influences integrin mediated cell adhesion and migration. In some embodiments of any of the aspects, the POI interacts with fibronectin. In some embodiments of any of the aspects, the POI interacts or co-localizes with Pentraxin 3 (PTX3). In some embodiments of any of the aspects, the POI interacts with fibrinogen during clot remodeling.

[0186] The engineered exosomes provided herein can be designed to activate, block, or modulate a given target polypeptide with the appropriate POI domain that binds to, interacts with, or modulates the function or expression of the target polypeptide.

[0187] In some embodiments, the present disclosure provides a fusion polypeptide comprising a signaling domain that specifically interacts with one or more extracellular or cell-surface molecules. In some embodiments, the signaling domain is configured to bind hyaluronan (HA) or other extracellular matrix (ECM) components, thereby localizing the fusion polypeptide to HA-rich or inflamed tissue environments. In various embodiments, the fusion polypeptide further comprises an HA-binding motif, such as a LINK module derived from an HA-binding protein, including but not limited to tumor necrosisfactor-stimulated gene 6 (TSG-6), cartilage link protein (HAPLN1), aggrecan, versican, brevican, neurocan, CD44, receptor for hyaluronan-mediated motility (RHAMM), Lymphatic Vessel Endothelial HA Receptor 1 (LYVE-1), or stabilin family receptors.

[0188] In certain embodiments, the signaling domain may be derived from, or retain binding specificity for one or more proteins or polysaccharides known to interact with fibronectin, thrombospondin- 1, thrombospondin-2, pentraxin-3 (PTX3), inter-a-inhibitor (lai) heavy chains, bikunin, chondroitin-4- sulfate, dermatan sulfate, heparin, heparan sulfate, and chemokines such as CXCL8 (IL-8) or CXCL12 (SDF-1), cartilage or neural proteoglycans, such as brevican, neurocan, phosphacan, or tenascins, or to ECM proteins including fibrillin, laminin, or tenascin-C.

[0189] In certain embodiments, the signaling domain may be derived from, or retain binding specificity for one or more proteins or polysaccharides known to interact with TSG-6. Exemplary binding partners of TSG-6 include, but are not limited to, fibronectin, thrombospondin- 1, thrombospondin-2, pentraxin-3 (PTX3), inter-a-inhibitor (lai) heavy chains, bikunin, chondroitin-4-sulfate, dermatan sulfate, heparin, heparan sulfate, and chemokines such as CXCL8 (IL-8) or CXCL12 (SDF-1). In certain embodiments, the fusion polypeptide may also bind to cartilage or neural proteoglycans, such as brevican, neurocan, phosphacan, or tenascins, or to ECM proteins including fibrillin, laminin, or tenascin-C.

[0190] In some embodiments, the signaling domain may include one or more structural motifs capable of interacting with sulfated glycosaminoglycans (GAGs) or with proteins containing RGD or collagen- like sequences, thereby facilitating the localization of the fusion polypeptide to extracellular matrices, connective tissues, or sites of inflammation. In some embodiments, the signaling domain of the fusion polypeptide is configured to modulate cellular responses such as cytokine production, macrophage polarization, or T-cell activation upon binding of the signaling domain to its respective target.

[0191] Accordingly, in various embodiments, the fusion polypeptide comprises a signaling domain operably linked to a domain from or mimicking an HA-binding or TSG-6-interacting protein, thereby enabling selective localization, receptor clustering, or signal modulation within HA-containing, GAG- rich, or inflamed tissue microenvironments.

[0192] Non-limiting examples of targets, (e.g., polypeptides, polysaccharides, etc.) include those listed in Table 2 (below).Table 2. Exemplary Target Polypeptides

[0193] The EVs provided herein further comprise at least one fusion polypeptide comprising a vesicle targeting domain. In various embodiments, the vesicle targeting domain provided herein is capable of binding or anchoring the fusion polypeptide provided herein to an extracellular vesicle, e.g., via targeting of the phospholipid bilayer membrane. In various embodiments, the vesicle targeting domain is a GPI domain (e.g., GPI linker, GPI anchor), fatty acetylation site, prenylation site, or transmembrane domain. In some embodiments, a vesicle targeting domain comprises a transmembrane domain from a tetraspanin. In some embodiments, sequences for vesicle targeting domains include transmembrane regions of CD9 (for example, transmembrane 2 or 3 of CD9, also known as CD9tm2 or CD9tm3, respectively). In some embodiments, a vesicle targeting domain is the transmembrane 2 domain from CD9, or CD9tm2. In another embodiment, a vesicle targeting domain comprises the transmembrane 3 domain from CD9, or CD9tm3. One of skill in the art can appreciate that the aforementioned refer to peptide or protein sites, wherein covalent lipid attachment supports embedding of the lipid in a cell membrane (e.g., phospholipid bilayer). Biochemical forces that anchor vesicle targeting domains to the EV phospholipid bilayer may include, but are not limited to, electrostatic forces, affinity for EVs through protein-protein interactions with natively resident proteins (e.g., CD81, CD63, CD9, ALIX, TSG101, ITGB1, CD98, CD298, MARCKS, PTGFRN, Lactadherin (MFGe8)), association or affinity for negatively or positively curved phospholipids, association or affinity for negatively or positively charged domains of resident membrane associated proteins, etc., or the like.

[0194] In some embodiments, the fusion polypeptide comprises one or more, two or more, three or more, four or more, five or more, or six or more vesicle targeting domains on the same polypeptide or nucleic acid construct encoding said polypeptide. For example, the fusion polypeptides provided herein can comprise TSG-6, or a fragment thereof (e.g., truncated tTSG-6), and CD9tm2.

[0195] In some embodiments, the fusion polypeptide is a prenylated protein. Prenylated proteins are proteins that have at least one prenylation site. In some embodiments, the fusion polypeptide comprises a vesicle targeting domain. In some embodiments, the vesicle targeting domain comprises a prenylation site. Prenylation occurs when a 15-carbon or 20-carbon, famesyl or geranylgeranyl isoprenoid, respectively, is covalently bound via a thioether bond to a cysteine at or near the carboxy terminus of a protein. In general, a prenylation site comprises an amino acid sequence CAAX (also referred to as a “CAAX box”), wherein C represents cysteine, A represents an aliphatic amino acid (glycine, alanine, valine, leucine, or isoleucine), and X represents alanine, methionine, serine, leucine, or glutamine. In some embodiments, the CAAX amino acid sequence is a CVIM motif from KRAS4B. In some embodiments, the CAAX box (e.g., CVIM motif from KRAS4B) is added at the C-terminus of the fusion polypeptide. In some embodiments, the CAAX box is famesylated (a type of prenylation) which is a post translational modification, wherein an isoprenyl group is added to the cysteine residue, and the VIM is cleaved via proteolysis.

[0196] In some embodiments the fusion polypeptide is a fatty acylated protein. In some embodiments the fusion polypeptide comprises a vesicle targeting domain. In some embodiments, the vesicle targeting domain is fatty acylated. The term “fatty acylated” refers to a protein that has undergone a process called fatty acylation, wherein a fatty acid is covalently attached to the protein. This modification increases the protein’s hydrophobicity, which helps anchor it to cell membranes and influences localization, interactions, and function. In some embodiments, the vesicle targeting domain is fatty acylated. Fatty acylated proteins are proteins that have been modified post-translationally by covalent attachment of one or more fatty acids, generally with a saturated fatty acid that comprises 14-carbon (e.g, myristic acid) via myristoylation or 16- carbons (e.g., palmitic acid) via palmitoylation. For example, proteins destined to become myristoylated begin with the amino acids Met-Gly-X-X-X followed by a serine or threonine at position 6 and lysine or arginine at position 7 and / or 8 wherein X can be any amino acid. The methionine is removed and a myristate is linked to the glycine via an amide bond. Palmitoylation herein means a posttranslational covalent attachment of fatty acids (e.g., palmitic acid) to cysteine (S-palmitoylation), serine and / or threonine (O- palmitoylation), and to the amino group of lysine (N-palmitoylation) of proteins. Palmitoylated proteins may be acylated by attachment of a thioester linkage to a sulfhydryl group of cysteine, or via a palmitate linked to the amino group of an N-terminal cysteine. Palmitoylation sites may be present near the N- or C- terminus of a protein.

[0197] In some embodiments, the vesicle targeting domain is a glycosylphosphatidylinositol (GPI) anchor. In some embodiments, the vesicle targeting domain is a transmembrane domain from a tetraspanin. In some embodiments, the vesicle targeting domains is a transmembrane region of CD9 (for example, transmembrane 2 or 3 of CD9, also known as CD9tm2 or CD9tm3, respectively). One preferred embodiment of a vesicle targeting domain is the transmembrane 2 domain from CD9, or CD9tm2. GPI anchors or CD9tm2 are exemplary means of stably anchoring a protein to an outer leaflet (e.g., exterior layer of a phospholipid bilayer) of a cell membrane. A GPI anchor comprises a glycan, a phosphoethanolamine linker, a phospholipid tail, and may be modified by various glycan sidechains. The glycan core comprises phosphoinositol, glucosamine, and mannose residues wherein said mannose residues may be modified for example, with phosphoethanolamine or carbohydrates. The phosphoethanolamine is amide-bonded to the carboxyl terminus of a protein during the process of GPI attachment. In some embodiments, the vesicle targeting domain may have affinity to EV resident proteins, e.g., CD81, CD63, CD9, ALIX, TSG101, ITGB1, CD98, CD298, MARCKS, PTGFRN, or Lactadherin (MFGe8).

[0198] Non-limiting examples of vesicle targeting domains that enhance fusion polypeptide structure and function on the extracellular vesicles are provided in Table 3 (below).Table 3. Vesicle Targeting Domain

[0199] In some embodiments of any of the aspects provided herein, the fusion polypeptide furthercomprises at least one linker (also referred to as a linker domain). The linker may be flexible, rigid, or cleavable. Further, the linker can be linked directly or via another linker (e.g., a peptide of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids) to the fusion polypeptides described herein. Linkers can be configured according to a specific need, e.g., based on at least one of the following characteristics. In some embodiments of any of the aspects, linkers can be configured to have a sufficient length and flexibility such that it can allow for a cleavage at a target site. In some embodiments of any of the aspects, linkers can be configured to allow multimerization of the fusion polypeptides provided herein. In some embodiments of any of the aspects, linkers can be configured to facilitate expression and purification of the fusion polypeptides or engineered extracellular vesicles provided herein.

[0200] In some embodiments of any of the aspects, a linker can be configured to have any length in a form of a peptide, peptidomimetic, an aptamer, a protein, a nucleic acid (e.g., DNA or RNA), or any combinations thereof. For example, in one embodiment, the linker may be a polypeptide linker.

[0201] In some embodiments of any of the aspects, the linker is an Fc linker. In some embodiments of any of the aspects, the linker comprises an Fc domain. In some embodiments, the linker can comprise any one of Fc, (i.e., Fc from IgGl, Fc from IgG2, Fc from IgG3, Fc from IgG4 (4Fc)) and sequences with at least 70%, 80%, or 90% homology with any of the foregoing. In a preferred embodiment, the linker comprises an Fc from IgGl. An exemplary nucleic acid sequence encoding an IgG consisting of an Fc linker is SEQ ID NO: 32. An exemplary amino acid sequence encoding an IgG consisting of an Fc linker is SEQ ID NO: 33.

[0202] An exemplary amino acid sequence of the linker comprises SEQ ID NO: 37, or a functional fragment thereof. An exemplary amino acid sequence of a linker is SEQ ID NO: 37. Another exemplary amino acid sequence of the linker is SEQ ID NO: 37, or a functional fragment thereof. Another exemplary amino acid sequence of a linker is SEQ ID NO: 45.

[0203] In some embodiments of any of the aspects, the polypeptide linker is a non-cleavable linker. In some embodiments of any of the aspects, a linker can be a chemical linker of any length.

[0204] In some embodiments of any of the aspects, the linker comprises a multimerization (e.g., dimerization) domain wherein one fusion polypeptide may connect with another fusion polypeptide at each fusion polypeptide’s respective multimerization domain. Multimerization of multiple fusion polypeptides will provide multiple fusion polypeptides within close proximity to one another to one or more a target receptor on the target cell, wherein the multiple fusion peptides will enhance target clustering on a target cell, for example target receptor clustering on the target cell. Clustering receptors on a target cell will result in enhanced signal transduction. Without receptor clustering a signal may be weaker or not occur all together. For example, Fc domain sequences presented herein dimerize resulting in two fusion polypeptides connected by a covalent bond via the two Fc domains on their respective fusion polypeptide. One embodiment of an Fc domain is the Fc domain from IgG4. In a preferred embodiment the Fc domain is from IgGl, herein labeled Fc. In certain embodiments Fc from anotherimmunoglobulin, (e.g, IgG2, IgG3, etc.) may be used.

[0205] In various embodiments, the polypeptide linker comprises Fc or Fc mutein. In one embodiment, the Fc is hlgGl nucleic acid sequence SEQ ID NO: 32 or amino acid sequence SEQ ID NO: 33, or a fragment thereof. In one embodiment, the Fc is hlgGl nucleic acid sequence SEQ ID NO: 32 or amino acid sequence SEQ ID NO: 33, or a functional fragment thereof. In one embodiment, the comprises nucleic acid sequence SEQ ID NO: 32 or amino acid sequence SEQ ID NO: 33, or a fragment thereof.

[0206] In one embodiment, the Fc mutein is nucleic acid sequence SEQ ID NO: 36 or amino acid sequence SEQ ID NO: 37, or a fragment thereof. In one embodiment, the Fc mutein is nucleic acid sequence SEQ ID NO: 32 or amino acid sequence SEQ ID NO: 33, or a functional fragment thereof. In one embodiment, the Fc mutein comprises nucleic acid sequence SEQ ID NO: 32 or amino acid sequence SEQ ID NO: 33, or a functional fragment thereof. In a preferred embodiment, the Fc mutein is nucleic acid sequence SEQ ID NO: 32. In a preferred embodiment, the Fc mutein is amino acid sequence SEQ ID NO: 33. In various embodiments, the Fc mutein has reduced or abolished Fc-mediated effector functions, for example, reduced or abolished Fc-Fc Receptor (FcR) mediated effector functions. In various embodiments, the FcR mutein has reduced or abolished FcvRI binding. In another embodiment, the hlgGl is modified one or more of the following mutations L234A, L235A, and P329G (Fc-LALAPG) (nucleic acid sequence SEQ ID NO: 36, and amino acid sequence SEQ ID NO: 37; of which the L234A, L235A, P329G substitutions are indicated in bold). In various embodiments, the Fc-LALAPG mutein has reduced or abolished Fc-mediated effector functions, for example, reduced or abolished FcR or FcvRI mediated effector functions. In various embodiments, the Fc-LALAPG mutein has reduced or abolished FcvRI binding and Fc dependent cellular phagocytosis. The structural features that provide reduced or abolished FcyRl binding and Fc dependent functional effects are described in Tilman Schlothauer, Sylvia Herter, Claudia Ferrara Koller, Sandra Grau-Richards, Virginie Steinhart, Christian Spick, Manfred Kubbies, Christian Klein, Pablo Umana, Ekkehard Mdssner, Novel human IgGl and IgG4 Fc-engineered antibodies with completely abolished immune effector functions, Protein Engineering, Design and Selection, Volume 29, Issue 10, October 2016, Pages 457-466, the contents of which is incorporated herein by reference in its entirety.

[0207] In some embodiments of any of the aspects, the linker provides a multimerization (e.g. , dimerization) domain, wherein one fusion polypeptide may connect with another fusion polypeptide at each fusion polypeptide’s respective multimerization domain. Specific amino acid sequences or multimerization domains of the fusion polypeptides facilitate multimerization of the fusion polypeptides. Multimerization of multiple fusion polypeptides will provide multiple fusion polypeptides within close proximity to one another to one or more a target protein, wherein the multiple fusion polypeptides will enhance interaction with one or more target protein.

[0208] For example, Fc domain sequences presented herein dimerize resulting in two fusion polypeptides connected by a covalent bond via the two Fc domains on their respective fusion polypeptide.One embodiment of an Fc domain is from IgG4, herein labeled 4Fc (nucleic acid sequence SEQ ID NO: 42 and amino acid sequence SEQ ID NO: 43). In other embodiments Fc may be from IgG heavy chain (IgG H) (nucleic acid sequence SEQ ID NO: 32 and amino acid sequence SEQ ID NO: 33). In certain embodiments Fc from another immunoglobulin known in the art, (e.g., IgG2, IgG3, etc.) may be used. In some embodiments, the nucleic acid sequence of IgG2 is SEQ ID NO: 38. In some embodiments, the amino acid sequence of IgG2 is SEQ ID NO: 39. In some embodiments, the nucleic acid sequence of IgG3 is SEQ ID NO: 40. In some embodiments, the amino acid sequence of IgG3 is SEQ ID NO: 41.

[0209] In some embodiments, the Fc domain sequences described herein facilitate dimerization, resulting in two fusion polypeptides joined by covalent disulfide bonds formed between the Fc regions of their respective fusion polypeptides. In one embodiment, an Fc domain is derived from an IgG4 heavy chain (herein referred to as 4Fc; nucleic acid sequence SEQ ID NO: 42 and amino acid sequence SEQ ID NO: 43).

