Surface engineered extracellular vesicles and therapeutic uses thereof

Abstract

The present invention provides surface engineered extracellular vesicles, compositions comprising the surface engineered extracellular vesicles, methods of making the surface engineered extracellular vesicles, and methods of using the surface engineered extracellular vesicles or the compositions.

Description

Technical Field

[0001] This application claims priority to U.S. patent application No. 63 / 345.040, filed on May 24, 2022, the entire contents of which are incorporated by reference into this application.

[0002] The present invention generally relates to surface engineered extracellular vesicles, compositions comprising the surface engineered extracellular vesicles, methods of preparing the surface engineered extracellular vesicles, and methods of using the surface engineered extracellular vesicles or the compositions. Background of the Invention

[0004] Considerable efforts have been made to utilize exosomes to deliver various therapeutic molecules to desired target cells for therapeutic purposes, examples of which include therapeutic proteins, membrane proteins, reporter proteins, enzymes, antibody fragments, cytokines, tumor necrosis factor superfamily (TNFSF) ligands, RNA binding proteins, Cas9, and vaccine antigens. Exosomes with therapeutic molecules on their surface have been proposed. Scaffolds that can display therapeutic molecules on the surface of exosomes have been proposed. Prostaglandin F2 receptor regulatory protein (PTGFRN) has been proposed as a scaffold for displaying various therapeutic molecules on the surface of exosomes. However, there is still a need for a new scaffold that can display therapeutic molecules on the surface of exosomes in a better manner. Summary of the invention

[0005] 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 includes a sequence represented by GaSb-X1-c-X2 (extracellular vesicle sorting motif, ESM), wherein X1 represents G, A, S or T; X2 represents G or S; a represents 3-4 amino acids; b represents 2-3 amino acids; and c represents 6-7 amino acids; G represents glycine; S represents serine; A represents alanine; and T represents threonine.

[0006] In some embodiments, the sequence GaSb-X1-c-X2 may have 15-17 amino acids. In some embodiments, the scaffold peptide may have 22-57 amino acids. In some embodiments, amino acids a, b and c may include V, G, L, I, A, T, S, C, F, W, Y and P, wherein 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 may represent 3-4 amino acids selected from V, G, L, I, T and A, wherein V represents valine, G represents glycine, L represents leucine, I represents isoleucine, T represents threonine, and A represents alanine. In some embodiments, a may represent VGL, IGL, VGLT, IGLT, VGLA or IGLA. In some embodiments, b may represent 2-3 amino acids selected from V, I, A and T, wherein V represents valine, I represents isoleucine, A represents alanine, and T represents threonine. In some embodiments, b may represent VI, AV, TVI or AVI. In some embodiments, c may represent 6-7 amino acids selected from L, S, C and I, wherein L represents leucine, S represents serine, C represents cysteine, and I represents isoleucine. In some embodiments, c may represent LLSCLI or ILLSCLI. In some embodiments, the sequence GaSb-X1-c-X2 may be one of the amino acid sequences shown in ESM SEQ ID NOS:1-100. In some embodiments, the scaffold peptide may also include KYPLLI at the N-terminus of the sequence GaSb-X1-c-X2, wherein K represents lysine, Y represents tyrosine, P represents proline, L represents leucine, and I represents isoleucine. In some embodiments, the scaffold peptide may also include DVLNAFKYPLLI at the N-terminus of the sequence GaSb-X1-c-X2, wherein 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 may also include YCSS at the C-terminus of the sequence GaSb-X1-c-X2, wherein Y represents tyrosine, C represents cysteine, and S represents serine.In some embodiments, the scaffold peptide may also include YCSSHWCCKKEVQETRRERRRLMSMEMD at the C-terminus of the sequence GaSb-X1-c-X2, wherein 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 may also include YCSSHWC at the C-terminus of the sequence GaSb-X1-c-X2, wherein Y represents tyrosine, C represents cysteine, S represents serine, H represents histidine, and W represents tryptophan. In some embodiments, the DNA construct may also include a DNA sequence encoding the amino acid sequence of the target protein. In some embodiments, the target protein may be a therapeutic protein. In some embodiments, the scaffold peptide may display the target protein at a desired position of an extracellular vesicle. In some embodiments, the desired location can be the inner surface or the outer surface of an extracellular vesicle.

[0007] Another aspect of the present invention provides a vector comprising the above DNA construct. A non-limiting example of the vector is an expression plasmid comprising a DNA sequence encoding a scaffold peptide.

[0008] The present invention provides a host cell comprising the above-mentioned vector on the other hand. Non-limiting examples of the host cell may include HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSC) and cells derived from HEK293 cells, CHO cells or MSC. In addition, non-limiting examples of the host cell may include mast cells, immune cells, natural killer cells, dendritic cells, macrophages, T lymphocytes, B lymphocytes, epithelial cells, human cardiac progenitor cells, adipose stem cells, umbilical cord blood mesenchymal stem cells and bone marrow mesenchymal stem cells.

[0009] Another aspect of the present invention provides an extracellular vesicle (EV) isolated from the above-mentioned host cell. In some embodiments, the scaffold peptide can be displayed at a desired position of the extracellular vesicle. For example, the scaffold peptide can be displayed on the inner surface of the extracellular vesicle, the outer surface of the extracellular vesicle, or both. In some embodiments, the extracellular vesicle may also include a target protein. In some embodiments, the scaffold peptide may be fused to the target protein. In some embodiments, the scaffold peptide may include an affinity tag with affinity for a binding agent. In some embodiments, the extracellular vesicle may also include a targeting portion. In some embodiments, the extracellular vesicle may also include a therapeutic substance.

[0010] When the extracellular vesicle comprises the scaffold peptide of the present invention, the extracellular vesicle may have the scaffold peptide displayed on the surface of the extracellular vesicle at a higher density than the extracellular vesicle comprising the 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 variants, and variants of fragments. When the extracellular vesicle comprises the scaffold peptide of the present invention, the extracellular vesicle may comprise a higher amount of target protein than the extracellular vesicle comprising the scaffold peptide different from the scaffold peptide of the present invention.

[0011] Another aspect of the present invention provides an extracellular vesicle comprising a scaffold peptide encoded by the above-mentioned DNA construct. In some embodiments, the scaffold peptide can be displayed at a desired position of the extracellular vesicle. For example, the scaffold peptide can be displayed on the inner surface of the extracellular vesicle, the outer surface of the extracellular vesicle, or both.

[0012] Another aspect of the present invention provides a pharmaceutical composition comprising the extracellular vesicles described above. In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. In some embodiments, the present invention provides the pharmaceutical composition described above for preventing, improving or treating diseases, disorders or conditions associated with the nervous, digestive, endocrine, skeletal, respiratory, integumentary, lymphatic, reproductive, muscular, excretory or immune systems.

[0013] Another aspect of the present invention provides a method for preventing, improving or treating a disease, disorder or condition associated with the nervous, digestive, endocrine, skeletal, respiratory, skin, lymphatic, reproductive, muscle, excretion or immune system, the method comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition to a subject in need. In some embodiments, the disease, disorder or condition may be at least one selected from the following group: certain infectious or parasitic diseases, tumors, blood or hematopoietic organ diseases, immune system diseases, endocrine, nutritional or metabolic diseases, mental, behavioral or neurodevelopmental disorders, sleep-wake disorders, nervous system diseases, visual system diseases, ear or mastoid diseases, circulatory system diseases, respiratory system diseases, digestive system diseases, skin diseases, musculoskeletal system or connective tissue diseases, urogenital system diseases, conditions related to sexual health, gynecological diseases, developmental abnormalities, certain conditions originating from the perinatal period, unclassified symptoms, signs or clinical findings, consequences of injury, poisoning or certain other external causes, external causes of morbidity or death.

[0014] Another aspect of the present invention provides a method for preparing a surface engineered extracellular vesicle for therapeutic use. In particular, the surface engineered extracellular vesicle is prepared using the above-mentioned DNA construct and / or the above-mentioned scaffold peptide. In some embodiments, the target protein (e.g., therapeutic protein) can be combined with the scaffold peptide to prepare a fusion protein, and the fusion protein can be displayed on the surface of the extracellular vesicle to prepare a surface engineered extracellular vesicle. The surface engineered extracellular vesicle prepared according to the method of the present invention using the above-mentioned scaffold peptide is superior to the surface engineered extracellular vesicle prepared using the method of some other scaffolds (e.g., PTGFRN) in many aspects. For example, the density of the therapeutic protein, scaffold or both displayed on the surface of the extracellular vesicle prepared according to the method of the present invention is higher than the density of the therapeutic protein, scaffold or both displayed on the surface of the extracellular vesicle prepared by using the method of some other scaffolds (e.g., PTGFRN). In addition, the surface engineered extracellular vesicle prepared according to the method of the present invention shows a higher therapeutic efficacy than the surface engineered extracellular vesicle prepared by using the method of some other scaffolds (e.g., PTGFRN). In addition, the scaffold peptide of the present invention is shorter than some other scaffolds (e.g., PTGFRN). Furthermore, the therapeutic protein displayed on the surface of the extracellular vesicles prepared by the method according to the present invention is more effective than the therapeutic protein displayed on the surface of the extracellular vesicles prepared by the method using some other scaffolds (e.g., PTGFRN).

[0015] Another aspect of the present invention provides the use of a composition comprising the extracellular vesicles described in the above claims as an active ingredient in the preparation of a preparation for preventing, improving or treating diseases, disorders or conditions related to the nervous, digestive, endocrine, skeletal, respiratory, integumentary, lymphatic, reproductive, muscular, excretory or immune systems.

