Surface-modified extracellular vesicles and their therapeutic use

Abstract

The present invention provides a surface-modified extracellular vesicle, a composition containing the surface-modified extracellular vesicle, a method for producing the surface-modified extracellular vesicle, and a method for using the surface-modified extracellular vesicle or the composition.

Description

Technical Field

[0001] This application claims priority based on U.S. Patent Application No. 63 / 345,040, filed on May 24, 2022, and the entire specification of said application is incorporated herein by reference.

[0002] The present invention relates to surface-modified extracellular vesicles, compositions containing surface-modified extracellular vesicles, methods for producing surface-modified extracellular vesicles, and methods of using surface-modified extracellular vesicles or compositions.

Background Art

[0003] Many efforts have been made to use exosomes to deliver various therapeutic molecules to desired target cells for therapeutic purposes, examples of which include therapeutic proteins, membrane proteins, protein reporters, enzymes, antibody fragments, cytokines, TNFSF (tumor necrosis factor superfamily) ligands, RNA-binding proteins, Cas9, and vaccine antigens. Exosomes having therapeutic molecules on their surface have been proposed. Scaffolds have been proposed that can display therapeutic molecules on the surface of exosomes. PTGFRN (Prostaglandin F2 receptor regulatory protein) has been proposed as a scaffold that can display various therapeutic molecules on the surface of exosomes. However, there is still a need for new scaffolds that can display therapeutic molecules on the surface of exosomes in a better way.

Summary of the Invention

Problems to be Solved by the Invention

[0004] One aspect of the present invention provides a DNA construct comprising a DNA sequence encoding a scaffold peptide, wherein the amino acid sequence of the scaffold peptide comprises a sequence represented by G-a-S-b-X1-c-X2 (extracellular vesicle sorting motif, ESM), X1 is G, A, S or T; X2 is G or S; a is 3 to 4 amino acids; b is 2 to 3 amino acids; c is 6 to 7 amino acids; G is glycine; S is serine; A is alanine; T is threonine.

[0005] In some embodiments, the sequence G-a-S-b-X1-c-X2 can have 15 to 17 amino acids. In some embodiments, the scaffold peptide can have 22 to 57 amino acids. In some embodiments, the amino acids a, b, and c can include V, G, L, I, A, T, S, C, F, W, Y, and P. Here, V represents valine, G represents glycine, L represents leucine, I represents isoleucine, A represents alanine, T represents threonine, S represents serine, C represents cysteine, F represents phenylalanine, W represents tryptophan, Y represents tyrosine, and P represents proline. In some embodiments, a can represent 3 to 4 amino acids selected from the group consisting of V, G, L, I, T, and A, where V represents valine, G represents glycine, L represents leucine, I represents isoleucine, T represents threonine, and A represents alanine. In some embodiments, a can represent VGL, IGL, VGLT, IGLT, VGLA, or IGLA. In some embodiments, b can represent 2 to 3 amino acids selected from the group consisting of V, I, A, and T, where V represents valine, I represents isoleucine, A represents alanine, and T represents threonine. In some embodiments, b can represent VI, AV, TVI, or AVI. In some embodiments, c can represent 6 to 7 amino acids selected from the group consisting of L, S, C, and I, where L represents leucine, S represents serine, C represents cysteine, and I represents isoleucine. In some embodiments, c can represent LLSCLI or ILLSCLI. In some embodiments, the sequence G-a-S-b-X1-c-X2 can be one of the amino acid sequences shown in ESM SEQ ID NOs: 1 to 100. In some embodiments, the scaffold peptide further includes KYPLLI at the N-terminus of the sequence G-a-S-b-X1-c-X2, where K represents lysine, Y represents tyrosine, P represents proline, L represents leucine, and I represents isoleucine.In some embodiments, the scaffold peptide further comprises DVLNAFKYPLLI at the N-terminus of the sequence represented by G-a-S-b-X1-c-X2, where D represents aspartic acid, V represents valine, L represents leucine, N represents asparagine, A represents alanine, F represents phenylalanine, K represents lysine, Y represents tyrosine, P represents proline, L represents leucine, and I represents isoleucine. In some embodiments, the scaffold peptide further comprises YCSS at the C-terminus of the sequence represented by G-a-S-b-X1-c-X2, where Y represents tyrosine, C represents cysteine, and S represents serine. In some embodiments, the scaffold peptide further comprises YCSSHWCCKKEVQETRRERRRLMSMEMD at the C-terminus of the sequence G-a-S-b-X1-c-X2, where Y represents tyrosine, C represents cysteine, S represents serine, H represents histidine, W represents tryptophan, K represents lysine, E represents glutamic acid, V represents valine, Q represents glutamine, T represents threonine, R represents arginine, L represents leucine, M represents methionine, and D represents aspartic acid. In some embodiments, the scaffold peptide further comprises YCSSHWC at the C-terminus of the sequence represented by G-a-S-b-X1-c-X2, where Y represents tyrosine, C represents cysteine, S represents serine, H represents histidine, and W represents tryptophan. In some embodiments, the DNA construct may further comprise a DNA sequence encoding the amino acid sequence of a target protein. In some embodiments, the target protein can be a therapeutic protein. In some embodiments, the scaffold peptide can display the target protein at a desired position in the extracellular endoplasmic reticulum. In some embodiments, the desired position can be the inner or outer surface of the extracellular endoplasmic reticulum.

[0006] A further aspect of the invention provides a vector comprising the DNA construct. Non-limiting embodiments of the vector are expression plasmids containing a DNA sequence encoding the scaffold peptide.

[0007] A further aspect of the present invention provides a host cell comprising the vector. Non-limiting embodiments of the host cell can include HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSC), cells derived from HEK293 cells, CHO cells, or MSC. Further, non-limiting embodiments of the host cell can include mast cells, immune cells, natural killer cells, dendritic cells, macrophages, T lymphocytes, B lymphocytes, epithelial cells, human cardiac progenitor cells, adipose-derived stem cells, umbilical cord blood-derived mesenchymal stem cells, and bone marrow-mesenchymal stem cells.

[0008] Another further aspect of the present invention provides an extracellular vesicle (EV) isolated from the host cell. In some embodiments, the scaffold peptide can be displayed at a desired position on the extracellular vesicle. For example, the scaffold peptide can be displayed on the inner surface, the outer surface, or both the inner and outer surfaces of the extracellular vesicle. In some embodiments, the extracellular vesicle can further comprise a target protein. In some embodiments, the scaffold peptide can be fused to the target protein. In some embodiments, the scaffold peptide can comprise an affinity tag having a binding agent. In some embodiments, the extracellular vesicle can further comprise a targeting moiety. In some embodiments, the extracellular vesicle can further comprise a therapeutic substance.

[0009] When the extracellular vesicles contain the scaffold peptide of the present invention, the extracellular vesicles can display the scaffold peptide at a high density on the surface of the extracellular vesicles as compared with the case where the extracellular vesicles contain a scaffold peptide different from the scaffold peptide of the present invention. Non-limiting examples of scaffold peptides different from the scaffold peptide of the present invention may include conventional extracellular vesicle proteins, fragments or variants thereof, fragments of such variants, and variants thereof. When the extracellular vesicles contain the scaffold peptide of the present invention, the extracellular vesicles may contain a larger amount of target protein as compared with the case where the extracellular vesicles contain a scaffold peptide different from the scaffold peptide of the present invention.

[0010] Another further aspect of the present invention provides extracellular vesicles containing the scaffold peptide encoded by the DNA construct. In some embodiments, the scaffold peptide can be displayed at a desired position on the extracellular vesicles. For example, the scaffold peptide can be displayed on the inner surface, the outer surface, or both of the extracellular vesicles.

[0011] Another further aspect of the present invention provides a pharmaceutical composition containing the extracellular vesicles. In some embodiments, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. In some embodiments, the present invention provides the above-mentioned pharmaceutical composition for preventing, improving, or treating diseases, disorders, or conditions related to the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system.

[0012] Another further aspect of the present invention provides a method for preventing, ameliorating, or treating a disease, disorder, or condition associated with the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system, which includes administering a therapeutically effective amount of a pharmaceutical composition to an individual in need thereof. In some embodiments, the disease, disorder, or condition can be at least one selected from the group consisting of certain infectious or parasitic diseases, neoplasms, diseases of the blood or blood-forming organs, immune system diseases, endocrine, nutritional or metabolic diseases, mental, behavioral or neurodevelopmental disorders, sleep-wake disorders, nervous system diseases, visual system diseases, diseases of the ear or mastoid process, circulatory system diseases, respiratory diseases, digestive system diseases, skin diseases, musculoskeletal or connective tissue diseases, urogenital system diseases, sexual health-related diseases, gynecological diseases, developmental disorders, certain diseases occurring during the perinatal period, symptoms, signs, or clinical findings not classified in other categories, injuries, poisonings, or other specific consequences, morbidity, or mortality due to external causes.

[0013] Another further aspect of the present invention provides a method for producing therapeutically surface-modified extracellular vesicles. In particular, the DNA construct and / or the scaffold peptide are used or are to be used in the preparation of surface-modified extracellular vesicles. In some embodiments, the target protein (e.g., a therapeutic protein) and the scaffold peptide can be conjugated to produce a fusion protein, and surface-modified extracellular vesicles can be prepared by displaying the fusion protein on the surface of the extracellular vesicles.

[0014] The surface-modified extracellular vesicles prepared by the method of using the scaffold peptide according to the present invention are superior in some aspects to the surface-modified extracellular vesicles prepared by the method of using other scaffolds (e.g., PTGFRN). For example, the surface of the extracellular vesicles prepared by the method according to the present invention displays a higher density of the therapeutic protein, scaffold, or both of interest than the surface of the extracellular vesicles prepared by the method of using several different scaffolds (e.g., PTGFRN).

[0015] Furthermore, the surface-modified extracellular vesicles produced by the method according to the present invention exhibit higher therapeutic efficacy than the surface-modified extracellular vesicles prepared by methods using several different scaffolds (e.g., PTGFRN). Furthermore, the scaffold peptide of the present invention is shorter than several different scaffolds (e.g., PTGFRN). Furthermore, the display of a therapeutic protein on the surface of the extracellular vesicles prepared by the method according to the present invention is more effectively displayed than the display of a therapeutic protein on the surface of the extracellular vesicles prepared by methods using several other scaffolds (e.g., PTGFRN).

[0016] Another further aspect of the present invention provides the use of a composition comprising the extracellular vesicles of the above claims as an active ingredient for manufacturing a preparation for preventing, improving, or treating a disease, disorder, or condition related to the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system.

[0017] The above and other aspects and embodiments of the present invention are discussed in more detail below.

Brief Description of the Drawings

[0018] The drawings illustrate various embodiments of the present invention for illustrative purposes only. Those skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods described herein can be used without departing from the principles of the present invention described herein.

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[0019] [Embodiments of the Invention] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. The following terms used herein have the meanings described below.

[0020] As used herein, the above terms "comprising" (and all forms of "comprise" and "comprises"), "having" (and all forms of "have" and "has"), "including" (and any form of "includes" and "include"), or "containing" (and all forms of "contains" and "contain") shall be inclusive or open-ended and shall not exclude additional, unrecited elements or method steps.