[0210] In other embodiments, the Fc domain is derived from an immunoglobulin heavy chain (IgG H) (nucleic acid sequence SEQ ID NO: 32 and amino acid sequence SEQ ID NO: 33), or from another immunoglobulin known in the art, such as IgG2 or IgG3. The IgG2 heavy chain sequences are provided as SEQ ID NO: 38 and SEQ ID NO: 39. The IgG3 heavy chain sequences are provided as SEQ ID NO: 40 and SEQ ID NO: 41. Fc regions useful in the present disclosure of any of the sequences presented herein may comprise functional fragments, domains, or variants thereof.

[0211] In certain embodiments, the Fc domain comprises a functional fragment or variant of an IgG heavy chain constant region, including sequences having one or more amino acid substitutions, deletions, or insertions that retain Fc-mediated dimerization, structural stability, or effector functions such as binding to Fc receptors or complement components. In particular embodiments, the Fc-derived sequences incorporated into the disclosed fusion polypeptides comprise SEQ ID NO: 36 or SEQ ID NO: 37, or functional fragments or variants thereof.

[0212] In certain embodiments, the Fc comprises amino acid substitutions, deletions, or insertions that modulate Fc function. In various embodiments, the Fc mutein has reduced or abolished Fc-mediated effector functions, for example, reduced or abolished Fc-Fc Receptor (FcR) mediated effector functions. In various embodiments, the FcR mutein has reduced or abolished FcyRl binding. In certain embodiments, the Fc comprises amino acid substitutions, deletions, or insertions that reduce, abolish, or abrogate binding of said Fc to Fc receptors (e.g., FcyR, FcR). In certain embodiments, the Fc comprises amino acid substitutions, deletions, or insertions that abrogate binding of said Fc to Fc receptors (e.g., FcyR, FcR), abolishing antibody directed cytotoxicity (ADCC) effector function. Fc comprising amino acid substitutions, deletions, or insertions that abrogate binding of said Fc to Fc receptors (e.g., FcyR, FcR), abolishing ADCC effector function are herein labelled Fc mutein. In certain embodiments the Fc mutein is an IgGl Fc mutein. In certain embodiments, the IgGl Fc mutein comprises amino acid substitutions, deletions, or insertions that abrogate binding of Fc receptors (e.g., FcyR, FcR), abolishingantibody directed cytotoxicity (ADCC) effector function. In certain embodiments, the IgGl Fc mutein comprises amino acid substitutions L234A, L235A, P329G (SEQ ID NO: 37) that abrogate binding of Fc receptors (e.g., FcyR, FcR), abolishing antibody directed cytotoxicity (ADCC) effector function. In certain embodiments, the IgG2 Fc mutein comprises amino acid that abrogate binding of Fc receptors (e.g., FcyR, FcR), abolishing ADCC effector function. In certain embodiments, the IgG3 Fc mutein comprises amino acid that abrogate binding of Fc receptors (e.g., FcyR, FcR), abolishing ADCC effector function. In certain embodiments, the IgG4 Fc mutein comprises amino acid that abrogate binding of Fc receptors (e.g., FcyR, FcR), abolishing ADCC effector function.

[0213] In some embodiments, the Fc mutein may be a Knob-into-Hole (KiH) Fc mutein. KiH Fc muteins refers to an Fc mutein wherein specific amino acid residues are mutated to enhance the interaction interface or stability of interaction or structural integrity of interactions between two Fc domains. The knob residue on one Fc chain fits into a complimentary hole in another Fc chain, resulting in enhanced interface interaction or stability of interaction or structural integrity of interactions between two Fc domains. KiH Fc muteins can be used to homodimerize two fusion polypeptides. KiH Fc muteins can be used to heterodimerize two fusion polypeptides. KiH Fc muteins can be used to form homodimers of two the same fusion polypeptides. KiH Fc muteins can be used to form heterodimers of two different fusion polypeptides. KiH Fc muteins can be used to homodimerize two fusion polypeptides. KiH Fc muteins can be used to heterodimerize two fusion polypeptides. KiH Fc muteins can be used to form homodimers of two of the same fusion polypeptides. KiH Fc muteins can be used to form heterodimers of two different fusion polypeptides.

[0214] Non-limiting examples of KiH muteins are T366W knob pairing with the hole mutations T336S, L368A, Y407V in Fc CH3 domains of Fc. The KiH Fc muteins T366W (knob) and T336S, L368A, Y407V (hole) can be used for Fc-Fc dimerization of two individual fusion polypeptides. A fusion polypeptide comprising a linker with an Fc mutein further comprising a T366W mutation may heterodimerize with a fusion polypeptide comprising a linker with an Fc mutein further comprising one or more hole (e.g., T336S, L368A, Y407) mutations. Another non-limiting example of Knob-into-Hole muteins are cysteine mutations S354C knob pairing with Y349C hole in CH3 domains of Fc (Vaks, L.; Litvak-Greenfeld, D.; Dror, S.; Shefet-Carasso, L.; Matatov, G.; Nahary, L.; Shapira, S.; Hakim, R.; Alroy, I.; Benhar, I. Design Principles for Bispecific IgGs, Opportunities and Pitfalls of Artificial Disulfide Bonds. Antibodies 2018, 7, 27. https: / / doi.org / 10.3390 / antib7030027). The KiH Fc muteins S354C knob and Y349C hole can be used for Fc-Fc dimerization of two individual fusion polypeptides. A fusion polypeptide comprising a linker with an Fc mutein further comprising a S354C mutation may heterodimerize with a fusion polypeptide comprising a linker with an Fc mutein further comprising Y349C hole mutation.

[0215] The KiH Fc muteins presented herein can be used to homodimerize two fusion polypeptides. The KiH Fc muteins presented herein can be used to heterodimerize two fusion polypeptides. The KiH Fcmuteins presented herein can be used to form homodimers of two the same fusion polypeptides. The KiH Fc muteins presented herein can be used to form heterodimers of two different fusion polypeptides, lire KiH Fc muteins presented herein can be used to homodimerize two fusion polypeptides. The KiH Fc muteins presented herein can be used to heterodimerize two fusion polypeptides. Tire KiH Fc muteins presented herein can be used to form homodimers of two of the same fusion polypeptides. The KiH Fc muteins presented herein can be used to form heterodimers of two different fusion polypeptides.

[0216] In some embodiments of any of the aspects, the linker provides a multimerization domain wherein one fusion polypeptide may connect with at least one other fusion polypeptide at each fusion polypeptide’s respective multimerization domain. Multimerization of multiple fusion polypeptides will provide multiple fusion polypeptides within close proximity to one another to one or more a target (e.g., target protein, polypeptide, polysaccharide, glycoprotein, etc.) wherein the multiple fusion peptides will enhance receptor clustering on the target cell. Clustering receptors on a target cell will result in enhanced signal transduction. Without receptor clustering a signal may be weaker or not occur all together. The multimerization domains may form heterotypic or homotypic multimers. The multimerization domains may multimerize in either a heterotypic or homotypic fashion. The interaction between multiple individual fusion polypeptides may be facilitated by the multimerization of the multimerization domain present on each respective fusion polypeptide.

[0217] In some embodiments of any of the aspects, the linker provides a dimerization domain wherein one fusion polypeptide may connect with at least one other fusion polypeptide at each fusion polypeptide’s respective dimerization domain. In some embodiments of any of the aspects, the linker provides a dimerization domain wherein one fusion polypeptide may connect with at least one other fusion polypeptide at each fusion polypeptide’s respective dimerization domain. Dimerization of multiple fusion polypeptides will provide multiple fusion polypeptides within close proximity to one another to one or more a target receptor on the target cell, wherein the multiple fusion peptides will enhance receptor clustering on the target cell. Clustering receptors on a target cell will result in enhanced signal transduction. Without receptor clustering a signal may be weaker or not occur all together. The dimerization domains may form heterodimers or homodimers. The dimerization domains can dimerize in either a heterotypic or homotypic fashion. The interaction between two fusion polypeptides may be facilitated by the homodimerization of the dimerization domain present on each respective fusion polypeptide.

[0218] A non-limiting example of a dimerization domain that can be used to dimerize two fusion polypeptides is a PDZ domain. PDZ domains presented herein may dimerize resulting in two fusion polypeptides connected via the two PDZ domains on their respective fusion polypeptide. Non-limiting examples of PDZ proteins in humans are AAG12, AHNAK, AHNAK2, AIP1, ALP, APBA1, APBA2, APBA3, ARHGAP21, ARHGAP23, ARHGEF11, ARHGEF12, CARDIO, CARD11, CARD14, CASK, CLP-36, CNKSR2, CNKSR3, CRTAM, DFNB31, DLG1, DLG2, DLG3, DLG4, DLG5, DVL1,DVL1L1, DVL2, DVL3, ERBB2IP, FRMPD1, FRMPD2, FRMPD2L1, FRMPD3, FRMPD4, GIPC1, GIPC2, GIPC3, GOPC, GRASP, GRIP1, GRIP2, HTRA1, HTRA2, HTRA3, HTRA4, IL 16, INADL, KIAA1849, LDB3, LIMK1, LIMK2, LIN7A, LIN7B, LIN7C, LM07, LNX1, LNX2, LRRC7, MAGI1, MAGE, MAGI3, MAGIX, MAST1, MAST2, MAST3, MAST4, MCSP, MLLT4, MPDZ, MPP1, MPP2, MPP3, MPP4, MPP5, MPP6, MPP7, MY018A, NHERF1, NOS1, PARD3, PARD6A, PARD6B, PARD6G, PDLIM1, PDLIM2, PDLIM3, PDLIM4, PDLIM5, PDLIM7, PDZD11, PDZD2, PDZD3, PDZD4, PDZD5A, PDZ6, PDZD7, PDZD8, PDZK1, PDZRN3, PDZRN4, PICK1, PPP1R9A, PPP1R9B, PREXI, SDCBP2, SHANK1, CHANK2, SHANK3, SHR00M2, SHR00M3, SHR00M4, SIPA1, SIPA1L1, SIPA1L2, SIPA1L3, SLC9A3R1, SLC9A3R2, SNTA1, SNTB1, SNTB2, SNTG1, SNTG2, SNX27, SPLA2, STXBP4, SYNJ2BP, SYNPO2, SYNPO2L, TAX1BP3, TIAM1, TIAM2, TJP1, TJP2, TJP3, TRPC4, TRPC5, USH1C, and WHRN.

[0219] PDZ domains presented herein may form heterodimers or homodimers. The PDZ domains can dimerize in either a heterotypic or homotypic fashion. The interaction between two fusion polypeptides may be facilitated by the homodimerization of the PDZ domain present on each respective fusion polypeptide. For example, SHANK1 PDZ domains form PDZ -PDZ homodimers (Im YJ, Lee JH, Park SH, Park SJ, Rho SH, Kang GB, Kim E, Eom SH. Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ -PDZ dimerization. J Biol Chem. 2003 Nov 28;278(48):48099-104. doi: 10.1074 / jbc.M306919200. Epub 2003 Sep 3. PMID: 12954649). Other examples of PDZ-PDZ homodmiers are GRIP 1 -GRIP 1 and GR1P2-GRIP2.

[0220] The interaction between two fusion polypeptides may be facilitated by the heterodimerization of the PDZ domain present on each respective fusion polypeptide. For example, NOS1 and GRIP1 heterodimerize with SNTA1 and PDZ6, respectively (Chang BH, Gujral TS, Karp ES, BuKhalid R, Grantcharova VP, MacBeath G. A systematic family-wide investigation reveals that -30% of mammalian PDZ domains engage in PDZ-PDZ interactions. Chem Biol. 2011 Sep 23;18(9): 1143-52. doi: 10.10I6 / j.chembiol.2011.06.013. PMID: 21944753; PMCID: PMC3442787). The PDZ dimerization domains presented herein can be used to homodimerize two fusion polypeptides. The PDZ dimerization domains presented herein can be used to heterodimerize two fusion polypeptides. PDZ domains can be used to form homodimers of two the same fusion polypeptides. PDZ domains can be used to form heterodimers of two different fusion polypeptides. The PDZ domains of the PDZ domain containing proteins presented herein can be used to homodimerize two fusion polypeptides. The PDZ domains of the PDZ domain containing proteins presented herein can be used to heterodimerize two fusion polypeptides. The PDZ domains of the PDZ domain containing proteins presented herein can be used to form homodimers of two of the same fusion polypeptides. The PDZ domains of the PDZ domain containing proteins presented herein can be used to form heterodimers of two different fusion polypeptides.

[0221] Another non-limiting example of a dimerization domain or motif that can be used to dimerize two fusion polypeptides is a coiled-coil domain. For example, the coiled-coil domain from myosin thatplays a role in myosin subunit association. A non-limiting example of a coiled-coil dimerization domain that can be used to dimerize two fusion polypeptides is a basic Leucine Zipper Domain (bZIP) domain. The leucine rich pattern in bZIP domains form coiled-coil structural motifs that enable protein-protein interactions and facilitate dimerization. Non-limiting examples of bZIP domain containing proteins are c- Fos, c-Jun, Activating Transcription Factor (ATF) Family (e.g., ATF1, ATF2, ATF3, ATF4, ATF5, and ATF6), cAMP Response Element-Binding (CREB) Family (e.g., CREB1, CREM, ATF1, ATF2, and ATF4), Nuclear Factor erythroid 2-related factor (Nrf) Family (e.g., Nrfl (NFE2L1), Nrf2 (NFE2L2), and Nrf3 (NFE2L3)), X-box binding protein 1 (XBP1), General Control Nondepressible 4 (GCN4), c-Myc- interacting zinc finger protein (ZIP) Family (e.g., ZIP1, ZIP2, ZIP3, ZIP4), CCAAT / enhancer-binding protein (CEBP) Family (e.g., CEBP CEBPa, CEBP , CEBPy, CEBP5, CEBPa, and CEBPQ. and Activating Transcription Factor 6 (ATF6). Proteins containing bZIP domain may form heterodimers or homodimers. The Coiled-coil domains of the Coiled-coil domain containing proteins presented herein can be used to homodimerize two fusion polypeptides. The Coiled-coil domains of the Coiled-coil domain containing proteins presented herein can be used to heterodimerize two fusion polypeptides. The Coiled- coil domains of the Coiled-coil domain containing proteins presented herein can be used to form homodimers of two of the same fusion polypeptides. The Coiled-coil domains of the Coiled-coil domain containing proteins presented herein can be used to form heterodimers of two different fusion polypeptides.

[0222] Another non-limiting example of a dimerization domain that can be used to dimerize two fusion polypeptides is a basic Helix-Loop-Helix (HLH) domain. HLH domains are protein structural motifs that facilitate protein-protein interactions and the formation of homodimers or heterodimers. Non-limiting examples of HLH domain containing proteins are c-Myc, Max, MyoD, E12 / E47 (E2A), Neurogenins (e.g., Ngnl, Ngn2, and Ngn3), Inhibitor of DNA binding (Id) proteins, Hypoxia-Inducible Factor 1 Alpha (HIF-la), Transcription Factor E3 (TFE3), Transcription Factor EB (TFEB), and Achaete-Scute Complex (AS-C) proteins. The interaction between two fusion polypeptides may be facilitated by the heterodimerization of the HLH domain present on each respective fusion polypeptide. For example, the HLH Domain of Myc heterodimerizes with the HLH domain of Max. The interaction between two fusion polypeptides may be facilitated by the homodimerization of the HLH domain present on each respective fusion polypeptide. For example, the HLH domain of MyoD, Neurogenins, TFE3, TFEB, and AS-C proteins can homodimerizes. The HLH domain of E2A may homodimerize or heterodimerize with other HLH proteins. The HLH domains of the HLH domain containing proteins presented herein can be used to homodimerize two fusion polypeptides. The HLH domains of the HLH domain containing proteins presented herein can be used to heterodimerize two fusion polypeptides. The HLH domains of the HLH domain containing proteins presented herein can be used to form homodimers of two the same fusion polypeptides The HLH domains of the HLH domain containing proteins presented herein can be used to form heterodimers of two different fusion polypeptides.

[0223] Another non-limiting example of dimerization domains that can be used to dimerize two fusion polypeptides are Src Homology 2 (SH2) and Src Homology 3 (SH3) domains of Src kinase and Grb2 adaptor protein, respectively. SH2 domains are structural motifs that mediate protein-protein interactions by binding phosphorylated tyrosine residues on target proteins, for example target proteins displaying the amino acid motif Y-X-X-M where Y represents tyrosine, M represents methionine, and X represents any amino acid. A non-limiting example of an SH2 domain containing proteins is Growth Factor Receptor- Bound Protein 2 (Grb2) that binds to tyrosine residues in receptor tyrosine kinases. SH3 domains are structural motifs that mediate protein-protein interactions by binding proline rich sequences on target proteins, for example target proteins displaying the amino acid motif P-X-X-P where P represents proline and X represents any amino acid. A non-limiting example of an SH3 domain containing proteins is Src Kinase that interacts with proline-rich residues in target proteins. The SH2 dimerization domains presented herein can be used to homodimerize two fusion polypeptides. The SH2 dimerization domains presented herein can be used to heterodimerize two fusion polypeptides. The SH3 dimerization domains presented herein can be used to homodimerize two fusion polypeptides. The SH3 dimerization domains presented herein can be used to heterodimerize two fusion polypeptides. SH2 domains can be used to form homodimers of two of the same fusion polypeptides. SH2 domains can be used to form heterodimers of two different fusion polypeptides. SH3 domains can be used to form homodimers of two of the same fusion polypeptides. SH3 domains can be used to form heterodimers of two different fusion polypeptides.