[0016] The above and other aspects and embodiments of the present invention are discussed in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 1 Shown are DNA constructs of some embodiments of the present invention, signal regulatory protein alpha (SIRPα) and CD81 protein expression of said constructs in extracellular vesicles, and semi-quantitative analysis of SIRPα protein expression normalized by CD81 in extracellular vesicles;

[0020] Figure 2Shown are DNA constructs of some embodiments of the present invention, SIRPα and CD81 protein expression of the constructs in extracellular vesicles, semi-quantitative analysis of SIRPα protein expression in extracellular vesicles normalized by CD81, and SIRPα and actin expression of the constructs in cell lysates;

[0021] Figure 3 Shown are DNA constructs of some embodiments of the present invention, SIRPα and CD81 protein expression of the constructs in extracellular vesicles, semi-quantitative analysis of SIRPα protein expression in extracellular vesicles normalized by CD81, and SIRPα and actin expression of the constructs in cell lysates;

[0022] Figure 4 Shown are DNA constructs of some embodiments of the present invention, expression of epidermal growth factor (EGF) and CD81 proteins of said constructs in extracellular vesicles, and semi-quantitative analysis of EGF protein expression normalized by CD81 in extracellular vesicles;

[0023] Figure 5 Shown are DNA constructs according to some embodiments of the present invention, and the EGF protein expression of the constructs in extracellular vesicles or cell lysates;

[0024] Figure 6 Shown are DNA constructs according to some embodiments of the present invention, and the EGF and CD81 protein expression of the constructs in extracellular vesicles or cell lysates;

[0025] Figure 7 DNA constructs of other embodiments of the present invention are shown;

[0026] Figure 8 Shown based on Figure 7 DNA constructs for SIRPα and CD81 protein expression in extracellular vesicles;

[0027] Fig. 9 Shows Figure 8 Relative SIRPα expression in extracellular vesicles normalized by CD81;

[0028] Fig.10 Shown based on Figure 7 DNA constructs for EGF and CD81 protein expression in extracellular vesicles;

[0029] Fig.11 Shows Fig.10 Relative EGF expression in extracellular vesicles normalized by CD81;

[0030] Fig.12Shown are DNA constructs according to some embodiments of the present invention, and the expression of SIRPα protein in extracellular vesicles by the constructs;

[0031] Fig.13 Shown are DNA constructs of some embodiments of the present invention, SIRPα and actin expression of the constructs in cell lysates, and SIRPα and CD81 protein expression of the constructs in extracellular vesicles;

[0032] Fig.14 Shows Fig.13 Relative SIRPα expression in extracellular vesicles of

[0033] Fig.15 HEK293 cells stably transduced with K-SIRPα-mV1(T11A / V7I) plasmid and transfected with control (pMX-U6), CD9 or CD81 short hairpin RNA (shRNA), and SIRPα, CD81, CD9 and Alix protein expression in extracellular vesicles from transfected stable HEK293 cells are shown; and

[0034] Fig.16 Shown are the results of a comparison of protein EV sorting efficiency by adding a small amount of amino acids before and after mV1 (T11A / V7I) with ESM. DETAILED DESCRIPTION OF THE INVENTION

[0036] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, the following terms have the meanings assigned to them as described below.

[0037] As used herein, the terms "comprising" (and any grammatical forms thereof), "having" (and any grammatical forms thereof), "including" (and any grammatical forms thereof), or "containing" (and any grammatical forms thereof) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0038] As used herein, the term "a" or "an" when used with the term "comprising" in the claims and / or specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more". The term "or" in the claims is used to mean "and / or" unless it is explicitly stated that only alternatives are referred to or the alternatives are mutually exclusive, although the present disclosure supports definitions that only refer to alternatives and "and / or". The use of the term "at least one" should be understood to include one and any number more than one, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or any integer contained therein. The term "at least one" can extend to 100 or 1000 or more, depending on the term to which it is attached; in addition, the number of 100 / 1000 should not be regarded as a limit, as higher limits can also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” should be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0039] As used herein, the term "combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if the order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. Those skilled in the art will understand that, unless the context indicates otherwise, there is generally no limit to the number of items or terms in any combination.

[0040] As used herein, the term "about" is used to indicate a value that includes the inherent variation of error for a composition, for methods for administering the composition, or for variation that exists between study subjects.

[0041] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a large extent. For example, the term "substantially" means that the subsequently described event or circumstance occurs in at least 90% of the cases, or in at least 95% of the cases, or in at least 98% of the cases.

[0042] As used herein, the term "DNA construct" refers to a DNA sequence cloned according to a standard cloning procedure used in genetic engineering, which relocates a DNA segment from its natural location to a different location where it will be reproduced. The cloning process includes cutting and isolating the desired DNA segment, inserting the DNA fragment into a vector molecule, and incorporating the recombinant vector into a cell, where multiple copies or clones of the DNA segment will be replicated. In some embodiments, the DNA construct disclosed herein may include a non-naturally occurring DNA molecule, which may be provided as an isolate or integrated into another DNA molecule, such as in an expression vector or a chromosome of a eukaryotic host cell.

[0043] As used herein, the term "vector" refers to 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 and has a known restriction site and the ability to replicate in a host. In general, the vector may include a promoter, an enhancer, a terminator, an SD sequence, a translation initiation and termination codon, and an origin of replication. If necessary, the vector may also include a selection marker for selecting cells into which the vector has been introduced. Such selection markers include: genes that are resistant to drugs such as ampicillin, tetracycline, kanamycin, chloramphenicol, neomycin, hygromycin, puromycin, and zeocin; markers that allow the use of enzyme activity such as β-galactosidase as an indicator for selection; and markers that allow the use of fluorescence emission as an indicator for selection, such as GFP. Selection markers that are selected using surface antigens such as EGF receptors and B7-2 as indicators can also be used. By using these selection markers, only cells into which the vector has been introduced can be selected, more specifically, only 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. The type of vector used in the present invention is not limited, and any vector can be used. In some embodiments, the vector is selected from the following group: 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, Fusion vector, pTARGET TM - carrier, -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, -vector, pQE-vector, pLEXY-vector, pRNA-vector, pPK-vector, pUMVC-vector, pLIVE-vector, pCRUZ-vector, Duet-vector, and other vectors or their derivatives.

[0044] As used herein, the term "extracellular vesicle" refers to a cell-derived vesicle comprising a membrane surrounding an internal space. Extracellular vesicles include all membrane-bound vesicles, whose diameter is smaller than the diameter of the cell from which they are derived. Typically, the diameter of an extracellular vesicle ranges from 20nm to 1000nm, and may contain various macromolecular cargoes, which are located in the internal space, displayed on the outer surface of the extracellular vesicle and / or span the membrane. The cargo may include small molecules, nucleic acids, proteins, carbohydrates, lipids, small molecules and / or combinations thereof. For example, but not limited to, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles of vesicles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Extracellular vesicles may come from living or dead bodies, transplanted tissues or organs and / or cultured cells.

[0045] As used herein, the term "exosome" refers to a cell-derived nanovesicle comprising a lipid bilayer membrane surrounding an internal space, and produced by the cell through direct plasma membrane budding or through late endosome fusion with the plasma membrane. The exosome comprises lipids or fatty acids and polypeptides, and optionally comprises a therapeutically active payload, a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA), a sugar (e.g., a monosaccharide, a polysaccharide or a polysaccharide) or other molecules. The exosome may be derived from a production cell and separated from the production cell based on its size, density, biochemical parameters or a combination thereof. Exosomes are a type of extracellular vesicle.

[0046] As used herein, the term "surface engineered extracellular vesicle" refers to an extracellular vesicle whose membrane is modified in its composition. For example, the density of the scaffold protein or peptide of the surface engineered extracellular vesicle on the surface of the extracellular vesicle may be higher than (or lower than) the corresponding density of the naturally occurring extracellular vesicle. According to an embodiment of the present invention, the surface engineered extracellular vesicle may be produced by a genetically engineered production cell or its progeny. For example, the surface engineered extracellular vesicle may be produced by a cell transformed or transfected by an exogenous sequence or DNA construct encoding a scaffold protein or peptide. In some embodiments, the production cell may be a cell transformed or transfected by an exogenous sequence or DNA construct encoding a scaffold protein or peptide and an exogenous sequence or DNA construct encoding a therapeutically active payload. In some embodiments, the exogenous sequence or DNA construct encoding a scaffold protein or peptide and the exogenous sequence or DNA construct encoding a therapeutically active payload can be introduced into the production cell by different vectors. In some embodiments, the exogenous sequence or DNA construct encoding a scaffold protein or peptide and the exogenous sequence or DNA construct encoding a therapeutically active payload can be introduced into the production cell by the same vector. In some embodiments, the scaffold protein or peptide and the therapeutically active payload can be a fusion protein. In some embodiments, the surface engineered extracellular vesicle can also include a targeting moiety, which can be used to target the extracellular vesicle to the desired organ, tissue or cell. Non-limiting examples of the targeting moiety include antibodies, antigen-binding fragments of antibodies, antigen-binding variants of antibodies, antigen-binding fragments of antigen-binding variants of antibodies, and antigen-binding variants of antigen-binding fragments of antibodies. In some embodiments, the surface engineered extracellular vesicle according to the embodiments of the present invention has better characteristics than surface engineered extracellular vesicles known in the art. For example, the surface engineered extracellular vesicle produced by the cell introducing the exogenous sequence or DNA construct encoding the scaffold protein or peptide of the present invention has a higher density of scaffold proteins or peptides on the extracellular vesicle surface compared with surface engineered extracellular vesicles known in the art (for example, using conventional extracellular vesicle proteins such as PTGFRN produced extracellular vesicles).

[0047] As used herein, the term "production cell" or "host cell" refers to a cell used to produce extracellular vesicles or surface engineered extracellular vesicles. Production cells include, but are not limited to, cells known to effectively produce extracellular vesicles, such as HEK293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and mesenchymal stem cells (MSC). The production cell may be transformed or transfected by one or more vectors containing exogenous sequences or DNA constructs. In some embodiments of the present invention, the production cell may be transformed or transfected by a single vector containing an exogenous sequence or DNA construct encoding a scaffold protein or peptide of the present invention. In some embodiments, the production cell may be transformed or transfected by a single vector containing an exogenous sequence or DNA construct encoding a scaffold protein or peptide of the present invention and an exogenous sequence or DNA construct encoding a therapeutically active payload. In some embodiments, the production cell may be transformed or transfected by a vector containing an exogenous sequence or DNA construct encoding a scaffold protein or peptide of the present invention and another vector containing an exogenous sequence or DNA construct encoding a therapeutically active payload. In some embodiments, the production cell may be transformed or transfected with at least one additional exogenous sequence or DNA construct encoding another protein or peptide (e.g., a targeting moiety). The additional exogenous sequence may be introduced into a vector containing an exogenous sequence or DNA construct encoding a scaffold protein or peptide of the present invention, an exogenous sequence or DNA construct encoding a therapeutically active payload, or both. In some embodiments, an exogenous sequence or DNA construct encoding a therapeutically active payload, an additional exogenous sequence or DNA construct encoding another protein or peptide, or both may be introduced into the production cell to regulate the endogenous gene expression of the production cell. In some embodiments, an exogenous sequence or DNA construct encoding a therapeutically active payload, an additional exogenous sequence or DNA construct encoding another protein or peptide, or both may be introduced into the production cell to produce an extracellular vesicle containing the therapeutically active payload, another protein or peptide, or both surface-engineered extracellular vesicles on the surface of the extracellular vesicle.