[0021] As used herein, the term "a" or "an" can mean "one" when used in conjunction with the term "comprising" in the claims and / or the specification, but is also consistent with the meaning of "one or more", "at least one", and "one or more than one". The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, but the present disclosure supports a definition that refers only to alternatives and "and / or". The use of the term "at least one" is understood to include one or more quantities including, but not limited to, the integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or those included therein. The term "at least one" can be extended up to a maximum of 100 or more than 1000 according to the appended terms and should not be considered to limit the quantity to 100 / 1000, as higher limits can also yield satisfactory results. Further, the use of "at least one of X, Y, and Z" is understood to include X alone, Y alone, and Z alone, as well as all combinations of X, Y, and Z.

[0022] As used herein, the term "a combination thereof" or "combinations thereof" means all permutations and combinations of the items listed prior to such terms. For example, "A, B, C, or a combination thereof" or "A, B, C, or combinations thereof" means including at least one of the following: A, B, C, AB, AC, BC, or ABC, and in certain contexts where order matters, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB can be included. Following this example, combinations where one or more items or terms are repeated, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are explicitly included. One of ordinary skill in the art will understand that, unless otherwise clear from the context, there is generally no limit to the number of items or terms included in a combination. As used herein, the term "about" refers to a value that includes the inherent error variation with respect to the value, the method used for administering the composition, or the variation existing among the subjects under study.

[0023] As used herein, the term "substantially" means that the event or situation described below occurs completely, or that the event or situation described below occurs to a significant degree or extent. For example, the term "substantially" means that the event or situation described below has a probability of at least 90% or more, at least 95% or more, or at least 98% or more.

[0024] As used herein, the term "DNA construct" refers to a DNA sequence cloned according to standard cloning procedures used in genetic engineering to relocate a DNA segment from its natural position to another site for reproduction. The cloning procedures include excision and isolation of the desired DNA segment, insertion of the DNA fragment into a vector molecule, and incorporation of the recombinant vector into a cell in which multiple copies or clones of the DNA segment are replicated. In some embodiments, the DNA constructs disclosed herein may include non-natural DNA molecules, which may be provided as an isolate or inserted into other DNA molecules, such as within an expression vector or within the chromosome of a eukaryotic host cell.

[0025] As used herein, the term "vector" means a carrier DNA molecule or DNA construct for introducing a desired gene into a host cell and amplifying and expressing the desired gene. Preferably, the vector has an auxotrophic gene, has known restriction sites, and the ability to replicate in the host. Generally, a vector may include a promoter, an enhancer, a terminator, an SD sequence, translation start and stop codons, and a replication origin. Optionally, the vector may further include a selectable marker for selecting cells into which the vector has been introduced. Such selectable markers include: drug resistance genes such as ampicillin, tetracycline, kanamycin, chloramphenicol, neomycin, hygromycin, puromycin, and zeocin; markers that can be selected using the activity of an enzyme such as galactosidase as an indicator; and markers such as GFP that can be selected using fluorescence emission as an indicator, and selectable markers that enable selection using surface antigens such as the EGF receptor and B7-2 as indicators. Using such selectable markers, only cells into which the vector has been introduced, more specifically cells into which the vector of the present invention has been introduced, can be selected. The vector may include a signal sequence for polypeptide secretion. There is no limitation on the type of vector used in the present invention, and any vector can be used.In some embodiments, the vector is selected from the group consisting of pET-vector, pBAD-vector, pK184-vector, pMONO-vector, pSELECT-vector, pSELECT-Tag-vector, pVITRO-vector, pVIVO-vector, pORF-vector, pBLAST-vector, pUNO-vector, pDUO-vector, pZERO-vector, pDeNy-vector, pDRIVE-vector, pDRIVE-SEAP-vector, HaloTag™ Fusion-vector, pTARGET™-vector, Flexi™-vector, pDEST-vector, pHIL-vector, pPIC-vector, pMET-vector, pPink-vector, pLP-vector, pTOPO-vector, pBud-vector, pCEP-vector, pCMV-vector, pDisplay-vector, pEF-vector, pFL-vector, pFRT-vector, pFastBac-vector, pGAPZ-vector, pIZ / V5-vector, pLenti6-vector, pMIB-vector, pOG-vector, pOpti-vector, pREP4-vector, pRSET-vector, pSCREEN-vector, pSecTag-vector, pTEF1-vector, pTracer-vector, pTrc-vector, pUB6-vector, pVAX1-vector, pYC2-vector, pYES2-vector, pZeo-vector, pcDNA-vector, pFLAG-vector, pTAC-vector, pT7-vector, Gateway™-vector, pQE-vector, pLEXY-vector, pRNA-vector, pPK-vector, pUMVC-vector, pLIVE-vector, pCRUZ-vector, Duet-vector, and other vectors, or derivatives thereof.

[0026] As used herein, the term "extracellular vesicle" means a cell-derived vesicle that includes a membrane surrounding an internal space. Extracellular vesicles include all membrane-bound vesicles having a diameter smaller than the cell from which they are derived. Generally, extracellular vesicles range in diameter from 20 nm to 1000 nm and may contain various macromolecular cargo on the outer surface of the extracellular vesicle and / or within the internal space spanning the membrane. The cargo may include small molecules, nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. For example, extracellular vesicles include, but are not limited to, apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., serial extrusion or treatment with an alkaline solution), vesiculated organelles, and vesicles obtained within living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles may be derived from living or dead organisms, transplanted tissues or organs, and / or cultured cells.

[0027] As used herein, the term "exosome" means a cell-derived nanovesicle that includes a lipid bilayer membrane surrounding an internal space, which is produced from the cell by direct plasma membrane budding or fusion of late endosomes with the plasma membrane. The exosome contains lipids or fatty acids and polypeptides and may selectively contain a therapeutic active payload, a receiver (e.g., a target residue), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, a polysaccharide, or a glycan), or other molecules. The exosome can be derived from a producer cell and can be separated from the producer cell based on its size, density, biochemical parameters, or combinations thereof. Exosomes are a type of extracellular vesicle.

[0028] As used herein, the term "surface-engineered extracellular vesicle" refers to an extracellular vesicle having a membrane whose composition has been modified. For example, the surface-engineered extracellular vesicle can have a scaffold protein or peptide on the surface of the extracellular vesicle at a higher (or lower) density than a naturally-occurring extracellular vesicle. According to embodiments of the present invention, the surface-engineered extracellular vesicle can be produced from a genetically-engineered producer cell or its progeny. For example, the surface-engineered extracellular vesicle can be produced from a cell transformed or transfected with a DNA construct encoding an exogenous sequence or a scaffold protein or peptide. In some embodiments, the producer cell can be a cell transformed or transfected with both an exogenous sequence or DNA construct encoding a scaffold protein or peptide and an exogenous sequence or DNA construct encoding a therapeutic payload. In some embodiments, the exogenous sequence or DNA construct encoding the scaffold protein or peptide and the exogenous sequence or DNA construct encoding the therapeutic payload can be incorporated into the producer cell by different vectors. In some embodiments, the exogenous sequence or DNA construct encoding the scaffold protein or peptide and the exogenous sequence or DNA construct encoding the therapeutic payload can be incorporated into the producer cell by the same vector. In some embodiments, the scaffold protein or peptide and the therapeutic payload can be a fusion protein. In some embodiments, the surface-engineered extracellular vesicle can further comprise a targeting moiety that can be used to target the extracellular vesicle to a desired organ, tissue, or cell. Non-limiting examples of the targeting moiety include an antibody, an antigen-binding fragment of an antibody, an antigen-binding variant of an antibody, an antigen-binding fragment of an antigen-binding variant of an antibody, and an antigen-binding variant of an antigen-binding fragment of an antibody.In some embodiments, the surface-modified extracellular vesicles according to the embodiments of the present invention have properties superior to those of surface-modified extracellular vesicles known in the art. For example, surface-modified extracellular vesicles produced by cells incorporated with the exogenous sequence or DNA construct encoding the scaffold protein or peptide of the present invention have a higher density of scaffold protein or peptide on the surface of the extracellular vesicles than surface-modified extracellular vesicles known in the art (for example, extracellular vesicles generated using existing extracellular vesicle proteins such as PTGFRN).

[0029] As used herein, the terms “producer cell” or “host cell” refer to cells used to generate extracellular endoplasmic reticulum or surface-modified extracellular endoplasmic reticulum. Producer cells include, but are not limited to, cells known to be effective for the production of extracellular endoplasmic reticulum, such as HEK293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and mesenchymal stem cells (MSCs). The producer cells can be transformed or transfected by one or more vectors containing an exogenous sequence(s) or DNA construct(s). In some embodiments of the invention, the producer cells can be transformed or transfected by a single vector containing an exogenous sequence or DNA construct encoding the scaffold protein or peptide of the invention. In some embodiments, the producer cells can be transformed or transfected by a single vector containing an exogenous sequence or DNA construct encoding the scaffold protein or peptide of the invention and an exogenous sequence or DNA construct encoding a therapeutically active payload. In some embodiments, the producer cells can be transformed or transfected by a vector containing an exogenous sequence or DNA construct encoding the scaffold protein or peptide of the invention and another vector containing an exogenous sequence or DNA construct encoding a therapeutically active payload. In some embodiments, the producer cells can be transformed or transfected with at least one or more additional exogenous sequences or DNA constructs encoding another protein or peptide (e.g., a target residue). The additional exogenous sequence can be introduced into a vector containing an exogenous sequence or DNA construct encoding the scaffold protein or peptide of the invention, an exogenous sequence or DNA construct encoding a therapeutically active payload, or both.In some embodiments, to regulate the endogenous gene expression of the producer cells, an exogenous sequence or DNA construct encoding a therapeutic active payload, an additional exogenous sequence or DNA construct encoding another protein or peptide, or both can be introduced into the producer cells. In some embodiments, to produce surface-modified extracellular vesicles comprising a therapeutic active payload, another protein or peptide, or both on the surface of the extracellular vesicles, an exogenous sequence or DNA construct encoding the therapeutic active payload, an additional exogenous sequence or DNA construct encoding another protein or peptide, or both can be introduced into the producer cells.

[0030] As used herein, the term "scaffold", "scaffold protein", or "scaffold peptide" means a protein or peptide that can target the surface of extracellular vesicles. In some embodiments, the scaffold protein or peptide can be located, disposed, or included within / on the membrane of extracellular vesicles. Scaffold proteins or peptides known in the art include tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosome-associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI anchor proteins, lactadherin, syndecan, synaptotagmin, apoptosis-linked gene 2-interacting protein X (ALIX), syntenin, PTGFRN, fragments or variants thereof, variants of the fragments, and fragments of the variants. The scaffold protein or peptide according to embodiments of the present invention includes an amino acid sequence containing G-a-S-b-X1-c-X2, where X1 represents G, A, S, or T; X2 represents G or S; a represents 3 to 4 amino acids; b represents 2 to 3 amino acids; c represents 6 to 7 amino acids, where G represents glycine, S represents serine, A represents alanine, and T represents threonine. In some embodiments of the present invention, the scaffold can be a non-mutated protein or peptide (i.e., a protein or peptide that is naturally targeted to the exosome membrane), a fragment of a non-mutated protein or peptide, a variant of a non-mutated protein or peptide, a fragment of a variant of a non-mutated protein or peptide, or a variant of a fragment of a non-mutated protein or peptide.In some embodiments, the scaffold can be a mutant protein or peptide (i.e., a protein or peptide modified to be targeted to the exosome membrane), a fragment of a mutant protein or peptide, a variant of a mutant protein or peptide, a fragment of a variant of a mutant protein or peptide, or a variant of a fragment of a mutant protein or peptide. In some embodiments, the scaffold can be fused to another residue, such as, for example, a flag tag, a therapeutic peptide, a targeting moiety, etc. In some embodiments, the scaffold can include a transmembrane protein, a peripheral protein, or a soluble protein. In some embodiments, the scaffold can be attached to the membrane of the extracellular vesicle by a linker.

