Methods of making and using extracellular vesicles

Abstract

Disclosed herein are methods of enhancing extracellular vesicle production.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. PCT / CN2022 / 102338 filed on June 29, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to methods for the manufacture and application of extracellular vesicles. In particular, the present invention provides methods for enhancing the production of extracellular vesicles in cells and programmable extracellular vesicle engineering. Background Art

[0004] Extracellular vesicles (EVs) are of natural origin and are secreted by all cells of both prokaryotes and eukaryotes. When secreted under normal and pathophysiological conditions, EVs facilitate intercellular communication processes by acting as cargo collections that transport essential cellular components, i.e., soluble proteins or active enzymes, lipids, and nucleic acids, such as mRNAs, microRNAs, long noncoding RNAs, and metabolites.

[0005] This transport capacity of EVs has prompted the exploration of using EVs to deliver drugs (e.g., therapeutic agents) to or within target cells. Compared with other well-known synthetic drug delivery carriers (such as liposomes, lipid nanoparticles, or viral vectors, etc.), EVs have many advantages as drug carriers because they are natural secretions of cells and can be used for short- or long-distance intercellular communication; they have tropism for specific organs or cells by binding to certain surface receptors; they have higher cargo transport efficiency due to their multiple cellular uptake pathways, including endocytosis, phagocytosis, microphagocytosis, or direct fusion with the receptor cell membrane; and the ability to avoid immune clearance due to the intrinsic nature of the carrier.

[0006] EVs are broadly divided into extranuclear particles and exosomes. The average diameter of exosomes typically ranges from about 40 to about 160 nm, which is smaller than red blood cells. Exosomes are also very efficient in crossing the blood-brain barrier, an ability that makes them more attractive in drug delivery for various types of brain diseases. However, large-scale production of exosomes is challenging due to the limited amount of exosomes naturally produced in cells.

[0007] Therefore, there is a need in the art for enhanced methods of manufacturing EVs, in particular for enhancing exosome production in mammalian cells. The present disclosure addresses these needs. Summary of the invention

[0008] Disclosed herein are methods for producing extracellular vesicles (EVs). In particular, the present disclosure provides methods for enhancing the exosome production process.

[0009] In one aspect, the present invention provides a method for enhancing the production of extracellular vesicles (EVs), the method comprising the following steps: a) genetically engineering a production cell to overexpress at least one or more polypeptides; b) obtaining a plurality of EVs from the production cell. In some embodiments, the polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group. In some embodiments, the polypeptide is derived from any one of the polypeptides in Table A. In some cases, the polypeptide is derived from a group consisting of proteins selected from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, and GPC6. In some embodiments, the polypeptide is derived from CD59. In some embodiments, the polypeptide is derived from CD55. In some embodiments, the polypeptide is selected from the group consisting of CD52, CD55, CD58, and CD59. In some embodiments, the polypeptide is CD59. In some embodiments, the polypeptide is CD55. In some embodiments, EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some embodiments, EVs are exosomes.

[0010] In some embodiments, the production cell is genetically engineered by transfection of a recombinant vector system. In some embodiments, the recombinant vector system includes a nucleic acid sequence encoding a polypeptide. In some embodiments, the recombinant vector system includes an expression control sequence operably connected to the nucleic acid sequence. In some embodiments, the nucleic acid sequence includes at least one fluorescent marker. In some embodiments, the expression control sequence is a promoter. In some embodiments, the recombinant vector system includes a selection marker. In some embodiments, the production cell is a genetically engineered stable cell line. In some embodiments, multiple EVs are collected by dialysis or ultracentrifugation. In some embodiments, multiple EVs are collected by ultracentrifugation.