[0048] As used herein, the term "scaffold", "scaffold protein" or "scaffold peptide" refers to a protein or peptide that can target the surface of an extracellular vesicle. In some embodiments, the scaffold protein or peptide may be located or positioned on or contained in the membrane of an extracellular vesicle. Scaffold proteins or peptides known in the art include tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosomal associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI anchor protein, lactadherin, synaptotagmin, apoptosis linker gene 2 interacting protein X (ALIX), adapter protein (syntenin), PTGFRN, fragments or variants thereof, variants of fragments, and fragments of variants. The scaffold protein or peptide according to an embodiment of the present invention comprises an amino acid sequence of GaSb-X1-c-X2, wherein X1 represents G, A, S or T; X2 represents G or S; a represents 3-4 amino acids; b represents 2-3 amino acids; c represents 6-7 amino acids, wherein G represents glycine, S represents serine, A represents alanine, and T represents threonine. In some embodiments of the present invention, the scaffold may be a non-mutated protein or peptide (i.e., a protein or peptide that naturally targets 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 may be a mutant protein or peptide (i.e., a protein or peptide modified to target 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 may be fused to another part, including, for example, a flag tag, a therapeutic peptide, a targeting moiety, and the like. In some embodiments, the scaffold may include a transmembrane protein, a peripheral protein, or a soluble protein. In some embodiments, the scaffold may be attached to the membrane of the extracellular vesicle via a linker.

[0049] The scaffold, fragment of the scaffold, variant of the scaffold, fragment of the variant of the scaffold, and variant of the fragment of the scaffold according to the embodiments of the present invention have the ability to specifically target the surface of the extracellular vesicle. In some embodiments, the scaffold, fragment of the scaffold, variant of the scaffold, fragment of the variant of the scaffold, and variant of the fragment of the scaffold according to the embodiments of the present invention can be located or positioned on or contained in the membrane of the extracellular vesicle / in. As used herein, the term "fragment" of a protein, peptide, or nucleic acid refers to a segment of the protein, peptide, or nucleic acid. As used herein, the term "variant" of a protein, peptide, or nucleic acid refers to a protein, peptide, or nucleic acid having at least one amino acid or nucleotide 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 in the protein, peptide, or nucleic acid. The term can be used interchangeably with the term "mutant".

[0050] According to some embodiments of the present invention, the fragments of the scaffold can retain 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 extracellular vesicles. According to some embodiments of the present invention, the variants of the scaffold can retain 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 extracellular vesicles. The fragments of the variants of the scaffold can retain 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 specifically target extracellular vesicles. The variants of the fragments of the scaffold can retain 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 specifically target extracellular vesicles.

[0051] According to some embodiments of the present invention, the scaffold variants may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity with the scaffold. According to some embodiments of the present invention, the scaffold fragment variants may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity with the scaffold fragment.

[0052] As used herein, the terms "target protein" and "target peptide" are used interchangeably, and refer to a protein or peptide of interest to be delivered, expressed or introduced into the surface, surface or interior of the membrane of an extracellular vesicle. In some embodiments, the target protein or target peptide can be delivered, expressed or introduced into the surface, surface or interior of the membrane of an extracellular vesicle by fusion with 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 a scaffold protein or scaffold peptide. In some embodiments, the target protein or target peptide can be fused to a scaffold protein or scaffold peptide by a linker peptide. In some embodiments, the target protein or target peptide can be a therapeutic protein, an antigen, a cytokine, a ligand, a receptor, an immunoglobulin, a marker polypeptide (e.g., a marker protein, such as green fluorescent protein, or an enzyme), an enzyme, an ion channel, etc., or a portion thereof. In some embodiments, the target protein or target peptide can be a therapeutic molecule or a bioactive molecule.

[0053] As used herein, the terms "biologically active molecule" and "therapeutic molecule" are used interchangeably and refer to an agent that is active in a biological system (e.g., a cell or a human subject), including but not limited to a protein, polypeptide or peptide, including but not limited to a structural protein, an enzyme, a cytokine (e.g., an interferon and / or an interleukin) antibiotic, a polyclonal or monoclonal antibody or an effective portion thereof, such as an Fv fragment, which antibody or a portion thereof may be natural, synthetic or humanized, a peptide hormone, a receptor, a signaling molecule or other protein; a nucleic acid as defined below, including but not limited to an oligonucleotide or a modified oligonucleotide, an antisense oligonucleotide or a modified antisense oligonucleotide, a cDNA, a genomic DNA, an artificial or natural chromosome (e.g., a yeast artificial chromosome) or a portion thereof, an RNA, including an mRNA, a tRNA, an rRNA, or a ribozyme, or a peptide nucleic acid (PNA); a virus or a virus-like particle; a nucleotide or ribonucleotide or a synthetic analog thereof, which may be modified or unmodified; an amino acid or an analog thereof, which may be modified or unmodified; a non-peptide (e.g., a steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. In certain aspects, the bioactive molecule includes a therapeutic molecule (e.g., an antigen), a targeting moiety (e.g., an antibody or its antigen-binding fragment), an adjuvant, an immunomodulator, or any combination thereof. In some embodiments, the bioactive molecule includes a macromolecule (e.g., a protein, an antibody, an enzyme, a peptide, a DNA, an RNA, or any combination thereof). In some embodiments, the bioactive molecule includes a small molecule (e.g., an antisense oligomer (ASO), a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), siRNA, STING, a drug, or any combination thereof). In some embodiments, the bioactive molecule is exogenous to the extracellular vesicle, i.e., it is not naturally present in the extracellular vesicle. In some embodiments, the bioactive molecule or therapeutic molecule may be a therapeutic protein or a therapeutic peptide.

[0054] As used herein, the term "joint" refers to any molecular structure that can conjugate a peptide or protein to another molecule (e.g., different peptides or proteins, small molecules, etc.). Suitable joints are well known to those skilled in the art, and include, but are not limited to, straight or branched carbon joints, heterocyclic carbon joints, or peptide joints (see, e.g., Chen et al., Advanced Drug Delivery Reviews, 2013, Vol. 65: 10, pp. 1357-1369). The joint can be connected to the carboxyl and amino terminal amino acids through its terminal carboxyl or amino group or through its reactive side chain groups. In addition, in some embodiments, the joint can be divided into a flexible joint or a rigid joint, and they can be cleavable (e.g., comprising one or more protease cleavable sites, which can be located in the joint sequence or at either end of the joint sequence. The joint).

[0055] As used herein, the term "payload" refers to a preparation that can act on a target (e.g., a target cancer cell) in contact with an extracellular vesicle. In some embodiments, the payload can be introduced into an extracellular vesicle. In some embodiments, the payload can be introduced into a production cell. Non-limiting examples of the payload include nucleotides, nucleic acids (e.g., DNAmRNA, miRNA, dsDNA, lncRNA, and siRNA), amino acids, polypeptides, lipids, carbohydrates, and small molecules. In a preferred embodiment, the payload can be a therapeutic or bioactive agent.

[0056] As used herein, the terms "isolate" in various grammatical forms or "purify" in various grammatical forms and "extract" in various grammatical forms are used interchangeably and refer to the state of a desired extracellular vesicle preparation (e.g., a plurality of known or unknown amounts and / or concentrations) that has undergone one or more purification processes, such as selection or enrichment of a desired extracellular vesicle preparation. In some embodiments, as used herein, isolation or purification is the process of removing, partially removing (e.g., a portion) extracellular vesicles from a sample containing production cells. In some embodiments, the isolated extracellular vesicle composition has no detectable adverse activity, or alternatively, the level or amount of adverse activity is at or below an acceptable level or amount. In other embodiments, the isolated extracellular vesicle composition has an amount and / or concentration of desired extracellular vesicles that is equal to or higher than an acceptable amount and / or concentration. In other embodiments, the isolated extracellular vesicle composition is enriched compared to the starting material (e.g., production cell preparation) from which the composition is obtained. Compared to the starting material, such enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.999%, 99.9999%, or greater than 99.9999%. In some embodiments, the isolated extracellular vesicle preparation is substantially free of residual biological products. In some embodiments, the isolated extracellular vesicle preparation is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological substances. Residual biological products can include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products can also mean that the extracellular vesicle composition contains no detectable production cells and only extracellular vesicles can be detected.

[0057] As used herein, the term "pharmaceutically acceptable" refers to compounds and compositions that are suitable for administration to humans and / or animals without producing adverse side effects such as toxicity, irritation and / or allergic response and with a reasonable benefit / risk ratio.

[0058] As used herein, the term "biological activity" refers to the ability to alter the physiological system of an organism without regard to how the active agent exerts its physiological effect.

[0059] As used herein, the terms "subject" and "patient" are used interchangeably and should be understood to cover 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.

[0060] As used herein, the term "treat," in its various grammatical forms, refers to methods of prophylactically and / or therapeutically alleviating, alleviating, or ameliorating symptoms of a disease or condition, preventing other symptoms, ameliorating or preventing underlying metabolic causes of symptoms, inhibiting a disease or condition, arresting the development of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, ameliorating symptoms caused by a disease or condition, or terminating symptoms of a disease or condition.

[0061] As used herein, the term "administering" composition in various grammatical forms refers to providing the composition to an object 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 other therapeutic agents. The method of administering such a composition may include, but is not limited to, intravenous administration, inhalation, oral administration, rectal administration, parenteral administration, intravitreal administration, subcutaneous administration, intramuscular administration, intranasal administration, skin administration, topical administration, ocular administration, oral administration, tracheal administration, bronchial administration, sublingual administration, or ocular administration.