[0031] The scaffolds, fragments of scaffolds, variants of scaffolds, fragments of variants of scaffolds, and variants of fragments of scaffolds according to embodiments of the present invention have the ability to specifically target the surface of extracellular vesicles. In some embodiments, the scaffolds, fragments of scaffolds, variants of scaffolds, fragments of variants of scaffolds, and variants of fragments of scaffolds according to embodiments of the present invention can be located, positioned, or incorporated within / onto the membrane of extracellular vesicles. As used herein, the term "a fragment" of a protein, peptide, or nucleic acid means a segment of a protein, peptide, or nucleic acid. As used herein, the term "variant" of a protein, peptide, or nucleic acid means a protein, peptide, or nucleic acid having at least one or more amino acids or nucleotides different from the protein, peptide, or nucleic acid. Variants of proteins, peptides, or nucleic acids include, but are not limited to, substitutions, deletions, frameshifts, or rearrangements of proteins, peptides, or nucleic acids. This term can be used interchangeably with the above term "mutant".

[0032] Scaffold fragments according to some embodiments of the present invention can possess at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the ability of the scaffold to specifically target the extracellular endoplasmic reticulum. Variants of the scaffold according to some embodiments of the present invention can possess at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the ability of the scaffold to be specifically targeted to the extracellular endoplasmic reticulum. Fragments of the scaffold variant can possess at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the ability of the scaffold variant to be specifically targeted to the extracellular endoplasmic reticulum. Variants of the scaffold fragment can possess at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the ability of the scaffold fragment to be specifically targeted to the extracellular endoplasmic reticulum.

[0033] Variants of the scaffold according to some embodiments of the present invention can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the scaffold. Variants of the scaffold fragment according to some embodiments of the present invention can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the scaffold fragment.

[0034] As used herein, the above terms "target protein" and "target peptide" can be used interchangeably and refer to a protein or peptide of interest that is delivered, expressed, or introduced onto the surface, on the surface, or within the membrane of an extracellular vesicle. In some embodiments, the target protein or target peptide can be delivered, expressed, or introduced onto the surface, on the surface, or within the membrane of an extracellular vesicle by fusion to a scaffold protein or scaffold peptide. In some embodiments, the target protein or target peptide can be fused to the N-terminus or C-terminus of the scaffold protein or scaffold peptide. In some embodiments, the target protein or target peptide can be fused to the scaffold protein or scaffold peptide via a linker peptide. In some embodiments, the target protein or target peptide can be, or a part of, a therapeutic protein, antigen, cytokine, ligand, receptor, immunoglobulin, labeled polypeptide (e.g., a labeled protein such as green fluorescent protein or an enzyme), enzyme, ion channel, etc. In some embodiments, the target protein or target peptide can be a therapeutic molecule or a biologically active molecule.

[0035] As used herein, the above terms "biologically active molecule" and "therapeutic molecule" can be used interchangeably and refer to an agent having activity in a biological system (e.g., a cell or a human individual), Proteins, polypeptides or peptides, including but not limited to structural proteins, enzymes, cytokines (such as interferons and / or interleukins), antibiotics, polyclonal or monoclonal antibodies, or active moieties such as Fv fragments (where antibodies or portions thereof can be natural, synthetic or humanized), peptide hormones, receptors, signaling molecules or other proteins; nucleic acids, including but not limited to oligonucleotides or modified oligonucleotides, antisense oligonucleotides, or modified antisense oligonucleotides, cDNA, genomic DNA, artificial or natural chromosomes (such as yeast artificial chromosomes) or portions thereof, mRNA, tRNA, rRNA, or ribozymes, or peptide nucleic acids (PNAs); viruses or virus-like particles; nucleotides or ribonucleotides or their synthetic analogs (modified or unmodified); amino acids or their analogs which may or may not be modified; non-peptide (such as steroid) hormones; proteoglycans; lipids; or carbohydrates. In certain embodiments, the biologically active molecule includes a therapeutic molecule (such as an antigen), a target residue (such as an antibody or its antigen-binding fragment), an adjuvant, an immunomodulatory agent, or any combination thereof. In some embodiments, the biologically active molecule includes macromolecules (such as proteins, antibodies, enzymes, peptides, DNA, RNA, or any combination thereof). In some embodiments, the biologically active molecule includes small molecules (such as antisense oligomers (ASO), phosphorodiamidate morpholino oligomers (PMO), peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO), siRNA, STING, pharmaceutical drugs, or any combination thereof). In some embodiments, the biologically active molecule is exogenous to the extracellular vesicle, i.e., a molecule not naturally found in the extracellular vesicle. In some embodiments, the biologically active molecule or therapeutic molecule can be a therapeutic protein or a therapeutic peptide.

[0036] As used herein, the term "linker" refers to any molecular structure that can bind a peptide or protein to another molecule (e.g., another peptide or protein, a small molecule, etc.). Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers (see, e.g., Chen et al., Advanced Drug Delivery Reviews, 2013, Vol 65:10, pp. 1357-1369). The linker can bind to the carboxyl and amino terminal amino acids via a terminal carboxyl or amino group or via a reactive side chain group. Further, in some embodiments, the linker can be classified as ductile or rigid and can be cleavable (e.g., include one or more protease-cleavable sites that can be located within the linker sequence or on the linker side at both ends of the linker sequence).

[0037] As used herein, the term "payload" refers to an agent that can act on a target (e.g., a target cancer cell) in contact with the extracellular vesicle. In some embodiments, the payload can be introduced into the extracellular vesicle. In some embodiments, the payload can be introduced into the producer cell. Non-limiting examples of the payload include nucleotides, nucleic acids (e.g., DNA, mRNA, miRNA, dsDNA, lncRNA, and siRNA), amino acids, polypeptides, lipids, carbohydrates, and small molecules. In a preferred embodiment, the payload can be a therapeutic or biologically active agent.

[0038] As used herein, the terms “isolate,” “isolated,” “isolating,” or “purify,” “purified,” “purifying,” “extracted,” and “extracting” are used interchangeably and mean the state of one or more purification processes, such as the selection of a desired extracellular vesicle preparation or a preparation of desired extracellular vesicles that is enriched (e.g., of a plurality of known or unknown amounts and / or concentrations). In some embodiments, as used herein, isolating or purifying is a process of removing or partially removing extracellular vesicles from a sample containing producer cells (e.g., fractionation). In some embodiments, the isolated extracellular vesicle composition has no detectable undesirable activity or has a level or amount of undesirable activity that is below an acceptable level or amount.

[0039] In other embodiments, the isolated extracellular vesicle composition has an amount and / or concentration of desired extracellular vesicles that is at or above an acceptable amount and / or concentration. In another embodiment, the isolated extracellular vesicle composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. Such enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or 99.9999% or more compared to the starting material. In some embodiments, the isolated extracellular vesicle preparation is substantially free of residual biological products. In some embodiments, the isolated extracellular vesicle preparation is free of 100%, 99%, 98%, 97%, 96%, or 95% contaminating biological material. Residual biological products can include non-biological materials (including chemicals) or undesirable nucleic acids, proteins, lipids, or metabolites. The absence of substantial residual biological products can mean that the extracellular vesicle composition contains no detectable producer cells and only extracellular vesicles are detectable.

[0040] As used herein, the term "pharmaceutically acceptable" means compounds and compositions suitable for administration to humans and / or animals without undue adverse side effects such as toxicity, irritation and / or allergic reactions corresponding to a reasonable benefit / risk ratio.

[0041] As used herein, the term "biologically active" means the ability to modify a physiological system of an organism, regardless of the manner in which the active agent has a physiological effect.

[0042] As used herein, the terms "subject" and "patient" are used interchangeably herein and are understood to include mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc.

[0043] As used herein, the terms "treat", "treating" or "treatment" mean methods such as alleviation, reduction or improvement of symptoms of a disease or condition, prevention of further symptoms, improvement or prevention of the underlying metabolic cause of the symptoms, suppression of a disease or condition, arrest of the onset of a disease or condition, reduction of a disease or condition, induction of regression of a disease or condition, alleviation of symptoms due to a disease or condition or interruption of prophylactic / therapeutic symptoms of a disease or condition.

[0044] As used herein, the terms "administration" or "administering" of a composition means providing the composition to an individual in need of treatment. According to embodiments of the present invention, the therapeutic composition can be administered alone or in combination with one or more additional therapeutic agents. Methods of administration of such compositions can include, but are not limited to, intravenous administration, inhalation administration, oral administration, rectal administration, parenteral administration, intravitreal administration, subcutaneous administration, intramuscular administration, nasal administration, dermal administration, topical administration, ophthalmic administration, buccal administration, tracheal administration, bronchial administration, sublingual administration, or optic nerve administration.

[0045] As used herein, the terms "therapeutic composition" and "pharmaceutical composition" refer to an active agent-containing composition that can be administered to a subject by any method known to those of skill in the art or by a method contemplated herein, where administration of the composition results in a therapeutic effect as described elsewhere herein. Further, the compositions of the present disclosure can be designed to provide delayed, controlled, extended, and / or sustained release using formulation techniques well known to those of skill in the art. The compositions of the present disclosure can be administered via known pharmaceutical formulations including tablets, capsules, liquids, inhalants, nasal spray solutions, suppositories, solutions, gels, emulsions, ointments, eye drops, ear drops, and the like.

[0046] As used herein, the term "effective amount" or "therapeutically effective amount" means administering an amount of the active ingredient described herein sufficient to reduce to some extent one or more of the symptoms of the disease or condition being treated. As a result, a decrease and / or reduction in the signs, symptoms or causes of the disease, or other desired changes in the biological system, may occur. For example, an "effective amount" for therapeutic use means the amount of a composition comprising exosomes surface-modified as disclosed herein necessary to provide a clinically significant reduction in the symptoms of the disease. The effective amount for a patient can vary depending on the type of patient, the size and health of the patient, the nature and severity of the disease being treated, the method of administration, the duration of treatment, the nature of co-treatments (if any), the particular formulation used, and the like. Accordingly, it is not possible to specify the exact effective amount in advance. However, the effective amount for a given situation can be determined by those of ordinary skill in the art using routine experimentation based on the information provided herein.

[0047] In one aspect, the present invention provides a DNA construct comprising a DNA sequence encoding a scaffold peptide, wherein the amino acid sequence of the scaffold peptide comprises a sequence represented by G-a-S-b-X1-c-X2, where X1 represents G, A, S, or T, X2 represents G or S; a represents 3 to 4 amino acids; b represents 2 to 3 amino acids; c represents 6 to 7 amino acids; G represents glycine; S represents serine; A represents alanine; and T represents threonine.

[0048] In some embodiments, in the sequence of G-a-S-b-X1-c-X2, X1 and X2 can be G and G, G and S, A and G, A and S, S and G, S and S, T and G, or T and S, respectively.