[0224] Another non-limiting example of a dimerization domain that can be used to dimerize two fusion polypeptides is a Sterile Alpha Motif (SAM) domain. SAM domains are structural motifs involved in protein-protein interactions through homotypic or heterotypic interactions or higher-order oligomers. Homotypic and heterotypic interactions refer to intermolecular interactions between the same versus different molecules, respectively. This concept can be extended to distinguish interactions between the same versus different motifs on molecules. Higher-order oligomers, that is, oligomers in which the number of monomers in a complex is broadly distributed and can be large, have important functions in signal transduction and cell fate decisions. Non-limiting examples of SAM domain containing proteins are TEL Transcription Factor (ETV6), Ephrin Receptor-Interacting Protein (EPB4.1L5), Traf2- and Nck- Interacting Kinase (TNIK), and Sterile Alpha and TIR Motif Containing 1 (SARM1) protein. The interaction between two fusion polypeptides may be facilitated by the homodimerization of the SAM domain present on each respective fusion polypeptide. For example, the SAM domain of TEL Transcription Factor (ETV6), Ephrin Receptor-Interacting Protein (EPB4. 1L5), Traf2- and Nck- Interacting Kinase (TNIK), and Sterile Alpha and TIR Motif Containing 1 (SARM1) protein mediate formation of homodimers.

[0225] The SAM domains of the SAM domain containing proteins presented herein can be used to homodimerize two fusion polypeptides. The SAM domains of the SAM domain containing proteins presented herein can be used to heterodimerize two fusion polypeptides. The SAM domains of the SAMdomain containing proteins presented herein can be used to form homodimers of two of the same fusion polypeptides. The SAM domains of the SAM domain containing proteins presented herein can be used to form heterodimers of two different fusion polypeptides.

[0226] Another non-limiting example of a dimerization domain that can be used to dimerize two fusion polypeptides is a Toll / Interleukin-1 Receptor (TIR) domain that facilitates homodimerization. TIR domains are characterized by a conserved a / p sandwich fold and present in IL18R1 (CDw218a), IL18RAP (CDw218b), IL1R1 (CD 121a), IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2, MYD88, SIGIRR (TIR8), TLRs (e.g., TLR1 (CD281), TLR10 (CD290), TLR2 (CD282), TLR3 (CD283), TLR4 (CD284), TLR5 (CD285), TLR6 (CD286), TLR7 (CD287), TLR8 (CD288), TLR9 (CD289) and SARM1 (MyD88-5). TIR domains provide interfaces that allow for the formation of homodimers. TIR domains provide interfaces that allow for the formation of homodimers of two TLRs. The interaction between two fusion polypeptides may be facilitated by the homodimerization of the TIR domain present on each respective fusion polypeptide. For example, the TIR domain of TLR2, TRL3, TLR4, TLR7, TLR8, and TLR9 proteins mediate formation of homodimers. The interaction between two fusion polypeptides may be facilitated by the heterodimerization of the TIR domain present on each respective fusion polypeptide. For example, the TIR domain of TLR1, TLR5, TLR6, and TLR10 proteins mediate formation of heterodimers.

[0227] The TIR domains of the TIR domain containing proteins presented herein can be used to homodimerize two fusion polypeptides. The TIR domains of the TIR domain containing proteins presented herein can be used to heterodimerize two fusion polypeptides. The TIR domains of the TIR domain containing proteins presented herein can be used to form homodimers of two of the same fusion polypeptides. The TIR domains of the TIR domain containing proteins presented herein can be used to form heterodimers of two different fusion polypeptides.

[0228] Another non-limiting example of a dimerization domain that can be used to dimerize two fusion polypeptides is a RING domain. RING domains can be used to form homodimers or heterodimers of two fusion polypeptides. Ring domains are typically 40-60 amino acids in length and characterized by a zinc- binding fold and primarily serve as a scaffold for ubiquitin ligase complexes, for example E2 and E3 ligases. Individual RING domains provide interfaces that allow for the formation of dimers between two separate RING domains. For example, the RING domains between two ubiquitin-conjugating (E2) enzymes homodimerize facilitating activation of the E2 enzyme to transfer ubiquitin from one E2 to a substrate protein. In another example, the RING domain in Casitas B-lineage Lymphoma (CBL) protein is essential for homodimerization of CBL leading to enhanced activation of CBL protein’s E3 ubiquitin ligase activity and regulation of protein degradation. In another example, homodimerization of the RING domain of individual MDM2 proteins homodimerizes two MDM2 proteins leading to enhancement of MDM2 ubiquitin ligase activity. A non-limiting example of a RING heterodimer is the heterodimerization of the RING domains from BRCA1 and BARD1 wherein the RING domain of BRCA1 heterodimerizeswith the RING domain of BARD 1 leading to E3 ubiquitin ligase activity. Another non-limiting example of a RING heterodimer is the heterodimerization of the RING domains from RING1A1 and BMI1 wherein the RING domain of RING1A1 heterodimerizes with the RING domain of BMIlleading to ubiquitin ligase activity of PRC 1. The RING dimerization domains presented herein can be used to homodimerize two fusion polypeptides. The RING dimerization domains presented herein can be used to heterodimerize two fusion polypeptides. RING domains can be used to form homodimers of two of the same fusion polypeptides. RING domains can be used to form heterodimers of two different fusion polypeptides.

[0229] Another non-limiting example of a dimerization domain that can be used to dimerize two fusion polypeptides is subdomain IIA of human serum albumin (HSA) domain. The HSA protein is primarily known to exist in a monomeric state, however, under certain conditions HSA dimerizes or multimerizes into higher-order oligomers. Conditions that influence dimerization or multimerization of HSA subdomain IIA monomers include heat, pressure, pH, and ionic strength wherein under favorable conditions hydrophobic patches facilitate protein-protein interactions and stabilization of HAS dimers or oligomers. Subdomain IIA of HSA dimerization domains presented herein can be used to homodimerize two fusion polypeptides. Subdomain IIA of HSA domain presented herein can be used to multimerize more than two fusion polypeptides. HSA domains can be used to form homodimers of two of the same fusion polypeptides. HSA domains can be used to form heterodimers of two different fusion polypeptides.

[0230] Another non-limiting example of a dimerization domain that can be used to dimerize two fusion polypeptides is the Regulator of G Protein Signaling (RGS) domain of G protein coupled receptors. RGS domains can be used to form homodimers or heterodimers of two fusion polypeptides. RGS domains are approximately 120 amino acids in length and adopt a conserved alpha-helical fold, and primarily serve as regulators of G protein signaling. RGS domains facilitate dimerization wherein the individual RGS domain of two RGS domain containing proteins provide interfaces that allow for the formation of dimers of the said two RGS domain containing proteins. Individual RGS domains provide interfaces that allow for the formation of dimers between two separate RGS domain containing proteins. For example, the RGS domain between two RGS4 has been shown to homodimerize. In another example, the RGS domain in RGS9-2 can form homodimers. Non-limiting examples of RGS domain containing proteins are AXIN1, AXIN2 GRK1, GRK2, GRK3, GRK4, GRK5, GRK6, GRK7 RGS1, RGS2, RGS3, RGS4, RGS5, RGS6, RGS7, RGS8, RGS9, RGS10, RGS11, RGS12, RGS13, RGS14, RGS16, RGS17, RGS18, RGS19, RGS20, RGS21 and SNX13. The RGS domains of the RGS domain containing proteins presented herein can be used to homodimerize two fusion polypeptides. The RGS domains presented herein can be used to heterodimerize two fusion polypeptides. The RGS domains of the RGS domain containing proteins presented herein can be used to form homodimers of two of the same fusion polypeptides. The RGS domains of the RGS domain containing proteins presented herein can be used to form heterodimers of two different fusion polypeptides. The RGS domains of the RGS domain containing proteins presented hereincan be used to homotrimerize three fusion polypeptides. The RGS domains of the RGS domain containing proteins presented herein can be used to heterotrimerize three fusion polypeptides. The RGS domains of the RGS domain containing proteins presented herein can be used to form homotrimers of three of the same fusion polypeptides. The RGS domains of the RGS domain containing proteins presented herein can be used to form heterotrimers of three different fusion polypeptides.

[0231] Additional non-limiting examples of linkers that can be used and their properties are further described in detail, e.g., in Chen X, Zaro JL, Shen WC. Fusion polypeptide linkers: property, design and functionality. A dv Drug Deliv Rev. 2013;65(10): 1357-1369. doi: 10.1016 / j.addr.2012.09.039; O'Shea EK, Lumb KJ, Kim PS. Peptide 'Velcro': design of a heterodimeric coiled coil. Curr Biol. 1993 Oct1 ;3(10):658-67. doi: 10.1016 / 0960-9822(93)90063-t. PMID: 15335856; and Muller KM, Arndt KM, Alber T. Protein fusions to coiled-coil domains. Methods Enzymol. 2000;328:261-82. doi: 10.1016 / s0076-6879(00)28402-4. PMID: 11075350, the contents of which are incorporated herein by reference in their entireties.

[0232] In some embodiments of any of the aspects provided herein, the linker comprises a polypeptide or a fragment thereof selected from the group consisting of: Table 4. In some embodiments of any of the aspects provided herein, the linker comprises a nucleic acid sequence encoding said linker selected from the group consisting of: Table 4. Non-limiting examples of nucleic acid sequences and amino acid sequences of linkers are provided in Table 4.Table 4 - Linkers

[0233] The engineered extracellular vesicle compositions provided herein can comprise variations in the configuration of the POI domain, linkers, and / or vesicle targeting domain. The specific combination and localization of these domains can enhance fusion polypeptide anchoring, function, or therapeutic effect, e.g., modulating inflammation.

[0234] Thus, in one aspect, provided herein is an engineered extracellular vesicle comprising: at least one fusion polypeptide comprising: (i) at least one POI domain or a fragment thereof, wherein the POI comprises a truncated TSG-6; and (ii) at least one vesicle targeting domain, wherein the POI domain is in an extracellular position relative to a lipid membrane of the extracellular vesicle. In another aspect, provided herein is an engineered extracellular vesicle comprising at least one fusion polypeptide comprising: (i) a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C terminal tail, wherein at least a part of the C-terminal of the TSG-6 is truncated; and (ii) at least one vesicle targeting domain, wherein the fusion polypeptide is displayed on a surface of an engineered extracellular vesicle.

[0235] In some embodiments, the POI domain or a fragment thereof is an N-terminal domain of the fusion polypeptide, wherein the POI comprises a truncated TSG-6 (e.g., tTSG-6). In some embodiments, the vesicle targeting domain or a fragment thereof is a C-terminal domain of the fusion polypeptide.

[0236] In another aspect, provided herein is an engineered extracellular vesicle comprising: at least onefusion polypeptide comprising: (i) at least one POI domain or a fragment thereof; and (ii) at least one vesicle targeting domain; wherein the POI is a truncated TSG-6 (e.g., tTSG-6) comprising a LINK domain, a CUB domain, and a C terminal tail; wherein at least a part of the C-terminal tail of the TSG-6 is truncated; wherein the POI domain is in an extracellular position relative to a lipid membrane of the extracellular vesicle; and wherein the vesicle targeting domain is a transmembrane domain relative to a lipid membrane of the extracellular vesicle.

[0237] In some embodiments, the POI domain or a fragment thereof is a C-terminal domain of the fusion polypeptide, wherein the POI comprises a truncated TSG-6 (e.g., tTSG-6). In some embodiments, the vesicle targeting domain or a fragment thereof is a N-terminal domain of the fusion polypeptide. In some embodiments, the vesicle targeting domain is in a luminal position relative to the lipid membrane of the extracellular vesicle.

[0238] In some embodiments, the linker is in an exterior position relative to the lipid membrane of the extracellular vesicle. In some embodiments, the linker is a transmembrane linker. In some embodiments, the linker is in a luminal position relative to the lipid membrane of the extracellular vesicle.

[0239] The engineered extracellular vesicle compositions provided herein can comprise one or more of the following fusion polypeptide sequences in Table 5.Table 5. Exemplary Full-Length Constructs

[0240] In some embodiments of any of the aspects, the fusion polypeptides provided herein comprise two or more POI domains. The specific combinations of POI domains can be used to regulate inflammatory immune responses. In one nonlimiting example, the POI comprises TSG-6 (SEQ ID NO: 1) or a fragment thereof. In another nonlimiting example, the POI comprises TSG-6 (nucleic acid SEQ ID NO: 2, amino acid sequence SEQ ID NO: 3) or a fragment thereof. In another nonlimiting example, the POI comprises TSG-6 (nucleic acid SEQ ID NO: 4, amino acid sequence SEQ ID NO: 5) or a fragment thereof.

[0241] In some embodiments, a truncated wild-type TSG-6 is approximately the same size as full- length TSG-6 as assessed by immunoblot, and remained associated with exosomes. In some embodiments, a portion of a C terminal tail is truncated from the wild type human TSG-6. In some embodiments, an entire portion of the C terminal tail is truncated from the wild type human TSG-6 (z.e., a truncated TSG-6 (tTSG-6)). In some embodiments, the tTSG-6 comprises an amino acid sequence as set forth in SEQ ID NO: 7 or is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the truncated portion comprises an amino acid sequence as set forth in SEQ ID NO: 9 or is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 8. In some embodiments, one or more cleavage-sensitive sites are present within the unstructured C-terminal tail. In some embodiments, a full- length TSG-6 protein comprises one or more cleavage-sensitive sites. In some embodiments, a truncated TSG-6 protein comprises one or more cleavage-sensitive sites. In some embodiments, a cleavagesensitive site at the C-terminal region of a truncated TSG-6 protein is removed. In some embodiments, a truncated TSG-6 protein lacks a region comprising amino acids D248-L277. In some embodiments, a truncated TSG-6 protein lacks a region comprising one or more amino acids of the full-length TSG-6 sequence, wherein the nucleic acid sequence encoding the truncated TSG-6 is SEQ ID NO: 18 and the corresponding amino acid sequence is SEQ ID NO: 19. In some embodiments, D248-L277 is encoded by the following nucleic acid sequence SEQ ID NO: 18 or a fragment thereof. In some embodiments, D248- L277 comprises SEQ ID NO: 19 or a fragment thereof.

[0242] In some embodiments, a truncated TSG-6 protein lacks one or more amino acids of the full- length TSG-6 sequence, wherein the nucleic acid sequence encoding the truncated portion of TSG-6 is SEQ ID NO: 18 and the corresponding amino acid sequence of the truncated portion of TSG-6 is SEQ ID NO: 19. In certain embodiments, the region spanning amino acids D248-L277 is encoded by SEQ ID NO: 18 or a functional fragment thereof, and the corresponding amino acid region D248-L277 comprises SEQ ID NO: 19 or a functional fragment thereof. In particular embodiments, the truncated TSG-6 protein lacks one or more regions of the full-length TSG-6 protein, including, for example, the C-terminal tail or any contiguous or non-contiguous subset thereof. Such truncations may include deletions of amino acids outside of the LINK domain, which is retained in all embodiments, and outside of the N-terminal signal peptide, which is naturally cleaved following translation and translocation. Unless otherwise indicated, the term “truncated TSG-6” encompasses proteins lacking one or more amino acids of the C-terminal tail, including functional fragments that retain the LINK domain and one or more biological activities of the full-length TSG-6 protein.Tumor Necrosis Factor (TNF)-Stimulated Gene-6 (TSG-6)

[0243] Microglia assume diverse phenotypes dependent on dynamic microenvironmental cues, such as inflammation and oxidative stress. Microglia are activated in the brain and retina after an inflammatory event and are associated with uncontrolled inflammation. Mesenchymal stem cells (MSCs) express molecules that can modulate microglial activation and activate tissue repair mechanisms to confer neuroprotection from inflammation. TNF-Stimulated Gene-6 protein (TSG-6) is one such molecule expressed by MSCs that functions as the primary mediator of the anti-inflammatory therapeutic effects.