[0011] In another aspect, the present invention provides a method for preparing a stable cell line for producing EVs. In some cases, the method comprises the following steps: a) transfecting EV production cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence of at least one polypeptide and a selection marker, wherein the polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group; b) screening and selecting transfected cells; c) culturing the selected cells. In some embodiments, the polypeptide is selected from the group consisting of proteins of CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, and GPC6. In some embodiments, the polypeptide is CD59. In some embodiments, the polypeptide is CD55. In some embodiments, the polypeptide is derived from the group consisting of proteins selected from CD52, CD55, CD58, and CD59. In some embodiments, the polypeptide is derived from CD59. In some embodiments, the polypeptide is derived from CD55. In some embodiments, the expression vector comprises an expression control sequence operably linked to the nucleic acid sequence. In some embodiments, the expression control sequence is a promoter. In some embodiments, the nucleic acid sequence comprises at least one fluorescent marker. In some embodiments, the selectable marker is the group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance, and histidinol resistance.

[0012] In some embodiments, the concentration of EVs obtained from the production cells is at least 2 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the production cells is 2 to 40 times higher than that of the control cells. In some embodiments, the production cells are mammalian cells. In some embodiments, the production cells are stem cells, mesenchymal stem cells (MSC), HEK 293F cells, HEK 293T cells, or any combination thereof.

[0013] In some embodiments, EVs are loaded with cargo molecules. In some cases, the cargo molecules include active pharmaceutical ingredients (APIs). In some embodiments, APIs include small molecule therapeutics. In some embodiments, the cargo molecules include polypeptides, proteins, lipids, nucleic acids, carbohydrates, lipids, metabolites, or any combination thereof. In some embodiments, nucleic acids include DNA. In some embodiments, nucleic acids include peptide nucleic acids (PNAs). In some embodiments, the nucleic acids include RNA. In some cases, wherein the RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, microRNA (miRNA), and long non-coding RNA (lncRNA). In some embodiments, proteins include antibodies or enzymes. In some embodiments, cargo molecules include antisense oligonucleotides. In some embodiments, cargo molecules include morpholino oligomers. In some embodiments, cargo molecules include one or more components of a gene editing system. In some embodiments, the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs).

[0014] In another aspect, the present disclosure provides a cell line manufactured according to any of the methods described herein. The present disclosure also provides a kit for enhancing EV production, comprising any of the production cells or stable cell lines described herein. The present disclosure also provides a composition comprising a plurality of EVs produced according to any of the EVs production methods described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0015] In some embodiments, the present invention provides a composition comprising extracellular vesicles (EVs) producing cells, wherein the EV producing cells are genetically engineered to overexpress at least one or more polypeptides, wherein the polypeptides are linked to a glycosyl-phosphatidyl-inositol (GPI) group. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention may be obtained by referring to the following detailed description, which sets forth exemplary embodiments using the principles of the present invention, and the accompanying drawings:

[0017] Figure 1AThe protein domains of CD55 and artificial CD55 are shown as described in Example 1. WT CD55 includes a signal peptide domain, a sushi domain, and a GPI anchor (top), and artificial CD55 includes the N-terminal and C-terminal domains of CD55 fused with mCherry (bottom).

[0018] Figure 1B CD59 (293T CD59 )、CD46(293T CD46 ) and CD55(293T CD55 ) Representative immunoblot images of CD59, CD46, CD55, and GAPDH (loading control) in HEK 293T cells overexpressing CD59.

[0019] Figure 2 Schematic diagram of the process of producing small extracellular vesicles (sEVs) as described in Example 2.

[0020] Figure 3A -B shows the results of the experiment described in Example 2 from WT, 293T CD46 、293T CD59 and 293T CD55 The concentration of extracellular vesicles (EVs) obtained from cells (particles / L) ( Figure 3A ) and from WT, 293T CD46 、293T CD59 and 293T CD55 The concentration of extracellular vesicles obtained from cells (ug / mL) Figure 3B ).

[0021] Figure 4A -C shows the characteristics of the obtained extracellular vesicles (EVs), including the particle size distribution as described in Example 2. Figure 4A Shown are nFCM scatter plot data for standard beads. Figure 4B nFCM histogram data for standard beads are shown. Figure 4C As described in Example 2, the CD46 、293T CD59 and 293T CD55 Size and distribution of extracellular vesicles (EVs) obtained in cells.