[0049] In some embodiments, G-a-S-b-X1-c-X2 may not be GVGLSTVIGLLSCLIG.

[0050] In some embodiments, the sequence G-a-S-b-X1-c-X2 can have 16 amino acids. For example, a can represent 3 amino acids, b can represent 3 amino acids, and c can represent 6 amino acids. Also, for example, a can represent 4 amino acids, b can represent 2 amino acids, and c can represent 6 amino acids. Further, for example, a can represent 3 amino acids, b can represent 2 amino acids, and c can represent 7 amino acids.

[0051] In some embodiments, the amino acids a, b, and c can include V, G, L, I, A, T, S, C, F, W, Y, and P, where V represents valine, G represents glycine, L represents leucine, I represents isoleucine, A represents alanine, T represents threonine, S represents serine, C represents cysteine, F represents phenylalanine, W represents tryptophan, Y represents tyrosine, and P represents proline.

[0052] In some embodiments, a can represent 3 to 4 amino acids selected from the group consisting of V, G, L, I, and A, where V can represent valine, G can represent glycine, L can represent leucine, I can represent isoleucine, and A can represent alanine. Non-limiting examples of 3 to 4 amino acids are as follows: VGL, VGI, VGT, VGA, VLG, VLI, VLT, VLA, VIG, VIL, VIT, VIA, VTG, VTL, VTI, VTA, VAG, VAL, VAI, VAT, GVL, GVI, GVT, GVA, GLV, GLI, GLT, GLA, GIV, GIL, GIT, GIA, GTV, GTL, GTI, GTA, GAV, GAL, GAI, GAT, LVG, LVI, LVT, LVA, LGV, LGI, LGT, LGA, LIV, LIG, LIT, LIA, LTV, LTG, LTI, LTA, LAV, LAG, LAI, LAT, IVG, IVL, IVT, IVA, IGV, IGL, IGT, IGA, ILV, ILG, ILT, ILA, ITV, ITG, ITL, ITA, IAV, IAG, IAL, IAT, TVG, TVL, TVI, TVA, TGV, TGL, TGI, TGA, ILV, TLG, TLI, TLA, TIV, TIG, TIL, TIA, TAV, TAG, TAL, TAI, AVG, AVL, AVT, AVI, AGV, AGL, AGI, AGT, ALV, ALG, ALI, ALT, AIV, AIG, AIL, AIT, ATV, ATG, ATL, ATI, VGLI, VGLT, VGLA, VGIL, VGIT, VGIA, VGTL, VGTI, VGTA, VGAL, VGAI, VGAT, IGVL, IGVT, IGVA, IGLV, IGLT, IGLA, IGTV, IGTL, IGTA, IGAV, IGAL, or IGAT.

[0053] In some embodiments, b can represent 2 to 3 amino acids selected from the group consisting of V, I, A, and T, where V represents valine, I represents isoleucine, A represents alanine, and T represents threonine. Non-limiting examples of 2 to 3 amino acids are as follows: VI, VA, VT, IV, IA, IT, AV, AI, AT, TV, TI, TA, VV, II, AA, TT, VIA, VIT, VAI, VAT, IVA, IVT, IAV, ITV, ITA, AVI, AVT, AIV, AIT, ATV, ATI, TVI, TVA, ITV, TIA, TAV, and TAI.

[0054] In some embodiments, c can represent 6 to 7 amino acids selected from the group consisting of L, S, C, and I, where L represents leucine, S represents serine, C represents cysteine, and I represents isoleucine. Non-limiting examples of 6 to 7 amino acids are as follows: LLSCLI, LLSCIL, LLSLCI, LLSLIC, LLCSLI, LLCSIL, LLCISL, LLCILS, LLSICL, LLSILC, LSLCLI, LSLCIL, LSLLCI, LSLLIC, LSLCLI, LSLCIL, ILLSCLI, ILLSCIL, ILLSLCI, ILLSLIC, ILLCSLI, ILLCSIL, ILLCISL, ILLCILS, ILLSICL, ILLSILC, ILSLCLI, ILSLCIL, ILSLLCI, ILSLLIC, ILSLCLI, ILSLCIL, LILSCLI, LILSCIL, LILSLCI, LILSLIC, LILCSLI, LILCSIL, LILCISL, LILCILS, LILSICL, LILSILC, LISLCLI, LISLCIL, LISLLCI, LISLLIC, LISLCLI, and LISLCII.

[0055] In some embodiments, the sequence G-a-S-b-X1-c-X2 can be one of the amino acid sequences represented by ESM SEQ ID NOs: 1 to 14. (ESM SEQ ID NO: 1) GVGLSTVIGLLSCLIG (ESM SEQ ID NO: 2) GIGLSTVIGLLSCLIG (ESM SEQ ID NO: 3) GVGLSAVIGLLSCLIG (ESM Sequence No. 4) GIGLSAVIGLLSCLIG (ESM Sequence No. 5) GILLSAVIGLLSCLIG (ESM Sequence No. 6) GIGLSLVIGLLSCLIG (ESM Sequence No. 7) GIGLSAVIGLLLCLIG (ESM Sequence No. 8) GIGLSAVIGLLSLLIG (ESM Sequence No. 9) GIGLSAVIALLSCLIG (ESM Sequence No. 10) GIGLSAVISLLSCLIG (ESM Sequence No. 11) GIGLSAVITLLSCLIG (ESM Sequence No. 12) GIGLSAVIGLLSCLIS (ESM Sequence No. 13) GIGLASVIGLLSCLIG (ESM Sequence No. 14) GIGLSAVGILLSCLIG

[0056] Non-limiting examples of the G-a-S-b-X1-c-X2 sequence are as follows. [Table 1] [Table 2]

[0057] In some embodiments, the scaffold peptide may further comprise KYPLLI at the N-terminus of the sequence G-a-S-b-X1-c-X2. Here, K represents lysine, Y represents tyrosine, P represents proline, L represents leucine, and I represents isoleucine.

[0058] In some embodiments, the scaffold peptide further comprises FKYPLLI, AFKYPLLI, NAFKYPLLI, LNAFKYPLLI, VLNAFKYPLLI or DVLNAFKYPLLI at the N-terminus of the sequence G-a-S-b-X1-c-X2, where D represents aspartic acid, V represents valine, L represents leucine, N represents asparagine, A represents alanine, F represents phenylalanine, K represents lysine, Y represents tyrosine, P represents proline, L represents leucine, and I represents isoleucine.

[0059] In some embodiments, the scaffold peptide further comprises YCSS at the C-terminus of the sequence G-a-S-b-X1-c-X2, where Y represents tyrosine, C represents cysteine, and S represents serine.

[0060] In some embodiments, the scaffold peptide can further comprise YCSSH, YCSSHW, YCSSHWC, YCSSHWCC, YCSSHWCCK, YCSSHWCCKK, YCSSHWCCKKE, YCSSHWCCKKEV, YCSSHWCCKKEVQ, YCSSHWCCKKEVQE, YCSSHWCCKKEVQET, YCSSHWCCKKEVQETR, YCSSHWCCKKEVQETRR, YCSSHWCCKKEVQETRRE, YCSSHWCCKKEVQETRRER, YCSSHWCCKKEVQETRRERR, YCSSHWCCKKEVQETRRERRR, YCSSHWCCKKEVQETRRERRRL, YCSSHWCCKKEVQETRRERRRLM, YCSSHWCCKKEVQETRRERRRLMS, YCSSHWCCKKEVQETRRERRRLMSM, YCSSHWCCKKEVQETRRERRRLMSME, YCSSHWCCKKEVQETRRERRRLMSMEM or YCSSHWCCKKEVQETRRERRRLMSMEMD at the C-terminus of the sequence G-a-S-b-X1-c-X2, where Y represents tyrosine, C represents cysteine, S represents serine, H represents histidine, W represents tryptophan, K represents lysine, E represents glutamic acid, V represents valine, Q represents glutamine, T represents threonine, R represents arginine, L represents leucine, M represents methionine, and D represents aspartic acid.

[0061] In some embodiments, the scaffold peptide further comprises YCSSHWC at the C-terminus of the sequence G-a-S-b-X1-c-X2, where Y represents tyrosine, C represents cysteine, S represents serine, H represents histidine, and W represents tryptophan.

[0062] In some embodiments, the scaffold peptide can be one of the amino acid sequences specified in SEQ ID NOs: 101 to 142.

[0063] In some embodiments, the DNA construct can further comprise a DNA sequence encoding the amino acid sequence of the target protein. In some embodiments, the target protein can be a therapeutic protein. In some embodiments, the target protein can be fused to the scaffold peptide.

[0064] In another aspect, the present invention provides a vector comprising the above DNA structure. The vector can be a plasmid, phage, virus, artificial chromosome, etc. Typical examples include commercially available plasmids (e.g., plasmids derived from commercially available plasmids, especially pUC, pcDNA, pBR, etc.). Other examples include vectors derived from viruses such as replication-deficient retroviruses, adenoviruses, AAVs, baculoviruses, or vaccinia viruses. The selection of the vector can be adjusted by those skilled in the art according to the recombinant host cell in which the corresponding vector is to be used. For example, without intending to limit the scope of the present invention, a vector capable of infecting or transfecting mammalian cells can be selected.

[0065] In another aspect, the present invention provides a host cell comprising the above vector. In some embodiments, the host cell is capable of producing extracellular vesicles comprising the above scaffold peptide on its surface. The cells can be cultured and maintained in any suitable medium such as RPMI, DMEM. The culture can be carried out in any suitable apparatus such as a dish, tube, flask. The vector can be introduced into the host cell by conventional methods such as naked DNA technology, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated transfection, physical or chemical agents or treatments, electroporation. In this regard, it should be noted that since transient infection alone is sufficient to express the gene (i.e., the DNA construct of the present invention), there is no need to generate stable cell lines or optimize infection conditions.

[0066] In another aspect, the present invention provides an extracellular vesicle comprising a scaffold peptide encoded by the DNA structure of the present invention described above. In some embodiments, the extracellular vesicle may be surface-modified. In some embodiments, extracellular vesicles that are surface-modified and / or lumen-modified can be generated by chemical and / or physical methods such as PEG-induced fusion and / or sonication fusion. In other embodiments, surface-modified extracellular vesicles are generated by genetic engineering. Extracellular vesicles produced from genetically modified producer cells or progeny of genetically modified cells may contain a modified membrane composition. In some embodiments, the genetically modified producer cells or progeny of genetically modified cells may contain one or more exogenous proteins (peptides) not naturally found in the cell. In certain embodiments, one or more exogenous proteins may be scaffold proteins or peptides such as the scaffold peptides disclosed herein. In some embodiments, the surface-modified extracellular vesicles may have a higher density of the scaffold peptides disclosed herein compared to the density of other scaffold proteins or peptides such as tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosome-associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI-anchored proteins, lactadherin, syndecan, synaptotagmin, apoptosis-linked gene 2 interacting protein X (ALIX), syntenin, PTGFRN, fragments or variants thereof, variants of the fragments, and fragments of the variants. For example, surface-modified extracellular vesicles can be prepared from host cells or producer cells transformed with an exogenous sequence encoding the DNA structure disclosed herein. Extracellular vesicles containing peptides or proteins expressed from the exogenous sequence (e.g., the DNA construct disclosed herein) may contain a modified membrane protein composition.