[0244] TSG-6 has anti-inflammatory properties and is not expressed under non-inflammatory conditions but is rapidly produced by many cell types in response to proinflammatory mediators such as, for example, TNFa, IL-ip, IFNy, and LPS. In some embodiments, a truncated TSG-6 or a fragment thereof has anti-inflammatory properties. A primary signaling target of TSG-6 is the adhesion molecule CD44, which is upregulated on immune and parenchymal cells during inflammation. CD44 is expressed by, for example, microglia, astrocytes, and Muller glia in the retina. CD44 is upregulated on neurons in an inflammatory event. Association of TSG-6 with hyaluronic acid (HA) enhances the CD44-HA interaction and modulates inflammatory signaling. In some embodiments, a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG-6) associates with hyaluronic acid (HA) to enhance CD44-HA interaction. In some embodiments, a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG-6) modulates inflammatory signaling in a target cell by enhancing CD44-HA interaction. In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG-6) enhances CD44-HA interaction in a target cell. In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG-6) modulatesinflammatory signaling in a target cell by enhancing CD44-HA interaction. Suppression ofNFKB signaling downstream of CD44 is a mechanism by which TSG-6 suppresses macrophage and microglial cells. In some embodiments, a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG- 6) suppresses NFKB signaling. In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG-6) suppresses NFKB signaling in a target cell. TSG-6 is necessary to suppress STAT3, downstream ofNFKB. In some embodiments, a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG-6) suppresses expression levels of STAT3. In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) or a fragment thereof (e.g., truncated tTSG-6) suppresses expression levels of STAT3 in a target cell. Moreover, siRNA- mediated depletion of TSG-6 in cytokine primed ASC-CCM abrogated the protective effect. Thus, microglial modulation through the TSG-6 / CD44 axis is a promising approach to mitigating neuroinflammation. In one aspect, treatment with an engineered extracellular vesicle (e.g., an exosome) or a population of engineered extracellular vesicles described herein can improve inflammation in a target cell about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to treatment with a placebo. In some embodiments, inflammation comprises neuroinflammation. In some embodiments, treatment with an engineered extracellular vesicle (e.g., an exosome) or a population of engineered extracellular vesicles described herein can reduce levels of CD44 in a target cell about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to treatment with a placebo. In some embodiments, treatment with an engineered extracellular vesicle (e.g., an exosome) or a population of engineered EVs described herein can reduce levels ofNFKB in a target cell about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to treatment with a placebo.

[0245] Development of a therapeutic approach leveraging TSG-6 has not been realized. Recombinant TSG-6 is typically produced in E. coli, Drosophila S2 cells, or mouse NS0 cells. However, none of these systems produce native glycosylation, which could impact its function. Glycosylated TSG-6 has been produced in CHO cells, but because of its strong tendency to bind and crosslink extracellular matrix and other ligands, substantial measures must be taken to reduce cell clumping and aggregation. The small size of TSG-6 (~30 kDa) compared to typical biologies like antibodies or Fc-fusion polypeptides (-100-150 kDa) is likely to result in lower tissue retention. Additionally, the serum half-life of recombinant human TSG-6 in vivo is less than 10 minutes, which may limit its therapeutic efficacy for systemic administration. In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) has an in vivo serum half-life increased by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to recombinant human TSG-6. In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) has an in vivo serum half-life increased by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to full-length human TSG-6. In contrast to recombinant biologies, whole secretome is variable and complex, comprised of amixture of exosomes, extracellular matrix components, and diverse soluble biomolecules, which complicates manufacturing and characterization. Finally, MSCs are primary human cells that face challenges in manufacturing, limited population doublings, and variability between donors. These factors create several translational hurdles, including the need to establish a consistent, scalable cell source and develop robust, cGMP -compliant manufacturing processes for both upstream and downstream operations.

[0246] A truncated TSG-6 described herein is displayed on the surface of exosomes, suggesting a novel approach to delivering TSG-6 therapeutically. The association with the surface of exosomes may enhance tTSG-6's bioactivity, as a multivalency of therapeutic proteins on exosomes can increase their affinity for target receptors. Additionally, exosome cargo and exosome surface associated proteins tend to have a longer tissue half-life compared to soluble recombinant counterparts owing to the large size and dense composition of exosomes. Truncated TSG-6 or a fragment thereof as described herein delivered via exosomes exhibits at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 orders of magnitude greater potency than TSG-6 delivered in its recombinant form. In some embodiments, tTSG-6 or a fragment thereof delivered via exosomes as described herein exhibits at least about two orders of magnitude greater potency than TSG-6 delivered in its recombinant form.

[0247] In some embodiments, an engineered extracellular vesicle expresses a fusion polypeptide comprising a signaling domain comprising a truncated TSG-6 (e.g., tTSG-6), wherein the truncated TSG- 6 or a fragment thereof comprises a CUB domain and / or a LINK domain. In one embodiment, a truncated TSG-6 or a fragment thereof comprises a CUB domain and a LINK domain. The CUB acronym is derived from the names of three proteins in which the domain was first discovered: Clr / Cls, Uegf, and BMP. The CUB domain is involved in interactions between TSG-6 and components of the extracellular matrix (ECM) and works in unison with the LINK domain. Like the CUB domain, the LINK domain is a structural domain that binds to the extracellular matrix (ECM) of cells, for example, binding to glycosaminoglycans (GAG), particularly hyaluronan (HA). HA is an important component of a cell's ECM structure and function. The TSG-6 LINK domain functions to bind to HA, and this binding allows TSG-6 to regulate cell migration, ECM organization, and inflammation. The LINK domain in TSG-6 can also bind to other ECM molecules such as chondroitin sulfate and proteoglycans (such as aggrecan). In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) has HA -binding activity that is modulated by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to full-length human TSG-6. In some embodiments, an engineered extracellular vesicle expressing a fusion polypeptide comprising a truncated TSG-6 (e.g., tTSG-6) has HA -binding activity that is increased by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to full-length human TSG-6.

[0248] In some embodiments, the fusion polypeptide binds to one or more extracellular matrix polysaccharide. Extracellular matrix polysaccharides include but is not limited to chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), or glycosaminoglycan (GAG).

[0249] In some embodiments, the fusion polypeptide binds to one or more glycosaminoglycan, e.g., hyaluronan (HA). Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are negatively- charged polysaccharide compounds. In some embodiments, the fusion polypeptide binds to a hyaluronan.

[0250] In some embodiments, the fusion polypeptide binds to or interacts with large chondroitin sulfate proteoglycan (e.g., versican), aggrecan, or thrombospondin- 1 (TSP-1). In some embodiments, the fusion polypeptide binds to or interacts with Inter-a-inhibitor (lai) / Heavy Chains (HC1, HC2, HC3).

[0251] In some embodiments, the fusion polypeptide binds to Tumor Necrosis Factor-a (TNF-a). In some embodiments, the fusion polypeptide binds to CXCL8 (IL-8). In some embodiments, the fusion polypeptide modifies or modulated HA-CD44 interactions, wherein HA binding is enhanced or inhibited. In some embodiments, the fusion polypeptide binds to CXCL12 (SDF-1). In some embodiments, the fusion polypeptide influences TLR2 / TLR4-mediated macrophage activation. In some embodiments, the fusion polypeptide interacts with fibronectin, Pentraxin 3 (PTX3), or fibrinogen.

[0252] In some embodiments, a LINK domain of the fusion polypeptide binds to one or more glycosaminoglycan (GAG), e.g., hyaluronan (HA). Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are negatively charged polysaccharide compounds.

[0253] In some embodiments, a GAG comprises a heparin / heparan sulfate (HSGAGs), a chondroitin sulfate / dermatan sulfate (CSGAGs), a keratan sulfate, or a hyaluronic acid (or hyaluronan or hyaluronate). In some embodiments, a LINK domain of the fusion polypeptide binds to a hyaluronan. Hyaluronan is a long unbranched polysaccharide made of repeating disaccharide units D-glucuronic acid (GlcA) and N- acetyl-D-glucosamine (GlcNAc) linked by P(1^3) and P( 1 — >4) glycosidic bonds. HA binds to proteins, for example, TSG-6, CD44, Versican, and inter-a-inhibitor. In some embodiments, the LINK domain of the fusion polypeptide binds to or interacts with versican, decorin, lubricin (PRG4), or mucins. In some embodiments, the LINK domain of the fusion polypeptide binds to or interacts with large chondroitin sulfate proteoglycan (e.g., versican), aggrecan, or thrombospondin- 1 (TSP-1). In some embodiments, the LINK domain of the fusion polypeptide binds to or interacts with Inter-a-inhibitor (lai) / Heavy Chains (HC1, HC2, HC3). In some embodiments, the LINK domain of the fusion polypeptide binds to CXCL8 (IL-8). In some embodiments, the LINK domain of the fusion polypeptide modifies or modulates HA- CD44 interactions, wherein HA binding is enhanced or inhibited. In some embodiments, the fusion polypeptide binds to CXCL12 (SDF-1). In some embodiments, the LINK domain of the fusion polypeptide influences TLR2 / TLR4-mediated macrophage activation. In some embodiments, the LINK domain of the fusion polypeptide interacts with fibronectin, Pentraxin 3 (PTX3), or fibrinogen.Methods of preparing extracellular vesicle compositions

[0254] In another aspect, provided herein is a method of preparing an engineered extracellular vesicle provided herein. Generally, the method comprises providing a population of cells expressing a vector construct encoding a fusion polypeptide comprising one or more vesicle targeting domain and one ormore signaling domain (also referred to as the POI domain) and one or more linker.

[0255] The extracellular vesicles provided herein can be isolated and purified form any biological source, e.g., cells. In some embodiments, the cells that produce the engineered provided herein can be from any viable source or organism (e.g., an animal, vertebrate, or mammal). In some embodiments, the cells that produce the engineered extracellular vesicles provided herein further comprising the fusion polypeptide provided herein can be from any viable source or organism (e.g., an animal, vertebrate, or mammal). In some embodiments, the cell described herein is from a human. In some embodiments, the cell described herein is a human embryonic kidney cell (e.g., HEK 293 cell). The cells described herein can be from any tissue isolated from an organism by methods known in the art. One of skill in the art can appreciate that the cell source of the EVs may alter the cellular protein expression and the native or endogenous cargo within the EV. It is contemplated herein that this can be leveraged for therapeutic effect depending on the disease or disorder being treated.

[0256] In some embodiments, the population of cells has been altered by exposure to environmental conditions (e.g., hypoxia), small molecule addition, presence or absence of exogenous factors (e.g., growth factors, cytokines) at the time, or substantially contemporaneous with, isolating the plurality of engineered extracellular vesicles in a manner altering the regulatory state of the cell. In various embodiments, the cells are human derived cells. In various embodiments, the cells are human derived GMP-grade cells. In various embodiments, the cells are human embryonic kidney (HEK) 293 cells (e.g., HEK 293F, HEK 293FT, HEK 293T, HEK 293S, HEK 293FTM, HEK 293SG, HEK 293SGGD, HEK 293H (293H), HEK 293E, HEK 293EBNA1-6E, HEK 293MSR, HEK 293A, or the like as known by one of ordinary skill in the art), Flp-In™-293 , mesenchymal stem cells (MSCs), umbilical cord MSCs (UC- MSCs), bone marrow MSCs, (BM-MSCs), placental MSCs (P-MSCs), umbilical cord blood MSCs (CD- MSCs), adipose tissue MSCs (A-MSCs), PER.C6, fibrosarcoma HT-1080, HuH7, or HeLa cell lines. In other embodiments, the cells are hematopoietic cells, human embryonic stem cells (hESCs) or hESC derived cells, induced pluripotent stem cells (iPSCs), endothelial progenitor cells (EPCs), or neural stem cells (NSCs), or from primary cells (e.g., human fibroblast cells isolated from juvenile foreskin or from adult skin). In other embodiments, the cells are immune cells, for example lymphocytes (T cells, B cells, and NK cells), neutrophils, or monocytes / macrophages. In other embodiments, the cells are immortalized cell lines including T cell lines such as Jurkat cells, 3T3 cells, A549 cells, HeLa cells, HEK 293 cells, Huh7 cells, OK cells, Ptk2 cells, Vero cells, or the like as known by one of ordinary skill in the art. In various embodiments, the cells are cardio derived cells. In various embodiments, the cells are primary cardiomyocytes, cardiac progenitor or cardio-sphere-derived cells (CDCs) (e.g., Capricor Therapeutics CAP-1002), iPSC-derived cardiomyocytes (iPSC-CMs) (e.g., NCardia Cor.4U, Cor.AT™, Cor.VA™, Fujifilm Cellular Dynamics iCell® Cardiomyocytes), MSC-derived cardiomyocyte-like cells, or immortalized cardiac cell lines (e.g., AC16, AC10). In other embodiments the cells are derived from a hamster cell line (e.g., BHK21, CHO cell etc.) or murine cell line (e.g., C127, NSO, Sp2 / 0, etc.), or canines cell line (e.g., MDCK, IPC-366, TLM-1,CMGD-2, etc.), or monkey cell line (e.g., Vero, etc. , or feline cell line (e.g., CRFK, TiHo-0906, CAT-MT etc.). The cells may be grown in planar culture or suspension culture.

[0257] In other embodiments the cells are derived from hamster cell line (e.g., BHK21, CHO cell etc.) or murine cell line (e.g., C127, NSO, Sp2 / 0, etc.), or canine cell line (e.g., MDCK, IPC-366, TLM-1, CMGD-2, etc.), or feline cell line (e.g., CRFK, TiHo-0906, CAT-MT etc.). The cells may be grown in planar culture or suspension culture. In other embodiments the cells are derived from a non-human primate or monkey cell line (e.g., Vero, etc.).

[0258] In some embodiments, the engineered fusion polypeptide is expressed in non-mammalian host cells that are capable of performing post-translational modifications, such as glycosylation, lipidation, folding, and membrane incorporation, suitable for human therapeutic use. The fusion polypeptide may be designed to localize to the plasma membrane or to be incorporated into extracellular vesicles such as exosomes, which are subsequently released into the culture medium. Exemplary non-mammalian expression systems include yeast and insect cells that have been demonstrated to produce vesicles with structural and compositional features analogous to mammalian exosomes. Yeast expression systems suitable for such applications include Pichia pastoris (also known as Komcigatciellci phaffii), Saccharomyces cerevisiae, and Hansenula polymorphci, which can be engineered to express humancompatible glycosylation machinery and to efficiently secrete vesicles containing membrane-associated proteins.

[0259] In certain embodiments, insect cell systems such as Spodoptera frugiperda Sf9 or Sf21 cells, and Trichoplusia ni High Five™ (BTI-Tn-5Bl-4) cells, may be employed, typically in combination with baculovirus expression vectors. These insect cell systems are capable of generating extracellular vesicles and can support post-translational processing, including disulfide bond formation and glycosylation. Glycoengineered insect cell lines, such as Sf9 Mimic™ cells expressing mammalian glycosyltransferases, may also be used to produce human-like glycan structures on exosome surface proteins. Exosomes or extracellular vesicles produced in such non-mammalian systems can be isolated and purified using standard methods known in the art, including ultracentrifugation, size exclusion chromatography, or tangential flow filtration.

[0260] In some embodiments, the method comprises providing a population of cells and culturing the cells in serum-free or un-concentrated conditioned medium. This includes, for example, engineered extracellular vesicles secreted into media as conditioned by a population of cells in culture, further including cell lines capable of serial passaging. In certain embodiments, the cells in culture are grown to about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% confluency when engineered extracellular vesicles are isolated.

[0261] In some embodiments, the methods provided herein further comprise contacting the cells provided herein with a nucleic acid vector encoding the at least one fusion polypeptide provided herein. A vector is a nucleic acid construct designed for delivery to a host cell or for transfer of genetic materialbetween different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer genetic material to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc. In some embodiments of any of the aspects, the vector is selected from the group consisting of a plasmid, a cosmid, and a viral vector.

[0262] In some embodiments, a vector is capable of driving expression of one or more sequences in a mammalian cell (e.g., the vector is a mammalian expression vector). Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, promoters can be derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein.

[0263] In some embodiments, the recombinant expression vector is capable of directing expression of the exogenous fusion polypeptide nucleic acid sequence preferentially in a particular cell type (e.g., via tissue-specific regulatory elements).

[0264] In some embodiments, the recombinant expression vector can comprise tissue-specific and inducible regulatory elements. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific), lymphoid-specific promoters, promoters of T cell receptors, and immunoglobulins, neuron-specific promoters (e.g., the neurofilament promoter), pancreas-specific promoters, and mammary gland-specific promoters (e.g., milk whey promoter). Developmentally regulated promoters are also encompassed, e.g., the murine hox promoters, the a-fetoprotein promoter.

[0265] In some embodiments, the at least one nucleic acid sequence described herein is delivered to the cell described herein via an integrating vector. Integrating vectors have their delivered genetic material (or a copy of it) permanently incorporated into a host cell chromosome. Non-integrating vectors remain episomal, which means the nucleic acid contained therein is not integrated into a host cell chromosome. Examples of integrating vectors include retroviral vectors, lentiviral vectors, hybrid adenoviral vectors, and herpes simplex viral vectors.

[0266] In some embodiments, the at least one nucleic acid sequence described herein is delivered to the cell described herein via a non-integrative vector. Non-integrative vectors include non-integrative viral vectors. Non-integrative viral vectors eliminate one of the primary risks posed by integrative retroviruses, as they do not incorporate their genome into the host DNA. One example is the Epstein Barr oriP / Nuclear Antigen- 1 (“EBNA1”) vector, which is capable of limited self-replication and known to function in mammalian cells. Containing two elements from Epstein-Barr virus, oriP and EBNA1, binding of the EBNA1 protein to the virus replicon region oriP maintains a relatively long-term episomal presence of plasmids in mammalian cells. This particular feature of the oriP / EBNAl vector makes it ideal for generation of integration-free host cells. Other non-integrative viral vectors include adenoviral vectors andthe adeno-associated viral (AAV) vectors. Another non-integrative viral vector is RNA Sendai viral vector, which can produce protein without entering the nucleus of an infected cell. The F-deficient Sendai virus vector remains in the cytoplasm of infected cells for a few passages but is diluted out quickly and completely lost after several passages (e.g., about 10 passages). This permits a self-limiting transient expression of a chosen heterologous gene or genes in a target cell. This aspect can be helpful, e.g., for the transient introduction of reprogramming factors, among other uses. As noted above, in some embodiments, the nucleic acid sequence described herein is expressed in the cells from a viral vector.