[0022] Figure 5 The results from WT, 293T CD59 、293T CD46 and 293T CD55 Representative immunoblots of cells for Alix, TSG101, PTGFRN, CD59, CD46, CD55, and GAPDH (loading control).

[0023] Figure 6 Demonstrated as described in Example 2 Figure 5 Representative TEM images of EVs acquired in .

[0024] Figure 7 As described in Example 2, the WT 、293T CD46 、293T CD59 and 293T CD55 GPI-anchored proteins enriched in cell-harvested EVs.

[0025] Fig. 8A -C shows representative immunoblot images of CD52 ( Fig. 8A ); from WT and 293T CD52 The concentration of extracellular vesicles (EVs) in cells (particles / L) ( Figure 8B ); and from 293T CD52 Morphology of cell-derived EVs ( Figure 8C ).

[0026] Fig. 9A -C shows representative immunoblot images of CD55 and CD59 ( Fig. 9A ); from WT, 293T CD55 、293T CD59 and 293T CD55&CD59 The concentration of extracellular vesicles (EVs) obtained from cells (particles / L) ( Fig. 9B );and from 293T CD55 、293T CD59 and 293T CD55&CD59 Morphology of cell-derived EVs ( Fig. 9C ).

[0027] Fig. 10A -C shows representative immunoblot images of CD55 ( Fig. 10A ); from 293F WT and 293F CD55 The concentration of extracellular vesicles (EVs) obtained from cells (particles / L) ( Fig. 10B ); and from 293F WT and 293F CD55 Morphology of cell-derived EVs ( Fig. 10C ).

[0028] Fig.11A -C shows representative immunoblot images of CD55 ( Fig.11A ); The concentration of extracellular vesicles (EVs) obtained from WT and CD55-overexpressing mesenchymal stem cells (MSCs) ( Fig. 11B ) and from MSC WT and MSC CD55 Morphology of cell-derived EVs ( Fig. 11C ).

[0029] Fig. 12A The protein domain structures of the HA-tagged CD55 (4) and truncated CD55 (3-0) as described in Example 4 are shown, which have a signal peptide domain, a sushi domain and / or a GPI anchor.

[0030] Fig. 12B -C shows the same as described in Example 4 Fig. 12A ( Fig. 12B ) in 293FWT and 293F4-0, and from 293F WT With 293F 4-0 The concentration of extracellular vesicles (EVs) obtained in producer cells.

[0031] Fig.12D The morphology of extracellular vesicles of the HA-tagged protein domain structures of CD55 (4) and truncated CD55 (3-0) as described in Example 4 is shown.

[0032] Fig.13A -D shows the protein domain structure of CD55 (top) and two CD55-based peptides (short and long) with a signal peptide domain, sushi domain, GPI anchor, and / or mCherry; Fig.13A ( Fig. 13B ) of WT and truncated constructs; and the concentration of extracellular vesicles (EVs) from producer cells ( Fig. 13C ); and cellular uptake of mCherry-loaded EVs as described in Example 5 ( Fig.13D ).

[0033] Fig.14A -E shows the protein domain structure of CD46 and two CD46-based peptides (short and long) with a signal peptide domain, an extracellular domain, a transmembrane domain, a cytoplasmic domain, and / or mCherry ( Fig.14A ); WT and CD46S and L polypeptide expression ( Fig. 14B ); the concentration of extracellular vesicles (EVs) from producer cells ( Fig. 14C ); Protein domain structure of CD46-based polypeptide encoding nanoluciferase (Nluc), near the N-terminus (outside) or C-terminus (inside) ( Fig.14D ); and immunoblot images of CD46 from production cells as described in Example 5 ( Fig.14E ).