[0067] In some embodiments, the scaffold peptides described herein that can immobilize cargos such as the exogenous biologically active molecules (e.g., those disclosed herein) or target proteins (or peptides) can be used to construct surface-modified extracellular vesicles.

[0068] The fusion protein can also be configured on the surface of the extracellular vesicle; for example, the scaffold peptides described herein can be fused to an affinity tag (e.g., His tag, GST tag, glutathione-S-transferase, S-peptide, HA, Myc, FLAG™ (Sigma-Aldrich), MBP, SUMO, and protein A) and used to purify or remove surface-modified extracellular vesicles using a binder specific to the affinity tag.

[0069] Fusion proteins having therapeutic activity can also be used to generate surface-modified extracellular vesicles. Thus, in some embodiments, the extracellular vesicles described herein can be engineered or modified to express a fusion protein and used to deliver one or more (e.g., two, three, four, five, or more) therapeutic molecules to a target. For example, the fusion protein can comprise a scaffold peptide and a therapeutic substance (e.g., a peptide or protein) described herein. In some embodiments, the therapeutic substance can be directly fused to the scaffold peptide described herein. In some embodiments, the therapeutic substance can be immobilized to the scaffold peptide described herein via a linker.

[0070] In some embodiments, the linker can be a peptide linker. In some embodiments, the peptide linker can comprise at least about 2 or more, at least about 3 or more, at least about 4 or more, at least about 5 or more, at least about 10 or more, at least about 15 or more, at least 20 or more, at least about 25 or more, at least about 30 or more, at least about 35 or more, at least about 40 or more, at least about 45 or more, at least about 50 or more, at least about 55 or more, at least about 60 or more, at least about 65 or more, at least about 70 or more, at least about 75 or more, at least about 80 or more, at least about 85 or more, at least about 90 or more, at least about 95 or more, or at least about 100 or more amino acids. In some embodiments, the peptide linker can be synthetic, i.e., synthetic that does not occur naturally. In some embodiments, the peptide linker can comprise a peptide (or polypeptide) (e.g., a peptide that occurs naturally or non-naturally) comprising an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids that does not naturally link or genetically fuse. For example, in some embodiments, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., including mutations such as additions, substitutions, or deletions). The linker can be cleavable (a "cleavable linker") and can facilitate the release of the exogenous biological active molecule. In some embodiments, the linker can comprise a non-cleavable linker.

[0071] In some embodiments, the biological active molecule (e.g., therapeutic peptide or protein) can be selected from the group consisting of natural peptides, recombinant peptides, synthetic peptides, and linkers to therapeutic substances. The therapeutic substance can be a nucleotide, amino acid, lipid, carbohydrate, or small molecule. The therapeutic peptide can be an antibody, enzyme, ligand, receptor, antimicrobial peptide, or a fragment or variant thereof. In some embodiments, the therapeutic peptide can be a nucleic acid-binding protein. The nucleic acid-binding protein can be Dicer, Argonaute protein, TRBP, or MS2 bacteriophage coat protein. In some embodiments, the nucleic acid-binding protein can further comprise one or more RNA or DNA molecules. The one or more RNAs can be miRNA, siRNA, antisense oligonucleotide, phosphorodiamidate morpholino oligomer (PMO), peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), guide RNA, lincRNA, mRNA, antisense RNA, dsRNA, or any combination thereof. In some embodiments, the biological active molecule can be part of a protein-protein interaction system. In some embodiments, the biological active molecule immobilized on the scaffold peptide described herein and expressed on the surface of extracellular vesicles can comprise an antigen. In certain embodiments, the antigen can comprise a tumor antigen. Non-limiting examples of tumor antigens are as follows: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha-folate receptor, CE7R, IL-3, cancer testis antigen (CTA), MART-1 gp100, TNF-related apoptosis-inducing ligand, brachyury (preferentially expressed antigen in melanoma (PRAME)), and any combination thereof.In some embodiments, the antigen can be derived from bacteria, viruses, fungi, protozoa, or any combination thereof. In some embodiments, the antigen can be derived from oncogenic viruses. In other embodiments, the antigen can be derived from the group including: Human gamma herpesvirus 4 (Epstein - Barr virus), Influenza A virus, Influenza B virus, Cytomegalovirus, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, HIV - 1, HIV - 2, Coronaviruses (e.g., MERS - CoV and SARS CoV), Filoviruses (e.g., Marburg and Ebola), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium species (e.g., vivax and falciparum), Chikungunya virus, Human Papilloma virus (HPV), Hepatitis B, Hepatitis C, Human herpesvirus 8, Herpes simplex virus 2 (HSV2), Klebsiella, Pseudomonas aeruginosa, Enterococcus, Proteus, Enterobacter, Actinobacillus, coagulase - negative staphylococci (CoNS), Mycoplasma, and all combinations thereof.

[0072] Non-limiting examples of other suitable biologically active molecules include anti-cancer agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modulators, pharmacologically active drugs such as neuroactive agents, and gene active molecules. Examples of suitable payloads for therapeutic agents include those described in the following sections of "The Pharmacological Basis of Therapeutics", Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition: drugs acting on synapses and neuroeffector junctions; drugs acting on the central nervous system; autacoids: drug therapy of inflammation; water, salts and ions; drugs affecting renal function and electrolyte metabolism; cardiovascular drugs; drugs affecting gastrointestinal function; drugs affecting uterine motility; chemotherapy of parasitic infections; chemotherapy of microbial diseases; chemotherapy of neoplastic diseases; drugs used for immunosuppression; drugs acting on hematopoietic organs; hormones and hormone antagonists; vitamins, dermatology; and toxicology, all incorporated herein by reference for all purposes. Suitable payloads also include toxins, biological and chemical agents (see, for example, Somania, SM (ed), Chemical Warfare Agents, Academic Press, New York (1992)).

[0073] In some examples, fusion proteins having target residues can be used. For example, the fusion protein can include a scaffold peptide and a target residue as described herein. The target residue can be used to target extracellular vesicles to a specific organ, tissue, or cell for treatment using extracellular vesicles. In certain embodiments, the target residue can bind to a marker (or target molecule) expressed in a cell or cell population. In certain embodiments, the marker can be expressed in multiple cell types, such as all antigen-presenting cells (e.g., dendritic cells, macrophages, and B lymphocytes). In some embodiments, the marker can be expressed only in a specific cell population (e.g., dendritic cells). Non-limiting embodiments of markers expressed in a specific cell population (e.g., dendritic cells) include C-type lectin domain family 9 member A (CLEC9A) protein, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immunoreceptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialoglycoprotein receptor 1 (DC-ASGPR), DC immunoreceptor 2 (DCIR2), dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), dectin-2, Bst-2 (CD317), and any combination thereof.Antibodies and antigen-binding fragments thereof include whole antibodies, polyclonal, monoclonal antibodies and recombinant antibodies, fragments thereof, including single-chain antibodies, humanized antibodies, mouse antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., scFv, (scFv)2, Fab, Fab′ and F(ab′)2, F(ab′)2, F(ab1)2, Fv, dAb and Fd fragments), diabodies, and antibody-related polypeptides. Antibodies and antigen-binding fragments thereof can further include bispecific antibodies and multispecific antibodies as long as they exhibit the desired biological activity or function.

[0074] In some embodiments, the extracellular vesicles can encapsulate a target protein (e.g., a therapeutic protein or a therapeutic substance (e.g., nucleotides, amino acids, lipids, carbohydrates, small molecules, and any combination thereof)).

[0075] In some embodiments, the extracellular vesicles described herein exhibit superior properties compared to extracellular vesicles known to those of skill in the art. For example, extracellular vesicles produced using the scaffold peptides described herein contain proteins with a higher concentration of modified surfaces than extracellular vesicles of the prior art, such as extracellular vesicles produced using conventional exosomal proteins. In some embodiments, the expression level of the modified protein is at least about 5% or more, at least about 10% or more, at least about 20% or more, at least about 30% or more, at least about 40% or more, at least about 50% or more, at least about 60% or more, at least about 70% or more, at least about 80% or more, at least about 90% or more, at least about 100% or more, at least about 150% or more, at least about 200% or more, or at least about 300% or more increased (i.e., concentrated) compared to the expression of the corresponding protein using conventional exosomal proteins. Further, in some embodiments, the biological activity of the extracellular vesicles of the present disclosure is greater than the biological activity of extracellular vesicles known to those of skill in the art. For example, surface-modified extracellular vesicles containing a therapeutic or biologically relevant exogenous sequence fused to the scaffold peptide described herein may have more desired modified properties than those fused to scaffolds known to those of skill in the art. Examples of scaffold proteins known to those of skill in the art include tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosome-associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI-anchored protein, lactadherin, syndecan, synaptotagmin, apoptosis-linked gene 2-interacting protein X (ALIX), syntenin, PTGFRN, fragments or variants thereof, variants of said fragments, and fragments of said variants, and any peptides having an affinity for such a protein or fragment thereof, but are not limited thereto.

[0076] In some embodiments, the surface-modified extracellular vesicles comprise a fusion protein comprising an exogenous sequence (e.g., encoding an exogenous bioactive molecule such as an antigen, an adjuvant, a target residue, and / or an immunomodulatory agent), and the scaffold peptides described herein have a higher density of fusion protein than similarly engineered extracellular vesicles that comprise an exogenous sequence conjugated to a conventional extracellular vesicle protein (e.g., CD9, CD63, CD81, PDGFR, GPI-anchored protein, lactadherin LAMP2, LAMP2B, syndecan, synaptotagmin, apoptosis-linked gene 2 interacting protein X (ALIX), syntenin, PTGFRN, fragments or variants thereof, variants of the fragments, and fragments or peptides binding thereto) known to those of skill in the art. In some embodiments, the fusion protein comprising the scaffold peptides described herein is present on the extracellular vesicle surface at a density that is about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1000-fold, or higher than the fusion protein on the surface of other extracellular vesicles similarly modified using a conventional extracellular vesicle protein.

[0077] In some embodiments, the extracellular vesicles described herein can be isolated from a host cell or producer cell comprising the vector described herein. When extracellular vesicles are produced from in vitro cell culture, various producer cells can be used in the present disclosure, such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSC), HT-1080 cells, MB-231 cells, large cells, PER.C6 cells, and CAP cells. A non-limiting example of a host or producer cell is HEK293 cells.

[0078] The producer cell (or host cell) can be genetically modified to contain one or more exogenous sequences in order to produce surface-modified endoplasmic reticulum. In some embodiments, the one or more exogenous sequences can encode the scaffold peptides described herein. In some embodiments, the one or more exogenous sequences can encode the exogenous biologically active molecules described herein. In some embodiments, the one or more exogenous sequences can encode both the scaffold peptides described herein and the exogenous biologically active molecules described herein. The genetically modified producer cell can contain exogenous sequences introduced by transient or stable modification. The exogenous sequences can be introduced into the producer cell as plasmids. The exogenous sequences can be stably integrated into the genomic sequence of the producer cell at a target site or any site. In some embodiments, stable cell lines can be generated for the production of surface-modified endoplasmic reticulum. Surface-modified endoplasmic reticulum containing scaffold peptides can be generated by introducing an exogenous sequence encoding the scaffold peptides described herein. Surface-modified endoplasmic reticulum containing a fusion protein containing an affinity tag that binds to the scaffold peptide can be generated by introducing an exogenous sequence encoding the affinity tag. As described herein, in some embodiments, an exogenous sequence encoding an exogenous biologically active molecule is introduced, and surface-modified endoplasmic reticulum containing a fusion protein containing an exogenous biologically active molecule bound to the scaffold peptide (e.g., directly or via a linker) can be generated.