[0267] A “viral vector” includes a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide described herein in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo.

[0268] The nucleic acids described herein can be delivered using any transfection reagent or other physical means that facilitates entry of nucleic acids into a cell. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, electroporation, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent- enhanced uptake of DNA. Lipofection comprises the use of lipofection reagents (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). Lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, can be prepared using standard methods.

[0269] An “agent that increases cellular uptake” is a molecule that facilitates transport of a molecule, e.g., nucleic acid, or peptide or polypeptide, or other molecule that does not otherwise efficiently transit the cell membrane across a lipid membrane. For example, a nucleic acid can be conjugated to a lipophilic compound (e.g., cholesterol, tocopherol, etc.), a cell penetrating peptide (CPP) (e.g., penetratin, TAT, SynlB, etc.), or a polyamine (e.g., spermine). The one or more nucleic acid sequences encoding the fusion polypeptides provided herein can be delivered to the cell by any method discussed above or known in the art.

[0270] In some embodiments of any of the aspects, the vectors provided herein comprise a nucleic acid modification by methods known in the art. In some embodiments, the cell can be genetically manipulated to express one or more vectors, each encoding one or more fusion polypeptide comprising one or more vesicle targeting domain, one or more signaling domain, and one or more linker. In certain embodiments, the population of cells has been genetically manipulated. This includes, for example, knockout (KO) or transgenic (TG) cell lines, wherein an endogenous gene has been removed and / or an exogenous genetic sequence introduced in a stable, persistent manner. In certain embodiments, this further includes transient knockdown of one or more genes and associated coding and non-coding transcripts within the populationof cells, via any number of methods known in the art, such as introduction of dsRNA, siRNA, microRNA, etc. This further includes transient expression of one or more genes and associated coding and non-coding transcripts within the population of cells, via any number of methods known in the art, such as introduction of a vector, plasmid, artificial plasmid, replicative and / or non-replicative virus, etc.

[0271] In certain embodiments the cell population has been manipulated to knockout the expression of one or more endogenous gene sequences that encode for metalloendopeptidases. In certain embodiments the cell population has been manipulated to knockout the expression of one or more endogenous gene sequences that code for metalloproteinases. In certain embodiments the cell population has been manipulated to knockout the expression of one or more endogenous gene sequences that encode for a disintegrin and metalloproteinase (ADAM). For example, the cell population can be manipulated to knock of the expression of one or more gene sequences that encode for ADAMI, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAMI 1, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, etc.

[0272] In certain embodiments the cell population has been manipulated to knockout the expression of one or more endogenous genes that encode for enzymes that hydrolyze the inositol phosphate linkage in proteins anchored by phosphatidylinositol glycans, thereby preventing the release of proteins attached to the plasma membrane via GPI anchors. For example, the cell population can be manipulated to knock of the expression of phosphatidylinositol-glycan-specific phospholipase D (GPLD1).

[0273] In certain embodiments, the population of cells has been genetically manipulated. This includes, for example, knock-in of an exogenous genetic sequence, wherein the exogenous genetic sequence is expressed in a stable, persistent manner. In certain embodiments the cell population has been manipulated to knock-in a recombinase recognition sequence (e.g., FRT, loxP, attB or attP), transgenic reporters such as antibiotic resistance genes, fluorescent or enzymatic reporter genes, etc. or the like known in the art.

[0274] In some embodiments, the method comprises a step of isolating the engineered extracellular vesicles provided herein. Particulates within the medium are removed by a series of specific centrifugation steps and the media is filtered.

[0275] Despite advantages of exosomes as a therapeutic platform, several challenges limit the utility and clinical translation of MSC-derived exosomes, including i) primary cultures of MSCs tend to lose potency and become senescent during large-scale expansion, necessitating the use of several allogeneic donor cells and thus contributing to heterogeneity; ii) MSCs can acquire numerous chromosomal abnormalities during long-term culture; iii) MSCs do not express TSG-6 without exogenous inflammatory stimuli like cytokines or LPS, which would complicate manufacturing. Emerging exosome engineering techniques and the use of immortalized cell lines offer an alternate solution for achieving high and consistent TSG-6 expression on exosome surfaces. HEK 293-derived exosomes are considered safe for therapeutic use, as they are produced from a well -characterized human cell line. The neutral phenotype of HEK 293-derived exosomes and the high exosome productivity of HEK 293 cells make them an idealplatform for drug delivery applications. Additionally, the fact that several biopharmaceuticals produced using HEK 293 cells have been approved for clinical studies by regulatory bodies such as the FDA and EMA supports the safety and clinical-grade quality of exosomes derived from these cells. Therefore, TSG-6-engineered HEK exosomes would strike a balance by effectively addressing the biophysical, pharmacodynamic, and regulatory challenges posed by other TSG-6 modalities.

[0276] The TSG-6-engineered exosomes described herein can mimic MSC-derived exosomes for neuroprotection. Exosomes offer a novel approach to therapeutic delivery, featuring distinct pharmacokinetic properties that improve tissue retention. They are capable of penetrating difficult-to- reach sensory tissue niches, which make them suited for therapeutic delivery to the CNS and neural retina. Another significant advantage of exosome-based biologies is their capacity to be lyophilized, allowing them to be provided “off-the-shelf’ as ready-to-use products. The exosomes as described herein can be generated in a well-characterized HEK 293 cell line with the goal of controlled, industrial-scale manufacturing. While still emerging, exosome technologies - encompassing genetic engineering and biomanufacturing - are sufficiently mature for clinical application.

[0277] In some embodiments, isolating the plurality of engineered extracellular vesicles includes precipitation, centrifugation, filtration, immuno-separation, tangential flow, liquid chromatography, and / or flow fractionation. For example, differential ultracentrifugation has become a technique wherein secreted exosomes are isolated from the supernatants of cultured cells. This approach allows for separation of exosomes from non-membranous particles, by exploiting their relatively low buoyant density. Size exclusion allows for their separation from biochemically similar, but biophysically different microvesicles, which possess larger diameters of up to about 1,000 nm. Differences in flotation velocity further allows for separation of differentially sized exosomes. In general, exosome sizes will possess a diameter ranging from about 30 nm to about 300 nm, including sizes of from about 30 to aboutl50 nm. Further purification may rely on specific properties of the particular exosomes of interest. This includes, for example, use of immunoadsorption with a protein of interest to select specific vesicles with exoplasmic or outward orientations.

[0278] Among methods such as, for example, differential centrifugation, discontinuous density gradients, immunoaffinity, ultrafiltration and liquid chromatography (e.g., fast protein liquid chromatography (FPLC), differential ultracentrifugation is the most commonly used for exosome isolation. This technique utilizes increasing centrifugal force from 2000xg to 10,000xg to separate the medium- and larger-sized particles and cell debris from the exosome pellet at 100,000xg. Centrifugation alone allows for significant separation / collection of exosomes from a conditioned medium, although it is insufficient to remove various protein aggregates, genetic materials, particulates from media and cell debris that are common contaminants. Enhanced specificity of exosome purification may deploy sequential centrifugation in combination with ultrafiltration, or equilibrium density gradient centrifugation in a sucrose density gradient, to provide for the greater purity of the exosome preparation (flotationdensity l.l-1.2g / ml) or application of a discrete sugar cushion in preparation.

[0279] Ultrafiltration can be used to purify exosomes without compromising their biological activity. Membranes with different pore sizes, such as 100 kDa molecular weight cut-off (MWCO) or 300 kDa MWCO and gel filtration to eliminate smaller particles, have been used to avoid the use of a nonneutral pH or non-physiological salt concentration. Currently available tangential flow filtration (TFF) systems are scalable (to >10,000L), allowing one to not only purify, but concentrate the exosome fractions, and such approaches are less time consuming than differential centrifugation. Liquid Chromatography can also be used to purify exosomes to homogeneously sized particles and preserve their biological activity as the preparation is maintained at a physiological pH and salt concentration.

[0280] Other chemical methods have exploit differential solubility of exosomes for precipitation techniques, addition to volume -excluding polymers (e.g., polyethylene glycols (PEGs)), possibly combined additional rounds of centrifugation or filtration. For example, a precipitation reagent, ExoQuick®, can be added to conditioned cell media to quickly and rapidly precipitate a population of exosomes, although re-suspension of pellets prepared via this technique may be difficult. Flow field-flow fractionation (F1FFF) is an elution-based technique that is used to separate and characterize macromolecules (e.g., proteins) and nano- to micro-sized particles (e.g., organelles and cells) and which has been successfully applied to fractionate exosomes from culture media.

[0281] Beyond these techniques relying on general biochemical and biophysical features, focused techniques may be applied to isolated specific exosomes of interest. This includes relying on antibody immunoaffinity to recognizing certain exosome-associated antigens. Conjugation to magnetic beads, chromatography matrices, plates or microfluidic devices allows isolating of specific exosome populations of interest as may be related to their production from a parent cell of interest or associated cellular regulatory state. Other affinity-capture methods use lectins which bind to specific saccharide residues on the exosome surface.

[0282] In some embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes centrifugation of the cells and / or media conditioned by the cells. In some embodiments, ultracentrifugation is used. In some embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells is via size-exclusion filtration. In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of, for example, discontinuous density gradients, immunoaffinity, ultrafiltration, tangential flow and / or liquid chromatography.

[0283] In certain embodiments, differential ultracentrifugation includes using centrifugal force from about 1000-2000xg, 2000-3000xg, 3000-4000xg, 4000-5000xg, 5000xg-6000xg, 6000-7000xg, 7000- 8000xg, 8000-9000xg, 9000-10,000xg, to 10,000xg or more to separate larger-sized particles from a plurality of engineered extracellular vesicles derived from the cells.

[0284] In other embodiments, isolating a plurality of engineered extracellular vesicles from thepopulation of cells includes use of filtration or ultrafiltration. In certain embodiments, a size exclusion membrane with different pore sizes is used. For example, a size exclusion membrane can include use of a filter with a pore size of 0.1-0.5 micron (pm), 0.5-1.0 pm, 1-2.5 pm, 2.5-5 pm, 5 or more pm. In certain embodiments, the pore size is about 0.2 pm. In certain embodiments, filtration or ultrafiltration includes size exclusion ranging from about 100-500 daltons (Da), 500-1 kilodalton (kDa), 1-2 kDa, 2-5 kDa, 5-10 kDa, 10-25 kDa, 25-50 kDa, 50-100 kDa, 100-250 kDa, 250-500 kDa, 500 or more kDa. In certain embodiments, the size exclusion is for about 2-5 kDa. In certain embodiments, the size exclusion is for about 3 kDa. In other embodiments, filtration or ultrafiltration includes size exclusion includes use of hollow fiber membranes capable of isolating particles ranging from about 100-500 daltons (Da), 500-1 kilodalton (kDa), 1-2 kDa, 2-5 kDa, 5-10 kDa, 10-25 kDa, 25-50 kDa, 50-100 kDa, 100-250 kDa, 250- 500 kDa, 500 or more kDa. In certain embodiments, the size exclusion is for about 2-5 kDa. In certain embodiments, the size exclusion is for about 3 kDa. In other embodiments, a molecular weight cut-off (MWCO) gel filtration capable of isolating particles ranging from 100-500 daltons (Da), 500-1 kilodalton (kDa), 1-2 kDa, 2-5 kDa, 5-10 kDa, 10-25 kDa, 25-50 kDa, 50-100 kDa, 100-250 kDa, 250-500 kDa, 500 or more kDa. In certain embodiments, the size exclusion is for about 2-5 kDa. In certain embodiments, the size exclusion is for about 3 kDa. In various embodiments, such systems are used in combination with variable fluid flow systems. In certain embodiments, a size exclusion membrane with different pore sizes is used to purify extracellular vesicles from a solution comprising undesirable proteins or nucleic acids.

[0285] In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of tangential flow filtration (TFF) systems are used purify and / or concentrate the exosome fractions. In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of liquid chromatography can also be used to purify engineered extracellular vesicles to homogeneously sized particles. In various embodiments, density gradients as used, such as centrifugation in a sucrose density gradient or application of a discrete sugar cushion in preparation.

[0286] In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of a precipitation reagent. For example, a precipitation reagent, ExoQuick®, can be added to conditioned cell media to quickly and rapidly precipitate a population of engineered extracellular vesicles. In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of volume -excluding polymers (e.g., polyethylene glycols (PEGs)) are used. In another embodiment, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of flow field-flow fractionation (F1FFF), an elution-based technique.

[0287] In certain embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of one or more capture agents to isolate one or more engineered extracellular vesicles possessing specific biomarkers or containing particular biological molecules. In oneembodiment, one or more capture agents include at least one antibody. For example, antibody immunoaffinity recognizing exosome-associated antigens is used to capture specific engineered extracellular vesicles. In other embodiments, the at least one antibody can be conjugated to a fixed surface, such as magnetic beads, chromatography matrices, plates or microfluidic devices, thereby allowing isolation of the specific exosome populations of interest. In other embodiments, isolating a plurality of engineered extracellular vesicles artificial synapses from the population of cells includes use of one or more capture agents that is not an antibody. This includes, for example, use of a “bait” molecule presenting an antigenic feature complementary to a corresponding molecule of interest on the exosome surface, such as a receptor or other coupling molecule. In one embodiment, the non-antibody capture agent is a lectin capable of binding to polysaccharide residues on the exosome surface.

[0288] In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of ion exchange chromatography. In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of anion exchange chromatography. In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of cation exchange chromatography. In certain embodiments, ion exchange chromatography comprises a chromatography resin with a functional group selected from the group consisting of diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), quaternary ammonium (Q), carboxymethyl (CM), sulfopropyl (SP), and methyl sulfate (S). In certain embodiments, ion exchange chromatography comprises a chromatography resin which may have properties of a weak acid, strong acid, weak base, or strong basic. In certain embodiments, ion exchange chromatography comprises a chromatography selected from the group consisting of DEAE cellulose, DEAE Sephadex, Mono Q, Mini Q, HiTrap Capto, Capto Core 700, HiPrep Q, QAE Sephadex, Q Sepharose, CM Cellulose, SP Sepharose, SOURCE S, EAH-Sepharose, sulfoxyethyl cellulose, CM Sephadex, and CM Sepharose. Isolating a plurality of engineered extracellular vesicles can be prepared by any of a variety of ion exchange chromatography techniques.

[0289] In other embodiments, isolating a plurality of engineered extracellular vesicles from the population of cells includes use of a nuclease enzyme (e.g., a DNase or RNase). For example, a working concentration of Benzonase® nuclease may be added to an extracellular vesicle sample preparation in the presence of a divalent cation, for example, 1-2 mM Mg2+, 2-5 mM Mg2+, 10-20 mM Mg2+, 20-50 mM Mg2+, 50-100 mM Mg2+, or more than 100 mM Mg2+.

[0290] Following isolation and purification of the engineered EVs provided herein, EVs can be further evaluated for the desired structural and functional properties by methods known in the art. For example, the engineered exosomes provided herein can be assayed for functional activity on a target cell using a cell -based bioassays (e.g., those commercially available, Promega DiscoverX®), ligand-receptor binding assays, vesicle flow cytometric assays, enzyme-linked immunosorbent assays, tunable resistive pulse sensing (TRPS), nanoparticle tracking analysis (NTA), surface plasmon resonance (SSPR), nucleotidesequencing, lipidomics, proteomics, colorimetric assays, fluorescence assays, luminescence assays, immunoblotting, radioimmunoassays, electron microscopy, or EV automated analysis (e.g., ExoView®). Compositions

[0291] Compositions comprising fusion polypeptide or engineered extracellular vesicles (EVs) displaying or comprising one or more fusion polypeptides described herein are provided. Such EV compositions may include a plurality of the engineered vesicles in combination with one or more pharmaceutically acceptable carriers, excipients, or stabilizers suitable for clinical administration. Pharmaceutically acceptable carriers include those compatible with the EVs and appropriate for administration to a subject without undue toxicity, irritation, or immunogenicity. Examples include buffers, isotonic agents, surfactants, lipids, sugars, amino acids, and polymers that maintain vesicle stability and biological activity.

[0292] Fusion polypeptides or engineered EVs comprising one or more fusion polypeptide described herein can be formulated in solid, liquid, or gel dosage forms, including solutions, suspensions, lyophilized powders, or sustained-release preparations. Formulations may be adapted for parenteral (e.g., intravenous, intramuscular, or subcutaneous), intranasal, ocular, or pulmonary delivery, or for localized administration to mucosal or tissue surfaces. In certain embodiments, the EVs can be delivered using a pulmonary or intranasal delivery device such as a nebulizer, metered-dose inhaler, or dry-powder inhaler. Aerosol or powder formulations may include propellants, lactose, or other inert carriers known in the art to facilitate dispersion and uptake of formulations (e.g., fusion polypeptide formulations, or vesicle or extracellular vesicle formulations).