[0034] Fig.15A -B shows the concentration of extracellular vesicles (EVs) from CD55, CD55 / CD59, and CD55 / CD46 short producer cells ( Fig.15A ); Representative immunoblot images of CD55 and mOrange from producer cells and EVs concentrations from 293F-based WT, CD55, CD55 / CD63mOrange, CD55 / CD81 mOrange, and CD55 / CD9mOrange producer cells as described in Example 5 ( Fig. 15B ).

[0035] Fig.16A -F shows the cell survival rate and percentage of extracellular vesicle production in 28F and 293F cells cultured for different days ( Fig.16A ); Cell status (density and viability) and concentration of extracellular vesicles produced in 293F cells grown in different cell culture media ( Fig. 16B ); Cell status (density and viability) and concentration of extracellular vesicles produced in 28F cells grown in different cell culture media ( Fig. 16C ); Percentage of extracellular vesicle production in 293F cells grown in the presence or absence of antiaggregants ( Fig.16D ); based on the cell status (density and viability) and the concentration of extracellular vesicles produced by different cell culture methods ( Fig.16E ); and the ratio of the permeate to the retentate after filtration using the test filter described in Example 6 ( Fig.16F ).

[0036] Fig.17 The downstream purification process as described in Example 6 is demonstrated.

[0037] Fig.18A -E shows the size distribution and percentage of extracellular vesicles (EVs) obtained by 28F ( Fig.18A );TEM image( Fig.18B ); Cryo-EM images ( Fig.18C ); nFCM diagram( Fig.18D ); CD55, Alix, TSG101, CD9, CD63, CD81 and CYC1 in cells and EVs were immunoblotted as described in Example 6 ( Fig.18E ).

[0038] Fig.19A -B shows the stability of extracellular vesicles (EVs) obtained at different temperatures or after lyophilization as described in Example 6 over time.

[0039] Fig. 20A-B shows the exterior of the siRNA as described in Example 6 ( Fig. 20A ) or internal ( Fig. 20B )EVs loading.

[0040] Fig.21 nFCM images of siRNA-loaded extracellular vesicles (EVs) with or without RNAse treatment as described in Example 6 are shown.

[0041] Fig.22A -C shows the RNA loading efficiency in extracellular vesicles (EVs) at different EV concentrations ( Fig.22A ); the ratio of EVs to RNA ( Fig. 22B ); and the voltage as described in Example 7 ( Fig. 22C ).

[0042] Fig.23 nFCM images showing HA loading in the lumen (inside) or outside of extracellular vesicles (EVs) as described in Example 7 are shown.

[0043] Fig.24A -F shows representative immunoblot images ( Fig.24A ); The response ratio of BLAM of cargo molecules loaded inside or outside of extracellular vesicles in the presence or absence of vesicular stomatitis virus glycoprotein (VSVG) ( Fig. 24B ); As described in Example 8, the target cells take up the CD63-( Fig.24C -D) or CD46-( Fig.24E -F) Representative immunoblots of BLAM and VSVG in cells or EVs with or without VSVG overexpression after EVs containing BLAM fusion protein.

[0044] Fig.25A -B shows the presence or absence of VSVG from the interior of extracellular vesicles (EVs) as described in Example 8 ( Fig.25A ) or external ( Fig.25B )The effect of released siRNA. DETAILED DESCRIPTION

[0045] definition

[0046] As used herein, the term "about" and its grammatical equivalents, when related to a reference numerical value, indicate a range of fluctuations above and below the numerical value, which may include a range of ±10% of the numerical value, such as a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the numerical value. For example, the number "about 10" includes the amount of 9 to 11. Unless otherwise stated, some embodiments herein contemplate numerical ranges. When a numerical range is provided, unless otherwise stated, the range includes the range endpoints. Unless otherwise stated, a numerical range includes all values ​​and subranges therein, as if expressly written.

[0047] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Accordingly, unless indicated otherwise, the numerical parameters set forth in this application are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

[0048] Unless otherwise stated, open terms such as "includes," "comprising," "including," "comprising," etc., are intended to be inclusive.