[0079] In some embodiments, the producer cell (or host cell) can be further modified to contain additional exogenous sequences. For example, the additional exogenous sequences may be introduced to regulate endogenous gene expression, or may produce extracellular vesicles containing a specific polypeptide as a payload. In some embodiments, the producer cell can be modified to contain two exogenous sequences, one encoding a scaffold peptide and the other encoding a payload. In some embodiments, the producer cell can be further modified to contain additional exogenous sequences that confer additional functions to the extracellular vesicles, such as specific targeting functions, delivery functions, enzymatic functions, or increases or decreases in in vivo half-life. In some embodiments, the producer cell can be modified to contain two exogenous sequences, one encoding a scaffold peptide and the other encoding a protein that confers additional functions to the extracellular vesicles.

[0080] In some embodiments, the producer cell (or host cell) can be modified to contain two exogenous sequences, each of which encodes a fusion protein on the surface of the extracellular vesicle. In some embodiments, the surface-modified extracellular vesicles from the producer cell have a higher density of scaffold peptides compared to native extracellular vesicles isolated from unmodified cells of the same or similar cell type. In some embodiments, the surface-modified extracellular vesicles contain scaffold peptides at a density that is about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1000-fold or more higher than native extracellular vesicles isolated from unmodified cells of the same or similar cell type.

[0081] More specifically, the surface-modified extracellular vesicles can be produced from cells transformed (or transfected) with one or more sequences encoding a scaffold. The one or more scaffold peptides described herein can be expressed in producer cells from other exogenous nucleic acids such as plasmids, exogenous sequences inserted into the genome, or synthetic messenger RNA (mRNA).

[0082] In some embodiments, the scaffold peptides described herein can be fused to one or more heterologous proteins (e.g., exogenous bioactive molecules). In some embodiments, one or more heterologous proteins can be fused to the N-terminus of the scaffold peptide. In some embodiments, one or more heterologous proteins can be fused to the C-terminus of the scaffold peptide. In some embodiments, one or more heterologous proteins can be fused to both the N-terminus and the C-terminus of the scaffold peptide.

[0083] In another aspect, the present invention provides a pharmaceutical composition comprising the extracellular vesicles described herein and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutically acceptable excipient or carrier can be determined by the particular composition being administered in part, as well as by the particular method used to administer the composition. Accordingly, suitable formulations of pharmaceutical compositions containing multiple extracellular vesicles are highly diverse (see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)). The pharmaceutical composition can generally be manufactured under sterile conditions in full compliance with all good manufacturing practice (GMP) regulations of the US Food and Drug Administration (FDA). In some embodiments, the pharmaceutical composition can include one or more therapeutic agents and the extracellular vesicles described herein. In certain embodiments, the extracellular vesicles can be administered in combination with one or more additional therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising the extracellular vesicles can be administered prior to the administration of an additional therapeutic agent. In some embodiments, the pharmaceutical composition comprising the extracellular vesicles can be administered after the administration of an additional therapeutic agent. In some embodiments, the pharmaceutical composition comprising the extracellular vesicles can be administered concomitantly with an additional therapeutic agent. In some embodiments, the above pharmaceutical composition can be used to prevent, ameliorate, or treat a disease, disorder, or condition associated with the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system.

[0084] In another aspect, the present invention provides the use of a composition comprising the above extracellular vesicles as an active ingredient for manufacturing a formulation for preventing, ameliorating, or treating a disease, disorder, or condition associated with the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system.

[0085] Hereinafter, embodiments of the present disclosure will be described in detail by the following examples. However, the present disclosure is not limited to the described embodiments. Rather, the above examples are provided to thoroughly and completely convey the concept of the present disclosure to those skilled in the art based on the content introduced in this specification.

Mode for Carrying Out the Invention

[0086] Example 1 Construction of Plasmid DNAs As described in U.S. Patent No. 11,319,630, which is incorporated herein by reference, a DNA sequence encoding the full-length PTGFRN protein and a DNA sequence encoding the mutant SIRPα protein were fused to produce a plasmid DNA, the K-SIRPα-Full Length PTGFRN vector. A DNA sequence encoding the transmembrane domain (TMD) of the PTGFRN protein and a DNA sequence encoding the mutant SIRPα protein were fused to produce a plasmid DNA, the K-SIRPα-PTGFRN TMD (V1) vector. See Figure 1.

[0087] DNA sequences encoding the TMD of the PTGFRN protein (i.e., WT) and DNA sequences encoding therapeutic proteins such as mutant SIRPα and mature form EGF were prepared. Additional mutant TMDs were prepared by substituting at least one or more amino acids of the TMD with other amino acids. More specifically, an amino acid sequence of a mutant TMD in which the 11th amino acid T was substituted with amino acid A (i.e., mV1(T11A)) was prepared, an amino acid sequence of a mutant TMD in which the 7th amino acid V was substituted with amino acid I (i.e., mV1)(V7I)) was prepared, an amino acid sequence of a mutant TMD in which the 7th amino acid V was substituted with amino acid I (i.e., mV1(T11A / V7I)) was produced, and a mutant TMD in which the 11th amino acid T was substituted with amino acid A was produced. See Figures 2 and 4.

[0088] DNA sequences encoding mutant TMD, K-SIRPα-mV1(T11A / V7I), and DNA sequences encoding mutant SIRPα protein were prepared. The DNA sequence encoding the TMD of the K-SIRPα-mV1(T11A / V7I) plasmid was replaced with the DNA sequence encoding the PDGFR TMD of the commercially available pDisplay vector (Catalog V66020 of Thermo Fisher Scientific) to prepare plasmid DNA, the K-SIRPα-PDGFR TMD vector. The DNA sequence encoding the signal peptide of K-SIRPα-mV1(T11A / V7I) was replaced with the DNA sequence encoding the signal peptide of stabilin-2 of human STAB2[NM_017564] (MMLQHLVIFCLGLVVQNFCSP) to prepare plasmid DNA, the S-SIRPα-mV1(T11A / V7I) vector. Refer to Figure 3.

[0089] To prepare various plasmid DNAs according to embodiments of the present invention, a commercially available DNA sequence encoding the entire EGF protein was used. More specifically, the DNA sequence (RC210817) encoding the entire EGF protein was purchased from Origin, Inc. The DNA sequences encoding the pro-region and the shedding region were removed from the EGF coding region of the RC210817 vector to prepare truncated EGF (tEGF) DNA. The DNA sequence encoding TMD and the CD of tEGF were replaced with the DNA sequence encoding PTGFRN TMD(V1) to prepare plasmid DNA, the EGF-V1 vector. The DNA sequence encoding the TMD of EGF-V1 was replaced with the DNA sequence encoding mV1(T11A) to prepare plasmid DNA, the EGF-mV1(T11A) vector. The DNA sequence encoding the TMD of EGF-V1 was replaced with the DNA sequence encoding mV1(V7I) to prepare plasmid DNA, the EGF-mV1(V7I) vector. The DNA sequence encoding the TMD of EGF-V1 was replaced with the DNA sequence encoding mV1(T11A / V7I) to prepare plasmid DNA, the EGF-mV1(T11A / V7I) vector. Refer to Figure 4.

[0090] In addition to the amino acids in the PTGFRN TMD version (V1) sequence upstream (i.e., DVLNAF) and downstream (i.e., HWCCKKEVQETRRERRRLMSMEMD), PTGFRN TMD version 2 (V2) was prepared. In the PTGFRN TMD version 1 (V1) sequence, in addition to the amino acids upstream (i.e., DVLNAF) and downstream (i.e., HWC), PTGFRN TMD version 3 (V3) was prepared. The DNA sequence encoding the TMD was replaced with the DNA sequence encoding the CD of mV2 (V7I) or mV3 (V7I) of tEGF to prepare a recombinant plasmid DNA, an EGF-mV2 (V7I) or EGF-mV3 (V7I) vector. The DNA sequence encoding the CD of the tEGF plasmid DNA was replaced with the DNA sequence encoding the CD of the PTGFRN protein to prepare a cleaved EGF plasmid DNA, a tEGF replacement CD vector. See Figures 5 and 6.

[0091] At least one or more amino acids of the TMD (Extracellular vesicle Sorting Motif, ESM) of mV1 (T11A / V7I) were replaced with other amino acids to prepare additional mutant TMDs (Figures 7-11). The amino acid sequence of the mutant TMD (Figure 12) was prepared by replacing one of the essential amino acids of the ESM encoded by the mV1 (T11A / V7I) DNA sequence with another amino acid. The amino acid sequence of the mutant TMD (Figures 13-14) was prepared by deleting or adding one or more amino acids from the ESM encoded by the mV1 (T11A / V7I) DNA sequence. See Figures 7-14.

[0092] A plasmid encoding the control plasmid (pMX-U6) or CD9 (pMx-U6-shCD9) or CD81 (pMx-U6-shCD81) was prepared and used to infect 293FT cells stably transfected with the K-SIRPα-mV1 (T11A / V7I) plasmid. See Figure 15.

[0093] DNA sequences encoding mutant TMD, K-SIRPα-mV1(T11A / V7I), and DNA sequences encoding mutant SIRPα protein were prepared. The DNA sequence encoding the TMD of K-SIRPα-mV1(T11A / V7I) was replaced with the DNA sequence encoding the PDGFR TMD of a commercially available pDisplay vector (Catalog V66020 of Thermo Fisher Scientific) to prepare plasmid DNA, K-SIRPα-PDGFR TMD vector. In the sequence K-SIRPα-mV1(T11A / V7I), the upstream sequence (i.e., DVLNAF) and downstream sequence (i.e., HWC) of mV1(T11A / V7I) were added to produce K-SIRPα-mV3(T11A / V7I). Refer to Figure 16.