[0062] As used herein, the terms "therapeutic composition" and "pharmaceutical composition" refer to a composition containing an active agent that can be administered to a subject by other means known in the art or contemplated herein, wherein the administration of the composition produces a therapeutic effect as described elsewhere herein. In addition, the compositions of the present disclosure can be designed to provide delayed, controlled, extended and / or sustained release using formulation techniques well known in the art. The compositions of the present disclosure can be administered by known pharmaceutical formulations, including tablets, pills, capsules, liquids, inhalants, nasal spray solutions, suppositories, solutions, gels, emulsions, ointments, eye drops, ear drops, etc. As used herein, the term "effective amount" or "therapeutically effective amount" refers to the administration of a sufficient amount of the active ingredient described herein, which will alleviate one or more symptoms of the disease or condition being treated to a certain extent. The result can be a reduction and / or alleviation of the signs, symptoms or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising the surface engineered exosomes disclosed herein required to provide a clinically significant reduction in disease symptoms. For a patient, the effective amount depends 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 concurrent treatment (if any), the specific formulation used, etc. Therefore, it is impossible to specify an exact effective amount in advance. However, one of ordinary skill in the art can determine the effective amount in a given situation using routine experiments based on the information provided herein.

[0063] On the one hand, the present invention provides a DNA construct comprising a DNA sequence encoding a scaffold peptide, wherein the amino acid sequence of the scaffold peptide includes a sequence represented by GaSb-X1-c-X2, wherein X1 represents G, A, S or T; X2 represents G or S; a represents 3-4 amino acids; b represents 2-3 amino acids; c represents 6-7 amino acids; G represents glycine; S represents serine; A represents alanine; and T represents threonine.

[0064] In some embodiments, in the sequence of GaSb-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.

[0065] In some embodiments, GaSb-X1-c-X2 may not be GVGLSTVIGLLSCLIG.

[0066] In some embodiments, the sequence GaSb-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. In addition, for example, a can represent 4 amino acids, b can represent 2 amino acids, and c can represent 6 amino acids. Furthermore, for example, a can represent 3 amino acids, b can represent 2 amino acids, and c can represent 7 amino acids.

[0067] In some embodiments, amino acids a, b and c may include V, G, L, I, A, T, S, C, F, W, Y and P, wherein 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.

[0068] In some embodiments, a may represent 3-4 amino acids selected from V, G, L, I and A, wherein V represents valine, G represents glycine, L represents leucine, I represents isoleucine, and A represents alanine. Non-limiting examples of the 3-4 amino acids include: 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,AVI,AVT,AGV,AGL,AGI,AGT,ALV,ALG,ALI,AL T, 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.

[0069] In some embodiments, b can represent 2-3 amino acids selected from V, I, A and T, wherein V represents valine, I represents isoleucine, A represents alanine, and T represents threonine. Non-limiting examples of the 2-3 amino acid sequence include: 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.

[0070] In some embodiments, c may represent 6-7 amino acids selected from L, S, C and I, wherein L represents leucine, S represents serine, C represents cysteine, and I represents isoleucine. Non-limiting examples of the 6-7 amino acids include: 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, LLSLCI, LILSLIC, LILCSLI, LILCSIL, LILCISL, LILCILS, LILSICL, LILSILC, LISLCLI, LISLCIL, LISLLCI, LISLLIC, LISLCLI, and LISLCII.

[0071] In some embodiments, the sequence GaSb-X1-c-X2 can be one of the amino acid sequences represented by ESM SEQ ID NO: 1-14.

[0072] (ESM SEQ ID NO:1)GVGLSTVIGLLSCLIG

[0073] (ESM SEQ ID NO:2)GIGLSTVIGLLSCLIG

[0074] (ESM SEQ ID NO:3)GVGLSAVIGLLSCLIG

[0075] (ESM SEQ ID NO:4)GIGLSAVIGLLSCLIG

[0076] (ESM SEQ ID NO:5)GILLSAVIGLLSCLIG

[0077] (ESM SEQ ID NO:6)GIGLSLVIGLLSCLIG

[0078] (ESM SEQ ID NO:7)GIGLSAVIGLLLCLIG

[0079] (ESM SEQ ID NO:8)GIGLSAVIGLLSLLIG

[0080] (ESM SEQ ID NO:9)GIGLSAVIALLSCLIG

[0081] (ESM SEQ ID NO:10)GIGLSAVISLLSCLIG

[0082] (ESM SEQ ID NO:11)GIGLSAVITLLSCLIG

[0083] (ESM SEQ ID NO:12)GIGLSAVIGLLSCLIS

[0084] (ESM SEQ ID NO:13)GIGLASVIGLLSCLIG

[0085] (ESM SEQ ID NO:14)GIGLSAVGILLSCLIG

[0086] Non-limiting examples of the sequence GaSb-X1-c-X2 include:

[0087] [Table 1]

[0088]

[0089]

[0090]

[0091]

[0092] In some embodiments, the scaffold peptide may further comprise KYPLLI at the N-terminus of the sequence GaSb-X1-c-X2, wherein K represents lysine, Y represents tyrosine, P represents proline, L represents leucine, and I represents isoleucine.

[0093] In some embodiments, the scaffold peptide may also include FKYPLLI, AFKYPLLI, NAFKYPLLI, LNAFKYPLLI, VLNAFKYPLLI or DVLNAFKYPLLI at the N-terminus of the sequence GaSb-X1-c-X2, wherein 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.

[0094] In some embodiments, the scaffold peptide may further comprise YCSS at the C-terminus of the sequence GaSb-X1-c-X2, wherein Y represents tyrosine, C represents cysteine, and S represents serine.

[0095] In some embodiments, the scaffold peptide may 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, wherein 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.

[0096] In some embodiments, the scaffold peptide may further comprise YCSSHWC at the C-terminus of the sequence GaSb-X1-c-X2, wherein Y represents tyrosine, C represents cysteine, S represents serine, H represents histidine, and W represents tryptophan.

[0097] In some embodiments, the scaffold peptide can be one of the amino acid sequences shown in SEQ ID NOs: 101-142.

[0098] In some embodiments, the DNA construct may further comprise a DNA sequence encoding an amino acid sequence of a target protein. In some embodiments, the target protein may be a therapeutic protein. In some embodiments, the target protein may be fused to a scaffold peptide.

[0099] On the other hand, the present invention provides a vector comprising the above-mentioned DNA construct. The vector can be a plasmid, a phage, a virus, an artificial chromosome, etc. Typical examples include plasmids, such as those from commercially available plasmids, particularly pUC, pcDNA, pBR, etc. Other examples are vectors from viruses, such as replication-defective retroviruses, adenoviruses, AAV, baculoviruses, or vaccinia viruses. Those skilled in the art can adjust the selection of the vector according to the recombinant host cell in which the vector should be used. Without limiting the scope of the present invention, for example, a vector that can transfect or infect mammalian cells can be selected.

[0100] On the other hand, the invention provides host cells comprising the above-mentioned vector. In some embodiments, the host cell can produce the extracellular vesicle comprising the above-mentioned scaffold peptide on its surface. The cell can be cultured and maintained in any suitable culture medium, such as RPMI, DMEM, etc. The culture can be carried out in any suitable device, such as culture plate, culture dish, culture tube, culture bottle, etc. The vector can be introduced into the host cell by any conventional method, such as by naked DNA technology, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated infection, physical or chemical agent or treatment, electroporation, etc. In this regard, it should be noted that transient transfection is enough to express the gene (i.e., DNA construct of the present invention), so it is not necessary to set up a stable cell line or optimize transfection conditions.

[0101] On the other hand, the present invention provides an extracellular vesicle comprising a scaffold peptide encoded by the DNA construct of the present invention described above. In some embodiments, the extracellular vesicle can be surface engineered. In some embodiments, the surface engineered and / or lumen engineered extracellular vesicle can be produced by chemical and / or physical methods, such as PEG induced fusion and / or ultrasonic fusion. In other embodiments, the surface engineered extracellular vesicle is produced by genetic engineering. The extracellular vesicle produced by the progeny of genetically modified production cells or genetically modified cells can contain a modified membrane composition. In some embodiments, the progeny of the genetically modified production cells or genetically modified cells can include one or more exogenous proteins (peptides) that are not naturally present in the cell. In some aspects, the one or more exogenous proteins can be scaffold proteins or peptides, such as scaffold peptides disclosed herein. In some embodiments, the surface engineered extracellular vesicles can have a higher density of scaffold peptides disclosed herein compared to other scaffold proteins or peptides, such as four transmembrane protein molecules (e.g., CD63, CD81, CD9, etc.), lysosomal associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI anchor protein, lactadherin, synaptotagmin, apoptosis linker gene 2 interacting protein X (ALIX), adapter protein (syntenin), PTGFRN, fragments or variants thereof, variants of the fragments, and fragments of the variants. For example, the surface engineered extracellular vesicles can be produced by host cells or production cells transformed with exogenous sequences encoding DNA constructs disclosed herein. Extracellular vesicles containing peptides or proteins expressed by exogenous sequences (e.g., DNA constructs described herein) can include modified membrane protein compositions.

[0102] In some embodiments, the scaffold peptides described herein that are capable of anchoring cargo or target proteins (or peptides), such as exogenous bioactive molecules (such as those disclosed herein), can be used to construct surface-engineered extracellular vesicles.

[0103] Fusion proteins can also be included on the surface of the extracellular vesicles; for example, with affinity tags (e.g., His tags, GST tags, glutathione-S-transferase, S-peptide, HA, Myc, FLAG TM The scaffold peptides described herein fused with AMP (Sigma-Aldrich Co.), MBP, SUMO and Protein A) can be used to purify or remove the surface engineered extracellular vesicles using a binding agent specific for the affinity tag.