[0049] The terms "agent," "active pharmaceutical ingredient (API)," "therapy," "therapeutic agent," and "drug" are used interchangeably herein and include an agent that has a pharmacological effect that induces a biological or medical response in an animal or human tissue or cell system desired by a researcher, veterinarian, general practitioner, or other physician, including altering a biological system at the molecular level (e.g., as an inhibitor, activator, or modulator of a protein), alleviating or symptom(s) of the disease or condition being treated; the agent may be a compound, a biological molecule with therapeutic activity (e.g., siRNAs, miRNAs, anti-miRNAs, shRNAs, etc., antibodies, antibody fragments that recognize specific epitopes), an anti-tumor drug, or a radiotherapeutic drug.

[0050] The term "cargo molecule" refers to any molecule or compound that has been or is about to be incorporated, encapsulated, fused or injected into a transfer cargo molecule (such as a vesicle, exosome, etc.), which can be a chemical or biological molecule with or without therapeutic activity.

[0051] The term "extracellular vesicle" is to be understood as having a well-known meaning in the art and refers to a vesicle containing a membrane-enclosed cytoplasmic portion released from cells in the microenvironment. These vesicles represent a heterogeneous population consisting of multiple types of vesicles, including "exosomes" and microvesicles, or apoptotic bodies, which can be distinguished based on size, antigenic composition and secretion pattern. The terms "therapeutic delivery vesicle" and "therapeutic cargo" are to be understood as referring to any type of vesicle, such as can be obtained from cells, such as microvesicles (any vesicle shed from the plasma membrane of the cell), exosomes (any vesicle obtained from the endosomal pathway), apoptotic bodies (obtained from apoptotic cells), microparticles (obtainable from, for example, platelets), extranuclear particles (obtained from, for example, neutrophils and monocytes in serum), prostate bodies (obtained from prostate cancer cells) and myosomes (obtained from myocardial cells), etc. Furthermore, the terms "cargo molecule delivery vesicle" and "delivery vesicle" should also be understood to possibly be associated with lipoprotein particles, such as LDL, VLDL, HDL and chylomicrons, as well as liposomes, lipid-like particles, liposomes, etc. Essentially, the present invention may relate to any type of lipid-based structure (vesicle or having any other type of suitable morphology) that can serve as a delivery or transport vehicle for cargo molecules.

[0052] The term "fusion" or "fusion polypeptide" as used herein refers to a recombinant protein of two or more polypeptides. For example, by connecting a nucleic acid sequence encoding a polypeptide to a nucleic acid encoding another polypeptide or protein domain, a fusion protein can be produced so that it constitutes a single open reading frame that can be translated into a single polypeptide containing all the expected proteins in the cell. The order of arrangement of the polypeptides can vary. The fusion polypeptide can include an epitope tag or a half-life extender. Epitope tags include biotin, FLAG tags, c-myc, hemagglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tags, GST, β-galactosidase, AU1, AU5 and avidin. Half-life extenders include Fc domains and serum albumin.

[0053] In the present disclosure, the term "linked", "anchored" or "associated" is understood as any interaction between two groups, for example, an interaction between a polypeptide and a GPI group or a mutual influence between a GPI-anchored polypeptide and a membrane. This includes enzyme interactions, ionic binding, covalent binding, non-covalent binding, hydrogen bonding, London forces, van der Waals forces, hydrophobic interactions, lipophilic interactions, magnetic interactions, electrostatic interactions, etc.

[0054] In the present disclosure, the term "loading" or "loading an extracellular vesicle" is understood as an activity or state that results in the vesicle including one or more molecules of interest that are not normally present in the interior, inside the vesicle, and / or on its membrane surface. In some embodiments, the cargo molecule is loaded into the lumen of the extracellular vesicle. In some embodiments, the cargo molecule is loaded onto the outer surface of the extracellular vesicle. In some embodiments, the cargo molecule is loaded within the membrane of the extracellular vesicle.

[0055] The term "nucleic acid molecule" refers to a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases. It includes chromosomal DNA and self-replicating plasmids, vectors, mRNA, tRNA, siRNA, etc. It can be recombinant, and when the nucleic acid is introduced into a cell, an exogenous polypeptide can be expressed therefrom.