[0094] The above plasmid was amplified and isolated according to the protocol of the Qiagen™ Plasmid Maxi kit. More specifically, 1 μl (0.1 μg) of plasmid DNA and 100 μl of competent cells DH5α were mixed in a 1.5 ml microcentrifuge tube. The plasmid DNA was introduced into the competent cells DH5α by heat shock. More specifically, the microcentrifuge tube containing the mixture of plasmid DNA and competent cells DH5α was heated at 42 °C for 45 seconds using a heat block. Thereafter, the heated microcentrifuge tube was placed on ice for 2 minutes. After cooling, 900 μl of antibiotic-free LB agar medium was added to the microcentrifuge tube. Subsequently, this microcentrifuge tube was incubated at 37 °C for 45 minutes on a 200 rpm shaker. After incubation, 100 μl from the microcentrifuge tube was spread onto an LB medium plate containing 100 μg / ml of ampicillin. All plates were incubated overnight at 37 °C. The next day, colonies were collected from the plate surface and incubated in 3 mL of LB medium containing 100 μg / ml of ampicillin at 37 °C for 8 hours. After incubation, 1 ml of the mixture of colonies and LB medium containing the antibiotic was transferred into a flask containing 500 ml of LB / ampicillin medium and incubated overnight at 37 °C. The bacterial cells were collected by centrifugation at 6000 xg for 15 minutes at 4 °C, and the bacterial pellet was resuspended in buffer P1 containing 100 μg / ml of RNase A. Buffer P2 was added, and after the sealed tube was strongly inverted 4 - 6 times to mix thoroughly, the resulting mixture was incubated at room temperature for 5 minutes. Cold buffer P3 was added, inverted 4 - 6 times, and immediately mixed thoroughly, and then the resulting mixture was incubated on ice for 20 minutes. After centrifugation at ≥20000 xg for 30 minutes at 4 °C, the supernatant containing the plasmid DNA was immediately collected. The supernatant was centrifuged again at ≥20000 xg for 15 minutes at 4 °C, and the supernatant containing the plasmid DNA was immediately recovered. After applying buffer QBT and emptying the column by gravity flow to equilibrate the QIAGEN-tip 500, the collected supernatant was applied to the QIAGEN-tip to enter the resin by gravity flow.After washing the QIAGEN-tip with buffer QC, the DNA was eluted with buffer QC. Isopropanol at room temperature was added to the eluted DNA for precipitation. The mixture was immediately mixed and centrifuged at ≥15,000 xg for 30 minutes at 4°C, and then the supernatant was carefully decanted. The DNA pellet was washed with 70% ethanol at room temperature and centrifuged at ≥15,000 xg for 10 minutes, and then the supernatant was carefully decanted without disturbing the pellet. The pellet was air-dried for 5 - 10 minutes, and then the final plasmid DNA was redissolved in an appropriate amount of buffer.

[0095] Example 2 Isolation of Extracellular Vesicles HEK293 cells (6×10 6 ) were incubated at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). When the cells were concentrated to about 80 - 90%, the cells were infected with plasmid DNA using a transfecting agent or infected with retrovirus for stable cell production.

[0096] In the case of transient infection, cells were infected with an infecting agent such as Lipofectamine 2000, Lipofectamine 3000, or polyethyleneimine (PEI). The cell medium was exchanged with DMEM, and a mixture of DNA and the infecting reagent was added to the cells. Next, the cells were incubated at 37°C with 5% CO2 for 24 hours. 24 hours after infection, the medium containing the infecting reagent and plasmid was exchanged with DMEM supplemented with 10% FBS and 1% antibiotic-antifungal agent. The transiently infected cells were incubated at 37°C with 5% CO2 for 24 hours. 24 hours after recovery, the medium was exchanged with DMEM medium supplemented with insulin-transferrin-selenium (Gibco). Serum-free cells were incubated at 37°C with 5% CO2 for 48 hours. See, for example, Gi Kim et al., Xenogenization of tumor cells by fusogenic exosomes in tumor microenvironment ignites and propagates antitumor immunity, SCIENCE ADVANCES, Vol 6, Issue 27 (July 1, 2020), which is incorporated herein by reference.

[0097] For stable cell production, Plat-E cells were used to produce retroviruses that packaged a retroviral vector containing the DNA sequence of interest and the DNA sequence of the puromycin-resistance gene. Specifically, Plat-E cells (2×10 6) was incubated in Dulbecco's modified Eagle's medium (DMEM) with 5% CO2 at 37°C, and 10% FBS was added. When the cells reached a concentration of 80 - 90%, the cells were infected with a retroviral vector encoding the target DNA sequence using Lipofectamine 2000. After 24 hours, the culture medium was replaced with DMEM supplemented with 10% FBS, and incubation was continued for an additional 24 hours. 48 hours after infection, the culture fluid containing virus particles was collected, centrifuged at 3000 rpm, and then filtered through a 0.45 μm filter for use in 293FT cell infection. See, for example, Park, SY., Yun, Y., Lim, JS. et al. Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration. Nat Commun 7, 10871 (2016), which is incorporated herein by reference.

[0098] To isolate extracellular vesicles, the cell supernatant was collected 48 hours after infection. The supernatant was centrifuged at 300 g for 10 minutes, 2000 g for 10 minutes, and 10000 g for 30 minutes. Next, the supernatant was filtered and concentrated using a tangential flow filtration (TFF) system or a 100 kDa Amicon Ultra-15 centrifugal filter device. Thereafter, the supernatant was centrifuged at 150000 g for 3 hours. The extracellular vesicle pellet was resuspended in PBS containing a protease inhibitor cocktail and stored at 4°C.

[0099] See, for example, Gi Kim et al., Xenogenization of tumor cells by fusogenic exosomes in tumor microenvironment ignites and propagates antitumor immunity, SCIENCE ADVANCES, Vol 6, Issue 27 (July 1, 2020), which is incorporated herein by reference.

[0100] Example 3 Characterization of Surface-Engineered Extracellular Vesicles

[0101] A Western blot assay was performed to identify the characteristics of surface-engineered extracellular vesicles. More specifically, the total protein amount in the extracellular vesicles was measured using a bicinchoninic acid (BCA) protein assay. Standard solutions were prepared, and 5 μl of bovine serum albumin at each concentration (2, 1, 0.5, 0.25, 0.125, and 0 mg / ml) was applied to a 96-well plate. The extracellular vesicle sample was diluted with PBS, and 5 μl of the resulting sample was applied to the 96-well plate. Reagent A (500113, Bio-Rad) and S (500114, Bio-Rad) were mixed at a ratio of 50 to 1, and 25 μl of the reagent mixture was applied to the 96-well plate. 200 μl of Reagent B (500115, Bio-Rad) was applied to the 96-well plate, and the plate was gently tapped. The samples were incubated for 15 minutes in the dark. The protein amount was measured using a microplate reader at a wavelength of 750 nm. Furthermore, the amount of extracellular vesicles was analyzed using Zetaview. After evaluating the alignment test using QC beads, the diluted samples were loaded. After setting to observe 150 - 200 particles, the number of extracellular vesicles was analyzed.

[0102] The purified extracellular vesicles were added to RIPA buffer together with a protease inhibitor cocktail (Calbiochem) to lyse the extracellular vesicles, and then mixed with SDS-PAGE sample buffer. The same amount of extracellular vesicle proteins was subjected to SDS-PAGE electrophoresis. After gel electrophoresis, the bands were transferred to a nitrocellulose membrane or a methanol-activated polyvinylidene difluoride (PVDF) membrane. After pre-blocking with 5% skim milk dissolved in Tris Buffered Saline (TBST) with added Tween-20 at room temperature for 1 hour, the membrane was incubated with the primary antibody at 4°C overnight. To detect protein expression, the CD81, SIRPα, EGF, and actin antibodies were used. The membrane was incubated with an HRP-conjugated secondary antibody, and then the blot was probed using a ChemiDoc imaging system (Bio-Rad) (Figs. 1-6, 8-10, and 12-15).

[0103] To measure protein expression, a capillary Western blot assay was performed. Extracellular vesicle samples were prepared using the EZ standard pack 1 (ProteinSimple, 96655). Four parts of the diluted protein were combined with one part of 5X fluorescent master mix. Each sample was denatured in a heat block at 95°C for 5 minutes. 3 μl of the sample was loaded into the appropriate wells of the cartridge. The sample was pre-blocked with antibody diluent 2 (ProteinSimple, 95905) for 10 minutes. Appropriate antibodies were diluted to the desired concentration and used to detect protein expression. The protein expression was detected by an anti-mouse secondary antibody (ProteinSimple, 96113). The sample was analyzed through Compass for SW (ProteinSimple).

[0104] As shown in Fig. 1, the expression levels of the SIRPα protein, which is known to play a role in promoting the elimination of diseased cells by phagocytes, were evaluated by utilizing both the entire PTGFRN and PTGFRN fragments that have been reported to exhibit effective protein expression on the surface of extracellular endoplasmic reticulum. As a result of the experiment, it was found that the K-SIRPα-PTGFRN TMD version 1 (V1), which is a plasmid that mainly utilizes the TMD of PTGFRN, has superior protein expression efficiency compared to the K-SIRPα-full length PTGFRN (K-SIRPα-Full Length PTGFRN), which is the entire PTGFRN. K-SIRPα-full length PTGFRN (K-SIRPα-Full Length PTGFRN) sequence (SEQ ID NO: 143):

[0105] K-SIRPα-PTGFRN TMD Version 1 (V1) sequence (SEQ ID NO: 144): METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGVGLSTVIGLLSCLIGYCSS

[0106] As shown in Fig. 2, in order to induce a motif with improved protein expression efficiency on the surface of the extracellular endoplasmic reticulum, random mutations of PTGFRN TMD version 1 (V1) were used in K-SIRPα-V1. K-SIRPα-mV1(T11A) sequence (SEQ ID NO: 145): METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGVGLSAVIGLLSCLIGYCSS K-SIRPα-mV1(V7I) sequence (SEQ ID NO: 146): METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGIGLSTVIGLLSCLIGYCSS K-SIRPα-mV1(T11A / V7I) sequence (SEQ ID NO: 147): METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGIGLSAVIGLLSCLIGYCSS

[0107] As shown in Fig. 3, to verify the superiority of the derived K-SIRPα-mV1 (T11A / V7I), a comparative experiment was conducted with PDGFR TMD, which is commonly used for the expression of desired proteins on the cell and extracellular vesicle surfaces. As a result of the experiment, it was confirmed that K-SIRPα-mV1 (T11A / V7I) exhibited a significantly higher protein expression efficiency on the extracellular vesicle surface compared to K-SIRPα-PDGFR TMD. Furthermore, the difference in protein expression efficiency on the EV surface when the Ig-kappa signal peptide of K-SIRPα-mV1 (T11A / V7I) was replaced with the signal peptide of the staphylin-2 protein was examined. As a result, it was shown that the efficient expression of the protein was maintained for mV1 (T11A / V7I) not only with the Ig-kappa signal peptide but also in combination with the staphylin-2 signal peptide. K-SIRPα-PDGFR TMD sequence (SEQ ID NO: 148):

[0108] METDTLLLWVLLLWVPGSTGDYPYDVPDYAGAQPARSMEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPVDEQKLISEEDLNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR S-SIRPα-mV1 (T11A / V7I) sequence (SEQ ID NO: 149): MMLQHLVIFCLGLVVQNFCSPGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGIGLSAVIGLLSCLIGYCSS

[0109] As shown in Fig. 4, to demonstrate the versatility of the motif, the fusion of the mature EGF protein, a regenerative factor, and SIRPα protein was evaluated by replacing them with mV1(T11A / V7I). The experimental results showed that, similar to SIRPα, when the 11th amino acid T of V1 was mutated to A and the 7th amino acid V of V1 was mutated to I in EGF, it had excellent protein expression efficiency. The double mutant EGF-mV1(T11A / V7I) showed improved protein expression efficiency compared to the single mutant and the wild-type PTGFRN TMD (V1). EGF-V1 sequence (SEQ ID NO: 150): MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELREFKYPLLIGVGLSTVIGLLSCLIGYCSS EGF-mV1(T11A) sequence (SEQ ID NO: 151): MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELREFKYPLLIGVGLSAVIGLLSCLIGYCSS EGF-mV1(V7I) sequence (SEQ ID NO: 152): MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELREFKYPLLIGIGLSTVIGLLSCLIGYCSS EGF-mV1(T11A / V7I) sequence (SEQ ID NO: 153): MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELREFKYPLLIGIGLSAVIGLLSCLIGYCSS