[0293] Fusion polypeptides or engineered EVs comprising fusion polypeptide described herein may be formulated with lipid or polymer-based carriers to enhance delivery or stability. Exemplary lipid-based systems include liposomes, micelles, emulsions, or lipid-drug complexes, whereas suitable polymer-based systems include nanoparticles, microspheres, and biodegradable or biocompatible polymers such as polyethylene glycol (PEG), polylactide, polyglycolide, poly(lactide-co-glycolide), or polyvinyl alcohol. Amphiphilic excipients such as polysorbates (e.g., Tween-series), PEGylated fatty acid esters (e.g., Gelucire, Labrasol), or lecithin can further stabilize fusion polypeptide or engineered extracellular vesicle dispersion.

[0294] In certain embodiments, the fusion polypeptide or engineered extracellular vesicle comprising fusion polypeptide described herein compositions described herein further comprise one or more excipients to enhance stability, tonicity, or dispersibility of the formulation. Suitable excipients include, but are not limited to, disaccharides such as trehalose, sucrose, and sucralose, which act as cryoprotectants and lyoprotectants to preserve vesicle integrity during freeze-drying or storage. Additional carbohydrate- based excipients can include mannitol, sorbitol, glucose, lactose, maltose, and dextran. In some embodiments, the compositions further comprise polyhydric alcohols such as glycerol, propylene glycol, or polyethylene glycol to maintain osmotic balance and prevent vesicle aggregation. Other suitableformulation excipients for fusion polypeptides or engineered extracellular vesicles comprising fusion polypeptides described herein include buffering agents (e.g., phosphate, HEPES, citrate), amino acids such as glycine, arginine, or histidine, and surfactants such as polysorbate 20, polysorbate 80, or poloxamer 188 to prevent vesicle fusion or surface adsorption. These excipients are known in the art to stabilize proteins or extracellular vesicle membranes, maintain particle size distribution, and preserve biological activity during long-term storage, reconstitution, or transport.

[0295] Therapeutic EV formulations can also include preservatives, antioxidants, or metal chelators to maintain long-term stability during storage. Lyophilized or frozen formulations may be reconstituted prior to administration, providing flexibility for clinical use. In some embodiments, fusion polypeptides or engineered EVs comprising a fusion polypeptide described herein are encapsulated in microcapsules, emulsions, or sustained-release polymer matrices (e.g., PLGA or hydrogel systems) to permit controlled release over extended durations.

[0296] Compositions of this type are known in the art to improve biodistribution, circulation time, and targeting efficiency of biologies, including exosome or liposome-based therapeutics. Accordingly, engineered EVs comprising a fusion polypeptide may be formulated using established pharmaceutical excipients and delivery systems to achieve suitable pharmacokinetic and pharmacodynamic properties for therapeutic use as known by one of ordinary skill in the art.Administration, Dosing, Efficacy

[0297] The engineered extracellular vesicle compositions described herein can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0298] Generally, application of engineered extracellular vesicles as therapy will take into account similar parameters as other therapeutic strategies, including concentration, timing of delivery, and sustained bioavailability at injury / disease site. Extracellular vesicle can be delivered via a number of routes: intravenous, intracoronary, and intramyocardial. Extracellular vesicles (e.g., exosomes), also allow for new delivery routes that were previously infeasible for cell therapy, such as inhalation or injection. These various approaches are described below, including injection, topical application, enteral administration, and pulmonary delivery.

[0299] The engineered EV compositions provided herein can be administered to a subject in need thereof by any appropriate route which results in an effective treatment in the subject. As used herein, the terms “administering,” and “introducing” are used interchangeably and refer to the placement of a composition provided herein into a subject by a method or route which results in at least partiallocalization of such compositions at a desired site, such as a site of inflammation or a tumor, such that a desired effect(s) is produced. The compositions can be administered to a subject by any mode of administration that delivers the composition systemically or to a desired surface or target, and can include, but is not limited to, injection, infusion, instillation, and inhalation administration. To the extent that the composition can be protected from inactivation in the gut, oral administration forms are also contemplated. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, retro-orbital, intravitreal, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebral, intratarsal, and intrastemal, intratumoral injection, and infusion or the like as known in the art.

[0300] A therapeutic dose of the compositions comprising engineered EVs as described herein may be delivered to a patient by means of controlled release, for example, but not limited to, implantable pump and implantable cannulas to provide continuous access to the venous or arterial system.

[0301] Topical application refers to applying or spreading a composition of the present invention onto surfaces on or in the body, both internally and / or externally, in a therapeutically effective amount for local and / or systemic treatment. Topical application may be epicutaneuos wherein a composition of the present invention may be directly applied onto a localized surface of the skin or mucous membranes. Topical application may include transdermal application wherein a composition of the present invention may be absorbed into the body to obtain systemic delivery and systemic distribution. Topical application formulations may include, but are not limited to, creams, foams, gels, lotions, solutions, ointments, dermal patch, transdermal patches, powder, solid, sponge, tape, vapor, paste, fdm, liposomes, balm, shampoo, spray, or tincture or the like or a combination thereof. A therapeutic dose of a composition of the present invention may be delivered vaginally (for example, a vaginal suppository, vaginal ring, douche, intrauterine device, intravesical infusion, and the like) or urethra or the like or a combination thereof.

[0302] Enteral administration refers to a composition of the present invention administered via the gastrointestinal tract in a therapeutically effective amount for local or systemic treatment. Enteral administration may include, but is not limited to, delivery of a composition of the present invention via the mouth, sublingual, esophagus, gastric (for example, the stomach), small intestines, large intestines or rectum. Oral delivery of the present invention may include, but is not limited to, the use of a capsule, pastille, pill, tablet, solution, gel, suspension, emulsion, syrup, elixir, tincture, mouthwash, lozenges, chewing gum, lollipop, cream, foam, solution, powder, solid, vapor, liposomes, spray, or tincture osmotic- controlled release oral delivery system, or the like. Gastric delivery may involve the use of a tube or nasal passage that leads directly to the stomach, for example, a percutaneous endoscopic gastrostomy tube. Gastric delivery may involve direct injection made through the abdominal wall. Rectal delivery may involve, but is not limited to, the use of a suppository, ointment, enema, murphy drip, or the like. A therapeutic does of the present invention may be delivered to a patient by means of controlled release, forexample, but not limited to, controlled release drug delivery pellet or pill.

[0303] Inhalation (e.g., pulmonary delivery, pulmonary administration, etc.) refers to delivery to the respiratory system through the respiratory route, including but not limited to, intranasal administration, oral administration, and oral inhalative administration (e.g., intratracheal instillation and intratracheal inhalation) of a therapeutically effective amount for local or systemic treatment. Pulmonary delivery of a therapeutically effective amount of a composition of the present invention may be achieved by dispersion, for example, by using a syringe. Pulmonary delivery of a composition of the present invention may be achieved by aerosol administration, wherein aerosol administration may deposit a therapeutically effective amount of the present invention by gravitational sedimentation, inertial impaction, or diffusion.

[0304] Intravenous delivery technique can occur through a peripheral or central venous catheter. As the simplest delivery mode, this technique avoids the risk of an invasive procedure. However, intravenous may be regarded as a comparatively inefficient and less localized delivery method, as a high percentage of infused cell exosomes may become sequestered in organs such as the lung, liver, or spleen. Such sequestration may result in few or no cellular exosomes reaching broader circulation or have unintended systemic effects following their distribution.

[0305] In certain embodiments, administration can include delivery to a tissue or organ site that is the same as the site of diseased and / or dysfunctional tissue. In certain embodiments, administration can include delivery to a tissue or organ site that is different from the site or diseased and / or dysfunctional tissue. In certain embodiments, the delivery is via inhalation or oral administration. In various embodiments, administration of engineered extracellular vesicles can include combinations of multiple delivery techniques.

[0306] In some embodiments, the compositions described herein are administered by aerosol administration, nebulizer administration, or tracheal lavage administration.

[0307] In some embodiments, the compositions described herein are administered in therapeutically effective amounts. Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the engineered EVs or fusion polypeptides provided herein), which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels of therapeutic engineered EVs in plasma can be measured, for example, by high performance liquid chromatography, enzyme linked immunosorbent assay (ELISA), flow cytometry,FACS sorting, western blot, mass spectroscopy, tunable resistive pulse sensing, ExoView®, qRT-PCR, next generation sequencing (NGS), or by any analysis technique known by one of ordinary skill in the art. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0308] The engineered EV compositions or pharmaceutical compositions described herein can be formulated, in some embodiments, with one or more additional therapeutic agents currently used to treat injury or a disorder. The effective amount of such other agents depends on the amount of an engineered EV in the formulation, the type of disorder or injury, and other factors discussed above. These are generally used in the same dosages and with administration routes as used herein before or about from about 1% to about 99% of the heretofore employed dosages.

[0309] The dosage ranges for the compositions described herein depend upon the potency and encompass amounts large enough to produce the desired effect. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition, health, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication. In some embodiments, the dosage ranges from 0.001 mg / kg body weight to 100 mg / kg body weight. In some embodiments, the dose range is from 5 pg / kg body weight to 100 pg / kg body weight. Alternatively, the dose range can be titrated to maintain serum levels between 0.1 pg / mL and 1000 pg / mL. For systemic administration, subjects can be administered a therapeutic amount, such as, e.g., 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more. These doses can be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until, for example, the infection is treated, as measured by the methods described above or known in the art. However, other dosage regimens can be useful.

[0310] In various embodiments, the quantities of engineered extracellular vesicles that are administered to achieve these effects range from 1 x 106to 1 x 107, 1 x 107to 1 x 108, 1 x 108to 1 x 109, 1 x 109to 1 x 1010, 1 x 1010to 1 x 1011, 1 x 1011to 1 x 1012, 1 x 1012to 1 x 1013, 1 x 1013to 1 x 1014, 1 x 1014to 1 x 1015, 1 x 1015or more engineered extracellular vesicles. In other embodiments, the numbers of engineered extracellular vesicles are relative to the number of cells used in a clinically relevant dose for a cell-therapy method. For example, defining an effective dose range, dosing regimen and route of administration, may be guided by studies using fluorescently labeled engineered extracellular vesicles, and measuring target tissue retention, which can be about >10X, >5 OX, or >100X background, as measured 5, 10, 15, 30, or 30 or more min as a screening criterion. In certain embodiments, >100X background measured at 30 mins is a baseline measurement for a low and high dose that is then assess for safety and bioactivity. In various embodiments, single doses are compared to two, three, four, four or more sequentially-applied doses. In various embodiments, the repeated or sequentially-applied doses are provided for treatment of an acutedisease and / or condition. In various embodiments, the repeated or sequentially-applied doses are provided for treatment of a chronic disease and / or condition. In other embodiments, administration of the engineered extracellular vesicles is adjunctive to standard therapy.

[0311] In other embodiments, administering a composition includes 1 x IO10or more engineered extracellular vesicles in a single dose. In various embodiments, exosome quantity may be defined by protein quantity, such as dosages including about 1-10, 10-25, 25-50, 50-75, 75-100, or 100 or more mg exosome protein. In other embodiments, a single dose is administered multiple times to the subject. In other embodiments, administering a composition consists of one or more of: injection, topical administration, enteral, intravenous, intra-arterial, or inhalation.

[0312] In various embodiments, exosome quantity may be defined by protein quantity, such as dosages including about 1-10, 10-25, 25-50, 50-75, 75-100, or 100 or more mg exosome protein. In various embodiments, administering a composition includes multiple dosages of the engineered extracellular vesicles. In various embodiments, the repeated or sequentially-applied doses are provided for treatment of an acute disease and / or condition. In various embodiments, the repeated or sequentially-applied doses are provided for treatment of a chronic disease and / or condition.

[0313] In other embodiments, administering a composition including a plurality of engineered extracellular vesicles to the subject is adjunctive to standard therapy.

[0314] The duration of a therapy using the methods described herein will continue for as long as medically indicated or until a desired therapeutic effect (e.g., those described herein) is achieved. In certain embodiments, the administration of the vaccine composition described herein is continued for about 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or for a period of years up to the lifetime of the subject.

[0315] Appropriate dosing regimens for a given composition can comprise a single administration or multiple doses. Subsequent doses may be given repeatedly at time periods, for example, about two weeks or greater up through the entirety of a subject's life, e.g., to provide a sustained preventative effect. Subsequent doses can be spaced, for example, about two weeks, about three weeks, about four weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, or about one year after a primary immunization.

[0316] The precise dose to be employed in the formulation will also depend on the route of administration and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the practitioner or physician will decide the amount of the engineered EV or composition thereof to administer to particular subjects.

[0317] In some embodiments, a therapeutic agent is administered to a subject in need thereof. In some embodiments, the therapeutic agent comprises a composition comprising a plurality of the fusion polypeptides or a plurality of the engineered extracellular vesicles as provided herein. In someembodiments, the therapeutic agent is administered by any appropriate route as described herein. In one embodiment, the therapeutic agent is administered intranasally. In another embodiment, the therapeutic agent is administered intravenously. In yet another embodiment, the therapeutic agent is administered orally.Methods of modulating inflammation and treating autoimmune diseases

[0318] The engineered extracellular vesicles and compositions thereof provided herein can be deployed in a therapeutic strategy against virtually any injury or disease, as providing a platform for altering biological signaling. This includes, for example, inflammation and immune signaling, which plays a role in virtually all injuries and diseases in living organisms.

[0319] Thus, described herein is a method of modulating inflammation, including selecting a subject afflicted with an inflammatory related disease and / or condition; and administering to the subject a composition of engineered extracellular vesicles described herein to the subject, wherein administration of the composition modulates inflammation. In one aspect, described herein is a method to treat an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject a composition including a fusion polypeptide described herein or engineered extracellular vesicles described herein to the subject.

[0320] In some embodiments, the composition comprises a fusion polypeptide comprising: a signaling domain comprising a TSG-6 protein comprising a LINK domain, and a CUB domain, and a C terminal tail, wherein the at least a part of the C-terminal of the TSG-6 is truncated; and at least one vesicle targeting domain, e.g., a tTSG-6 fusion polypeptide construct described herein; and a linker domain, e.g., an Fc domain. In some embodiments, the composition comprises a plurality of fusion polypeptides wherein each fusion polypeptide comprises a signaling domain comprising a TSG-6 protein comprising a LINK domain, and a CUB domain, and a C terminal tail, wherein the at least a part of the C-terminal of the TSG-6 is truncated; at least one vesicle targeting domain, and a linker domain, e.g., an Fc domain. In some embodiments, the composition comprises an engineered extracellular vesicle wherein at least one fusion polypeptide described herein is displayed on the surface of the engineered extracellular vesicle, e.g., an engineered extracellular vesicle described herein. In some embodiments, the composition comprises a plurality of engineered extracellular vesicles wherein at least one fusion polypeptide described herein is displayed on the surface of the each engineered extracellular vesicle.

[0321] As used herein, the term “inflammation” or “inflamed” or “inflammatory” refers to activation or recruitment of the immune system or immune cells (e.g., T cells, B cells, macrophages). A tissue that has inflammation can become, for example, reddened, white, swollen, hot, painful, sensitivity, exhibit a loss of function, or have a fdm or mucus. Inflammation typically occurs following injury, infection by a microorganism, exposure to a substance (e.g., a toxin, chemical, or dust) or autoimmune dysfunction. Onset of inflammation may be rapid (e.g., immediately following injury) or slow (e.g., repeated exposureto an irritant such as a chemical over time) with a duration of minutes, hours, days, months, years, or an individual’s life.

[0322] Inflammation related disease or condition may be acute, for example, septicemia. Inflammation related disease or condition may be chronic, for example, chronic obstructive pulmonary disease. In other embodiments, the inflammatory condition is an autoimmune disease wherein the autoimmune disease and / or condition is one or more of: polymyositis, dermatomyositis, Graves’ disease, Hashimoto’s thyroiditis, myasthenia gravis, vasculitis, multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, inflammatory bowel disease, Crohn’s disease, hyperthyroidism, autoimmune adrenal insufficiency, Sjogren syndrome, type I diabetes mellitus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, ulcerative colitis, uveitis, polyarteritis nodosa, relapsing polychondritis, Behcet’s disease, reactive arthritis, ankylosing spondylitis, Guillain-Barre syndrome, or optic neuropathy. In other embodiments, the disease and / or condition is chronic obstructive pulmonary disease, rheumatoid arthritis, uveoretinitis, psoriasis, and eczema. In other embodiments, the disease and / or condition is irritable bowel disease, multiple sclerosis or lupus.