[0104] Fusion proteins with therapeutic activity can also be used to produce surface engineered extracellular vesicles. Therefore, in some embodiments, the extracellular vesicles disclosed herein can be engineered or modified to express fusion proteins, and can be used to deliver one or more (e.g., two, three, four, five or more) therapeutic molecules to a target. For example, the fusion protein may include a scaffold peptide and a therapeutic substance (e.g., a peptide or protein) as described herein. In some embodiments, the therapeutic substance can be directly fused to the scaffold peptide as described herein. In some embodiments, the therapeutic substance can be anchored to the scaffold peptide as described herein via a joint.

[0105] In some embodiments, the linker may be a peptide linker. In some embodiments, the peptide linker may comprise at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids. In some embodiments, the peptide linker may be synthetic, i.e., non-naturally occurring. In some embodiments, the peptide linker may include a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) comprising an amino acid sequence that connects or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence that is not naturally connected or genetically fused to it in nature. For example, in some embodiments, the peptide linker can include a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation such as an addition, substitution, or deletion). The linker can be easily cleaved (a "cleavable linker"), thereby facilitating the release of an exogenous bioactive molecule. In some embodiments, the linker can include a non-cleavable linker.

[0106] In some embodiments, the bioactive molecule (e.g., therapeutic peptide or protein) can be selected from natural peptides, recombinant peptides, synthetic peptides and therapeutic material joints. The therapeutic material can be a nucleotide, an amino acid, a lipid, a carbohydrate or a small molecule. The therapeutic peptide can be an antibody, an enzyme, a ligand, a receptor, an 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 phage coat protein. In some embodiments, the nucleic acid binding protein can additionally include one or more RNA or DNA molecules. The one or more RNAs can be miRNA, siRNA, antisense oligonucleotides, phosphorodiamidate morpholino oligomers (PMO), peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO), guide RNA, lincRNA, mRNA, antisense RNA, dsRNA or any combination thereof. In some embodiments, the bioactive molecule can be part of a protein-protein interaction system. In some embodiments, the bioactive molecule that can be anchored to the scaffold peptide described herein and expressed on the surface of an extracellular vesicle can include an antigen. In certain embodiments, the antigen can include a tumor antigen. Non-limiting examples of tumor antigens include: 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, α-folate receptor, CE7R, IL-3, testicular cancer antigen (CTA), MART-1gp100, TNF-related apoptosis-inducing ligand, Brachyury (antigen preferentially expressed in melanoma (PRAME)), and any combination thereof. In some embodiments, the antigen may be derived from bacteria, viruses, fungi, protozoa, or any combination thereof. In some embodiments, the antigen may be derived from an oncogenic virus.In a further embodiment, the antigen may be derived from the following group: human gammaherpesvirus 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 virus and Ebola virus), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium species (e.g., Plasmodium vivax and Plasmodium falciparum), Chikungunya virus, human papillomavirus (HPV), hepatitis B, hepatitis C, human herpes virus 8, herpes simplex virus 2 (HSV2), Klebsiella, Pseudomonas aeruginosa, Enterococcus, Proteus, Enterobacter, Actinomyces, coagulase-negative staphylococci (CoNS), Mycoplasma, and any combination thereof.

[0107] Non-limiting examples of other suitable biologically active molecules include pharmacologically active drugs and genetically active molecules, including anti-tumor agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modulators, and neuroactive agents. Examples of suitable payloads for therapeutic agents include those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; 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 Blood-Forming organs; Hormones and HormoneAntagonists;Vitamins, Dermatology;And Toxicology, all of which are incorporated herein by reference. Suitable payloads also include toxins, biological and chemical warfare agents, such as described in Somani, SM (ed.), ChemicalWarfare Agents, Academic Press, New York (1992)).

[0108] In some embodiments, a fusion protein with a targeting portion can be used. For example, a fusion protein can include a scaffold peptide and a targeting portion as described herein. The targeting portion can be used to target the extracellular vesicle to a specific organ, tissue or cell to treat with the extracellular vesicle. In certain embodiments, the targeting portion can be combined with a marker (or target molecule) expressed on a cell or cell population. In certain embodiments, the marker can be expressed on a variety of cell types, such as on all antigen presenting cells (e.g., dendritic cells, macrophages and B lymphocytes). In some embodiments, the marker can be expressed only on a specific cell population (e.g., dendritic cells). Non-limiting examples of markers expressed on specific cell populations (e.g., dendritic cells) include C-type lectin domain family 9 member A (CLEC9A) protein, dendritic cell-specific intercellular adhesion molecule-3-binding non-integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immune receptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialoglycoprotein receptor (DC-ASGPR), DC immune receptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), and any combination thereof. In some embodiments, the targeting moiety may be an antibody or an antigen-binding fragment thereof. Antibodies and antigen-binding fragments thereof include whole antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies and fragments thereof, and may also include single-chain antibodies, humanized antibodies, murine 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(ab1)2, Fv, dAb and Fd fragments), diabodies, and antibody-related polypeptides. Antibodies and antigen-binding fragments thereof may include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0109] In some embodiments, the extracellular vesicles can encapsulate a target protein (eg, a therapeutic protein or a therapeutic substance, such as nucleotides, amino acids, lipids, carbohydrates, small molecules, and any combination thereof).

[0110] In some embodiments, the extracellular vesicles described herein exhibit superior properties compared to extracellular vesicles known in the art. For example, the extracellular vesicles produced using the scaffold peptides described herein contain modified proteins, and the enrichment of these proteins on their surface is higher than that of the extracellular vesicles in the prior art (e.g., extracellular vesicles produced using conventional exosome proteins). In some embodiments, the expression level of the modified protein is increased (i.e., enriched) by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to the corresponding protein expression using conventional exosome proteins. In addition, in some embodiments, the biological activity of the extracellular vesicles disclosed herein is greater than the biological activity of the extracellular vesicles known in the art. For example, surface engineered extracellular vesicles comprising therapeutic or biologically relevant exogenous sequences fused to scaffold peptides as described herein can have more desired engineered properties than fusions with scaffolds known in the art. Examples of scaffold proteins known in the art include, but are not limited to, tetraspanins (e.g., CD63, CD81, CD9, etc.), lysosomal associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI anchors, lactadherins, synaptotagmins, apoptosis linker gene 2 interacting protein X (ALIX), syntenin, PTGFRN, fragments or variants thereof, variants of the fragments, fragments of the variants, and peptides with affinity for these proteins or their fragments.

[0111] In some embodiments, surface engineered extracellular vesicles comprising fusion proteins containing exogenous sequences (e.g., encoding exogenous biologically active molecules such as antigens, adjuvants, targeting moieties and / or immunomodulators) and scaffold peptides described herein have a higher fusion protein density than similarly engineered extracellular vesicles comprising exogenous sequences conjugated to conventional extracellular vesicle proteins known in the art (e.g., CD9, CD63, CD81, PDGFR, GPI anchors, lactadherin LAMP2, LAMP2B, synaptotagmin, ALIX, adaptor proteins, PTGFRN, fragments or variants thereof, variants of said fragments, and fragments of said variants, or peptides bound thereto). In some embodiments, the density of fusion proteins containing the scaffold peptides described herein on the surface of extracellular vesicles is about 2 times, about 4 times, about 8 times, about 16 times, about 32 times, about 64 times, about 100 times, about 200 times, about 400 times, about 800 times, about 1,000 times or more higher than that of fusion proteins on the surface of other extracellular vesicles similarly modified using conventional extracellular vesicle proteins.

[0112] In some embodiments, the extracellular vesicles described herein can be isolated from a host cell or a production cell comprising a vector described herein. When the extracellular vesicles are produced from in vitro cell culture, various production cells, such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSC), HT-1080 cells, MB-231 cells, Raji cells, PER.C6 cells and CAP cells, can be used for the present disclosure. A non-limiting example of the host or production cell is a HEK293 cell.

[0113] The production cell (or host cell) may be genetically modified to contain one or more exogenous sequences to produce surface engineered extracellular vesicles. In some embodiments, the one or more exogenous sequences may encode the scaffold peptides described herein. In some embodiments, the one or more exogenous sequences may encode the exogenous biologically active molecules described herein. In some embodiments, the one or more exogenous sequences may encode the scaffold peptides described herein and the exogenous biologically active molecules described herein. The genetically modified production cell may contain exogenous sequences introduced by transient or stable transformation. The exogenous sequence may be introduced into the production cell as a plasmid. The exogenous sequence may be stably integrated into the genomic sequence of the production cell, at a target site or a random site. In some embodiments, a stable cell line may be generated to produce surface engineered extracellular vesicles. Exogenous sequences encoding the scaffold peptides described herein may be introduced to produce surface engineered extracellular vesicles containing the scaffold peptides. Exogenous sequences encoding affinity tags may be introduced to produce surface engineered extracellular vesicles containing fusion proteins, the fusion proteins comprising affinity tags attached to the scaffold peptides. As described herein, in some embodiments, exogenous sequences encoding exogenous biologically active molecules can be introduced to produce surface-engineered extracellular vesicles containing fusion proteins comprising the exogenous biologically active molecule attached (e.g., directly attached or attached through a linker) to a scaffold peptide.

[0114] In some embodiments, the production cell (or host cell) can be further modified to include additional exogenous sequences. For example, additional exogenous sequences can be introduced to regulate endogenous gene expression, or to produce extracellular vesicles including a certain polypeptide as a payload. In some embodiments, the production cell can be modified to include two exogenous sequences, one encoding a scaffold peptide and the other encoding a payload. In some embodiments, the production cell can be further modified to include additional exogenous sequences that confer additional functions to the extracellular vesicle, such as specific targeting capabilities, delivery functions, enzymatic functions, increased or decreased in vivo half-life, etc. In some embodiments, the production cell can be modified to include two exogenous sequences, one encoding a scaffold peptide and the other encoding a protein that confer additional functions to the extracellular vesicle.

[0115] In some embodiments, the production cell (or host cell) may be modified to include two exogenous sequences, each of which encodes a fusion protein on the surface of the extracellular vesicle. In some embodiments, the surface engineered extracellular vesicles from the production cells have a higher scaffold peptide density than the natural extracellular vesicles isolated from unmodified cells of the same or similar cell type. In some embodiments, the density of the scaffold peptides contained in the surface engineered extracellular vesicles is about 2 times, about 4 times, about 8 times, about 16 times, about 32 times, about 64 times, about 100 times, about 200 times, about 400 times, about 800 times, about 1,000 times or higher than that of the natural extracellular vesicles isolated from unmodified cells of the same or similar cell type.