[0110] As shown in Fig. 5, it was shown that the EGF expression of the EGF-mutated PTGFRN TMD version 2 (V7I) was higher than that of tEGF, and it was confirmed that the EGF expression of the EGF-mutated PTGFRN TMD version 3 (V7I) was similar to or higher than that of EGF-mV2 (V7I). Conversely, it was shown that the tEGF-substituted CD lacking the PTGFRN TMD mutation had a very low EGF expression efficiency on the surface of the extracellular endoplasmic reticulum. These results suggest that the TMD of the PTGFRN protein plays an important role in displaying the therapeutic protein on the surface of the extracellular endoplasmic reticulum. tEGF sequence (SEQ ID NO: 154):

[0111] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRVIVVAVCVVVLVMLLLLSLWGAHYYRTQKLLSKNPKNPYEESSRDVRSRRPADTEDGMSSCPQPWFVVIKEHQDLKNGGQPVAGEDGQAADGSMQPTSWRQEPQLCGMGTEQGCWIPVSSDKGSCPQVMERSFHMPSYGTQTLEGGVEKPHSLLSANPLWQQRALDPPHQMELTQ EGF-mutated PTGFRN TMD version (Version) 2 (V7I) sequence (SEQ ID NO: 155): MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELREFDVLNAFKYPLLIGIGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD EGF-mutated PTGFRN TMD version (Version) 3 (V7I) sequence (SEQ ID NO: 156): MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELREFDVLNAFKYPLLIGIGLSTVIGLLSCLIGYCSSHWC tEGF replacement CD sequence (SEQ ID NO: 157): MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRVIVVAVCVVVLVMLLLLSLWGAHYYRTQEFHWCCKKEVQETRRERRRLMSMEMD

[0112] As shown in Figure 6, it was observed that the EGF expression of EGF-mV1 (T11A / V7I) and EGF-mV3 (V7I) was higher than that of EGF-mV2 (V7I) containing both the TMD and CD of the PTGFRN protein. This finding suggests that the CD of the PTGFRN protein is not required for the classification or targeting of extracellular vesicles.

[0113] As shown in Figures 7-11, to determine the important sequences for the extracellular vesicle surface expression of the desired protein in the mutant TMD (derived from ESM, mV1 (T11A / V7I)), the plasmid was generated by introducing a single mutation from ESM to L, aiming to induce the important TMD amino acid sequence pattern for the extracellular vesicle classification of the introduced protein (Figure 7). When the 6th amino acid G, 10th amino acid S, 14th amino acid G, 21st amino acid G, or a combination thereof was substituted with other amino acids, it was confirmed that the SIRPα or EGF protein expression of the tested DNA decreased significantly. The above results indicate that the G-S-G-G pattern is essential for the classification of protein E extracellular vesicles.

[0114] As shown in Fig. 12, to confirm that important amino acids of ESM, specifically the 6th amino acid G, 10th amino acid S, 14th amino acid G, and 21st amino acid G, are replaced with other amino acids other than L, the important amino acids were changed to four different amino acids, and the extracellular vesicle selection efficacy of the introduced protein was evaluated. As a result of the experiment, it was confirmed that G may be present at the 6th amino acid position, S at the 10th amino acid position, G, A, S, or T at the 14th amino acid position, and G or S at the 21st amino acid position.

[0115] As shown in Figs. 13 to 14, to evaluate the possible number of amino acids that can be presented between important amino acids in the derived G-S-G-G sequence, the EV selection efficacy of the protein was evaluated. The number of amino acids between the 6th G and the 10th S was designated as "a", the number of amino acids between the 10th S and the 14th G as "b", and the number of amino acids between the 14th G and the 21st G as "c". Six plasmids with the numbers of amino acids a, b, and c changed were generated compared to the original sequence, and the EV selection efficacy of the protein was evaluated. As a result of the experiment, it was found that it is possible to have 3 to 4 amino acids in the case of "a", 2 to 3 amino acids in the case of "b", and 6 to 7 amino acids in the case of "c".

[0116] As shown in Fig. 15, when EVs obtained from the DNA construct (K-SIRPα-mV1(T11A / V7I)) according to the embodiment of the present invention were infected with CD9 or CD81 shRNA, it was confirmed that SIRPα expression was significantly decreased. These results suggest that CD9 and CD81 proteins are related to the EV surface expression mechanism of the protein introduced by ESM.

[0117] As shown in Fig. 16, several amino acids were added before and after mV1(T11A / V7I) while retaining ESM to compare the protein EV sorting efficiency. As a result of the above experiment, both mV1(T11A / V7I) and mV3(T11A / V7I) showed excellent SIRPα protein expression efficiency on the EV surface, but mV3(T11A / V7I) showed slightly higher EV selection efficiency. K-SIRPα-mV3(T11A / V7I) sequence (SEQ ID NO: 158): METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFDVLNAFKYPLLIGIGLSAVIGLLSCLIGYCSSHWC

[0118] The present disclosure is described herein with respect to specific embodiments, and although its aspects can be more fully understood and recognized, the present invention is not intended to be limited to these specific embodiments. On the contrary, all alternatives, modifications, and equivalents are intended to be included within the scope of the present disclosure as defined herein. Accordingly, the foregoing examples, including specific embodiments, function as illustrations of the practice of the inventive concept of the present disclosure, and the specific matters shown are understood as examples and for illustrative discussion of specific embodiments only, and are provided to provide what is considered to be the most useful and easily understood explanation of the principles and conceptual aspects of the present invention, as well as the procedures.

[0119] Modifications can be made to the formulations of the various compositions described herein, the methods described herein, or the steps or order of steps of the methods described herein without departing from the spirit and scope of the present invention. Further, while various embodiments of the present invention are described in the following claims, the present invention is not intended to be limited to these specific claims.

Claims

**Claim 1** A DNA construct comprising a DNA sequence encoding a scaffold peptide, wherein the amino acid sequence of the scaffold peptide comprises a sequence represented by G-a-S-b-X1-c-X2: Herein, X1 is G, A, S, or T; X2 is G or S; a is 3 to 4 amino acids; b is 2 to 3 amino acids; c is 6 to 7 amino acids; G is glycine; S is serine; A is alanine; T is threonine. **Claim 2** The DNA construct according to claim 1, wherein the sequence represented by G-a-S-b-X1-c-X2 has 15 to 17 amino acids. **Claim 3** The DNA construct according to claim 1, wherein the scaffold peptide has 22 to 57 amino acids. **Claim 4** The DNA construct according to claim 1, wherein a, b, and c include V, G, L, I, A, T, S, C, F, W, Y, and P, where V is valine, G is glycine, L is leucine, I is isoleucine, A is alanine, T is threonine, S is serine, C is cysteine, F is phenylalanine, W is tryptophan, Y is tyrosine, and P is proline. **Claim 5** The DNA construct according to claim 1, wherein a is 3 to 4 amino acids selected from the group consisting of V, G, L, I, T, and A, where V is valine, G is glycine, L is leucine, I is isoleucine, T is threonine, and A is alanine. **Claim 6** The DNA construct according to claim 5, wherein a is VGL, IGL, VGLT, IGLT, VGLA, or IGL A. **Claim 7** The DNA construct according to claim 1, wherein b is 2 to 3 amino acids selected from the group consisting of V, I, A, and T, where V is valine, I is isoleucine, A is alanine, and T is threonine. **Claim 8** The DNA construct according to claim 7, wherein b is VI, AV, TVI, or AVI. **Claim 9** The DNA construct according to claim 1, wherein c is 6 to 7 amino acids selected from the group consisting of L, S, C, and I, where L is leucine, S is serine, C is cysteine, and I is isoleucine. **Claim 10** The DNA construct according to claim 9, wherein c is LLSCLL or ILLSCLL.

11. The DNA construct according to claim 1, wherein the sequence represented by G-a-S-b-X1-c-X2 is any one of the ESMs of SEQ ID NOs: 1 to 100.

12. The DNA construct according to claim 1, wherein the scaffold peptide further contains KYPLLI at the N-terminus of the sequence represented by G-a-S-b-X1-c-X2, where K is lysine, Y is tyrosine, P is proline, L is leucine, and I is isoleucine.

13. The DNA construct according to claim 1, wherein the scaffold peptide further contains DVLNAFKYPLLI at the N-terminus of the sequence represented by G-a-S-b-X1-c-X2, where D is aspartic acid, V is valine, L is leucine, N is asparagine, A is alanine, F is phenylalanine, K is lysine, Y is tyrosine, P is proline, L is leucine, and I is isoleucine.

14. The DNA construct according to claim 1, wherein the scaffold peptide further contains YCSS at the C-terminus of the sequence represented by G-a-S-b-X1-c-X2, where Y is tyrosine, C is cysteine, and S is serine.

15. The DNA construct according to claim 1, wherein the scaffold peptide further contains YCSSHW at the C-terminus of the sequence represented by G-a-S-b-X1-c-X2, where Y is tyrosine, C is cysteine, S is serine, H is histidine, and W is tryptophan.

16. The DNA construct according to claim 1, further comprising a DNA sequence encoding the amino acid sequence of a target protein.

17. The DNA construct according to claim 16, wherein the target protein is a therapeutic protein.

18. A vector comprising the DNA construct according to claim 1.

19. A host cell comprising the vector according to claim 18.

20. An extracellular vesicle isolated from the host cell according to claim 19, wherein the scaffold peptide is present at a desired position of the extracellular vesicle.

21. An endoplasmic reticulum extracellular vesicle comprising a scaffold peptide encoded by the DNA construct according to claim 1.

22. The extracellular vesicle according to claim 20 or 21, characterized in that it further comprises another extracellular peptide, which comprises CD9, CD63, CD81, PDGFR, PTGFRN, GPI-anchored protein, lactadherin, syndecan, synaptotagmin, ALIX (apoptosis-linked gene 2-interacting protein X), syntenin, LAMP2, LAMP2B, fragments or variants thereof, variants of said fragments, and fragments of said variants.

23. The extracellular vesicle according to claim 20 or 21, further characterized by further comprising a target protein.

24. The extracellular vesicle according to claim 23, characterized in that the target protein is a therapeutic protein.

25. The extracellular vesicle according to claim 23, characterized in that the scaffold peptide is fused to the target protein.

26. The extracellular vesicle according to claim 20 or 21, characterized in that the scaffold peptide comprises an affinity tag having an affinity for a binding agent.

27. The extracellular vesicle according to claim 20 or 21, characterized in that the scaffold peptide further comprises a targeting moiety.

28. The extracellular vesicle according to claim 20 or 21, further characterized by further comprising a therapeutic substance.

29. The extracellular vesicle according to claim 28, characterized in that the therapeutic substance is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, small molecule compounds, and combinations thereof.

30. The extracellular vesicle according to claim 28, characterized in that the therapeutic substance is fused to the scaffold peptide and / or encapsulated in the extracellular vesicle.

31. A pharmaceutical composition comprising the extracellular vesicle according to claim 20 or 21 and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition according to claim 31 for preventing, improving, or treating a disease, disorder, or condition related to the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system.

33. A method of preventing, improving, or treating a disease, disorder, or condition related to the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 31 to an individual in need thereof.

34. Use of the extracellular vesicles according to claim 20 or 21 for manufacturing a preparation for preventing, improving, or treating a disease, disorder, or condition related to the nervous system, digestive system, endocrine system, skeletal system, respiratory system, integumentary system, lymphatic system, genital system, muscular system, excretory system, or immune system.

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