[0056] In the present disclosure, the term "polypeptide" or "peptide" is understood to be an amino acid sequence consisting of amino acids linked by peptide bonds. The term can be used interchangeably with "protein" in the broadest sense to refer to a molecule of two or more amino acids, amino acid analogs or peptidomimetics. In some embodiments, the amino acids are linked by peptide bonds. In some embodiments, the amino acids are linked by other types of bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0057] In some cases, the peptides or polypeptides of the present disclosure include at least two amino acid residues and are less than about 50 amino acids in length (e.g., 40 amino acids, 30 amino acids, 20 amino acids, or any number therein). In some cases, the peptides or polypeptides of the present disclosure include at least 50 amino acids, 100 amino acids, 150 amino acids, or more. In some embodiments, the peptides or polypeptides have counterions. In some embodiments, the peptides or polypeptides include N- and / or C-terminal modifications, such as to reduce degradation or to have a blocking modification of a GPI group attached post-translationally.

[0058] The terms "purified," "isolated," and "obtained" are used interchangeably and are intended to mean removed from its natural environment. The term purified or isolated does not require absolute purity or isolation; rather, it is a relative term.

[0059] The term "vector" is a nucleic acid molecule, preferably self-replicating, which transfers and / or replicates an inserted nucleic acid molecule (e.g., a transgene or an exogenous nucleic acid) into and / or between host cells. It includes plasmids or viral chromosomes, into whose genome recombinant DNA fragments are inserted, for introducing recombinant DNA or transgenes into the polypeptides of the present disclosure.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative only and not restrictive.

[0061] Overview

[0062] Disclosed herein are methods for enhancing the production of extracellular vesicles (EVs) derived from or secreted from genetically engineered mammalian EV-producing cells. The EVs disclosed herein can be combined with a sufficient amount of one or more therapeutic agents or any molecules of interest to deliver an effective amount of the therapeutic agent or any molecule of interest to a target site.

[0063] To date, various manufacturing strategies have been considered to increase the production of EVs in cells. Some of these strategies include hypoxia induction, overexpression of four transmembrane proteins, and overexpression of hypoxia-inducible factor-1α. Genetic modification of genes in some exosome-producing cells (such as Nad B, SCD4, STEAP3) has been considered before to produce EVs in cells, although the overall effect of overexpressing these genes was not significant. Here, the present disclosure provides efficient and superior EV manufacturing methods that enhance the production of EVs in exosome-producing cells.

[0064] Methods to enhance the production of extracellular vesicles (EVs)

[0065] In one aspect, the present invention provides a method for enhancing the production of extracellular vesicles (EVs), the method comprising the following steps: a) genetically engineering a production cell to overexpress at least one or more polypeptides; b) obtaining multiple EVs from the production cell.

[0066] In some embodiments, EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some embodiments, EVs are exosomes.

[0067] In some embodiments, the production cell is genetically modified to contain one or more polypeptides. In some embodiments, the production cell naturally contains one or more polypeptides, and the exosomes derived therefrom also contain polypeptides. The level of any desired polypeptide can be modified directly on the exosomes (e.g., by contacting the complex with a recombinantly produced polypeptide to insert or bind to the membrane of the complex). Alternatively, or in addition, the level of any desired polypeptide can be modified directly on the production cell (e.g., by contacting the complex with a recombinantly produced polypeptide to insert or bind to the cell membrane). Alternatively, the production cell can be modified by transfecting an exogenous nucleic acid into the production cell to express the desired polypeptide. The polypeptide may already exist naturally on the production cell, in which case the exogenous structure can lead to overexpression of the polypeptide and increase the concentration of the polypeptide in or on the production cell. Alternatively, the naturally expressed protein can be removed from the production cell (e.g., by inducing gene silencing in the production cell). The polypeptide can confer different functions to the exosomes (e.g., specific targeting capabilities, delivery functions (e.g., fusion molecules), enzyme functions, increase or decrease in in vivo half-life, etc.).