[0116] More specifically, surface engineered extracellular vesicles can be produced by cells transformed (or transfected) with sequences encoding one or more scaffolds. Any of the one or more scaffold peptides described herein can be expressed in production cells from plasmids, exogenous sequences inserted into the genome, or other exogenous nucleic acids such as synthetic messenger RNA (mRNA).

[0117] 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, the one or more heterologous proteins can be fused to the N-terminus of the scaffold peptide. In some embodiments, the one or more heterologous proteins can be fused to the C-terminus of the scaffold peptide. In some embodiments, the one or more heterologous proteins can be fused to the N-terminus and C-terminus of the scaffold peptide.

[0118] In another aspect, the present invention provides a pharmaceutical composition comprising an extracellular vesicle as described herein and a pharmaceutically acceptable carrier and / or excipient. A pharmaceutically acceptable excipient or carrier may be determined in part by the specific composition administered and the specific method for administering the composition. Therefore, there are a variety of suitable formulations for pharmaceutical compositions comprising numerous extracellular vesicles (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)). The pharmaceutical composition can generally be prepared aseptically and fully complies with all good manufacturing practices (GMP) regulations of the U.S. Food and Drug Administration. In some embodiments, the pharmaceutical composition may comprise one or more therapeutic agents and an extracellular vesicle as described herein. In certain embodiments, the extracellular vesicle may be co-administered with one or more other therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising the extracellular vesicle may be administered before administering the other therapeutic agents. In some embodiments, the pharmaceutical composition comprising the extracellular vesicle may be administered after administering the other therapeutic agents. In some embodiments, the pharmaceutical composition comprising the extracellular vesicle may be administered simultaneously with the other therapeutic agents. In some embodiments, the pharmaceutical compositions described above can be used to prevent, ameliorate or treat diseases, disorders or conditions associated with the nervous, digestive, endocrine, skeletal, respiratory, integumentary, lymphatic, reproductive, muscular, excretory or immune systems.

[0119] In another aspect, the present invention provides a composition comprising the above-mentioned extracellular vesicles as an active ingredient for the preparation of a preparation for preventing, improving or treating diseases, disorders or conditions related to the nervous, digestive, endocrine, skeletal, respiratory, integumentary, lymphatic, reproductive, muscular, excretory or immune systems.

[0120] Embodiments of the present disclosure will be described in detail below in conjunction with the following examples. However, the present disclosure is not limited to the explained examples. On the contrary, the examples are provided to fully convey the concepts of the present disclosure to those skilled in the art to fully and completely introduce the contents introduced herein. Example

[0121] Example 1: Construction of plasmid DNA

[0122] The DNA sequence encoding the full-length PTGFRN protein is fused to the DNA sequence encoding the mutant SIRP-α protein to prepare a plasmid DNA, i.e., a K-SIRPα-full-length PTGFRN vector, as described in U.S. Pat. No. 11,319,360, which is incorporated herein by reference. The DNA sequence encoding the transmembrane domain (TMD) of the PTGFRN protein is fused to the DNA sequence encoding the mutant SIRP-α protein to prepare a plasmid DNA, i.e., a K-SIRPα-PTGFRN TMD (V1) vector. See Figure 1 .

[0123] Prepare a DNA sequence encoding the TMD of the PTGFRN protein (i.e., WT) and a DNA sequence encoding a therapeutic protein such as a mutant SIRP and a mature form of EGF. Replace at least one amino acid of the TMD with another amino acid to prepare another mutant TMD. More specifically, an amino acid sequence of a variant TMD in which the amino acid T at position 11 is replaced by the amino acid A (i.e., mV1(T11A)), an amino acid sequence of a variant TMD in which the amino acid V at position 7 is replaced by the amino acid I (i.e., mV1(V7I)), an amino acid sequence of a variant TMD in which the amino acid V at position 7 is replaced by the amino acid I and the amino acid T at position 11 is replaced by the amino acid A (i.e., mV1(T11A / V7I)) are prepared. See. Figure 2 and Figure 4 .

[0124] Prepare a DNA sequence encoding a variant TMD (K-SIRPα-mV1 (T11A / V7I)) and a DNA sequence encoding a mutant SIRP-α protein. Replace the DNA sequence encoding the TMD of the K-SIRPα-mV1 (T11A / V7I) plasmid with a DNA sequence encoding the PDGFR TMD of the commercially available pDisplay vector (Thermo Fisher Scientific product catalog V66020) to prepare a plasmid DNA, namely, a K-SIRPα-PDGFR TMD vector. Replace the DNA sequence encoding the K-SIRPα-mV1 (T11A / V7I) signal peptide with a DNA sequence encoding the stabilin-2 signal peptide (MMLQHLVIFCLGLVVQNFCSP) from human STAB2 [NM_017564] to prepare a plasmid DNA, namely, a S-SIRPα-mV1 (T11A / V7I) vector. See Figure 3 .

[0125] According to an embodiment of the present invention, various plasmid DNAs are prepared using commercially available DNA sequences encoding the entire EGF protein. More specifically, the DNA sequence encoding the entire EGF protein (RC210817) is purchased from Origin, Inc. The DNA sequences encoding the pro-region and the fall-off region are removed from the EGF coding region of the RC210817 vector to prepare truncated EGF (tEGF) DNA. The DNA sequences encoding the TMD and CD of tEGF are replaced with the DNA sequences encoding PTGFRN TMD (V1) to prepare plasmid DNA, i.e., EGF-V1 vector. The DNA sequence encoding the TMD of EGF-V1 is replaced with the DNA sequence encoding mV1 (T11A) to prepare plasmid DNA, i.e., EGF-mV1 (T11A) vector. The DNA sequence encoding the TMD of EGF-V1 is replaced with the DNA sequence encoding mV1 (V7I) to prepare plasmid DNA, i.e., 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 a plasmid DNA, namely, EGF-mV1 (T11A / V7I) vector. Figure 4 .

[0126] In the PTGFRN TMD version (V1) sequence, amino acids were added upstream (i.e., DVLNAF) and downstream (i.e., HWCCKKEVQETRRERRRLMSMEMD) to prepare PTGFRN TMD version 2 (V2). In the PTGFRN TMD version (V1) sequence, amino acids were added upstream (i.e., DVLNAF) and downstream (i.e., HWC) to prepare PTGFRN TMD version 3 (V3). The DNA sequences encoding the TMD and CD of tEGF were replaced with the DNA sequences encoding mV2 (V7I) or mV3 (V7I) to prepare recombinant plasmid DNA, i.e., EGF-mV2 (V7I) or EGF-mV3 (V7I) vectors. 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 truncated EGF plasmid DNA, i.e., tEGF-substituted CD vector. See Figure 5 and Figure 6 .

[0127] At least one amino acid of the TMD (extracellular vesicle sorting motif, ESM) of mV1 (T11A / V7I) was replaced with another amino acid to prepare another variant TMD ( Figure 7-11 The amino acid sequence of the variant TMD was prepared by replacing one of the essential amino acids in the ESM encoded by the mV1 (T11A / V7I) DNA sequence with another amino acid ( Fig.12The amino acid sequence of the variant TMD is prepared by deleting or adding one or more amino acids in the ESM encoded by the mV1 (T11A / V7I) DNA sequence ( Figure 13-14 ).See Figure 7-14 .

[0128] Control plasmid (pMX-U6) or plasmids encoding CD9 (pMx-U6-shCD9) or CD81 (pMx-U6-shCD81) were prepared for transfection into 293FT cells stably transduced with K-SIRPα-mV1(T11A / V7I) plasmid. Fig.15 .

[0129] Prepare a DNA sequence K-SIRPα-mV1 (T11A / V7I) encoding a variant TMD and a DNA sequence encoding a mutant SIRP-α protein. Replace the DNA sequence encoding the TMD of K-SIRPα-mV1 (T11A / V7I) with the DNA sequence encoding the PDGFR TMD of the commercially available pDisplay vector (Thermo Fisher Scientific product catalog V66020) to prepare a plasmid DNA, i.e., a K-SIRPα-PDGFR TMD vector. In the K-SIRPα-mV1 (T11A / V7I) sequence, add the upstream (i.e., DVLNAF) and downstream (i.e., HWC) sequences of mV1 (T11A / V7I) to prepare K-SIRPα-mV3 (T11A / V7I). See Fig.16 .

[0130] The above plasmids were The protocol of the Plasmid Maxi kit was used for amplification and separation. 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. Specifically, the microcentrifuge tube containing the mixture of plasmid DNA and competent cells DH5α was heated at 42°C for 45 seconds using a heating block. Subsequently, 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. Then, the microcentrifuge tube was incubated at 37°C for 45 minutes on an oscillator at 200 rpm. After incubation, 100 μl was taken from the microcentrifuge tube and spread on an LB medium plate containing 100 μg / ml ampicillin. All plates were incubated overnight at 37°C. The next day, a colony was taken from the surface of the plate and incubated at 37°C for 8 hours in 3 ml of LB medium containing 100 μg / ml ampicillin. After incubation, 1 ml of the colony and the LB medium mixture containing antibiotics was transferred to a culture flask containing 500 ml of LB / ampicillin medium and incubated at 37°C overnight. The bacterial cells were harvested by centrifugation at 6000 × g for 15 minutes at 4°C, and the bacterial pellet was resuspended in buffer P1 with 100 μg / ml RNase A. Buffer P2 was added, the sealed tube was inverted 4-6 times to mix well, and the resulting mixture was incubated at room temperature for 5 minutes. Cooled buffer P3 was added, and it was immediately inverted 4-6 times to mix well, and the resulting mixture was incubated on ice for 20 minutes. After centrifugation at ≥20,000 × g for 30 minutes at 4°C, the supernatant containing plasmid DNA was immediately collected. After the supernatant was centrifuged again at ≥20,000×g for 15 minutes at 4°C, the supernatant containing plasmid DNA was immediately collected. After the column was emptied by balancing the QIAGEN-tip 500 with buffer QBT and draining by gravity flow, the collected supernatant was applied to the QIAGEN-tip and entered the resin by gravity flow. After washing the QIAGEN-tip with buffer QC, the DNA was eluted with buffer QC. Room temperature isopropanol was added to the eluted DNA to precipitate the eluted DNA. Immediately after mixing, centrifuge at ≥15,000×g for 30 minutes at 4°C and carefully pour off the supernatant. After washing the DNA precipitate with room temperature 70% ethanol and centrifuging at ≥15,000×g for 10 minutes, the supernatant was carefully poured off without stirring the precipitate. After air-drying the precipitate for 5-10 minutes, the final plasmid DNA was redissolved in an appropriate volume of buffer.