[0323] In some embodiments, the inflammation related disease, or condition, or inflammatory disorder is caused by an injury, e.g., a physical injury. In some embodiments, the injury is caused by trauma, e.g., physical trauma such as a blast injury, concussion. In some embodiments, the injury can be caused for example by a blast, laceration, a crash, a collision, an impact. In some embodiments, the injury can be for example, an impact injury, a concussion, a traumatic brain injury. In some embodiments, the injury causes a trauma to the brain. In some embodiments, the inflammatory disorder is caused by a trauma to an organ in the subject’s body, e.g., brain, eye, lungs, heart, intestine, liver, kidney, stomach, ear, nose. In some embodiments, the inflammatory disorder is caused by a trauma to the head. In some embodiments, the inflammatory disorder is a neurotrauma. In some embodiments, the inflammatory disorder can cause damage to an organ in the body, e.g., nervous damage, optical damage, olfactory damage, auditory damage, sensory damage. In some embodiments, the inflammatory disorder is associated with nervous system injury. In some embodiments, the inflammatory disorder is caused by a trauma to a system in the subject’s body, e.g., neural system, digestive system, cardiovascular system, renal system. In some embodiments, the inflammatory disorder affects multiple organs of the body, e.g., brain, eye, lungs, heart, intestine, liver, kidney, stomach, ear, nose. In some embodiments, the inflammatory disorder is associated with secondary disorders such as glaucoma, dementia. In some embodiments, the inflammatory disorder is associated with visual neurotrauma such as optical blast injury. In some embodiments, the inflammatory disorder is associated with cerebral hemorrhage, swelling, and / or diffuse damage to neurons and their connecting fibers. In some embodiments, the inflammatory disorder is associated with a primary injury phase. In some embodiments, the inflammatory disorder is associated with a secondary injury phase. In some embodiments, the inflammatory disorder is associated with a tertiary injury phase.

[0324] In some embodiments, the inflammatory disorder is atraumatic brain injury (TBI). In some embodiments, the traumatic brain injury is mild. In some embodiments, the traumatic brain injury is severe. In some embodiments, a subject having the inflammatory disorder, e.g., a TBI experience function loss in other organs such as eye, ear. In some embodiments, a subject having the inflammatory disorder, e.g., a TBI experience visual impairments, including double vision, blurred vision, light sensitivity, and peripheral vision loss.

[0325] In some embodiments, the inflammation related disease, or condition, or inflammatory disorder is a neuroinflammatory disorder. In some embodiments, the neuroinflammation can be caused by but are not limited to age related degeneration, stroke, e.g., ischemic stroke, Alzheimer’s Disease (AD), amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease), Parkinson’s disease (PD), multiple sclerosis (MS), traumatic brain injury (TBI), prions.

[0326] In some embodiments, the inflammation related disease, or condition, or inflammatory disorder is associated or caused by neurodegeneration. In some embodiments, the neuroinflammation can be caused by but are not limited to age related degeneration, stroke, e.g., ischemic stroke, Alzheimer’s Disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), multiple sclerosis (MS), or prions.

[0327] In some embodiments, the inflammation related disease, or condition, or inflammatory disorder is associated or caused by a cancer. In some embodiments, the cancer is caused by an inflammatory disorder. In some embodiments, the cancer can be a hematologic cancer, a solid tumor, a soft tissue tumor, or a metastatic lesion. The cancer described herein can be a stage I cancer, a stage II cancer, a stage III cancer, or a stage IV cancer. As used herein, the term “tumor” refers to the physiological condition in mammals characterized by deregulated cell growth. In some embodiments, the cancer can be but is not limited to brain cancer, e.g., Glioblastoma (GBM), spinal cord cancer, lung cancer, colorectal cancer, prostate cancer, esophageal cancer, liver cancer, renal cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, hepatocellular cancer, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, nasopharyngeal cancer, Epstein Barr driven cancers, Human Papilloma virus driven cancers or soft tissue sarcoma. In some embodiments, the cancer can be a brain cancer such as GBM, Gliomas, Medulloblastoma, Meningioma, Brain metastases.

[0328] In one aspect, administration of an exosome or a population of engineered EVs described herein can reduce inflammation in a subject in need thereof by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to treatment with a placebo. In one aspect, administration of an exosome or a population of engineered EVs described herein can reduce neuroinflammation in a subject in need thereof by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to treatment with a placebo. In some embodiments, administration of an exosome or a population of engineered EVs described herein can reduce levels of an inflammatory marker in a subjectin need thereof by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, such that levels of the inflammatory marker are considered clinically normal, compared to administration with a placebo. In some embodiments, administration of an exosome or a population of engineered EVs described herein can reduce levels of CD44 in a subject in need thereof by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, such that CD44 levels are considered clinically normal, compared to administration with a placebo. In some embodiments, treatment with an exosome or a population of engineered EVs described herein can reduce levels of NFKB in a target cell about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, such that NFKB levels are considered clinically normal, compared to administration with a placebo.

[0329] In other embodiments, the inflammatory related disease and / or condition is an ocular disease. As used herein, the terms “ocular disease”, “eye disorder” and “eye disease” are used interchangeably and refer to a disease or disorder that affects the health and / or vision of either one or both eyes or the general area of the eye(s), eye lid(s), or area surrounding or in near proximity to the eye(s). Eye disease may include, but are not limited to, macular degeneration (e.g., age-related macular degeneration), cataracts, diabetic retinopathy, diabetic macular edema, eye floaters, eye flashes, glaucoma, amblyopia, strabismus, retinitis (e.g., CMV retinitis), color blindness, keratoconus, retinal detachment, eyelid twitching, ocular hypertension, blepharitis, uveitis, Bietti’s crystalline dystrophy, blepharospasm, cornea and corneal diseases, dry eye, histoplasmosis, macular hole, macular pucker, conjunctivitis, presbyopia, retinoblastoma, retinitis pigmentosa, retinopathy, Stargardt disease, Usher syndrome, uveal Coloma, and vitreous detachment, or the like. In some embodiments, the inflammatory disorder comprises inflammation due to injury, inflammation due to trauma (e.g., physical trauma), and / or neuroinflammation. In some embodiments, the inflammatory disorder comprises inflammation from trauma such as laceration, crush or impact injury, shock, loss of blood or oxygen flow, infection, chemical or heat exposure, poison or venom exposure, drug overuse or overexposure, and the like. In some embodiments, the inflammatory disorder comprises inflammation from trauma is an optical blast injury. In some embodiments, inflammation can comprise acute inflammation, chronic inflammation, and / or subacute inflammation. In some embodiments, acute inflammation comprises redness, swelling, pain, and heat. In some embodiments, chronic inflammation is a long-term inflammation that can last for a long period of time, such as, for example, weeks, months, or years. In some embodiments, subacute inflammation comprises the period between acute and chronic inflammation that can last between about 2 and about 6 weeks.

[0330] Described herein is a method for treatment including, selecting a subject in need of treatment, administering a composition including a plurality of engineered extracellular vesicles to the individual, wherein administration of the composition treats the subject. In certain embodiments, the subject is in need to treatment for a disease and / or condition involving tissue damage or dysfunction.

[0331] Described herein is a method of treating an autoimmune disease, inflammation, inflammatorydisease or condition, or cancer in a subject, the method comprising administering to a subject an engineered EV or composition thereof as provided herein to the subject.

[0332] Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a clinical or biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for a disease or disorder. It will be understood, however, that the total usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated.

[0333] Non-limiting examples of clinical tests that can be used to assess autoimmune diseases, inflammatory conditions, or inflammation parameters include blood tests, skin biopsy, MRI, eye examination, ocular pressure tests, etc. Where necessary or desired, animal models of injury or disease can be used to gauge the effectiveness of a particular composition as described herein.

[0334] In various embodiments, administration of the plurality of engineered extracellular vesicles alters gene expression in the damaged or dysfunctional tissue, improves viability of the damaged tissue, and / or enhances regeneration or production of new tissue in the individual. In various embodiments, administration of the plurality of engineered extracellular vesicles alters gene expression in the damaged or dysfunctional tissue, improves viability of the damaged tissue, and / or enhances regeneration or production of new tissue in the individual.

[0335] In various embodiments, the damaged or dysfunctional tissue is in need of repair, regeneration, or improved function due to an acute event. Acute events include, but are not limited to, trauma such as laceration, crush or impact injury, shock, loss of blood or oxygen flow, infection, chemical or heat exposure, poison or venom exposure, drug overuse or overexposure, and the like. Other sources of damage also include, but are not limited to, injury, age-related degeneration, cancer, and infection. In several embodiments, the regenerative cells used to prepare the engineered EVs provided herein are from the same tissue type as is in need of repair or regeneration. In several other embodiments, the regenerative cells are from a tissue type other than the tissue in need of repair or regeneration. In some embodiments, the engineered EVs provided herein are derived from the subject being treated. In some embodiments, the engineered EVs are derived from a donor subject.

[0336] In other embodiments, the damaged or dysfunctional tissue is in need of repair, regeneration, or improved function due to damage from chronic disease.EXAMPLES AND DEATAILED DESCRIPTIONS OF THE DRAWINGS The following examples are provided by way of illustration, not limitation.Example 1. TSG-6 is an exosome cargo protein.

[0337] TSG-6 is a secreted protein that naturally lacks a membrane-anchoring motif, such as aglycosylphosphatidylinositol (GPI) anchor or transmembrane domain. Despite this, native TSG-6 has been observed to co-purify with mesenchymal stem cell (MSC)-derived extracellular vesicles, including exosomes, where it contributes to their therapeutic activity in preclinical models of colitis, wound healing, mild traumatic brain injury, and spinal cord ischemia-reperfusion injury.

[0338] We sought to confirm these findings. Thus, bone marrow MSCs were cultured in complete media containing fetal bovine serum. The cells were subsequently incubated in serum free basal media with 10 ng / ml each TNF and IFN. The cells were subsequently incubated in serum free basal media incubated in serum-free medium, which was harvested, filtered through a 0.45 pm PES filter, concentrated, and buffer-exchanged into Dulbecco’s phosphate-buffered saline (DPBS) using Amicon Ultra- 15 centrifugal filters with a 10 kDa molecular weight cutoff. Extracellular vesicles were separated from smaller biomolecules using qEVoriginal 35 nm size -exclusion chromatography (SEC) columns (Izon Science) essentially according to the manufacturer’s instructions, the contents of which are incorporated herein by reference (support.izon.com / qev-columns#user-guides) (FIG. 1). Following the column void volume (fractions 1-6), fractions 7-18 were collected for analysis.

[0339] Dot blot analysis was performed on fractions 7-18 using a PVDF-FL membrane under vacuum suction in a 96-well Bio-Dot Microfdtration Apparatus (Bio-Rad). Blots were probed with antibodies recognizing putative EV protein (ALIX), putative non -EV protein TIMP1 and TSG-6, followed by appropriate fluorescent secondary antibodies, or stained for total protein using Li-Cor Revert 700 total protein stain. Membranes were imaged and analyzed with a Li-Cor Odyssey fluorescent imager. Results demonstrated that fractions 7, 8, and 9 contained the highest levels of ALIX and lowest levels of TIMP1, indicating that these fractions are enriched for extracellular vesicles (FIG. IB). TSG-6 co-purified primarily with EV-enriched fractions containing ALIX, while a soluble control protein of similar molecular weight, TIMP1, was detected exclusively in non-EV fractions (FIG. IB). These results indicate a preferential association of TSG-6 with exosomes in MSC-conditioned medium.

[0340] The requirement for cytokine priming to induce TSG-6 expression in MSCs presents both biological and technical limitations for the scalable manufacturing and rigorous purification of TSG-6- containing EV therapeutics. To overcome this limitation, a nucleotide sequence of TSG-6 comprising SEQ ID NO: 2 was cloned into a site-directed recombination expression vector and stably expressed in Flp-In™-293 cells to produce TSG-6 protein SEQ ID NO: 3, (herein also referred to as construct 0). Cells were expanded in serum containing medium then transferred to basal media lacking serum. The conditioned media harvested, filtered through a 0.45 pm PES filter, concentrated, and buffer-exchanged into Dulbecco’s phosphate-buffered saline (DPBS) using Amicon Ultra-15 centrifugal filters with a 10 kDa molecular weight cutoff. Western blot analysis of SEC fractions of CCM demonstrated that TSG-6 co-purified with the EV marker ALIX, confirming that TSG-6 association with EVs can occur independently of MSC origin (FIG. 1C).

[0341] The non-covalent nature of native TSG-6 association with exosomes poses challenges forconsistent surface display. Accordingly, we designed TSG-6 fusion polypeptides incorporating vesicletargeting domains and optional Fc linkers, as described in Example 2 (FIG. 2). These constructs enable stable anchoring of bioactive TSG-6 on the EV surface, facilitating reproducible production of engineered EVs with enhanced therapeutic potential.Example 2. Design of TSG-6 fusion polypeptides with extracellular vesicle-targeting domains.

[0342] Engineered extracellular vesicles are designed to modulate biological signaling pathways by delivering signaling proteins or domains to recipient cells. Such engineered vesicles may be configured to promote or inhibit signaling activity (e.g., agonist or antagonist function) depending on the incorporated signaling domain. The engineered extracellular vesicles retain characteristic features of extracellular vesicles, including a lipid bilayer membrane, expression of canonical tetraspanins (e.g, CD9, CD63, CD81), and a typical particle size distribution of approximately 30-150 nm.

[0343] In certain embodiments, the vesicles comprise a fusion polypeptide that includes at least one vesicle-targeting domain and at least one signaling domain (e.g., TSG-6 or a functional fragment thereof). The fusion polypeptide may further include a linker domain, such as an Fc linker, positioned between the vesicle-targeting and signaling domains to provide structural flexibility, promote proper folding, or facilitate purification (FIG. 2A).

[0344] Vesicle -targeting domains are peptide or protein sequences that promote localization or anchoring of the fusion polypeptide to the membrane of extracellular vesicles. Representative vesicletargeting domains include, but are not limited to, sequences derived from MFGe8 (Lactadherin), PTGFRN, Lamp2b, CD81, CD82, CD63, CD9, one or more transmembrane domains of CD9 (e.g., CD9tm2), MARCKS, KRAS4B, CD55, CD59, or other functionally equivalent sequences known to one of ordinary skill in the art. High efficiency of expression and vesicle incorporation has been observed when fusion polypeptides include the second transmembrane domain of CD9 (e.g. CD9tm2 domain).

[0345] Optionally, sequences directing protein lipidation may be incorporated adjacent to or within the vesicle-targeting domain to enhance association with the vesicle lipid bilayer. Nonlimiting examples of lipidation include N-myristoylation, S-palmitoylation, S -prenylation, and glycosylphosphatidylinositol (GPI) anchoring. In certain embodiments, a CAAX box motif (e.g., CVIM), which directs post- translational prenylation of the cysteine residue, was appended to the C-terminal end of the vesicle targeting domain of the fusion polypeptide sequence to generate Construct 2 (SEQ ID NO: 49), promoting membrane anchoring through famesylation.

[0346] Linker regions, such as Fc linkers, may optionally be included between the vesicle-targeting and signaling domains to enhance flexibility, improve folding, or simplify purification. Disclosed herein are compositions comprising extracellular vesicles (EVs), including exosomes, that express engineered TSG- 6 fusion polypeptides designed to exhibit enhanced stability and increased expression on the vesicle surface. FIG. 2B illustrates various embodiments of fusion polypeptide constructs described herein forEV surface display. Stars indicate putative proteolytic cleavage sites. Construct 3 (SEQ ID NO: 51) was generated from Construct 2 (SEQ ID NO: 49) by truncating TSG-6 residues D248-L277. Construct 4 (SEQ ID NO: 55) further lacks the CUB domain. Computationally predicted molecular weights for each construct are shown on the right.

[0347] In certain embodiments, the fusion polypeptide further comprises an Fc linker domain engineered to reduce or eliminate Fc-mediated effector functions. For example, the Fc linker may include amino acid substitutions known as LALA-PG mutations (L234A, L235A, P329G) that disrupt binding to Fey receptors and Clq, thereby silencing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Inclusion of these mutations provides enhanced biochemical stability and minimizes unintended immune activation while retaining the structural and dimerization properties of the Fc region.

[0348] In certain embodiments, the Fc domain also promotes heterodimerization of fusion polypeptides, facilitating formation of multimeric or bivalent TSG-6 display configurations, as illustrated in FIG. 2C. Additional constructs, including Construct 3 (truncated TSG-6, AD248-L277, SEQ ID NO: 51) and Construct 4 (further truncation removing the CUB domain, SEQ ID NO: 55), were designed to evaluate the impact of sequence modifications on proteolytic stability and EV display.Example 3. Isolation and characterization of Construct 1 (SEQ ID NO: 47) EVs.

[0349] Glycosylphosphatidylinositol (GPI)-anchored proteins are naturally enriched on exosomes, and short GPI anchors have been shown to enable exosomal display of functional proteins, such as EGFR nanobodies. To evaluate exosome display of TSG-6, a fusion polypeptide comprising TSG-6, an immunoglobulin Fc domain containing LALA-PG silencing mutations, and an engineered C-terminal GPI sequence derived from CD55 (Construct 1, (SEQ ID NO: 47), FIG. IB, FIG. 3A) was expressed in Flp- In™-293 cells.

[0350] Conditioned medium from the engineered 293 cells was fractionated by size-exclusion chromatography (SEC), and fractions 7-18 were analyzed by dot blot immunoblotting. TSG-6 was observed to co-purify with exosome marker ALIX, which was most abundant in the first three fractions (fractions 7-9) eluted after the column void volume (FIG. 3A). These results indicate that the fusion polypeptide encoded by Construct 1 (SEQ ID NO: 47) associates with extracellular vesicles. However, detectable TSG-6 was also present in non-vesicle fractions (fractions 10-18), suggesting partial cleavage or release of the fusion polypeptide from EVs during production or purification.