[0068] In some cases, the polypeptide includes but is not limited to LFA-3, NCAM, PH-20, CD9, CD14, CD16b, CD40, CD46, CD47, CD52, CD55 (DAF), CD58, CD59, CD63, CD81, CD109, GPC1, GPC4, GPC6, CD133, Thy-1, Qa-2, integrin, selectin, lectin, cadherin, carcinoembryonic antigen (CEA), scrapie prion protein, folate binding protein, or any polypeptide listed in Table A, or any combination thereof.

[0069] In some embodiments, the EVs of the present disclosure are exosomes, and their surface includes one or more polypeptides selected from LFA-3, NCAM, PH-20, CD9, CD14, CD16b, CD40, CD46, CD47, CD52, CD55 (DAF), CD58, CD59, CD63, CD81, CD109, GPC1, GPC4, GPC6, CD133, Thy-1, Qa-2, carcinoembryonic antigen (CEA), scrapie prion protein, folate binding protein, or any polypeptide listed in Table A, or any combination thereof. In some embodiments, the exosomes are modified to include one or more polypeptides.

[0070] In some embodiments, the production cell is a mammalian cell. In some embodiments, the production cell is selected from the group consisting of human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080 cells, and human embryonic retinal PER. C6 cells, kidney / B cell hybrid HKB-11 cells, primary human amniotic membrane cells CAP cells, human mesenchymal stem cells (MSC) or liver cancer HuH-7 human cells. In some embodiments, the production cell is HEK293-H, HEK293-T, HEK293-EBNA1 or HEK293-F. In some embodiments, the production cell is genetically engineered to provide transient overexpression of the polypeptide. In some embodiments, the production cell is a genetically engineered stable cell line that constitutively overexpresses the polypeptide.

[0071] In some embodiments, the polypeptide includes a glycosyl-phosphatidyl-inositol (GPI) group. In some embodiments, the GPI group is added to the C-terminus of the polypeptide after translation. GIP is a lipid moiety that includes a phosphoethanolamine linker, a glycan core, and a phospholipid tail. In some embodiments, the GPI group is covalently attached to the polypeptide as a post-translational modification marker to allow for distribution within lipid rafts, signal transduction, cell communication, or apical membrane targeting. In some embodiments, the addition of the GPI group allows the modified polypeptide to be anchored in the outer leaflet of the membrane region. In some embodiments, the GPI group-anchored polypeptide is sorted into exosomes. In some embodiments, the GPI-anchored polypeptide is exposed on the surface of the exosome.

[0072] In some cases, the GPI anchor polypeptide is any one of the polypeptides listed in Table A. In some embodiments, the GPI anchor polypeptide is any one or more polypeptides selected from Table A.

[0073] In some embodiments, the production cell is genetically engineered to overexpress any one of the polypeptides listed in Table A or its functional polypeptide fragments, the amount or copy number of which is sufficient to remain in the circulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or longer. In some embodiments, the production cell is genetically engineered to overexpress the polypeptides or their functional polypeptide fragments in Table A, the amount, copy number and / or ratio of which is sufficient to remain in the circulation for 15 days, 21 days, 30 days, 45 days, 60 days, 100 days, 120 days or longer.

[0074] In some embodiments, the GPI-anchored polypeptide is selected from the group consisting of CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, and GPC6.

[0075] In some embodiments, the GPI-anchored polypeptide is CD55, a 70 kDa membrane protein also known as complement decay accelerating factor or DAF. CD55 recognizes the C4b and C3b fragments of the complement system, which are produced during the activation of C4 (classical complement pathway and lectin pathway) and C3 (alternative complement pathway). CD55 can block the formation of the membrane attack complex or prevent the lysis of the complement cascade. In some embodiments, the production cell is genetically engineered to overexpress the CD55 polypeptide or its functional polypeptide fragment in an amount or copy number sufficient to remain in the circulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or more. In some embodiments, the producer cells are genetically engineered to overexpress a CD55 polypeptide or a functional polypeptide fragment thereof in an amount, copy number and / or ratio sufficient to remain in the circulation for 15 days, 21 days, 30 days, 45 days, 60 days, 100 days, 120 days or longer.