[0131] Example 2: Isolation of extracellular vesicles

[0132] HEK293 cells (6×10 6) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. When the cells reached 80-90% confluence, plasmid DNA was transfected into the cells using a transfection agent, or the cells were infected with a retrovirus to generate stable cells.

[0133] In the case of transient transfection, cells are transfected using a transfection agent such as Lipofectamine 2000, Lipofectamine 3000 or polyethyleneimine (PEI). Cell culture medium is replaced with DMEM, and a mixture of DNA and transfection reagent is added to the cells. The cells are then cultured for 24 hours at 37°C, 5% CO2. 24 hours after transfection, the culture medium containing the transfection agent and plasmid is replaced with DMEM with the addition of 10% FBS and 1% antibiotic-antimycotic. The cells of transient transfection are incubated at 37°C, 5% CO2 for 24 hours. After recovery 24 hours, the culture medium is replaced with the DMEM culture medium with the addition of insulin-transferrin-selenium (Gibco). Serum-free cells are incubated at 37°C, 5% CO2 for 48 hours. See, e.g., 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.

[0134] In the case of stable cell production, Plat-E cells were used to produce retroviruses that packaged a retroviral vector containing a DNA sequence of interest and a puromycin resistance gene. More specifically, Plat-E cells (2×10 6) were incubated at 37°C, 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. When the cells reached 80-90% confluence, the cells were transfected with a retroviral vector encoding a DNA sequence of interest using Lipofectamine 2000. After 24 hours, the culture medium was replaced with DMEM supplemented with 10% FBS and incubated for another 24 hours. 48 hours after transfection, the culture medium containing viral particles was collected, centrifuged at 3,000 rpm, filtered with a 0.45 μm filter, and used for 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.

[0135] To isolate extracellular vesicles, cell supernatants were harvested 48 hours after transfection. The supernatant was centrifuged at 300g for 10 minutes, at 2000g for 10 minutes, and at 10,000g for 30 minutes. The supernatant was then filtered and concentrated using a tangential flow filtration (TFF) system or a 100kDa Amicon Ultra-15 centrifugal filter device. Afterwards, the supernatant was centrifuged at 150,000g for 3 hours. The extracellular vesicle precipitate was resuspended with PBS containing a protease inhibitor cocktail and stored at 4°C. 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.

[0136] Example 3: Characterization of surface engineered extracellular vesicles

[0137] The surface engineered extracellular vesicles were characterized by Western Blot assay. More specifically, the amount of total protein in the extracellular vesicles was measured using the bipicolinic acid (BCA) protein assay. A standard solution was prepared and 5 μl of each concentration of bovine serum albumin (2, 1, 0.5, 0.25, 0.125, and 0 mg / ml) was added to a 96-well plate. The extracellular vesicle sample was diluted with PBS and 5 μl of the resulting sample was added to a 96-well plate. Reagent A (500113, Bio-Rad) and S (500114, Bio-Rad) were mixed in a ratio of 50:1. 25 μl of the reagent mixture was added to a 96-well plate. 200 μl of reagent B (500115, Bio-Rad) was added to the 96-well plate and the plate was gently tapped. The sample was incubated away from light for 15 minutes. The amount of protein was measured at a wavelength of 750 nm using a microplate reader. In addition, the number of extracellular vesicles was analyzed using Zetaview. After the comparison test using QC beads, diluted samples were added. 150-200 particles were set for observation and then the number of extracellular vesicles was analyzed.

[0138] The purified extracellular vesicles were added to RIPA buffer with a protease inhibitor cocktail (Calbiochem) to lyse the extracellular vesicles and mixed with SDS-PAGE sample buffer. Equal amounts of extracellular vesicle proteins were subjected to SDS-PAGE electrophoresis. After gel electrophoresis, the bands were transferred to nitrocellulose membranes or methanol-activated polyvinylidene fluoride (PVDF) membranes. After pre-blocking with 5% skim milk dissolved in Tris-buffered saline (TBST) supplemented with Tween-20 for 1 hour at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. CD81, SIRPα, EGF, and actin antibodies were processed to detect protein expression. The membrane was incubated with HRP-conjugated secondary antibodies, and the blots were then probed using the ChemiDoc imaging system (Bio-Rad) ( Figure 1-6 , 8-10 and 12-15).

[0139] Capillary Western blotting was performed to measure protein expression. Extracellular vesicle samples were prepared using EZ Standard Pack 1 (ProteinSimple, 96655). Four diluted proteins were mixed with a 5X Fluorescent Master Mix. Each sample was denatured at 95°C for 5 minutes by a heating block. 3 μl of sample was loaded into the appropriate well of the box. The sample was pre-blocked with Antibody Diluent 2 (ProteinSimple, 95905) for 10 minutes. The appropriate antibody was diluted to the desired concentration and used to detect protein expression. Protein expression was detected with anti-mouse secondary antibody (ProteinSimple, 96113). Samples were analyzed by Compass for SW (ProteinSimple).

[0140] like Figure 1 As shown, to evaluate the expression level of SIRPα protein, which is known for its role in promoting the clearance of pathological cells by phagocytes, complete PTGFRN (PTGFRN has been reported to show efficient protein expression on the surface of EVs) and PTGFRN fragments were used for evaluation. The experimental results showed that the plasmid using mainly the TMD of PTGFRN (K-SIRPα-PTGFRN TMD version 1 (V1)) showed higher protein expression efficiency compared with the complete PTGFRN (K-SIRPα-full-length PTGFRN).

[0141] K-SIRPα-full-length PTGFRN sequence (SEQ ID NO: 143):

[0142]

[0143] K-SIRPα-PTGFRN TMD version 1 (V1) sequence (SEQ ID NO: 144):

[0144] METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGVGLSTVIGLLSCLIGYCSS

[0145] like Figure 2 As shown, in order to obtain a motif with higher protein expression efficiency on the surface of EVs, a randomly mutated PTGFRN TMD version 1 (V1) was used in K-SIRPα-V1. The experimental results showed that a double mutation (K-SIRPα-mV1 (T11A / V7I)) in which the 11th amino acid T of V1 was mutated to A and the 7th amino acid V of V1 was mutated to I exhibited higher protein expression efficiency compared with the single mutation and the basic wild-type PTGFRN TMD (V1).

[0146] K-SIRPα-mV1(T11A) sequence (SEQ ID NO: 145):

[0147] METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGVGLSAVIGLLSCLIGYCSS

[0148] K-SIRPα-mV1(V7I) sequence (SEQ ID NO: 146):

[0149] METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGIGLSTVIGLLSCLIGYCSS

[0150] K-SIRPα-mV1(T11A / V7I) sequence (SEQ ID NO: 147):

[0151] METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGIGLSAVIGLLSCLIGYCSS

[0152] like Figure 3 As shown, in order to verify the superiority of the obtained K-SIRPα-mV1(T11A / V7I), a comparative experiment was performed using PDGFR TMD, which is commonly used for the desired protein expression on the surface of cells and EVs. The experimental results confirmed that K-SIRPα-mV1(T11A / V7I) exhibited higher protein expression efficiency on the EV surface compared with K-SIRPα-PDGFR TMD. In addition, the difference in protein expression efficiency on the EV surface when the Ig-κ signal peptide of K-SIRPα-mV1(T11A / V7I) was replaced by the signal peptide of stabilin-2 protein was also examined. The results showed that mV1(T11A / V7I) can maintain effective protein expression not only when using the Ig-κ signal peptide, but also when using the stabilin-2 signal peptide.

[0153] K-SIRPα-PDGFR TMD sequence (SEQ ID NO:148):

[0154] METDTLLLWVLLLWVPGSTGDYPYDVPDYAGAQPARSMEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNN MDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPVDEQKLISEEDLNAVGQDTQEVIVVPHSLPFKVVISAILALVVLTIISLIILIMLWQKKPR

[0155] S-SIRPα-mV1 (T11A / V7I) sequence (SEQ ID NO: 149):

[0156] MMLQHLVIFCLGLVVQNFCSPGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFKYPLLIGIGLSAVIGLLSCLIGYCSS

[0157] like Figure 4 As shown, to demonstrate the versatility of this motif, the fusion of mature regeneration factor EGF protein with mV1 (T11A / V7I) instead of SIRPα protein was evaluated. The experimental results showed that, similar to SIRPα, EGF also showed better protein expression efficiency when the 11th amino acid T in V1 was mutated to A and the 7th amino acid V in V1 was mutated to I. The double mutant EGF-mV1 (T11A / V7I) showed higher protein expression efficiency compared with the single mutation and basic wild-type PTGFRN TMD (V1).

[0158] EGF-V1 sequence (SEQ ID NO:150):

[0159] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNC VVGYIGERCQYRDLKWWELREFKYPLLIGVGLSTVIGLLSCLIGYCSS

[0160] EGF-mV1(T11A) sequence (SEQ ID NO:151):

[0161] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNC VVGYIGERCQYRDLKWWELREFKYPLLIGVGLSAVIGLLSCLIGYCSS

[0162] EGF-mV1(V7I) sequence (SEQ ID NO:152):

[0163] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNC VVGYIGERCQYRDLKWWELREFKYPLLIGIGLSTVIGLLSCLIGYCSS

[0164] EGF-mV1 (T11A / V7I) sequence (SEQ ID NO: 153):

[0165] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNC VVGYIGERCQYRDLKWWELREFKYPLLIGIGLSAVIGLLSCLIGYCSS

[0166] like Figure 5 As shown, the EGF mutant PTGFRN TMD version 2 (V7I) was found to have higher EGF expression than tEGF, while the EGF mutant PTGFRN TMD version 3 (V7I) had similar or higher EGF expression than EGF-mV2 (V7I). In contrast, both tEGF-substituted CDs lacking the PTGFRNTMD variants showed very low EGF expression efficiency on the EV surface. These findings suggest that the TMD of the PTGFRN protein plays a crucial role in displaying therapeutic proteins on the EV surface.