[0351] EV identity in the second collected fraction (fraction 8) was further confirmed by nanoparticle tracking analysis (NTA), which demonstrated a size distribution consistent with exosomes (FIG. 3B and FIG. 3C), and by analysis of canonical exosome epitopes using bead-based flow cytometry (MACSPlex EV Kit, Miltenyi) (FIG. 3D). Construct 1 (SEQ ID NO: 47) EVs exhibited comparable particle size and expression of EV markers relative to control EVs isolated from unmodified Flp-In™-293 cells (FIGS.3B-D) Median fluorescence intensity (MFI) of a tetraspanin antibody cocktail (CD9 / CD63 / CD81) is plotted on the Y -axis for each capture bead population on the X-axis, reflecting relative abundance of surface antigens within the EV preparation (FIG. 3D).

[0352] To further validate the presence and distribution of canonical tetraspanin markers (CD63, CD81, and CD9) on individual EVs, single-particle immunofluorescence analysis was performed using the ExoView R100 platform (FIG. 4). EVs were captured on antibody-functionalized surfaces and probed with fluorescently labeled antibodies against the tetraspanins. Fluorescence intensity and co-localization analysis confirmed that both control and Construct 1 (SEQ ID NO: 47) EVs expressed similar levels of CD63, CD81, and CD9, consistent with authentic exosome populations.

[0353] These data support the utility of the GPI-anchored TSG-6 fusion for exosome association, while also highlighting the potential for further optimization to minimize release of the polypeptide from the vesicle surface.Example 4. Engineering 293H Cells for Production of TSG-6-Containing Extracellular Vesicles

[0354] Flp-In™-293 cells require serum for adherent planar growth, which introduces potential contaminating vesicles and xenogeneic components, and limits scalability due to adherence requirements. To overcome these limitations, 293H cells were genetically engineered to create a stable, isogenic 293H cell line with an integrated fusion polypeptide sequence. This system allows high-level, reproducible expression of engineered fusion polypeptides in a serum-free suspension culture format, facilitating scalable engineered extracellular vesicle production.

[0355] Using this platform, 293H cells were stably engineered to produce either control EVs (lacking a fusion polypeptide), EVs expressing TSG-6 fusion protein Construct 1 (SEQ ID NO: 47), or EVs expressing truncated TSG-6 (tTSG-6) fusion protein Construct 3 (SEQ ID NO: 51). Cells were expanded in suspension culture over approximately seven days, and cell growth and metabolic activity were monitored throughout the culture period.

[0356] Cell proliferation, measured as cell concentration (cells / mL) over time (FIG. 5A), demonstrated that engineered 293H cells exhibited growth kinetics indistinguishable from those of parental, unmodified cells. Similarly, glucose consumption in the culture medium (FIG. 5B) proceeded at equivalent rates in engineered and parental cells, indicating that metabolic activity was unaffected by the introduction of TSG-6 fusion polypeptide constructs.

[0357] Simultaneous monitoring of cell density and glucose concentration for cells expressing Construct 2 (SEQ ID NO: 49) (FIG. 5C) confirmed that stable integration of the fusion constructs did not impair normal growth or metabolism.

[0358] These observations indicate that engineering 293H cells to express fusion polypeptides presented herein does not introduce detectable defects in proliferation or metabolic activity. Consequently, extracellular vesicles produced from engineered cells are expected to exhibit similarquality, composition, and functional properties to EVs derived from parental 293H cells, supporting their suitability for therapeutic applications.Example 5. Size- Exclusion Chromatography Analysis of TSG-6 Distribution in Engineered Extracellular Vesicles

[0359] To evaluate the association of TSG-6 with extracellular vesicles (EVs) produced by engineered 293H cell lines, conditioned medium (CCM) from parental and fusion polypeptide-expressing 293H cells was concentrated and subjected to size-exclusion chromatography (SEC) using qEV columns. For initial experiments (FIGS. 6A-6E), qEVoriginal columns (35 nm pore size) were employed, allowing separation of EVs from smaller proteins and biomolecules. For experiments requiring larger load volumes (FIGS. 6F-6G), qEV 10 columns were used to increase fraction size and protein recovery.

[0360] Concentrated CCM from unmodified 293H cells (control EVs) and from cells engineered to express TSG-6 constructs (Constructs 0-3) was applied to the SEC columns, and sequential fractions were collected according to the manufacturer’s instructions (support.izon.com / qev-columns#user-guides). Fractions were analyzed for total protein content using a Qubit protein assay and for TSG-6 signal using dot blot analysis. In selected experiments (FIGS. 6D and 6E), membranes were also probed for ALIX, a canonical EV marker, to confirm the presence of extracellular vesicles in early fractions.

[0361] Results from qEVoriginal fractionation (FIGS. 6A-6E) showed that TSG-6 from engineered cell lines primarily eluted in early fractions (fractions 7-9), consistent with the elution profile of EVs. In FIG. 6D and FIG. 6E, the TSG-6 signal overlapped with ALIX, confirming that these fractions are enriched for extracellular vesicles. Later fractions contained minimal TSG-6, indicating little non-vesicle- associated protein.

[0362] Larger-scale fractionations using qEV 10 columns (FIGS. 6F-6G) confirmed that TSG-6 signal was concentrated in early fractions, consistent with EV incorporation. Dot blot analysis across duplicate fractionations demonstrated that TSG-6 consistently co-purified in EV-enriched fractions, further supporting the robust association of the fusion polypeptides with extracellular vesicles.

[0363] Collectively, these data demonstrate that TSG-6 from engineered 293H cell lines predominantly associates with EVs. SEC purification effectively separates EV-associated TSG-6 from non-vesicle protein, supporting the production of EV preparations with reproducible TSG-6 display.Example 6. Evaluation of Proteolytic Stability of TSG-6 Fusion Polypeptides on Extracellular Vesicles

[0364] SEC-purified extracellular vesicles from engineered 293H cells expressing various TSG-6 fusion constructs were analyzed under reducing conditions to assess the integrity of the fusion polypeptides (FIG. 7). During production in chemically defined medium (CDM), substantial cleavage of Construct 1 (SEQ ID NO: 47) and Construct 2 (SEQ ID NO: 49) fusion proteins was observed, as evidenced by reduced full-length fusion protein bands and the appearance of smaller fragments onWestern immunoblots (FIGS. 7A-B). Addition of a broad-spectrum protease inhibitor cocktail to conditioned media at the time of harvest did not prevent cleavage, indicating that proteolytic processing occurred during cell cultivation rather than post-harvest. Furthermore, expression of Construct 1 (SEQ ID NO: 47) on EVs was reduced when cells were cultured in CDM compared to media containing fetal bovine serum (FBS), highlighting the influence of culture conditions on fusion protein display.

[0365] To improve exosome expression and proteolytic stability, Construct 1 (SEQ ID NO: 47) was reengineered by replacing the C-terminal glycosylphosphatidylinositol (GPI) anchor with a sequence encompassing the second transmembrane domain of the exosomal tetraspanin CD9 (CD9tm2) additionally amino acids VIM as shown in SEQ ID NO: 27 were appended to the C-terminal cysteine (generating a CVIM motif) to direct famesylation, generating Construct 2 (SEQ ID NO: 49). This modification significantly enhanced surface expression of the fusion polypeptide on EVs, although cleavage of TSG-6 persisted (FIGS. 7A-B).

[0366] Further engineering involved truncation of residues D248-L277 in TSG-6 to generate Construct 3 (SEQ ID NO: 51), a truncated TSG-6 fusion protein. Western immunoblot analysis demonstrated that this truncation completely abolished proteolytic cleavage, resulting in intact TSG-6 fusion protein on EVs (FIGS. 7A-C). In contrast, additional truncation to remove the entire CUB domain generated Construct 4 (SEQ ID NO: 55), which unexpectedly exhibited notable cleavage susceptibility, similar to Constructs 1 and 2. These findings highlight that the stability of Construct 3 (SEQ ID NO: 51) was not predictable based solely on sequence truncation and was determined empirically to be a preferred embodiment of the fusion polypeptide.

[0367] EVs derived from cells expressing Construct 3 (SEQ ID NO: 51) exhibited robust, full-length TSG-6 display with expression levels comparable to or exceeding those of parental constructs and control EVs. These results demonstrate that rational engineering of the TSG-6 fusion polypeptide, including replacement of the GPI anchor with a tetraspanin transmembrane domain (e.g., CD9tm2), strategic truncation of a cleavage -prone segment, and evaluation of alternative lipidation motifs (e.g., CVIM- directed famesylation), enables stable, full-length display of TSG-6 on extracellular vesicles. The data further support the production of EVs with improved reproducibility, enhanced stability, and resistance to proteolytic cleavage, which is critical for therapeutic development.Example 7. Nanoparticle Characterization of Engineered TSG-6 EVs

[0368] To determine whether expression of TSG-6 fusion polypeptides influenced extracellular vesicle (EV) production or physical properties, SEC-purified EV fractions were analyzed using Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). EVs were isolated from concentrated conditioned medium of unmodified 293H cells (Control EVs) or 293H cells engineered to express TSG-6 fusion protein Construct 1 (SEQ ID NO: 47) or Construct 3 (SEQ ID NO: 51), as described herein.

[0369] As shown in FIG. 8A, NTA analysis of SEC-purified EV fractions (fractions 7-9) demonstrated that at equivalent total protein concentrations, Control EVs, Construct 1 (SEQ ID NO: 47) EVs, and Construct 3 (SEQ ID NO: 51) EVs exhibited comparable particle numbers and similar unimodal size distributions centered at approximately 100-120 nm, consistent with the expected range for exosomes. These data indicate that incorporation of TSG-6 fusion polypeptides did not substantially alter vesicle size, heterogeneity, or overall yield.

[0370] Independent analysis by TRPS (FIG. 8B) confirmed these findings. TRPS measurements of Control EVs and Construct 3 (SEQ ID NO: 51) EVs showed highly similar particle concentrations and size profiles at matched total protein inputs, further validating that engineered TSG-6 expression does not affect EV morphology or abundance.

[0371] Collectively, the NTA and TRPS results demonstrate that the genetic modification of producer 293H cells to express TSG-6 fusion polypeptides does not impair EV biogenesis, particle yield, or size distribution. These findings confirm that engineered TSG-6 EVs maintain physical characteristics comparable to those of unmodified EVs, supporting their suitability for scalable production and consistent therapeutic performance.Example 8. Proteomic characterization of engineered extracellular vesicles

[0372] To evaluate the protein composition and purity of extracellular vesicles (EVs) expressing engineered TSG-6 fusion polypeptides, we performed comprehensive proteomic profiling of SEC-purified EVs isolated from 293H cells expressing either no transgene (Control EVs), the TSG-6-Fc-GPI fusion protein (Construct 1 (SEQ ID NO: 47) EVs), or the truncated TSG-6-Fc-CD9tm2 fusion protein (Construct 3 (SEQ ID NO: 51) EVs).

[0373] For each sample, 10 pg of total EV protein was precipitated on ice for 1 h with trichloroacetic acid (final concentration 20%), centrifuged at 14,000 x g for 30 min, and washed twice with ice-cold acetone. Pellets were air-dried and stored at -20 °C until further processing. Proteins were solubilized in 8 M urea, 100 mM Tris (pH 8.5), and digested using the protein aggregation capture (PAC) method (Batth et al., 2019). The resulting peptides were analyzed by online reversed-phase liquid chromatography coupled to a mass spectrometer operating in data-independent acquisition (DIA) mode. MS / MS spectra were processed with the DIA-NN algorithm (Demichev et al., 2020; Guzman et al., 2024), and identifications were filtered using a < 1% false discovery rate (FDR). Gene Ontology (GO) cellular component enrichment analysis was performed using ShinyGO 0.85 to assess sample composition.

[0374] As shown in FIGS. 9A-9C, GO enrichment analysis of proteins identified in Control EVs, Construct 1 (SEQ ID NO: 47) EVs, and Construct 3 (SEQ ID NO: 51) EVs revealed highly similar enrichment of canonical exosome-associated terms (e.g., extracellular exosome, extracellular vesicle, extracellular organelle), confirming both EV enrichment and expected cellular origin of the preparations.

[0375] A comparative analysis of total proteins identified by LC-MS revealed a substantial overlap among all three samples, as illustrated by the Venn diagram in FIG. 9D. The majority of detected proteins were shared among Control, Construct 1 (SEQ ID NO: 47), and Construct 3 (SEQ ID NO: 51) EVs, consistent with the preservation of core exosome composition despite genetic modification of producer cells.

[0376] Quantitative comparison of protein intensities between Control EVs and Construct 3 (SEQ ID NO: 51) EVs (FIG. 9E) demonstrated that inclusion of the truncated TSG-6 fusion polypeptide did not alter the overall proteome, aside from the expected appearance of TSG-6 peptides in Construct 3 (SEQ ID NO: 51) EVs. A heatmap of selected proteins (FIG. 9F) further confirmed the presence of TSG-6 in Construct 3 (SEQ ID NO: 51) EVs but not Control EVs and verified relatively high levels of established exosome markers (e.g, Syntenin-1, CD81, ALIX) and low or absent levels of non-EV proteins (e.g., APOA1, GM130, Bcl-2).

[0377] Collectively, these results demonstrate that (i) EVs produced by 293H cells expressing engineered TSG-6 fusion polypeptides retain the canonical proteomic signature of exosomes, (ii) TSG-6 fusion polypeptides are efficiently incorporated into the vesicle fraction, and (iii) the genetic engineering strategy does not substantially alter global EV composition. These findings establish the biochemical integrity of engineered EVs and support the utility of Construct 3 (SEQ ID NO: 51) as a stable and the preferred embodiment of exosome-anchored configuration of TSG-6.

Claims

CLAIMSWhat is claimed is:

1. A fusion polypeptide comprising: i. a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C terminal tail; and ii. at least one linker; and iii. at least one vesicle targeting domain.

2. The fusion polypeptide of claim 1, wherein the fusion polypeptide is displayed on a surface of an engineered extracellular vesicle.

3. The fusion polypeptide of claim 1 or 2, wherein at least a part of the C-terminal of the TSG-6 is truncated4. The fusion polypeptide of any one of the claims 1-3, wherein the entire C-terminal tail of the TSG-6 protein is truncated.

5. The fusion polypeptide of any one of claims 1-4, wherein the truncated TSG-6 protein lacks a fragment comprising an amino acid sequence as set forth in SEQ ID NO: 17.

6. The fusion polypeptide of any preceding claims 1-5, wherein the vesicle targeting domain is selected from a Glycosylphosphatidylinositol (GPI) anchor, a fatty acetylation site, a prenylation site, and a transmembrane domain.

7. The fusion polypeptide of claim 6, wherein the transmembrane domain is a CD9 transmembrane domain.

8. The fusion polypeptide of claim 7, wherein the CD9 transmembrane domain is a CD9 transmembrane 2 domain.

9. The fusion polypeptide of any preceding claims, wherein the vesicle targeting domain comprises an S-Palmitoylation site and an S-Famesylation site.

10. The fusion polypeptide of any preceding claims, wherein the fusion polypeptide comprises a linker between the signaling domain and the vesicle targeting domain, wherein the N terminus ofthe linker is linked to the C terminus the signaling domain and the C terminus of the linker is linked to the N terminus of the vesicle targeting domain.

11. The fusion polypeptide of claim 10, wherein the linker is selected from the group consisting of Fc domains, Fc from IgGl, Fc from IgG2, Fc from IgG3, Fc from IgG4 (4Fc), and sequences with at least 70%, 80%, or 90% homology with any of the foregoing.

12. The fusion polypeptide of claim 11, wherein the Fc domain comprises one or more mutations.

13. The fusion polypeptide of any preceding claim, wherein the TSG-6 protein comprises a sequence as set forth in SEQ ID NOS: 3, 5, 7, 9, 11, or 13.

14. The fusion polypeptide of any preceding claim, wherein the fusion polypeptide comprises a sequence as set forth in SEQ ID NOS: 47, 49, 51, 53, 55, or 57.

15. A nucleic acid encoding a fusion polypeptide of any preceding claims.

16. An engineered extracellular vesicle comprising a fusion polypeptide of any preceding claims.

17. The engineered extracellular vesicle of claim 16, wherein the engineered extracellular vesicle is an exosome.

18. The engineered extracellular vesicle of claim 16 or 17, wherein at least a portion of the vesicle targeting domain of the fusion polypeptide is embedded in a phospholipid bilayer of the engineered extracellular vesicle, and the signaling domain of the fusion polypeptide is in an extracellular position relative to a lipid membrane of the engineered extracellular vesicle.

19. A composition comprising a fusion polypeptide of any one of claims 1-14, the nucleic acid of claim 15, or the engineered extracellular vesicle of any of claims 16-18.

20. A fusion polypeptide comprising: i. a signaling domain comprising a TSG-6 protein comprising a LINK domain, a CUB domain, and a C terminal tail; and ii. at least one linker; and iii. at least one vesicle targeting domain; and the fusion polypeptide is displayed on a surface of an engineered extracellular vesicle.

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