[0167] tEGF sequence (SEQ ID NO:154):

[0168] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRVIVVAVCVVVLVMLLLLSLWGAHYYRTQKLLSKNPKNPYEESSRDVRSRR PADTEDGMSSCPQPWFVVIKEHQDLKNGGQPVAGEDGQAADGSMQPTSWRQEPQLCGMGTEQGCWIPVSSDKGSCPQVMERSFHMPSYGTQTLEGGVEKPHSLLSANPLWQQRALDPPHQMELTQ

[0169] EGF-mutant PTGFRN TMD version 2 (V7I) sequence (SEQ ID NO: 155):

[0170] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELREFDVLNAFKYPLLIGIGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD

[0171] EGF-mutant PTGFRN TMD version 3 (V7I) sequence (SEQ ID NO: 156):

[0172] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNC VVGYIGERCQYRDLKWWELREFDVLNAFKYPLLIGIGLSTVIGLLSCLIGYCSSHWC

[0173] tEGF replaced CD sequence (SEQ ID NO: 157):

[0174] MLLTLIILLPVVSKFSFVSLSANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRVIVVAVCVVVLVMLLLLSLWGAHYYRTQEFHWCCKKEVQETRRERRRLMSMEMD

[0175] like Figure 6 As shown, higher EGF expression was observed in EGF-mV1(T11A / V7I) and EGF-mV3(V7I) than in EGF-mV2(V7I), which contains the TMD and CD of the PTGFRN protein. This finding suggests that the CD of the PTGFRN protein is not essential for EV sorting or targeting.

[0176] like Figure 7-11 To determine the key sequences for EV surface expression of the desired protein in a variant TMD (ESM, derived from mV1(T11A / V7I)), a plasmid was generated for L by making single mutations in ESM, with the goal of obtaining TMD amino acid sequence patterns that are important for EV sorting of the introduced protein ( Figure 7 ). It was identified that when amino acid G at position 6, S at position 10, G at position 14, G at position 21, or any combination thereof was replaced by other amino acids, SIRPα or EGF protein expression of the tested DNA was significantly reduced. The results indicate that the GSGG pattern is essential for protein EV sorting.

[0177] like Fig.12As shown, in order to verify whether the key amino acids in ESM, especially amino acid G at position 6, amino acid S at position 10, amino acid G at position 14, and amino acid G at position 21, can be replaced by amino acids other than L, the EV sorting effect of the introduced protein was evaluated by changing each key amino acid to four different amino acids. The experimental results showed that G can be present at the amino acid position 6, S can be present at the amino acid position 10, G, A, S, or T can be present at the amino acid position 14, and G or S can be present at the amino acid position 21.

[0178] like Figure 13-14 As shown, in order to evaluate the number of amino acids that may exist between key amino acids in the resulting GSGG sequence, the EV sorting efficiency of the protein was evaluated. The number of amino acids between G at position 6 and S at position 10 was designated as "a", the number of amino acids between S at position 10 and G at position 14 was designated as "b", and the number of amino acids between G at position 14 and G at position 21 was designated as "c". Six different plasmids with different numbers of a, b, and c amino acids were generated compared to the original sequence, and the EV sorting efficiency of the protein was evaluated. The experimental results show that "a" can have 3-4 amino acids, "b" can have 2-3 amino acids, and "c" can have 6-7 amino acids.

[0179] like Fig.15 As shown, it was identified that the expression of SIRPα on EVs obtained from the DNA construct (K-SIRPα-mV1 (T11A / V7I)) in an embodiment of the present invention was significantly reduced after transfection with CD9 or CD81 shRNA. These results indicate that CD9 and CD81 proteins are involved in the EV surface expression mechanism of proteins introduced through ESM.

[0180] like Fig.16 As shown, the effect of adding a small amount of amino acids to mV1 (T11A / V7I) on protein EV sorting efficiency was compared in the presence of ESM. The experimental results showed that both mV1 (T11A / V7I) and mV3 (T11A / V7I) showed excellent SIRPα protein expression efficiency on the EV surface, but mV3 (T11A / V7I) showed a slightly higher EV sorting efficiency.

[0181] K-SIRPα-mV3 (T11A / V7I) sequence (SEQ ID NO: 158):

[0182] METDTLLLWVLLLWVPGSTGDGSEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPEFDVLNAFKYPLLIGIGLSAVIGLLSCLIGYCSSHWC

[0183] Although the present disclosure is described herein in conjunction with certain embodiments so that its various aspects can be more fully understood and appreciated, the present disclosure is not limited to these specific embodiments. On the contrary, the present disclosure is intended to include all alternatives, modifications and equivalents within the scope of the present disclosure defined herein. Therefore, the above-mentioned examples including specific embodiments will be used to illustrate the practice of the inventive concept of the present disclosure, it should be understood that the details shown are only used as examples and for illustrative discussion of specific embodiments, and are intended to provide the description of procedures that are considered to be the most useful and easiest to understand and the principle and concept aspects of the present disclosure.

[0184] The formulations of the various compositions described herein, the methods described herein, or the steps or order of steps of the methods described herein can be modified without departing from the spirit and scope of the present disclosure. In addition, although various embodiments of the present disclosure are described in the claims herein, the present disclosure is not limited to these specific claims.

Claims

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 GaSb-X1-c-X2, in: X1 represents G, A, S or T; X2 represents G or S; a represents 3-4 amino acids; b represents 2-3 amino acids; c represents 6-7 amino acids; G stands for glycine; S stands for serine; A represents alanine; and T stands for threonine.

2. A DNA construct according to claim 1, wherein the sequence GaSb-X1-c-X2 has 15-17 amino acids.

3. The DNA construct of claim 1, wherein the scaffold peptide has 22-57 amino acids.

4. A 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, wherein 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.

5. The DNA construct according to claim 1, wherein a represents 3-4 amino acids selected from V, G, L, I, T and A, wherein V represents valine, G represents glycine, L represents leucine, I represents isoleucine, T represents threonine and A represents alanine.

6. The DNA construct according to claim 5, wherein a represents VGL, IGL, VGLT, IGLT, VGLA or IGLA.

7. The DNA construct according to claim 1, wherein b represents 2-3 amino acids selected from V, I, A and T, wherein V represents valine, I represents isoleucine, A represents alanine, and T represents threonine.

8. A DNA construct according to claim 7, wherein b represents VI, AV, TVI or AVI.

9. The DNA construct according to claim 1, wherein c represents 6-7 amino acids selected from L, S, C and I, wherein L represents leucine, S represents serine, C represents cysteine, and I represents isoleucine.

10. The DNA construct according to claim 9, wherein c represents LLSCLI or ILLSCLI.

11. The DNA construct according to claim 1, wherein the sequence GaSb-X1-c-X2 is any one of ESM SEQ ID NOs: 1-100.

12. The DNA construct according to claim 1, wherein the scaffold peptide further comprises KYPLLI at the N-terminus of the sequence GaSb-X1-c-X2, wherein K represents lysine, Y represents tyrosine, P represents proline, L represents leucine, and I represents isoleucine.

13. The DNA construct according to claim 1, wherein the scaffold peptide further comprises DVLNAFKYPLLI at the N-terminus of the sequence GaSb-X1-c-X2, wherein 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.

14. The DNA construct according to claim 1, wherein the scaffold peptide further comprises YCSS at the C-terminus of the sequence GaSb-X1-c-X2, wherein Y represents tyrosine, and C represents cysteine, and S represents serine.

15. The DNA construct according to claim 1, wherein the scaffold peptide further comprises YCSSHWC at the C-terminus of the sequence GaSb-X1-c-X2, wherein Y represents tyrosine, C represents cysteine, S represents serine, H represents histidine, and W represents tryptophan. The DNA construct according to claim 1 , further comprising a DNA sequence encoding an amino acid sequence of a target protein.

17. The DNA construct of 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 location of the extracellular vesicle.

21. Extracellular vesicles comprising a scaffold peptide encoded by a DNA construct according to claim 1.

22. The extracellular vesicle according to claim 20 or 21, wherein the other extracellular peptide comprises CD9, CD63, CD81, PDGFR, PTGFRN, GPI anchor protein, lactadherin, syndecan, synaptotagmin, apoptosis linker gene 2 interacting protein X (ALIX), adaptor protein, LAMP2, LAMP2B, fragments or variants thereof, variants of the fragments and fragments of the variants.

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

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

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

26. The extracellular vesicle of claim 20 or 21, wherein the scaffold peptide comprises an affinity tag having affinity for a binding agent.

27. The extracellular vesicle of claim 20 or 21, wherein the scaffold peptide further comprises a targeting moiety.

28. The extracellular vesicle of claim 20 or 21, wherein the extracellular vesicle further comprises a therapeutic substance.

29. The extracellular vesicle of claim 28, wherein the therapeutic substance is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, small molecules, and any combination thereof.

30. The extracellular vesicle of claim 28, wherein the therapeutic substance is fused with the scaffold peptide and / or encapsulated in the extracellular vesicle.

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

32. The pharmaceutical composition according to claim 31, for preventing, ameliorating or treating diseases, disorders or conditions associated with the nervous, digestive, endocrine, skeletal, respiratory, integumentary, lymphatic, reproductive, muscular, excretory or immune systems.

33. A method for preventing, ameliorating or treating a disease, disorder or condition associated with the nervous, digestive, endocrine, skeletal, respiratory, integumentary, lymphatic, reproductive, muscular, excretory or immune systems, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 31.

34. Use of a composition comprising the extracellular vesicles of claim 20 or 21 as an active ingredient in the preparation of a preparation for preventing, improving or treating diseases, disorders or conditions related to the nervous, digestive, endocrine, skeletal, respiratory, integumentary, lymphatic, reproductive, muscular, excretory or immune systems.

Images