[0076] In some embodiments, the GPI-anchored polypeptide is CD59, also known as MAC inhibitory protein (MAC-IP), membrane inhibitor of reactive lysis (MIRL), protective protein or HRF, a protein attached to the host cell by a glycophosphatidylinositol (GPI) anchor. In some embodiments, the production cell is genetically engineered to overexpress the CD59 polypeptide or its functional polypeptide fragment in an amount or copy number sufficient to remain in the circulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or longer. In some embodiments, the production cell is genetically engineered to overexpress the CD59 polypeptide or its functional polypeptide fragment in an amount, copy number and / or ratio sufficient to remain in the circulation for 15 days, 21 days, 30 days, 45 days, 60 days, 100 days, 120 days or longer.

[0077] In some embodiments, the GPI anchor polypeptide is CD52. CD52 is expressed at high levels on T and B lymphocytes, at low levels on monocytes, and not on granulocytes and myeloid precursor cells. In some embodiments, the production cells are genetically engineered to overexpress the CD52 polypeptide or its functional polypeptide fragment, the amount or copy number of which is sufficient to stay in the circulation for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or longer. In some embodiments, the production cells are genetically engineered to overexpress the CD52 polypeptide or its functional polypeptide fragment, the amount, copy number and / or ratio of which is sufficient to stay in the circulation for 15 days, 21 days, 30 days, 45 days, 60 days, 100 days, 120 days or longer.

[0078] In some embodiments, the GPI-anchored polypeptide is selected from the group consisting of CD52, CD55, CD58, and CD59. In some embodiments, the polypeptide comprises a glycosyl-phosphatidyl-inositol (GPI) group, an extracellular domain, a transmembrane domain, a cytoplasmic domain, or a combination thereof.

[0079] In some embodiments, the production cells are genetically engineered by a transfection recombinant vector system to overexpress a polypeptide. The term "transfection" or "presumed transfection" used herein refers to a method for introducing exogenous genetic material into a host cell (e.g., transfection of mammalian cells by lentivirus), wherein the host cell can be transiently transfected or stably transfected. The genetic material can be an expression vector comprising a gene of interest (e.g., a recombinant GPI anchored polypeptide) or a polynucleotide sequence encoding siRNA or shRNA. The genetic material can also refer to the introduction of a viral nucleic acid sequence in the manner of a natural virus. The viral nucleic acid sequence does not need to exist in the form of a naked nucleic acid sequence, but can be packaged in a viral protein envelope.

[0080] Transfection of eukaryotic host cells with polynucleotides or expression vectors to produce genetically modified cells or transgenic cells can be performed by any method known in the art (see, for example, Sambrook J et al., 1989. Molecular cloning: A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press). Transfection methods include, but are not limited to, liposome-mediated transfection, calcium phosphate coprecipitation, electroporation, nuclear transfection, nuclear poration, microporation, polycation (such as DEAE-dextran)-mediated infection, protoplast fusion, viral infection, and microinjection. The transformation may result in transient or stable transformation of the host cell. In some embodiments, transfection is a stable transfection. In some embodiments, transfection is a transient transfection. It is preferred to provide a transfection method that provides optimal transfection frequency and heterologous gene expression in a specific host cell line and type. Suitable methods can be determined by conventional procedures. For stable transfectants, the structure is either integrated into the genome of the host cell, or integrated into an artificial chromosome / mini-chromosome, or located in the ectoderm so as to be stably maintained in the host cell. Therefore, the stably transfected sequence is actually retained in the genome of the cell and its daughter cells. Typically, this involves the use of a selective marker gene, and integrating the gene of interest or the polynucleotide sequence encoding the RNA with the selective marker gene. Screen and select cells with this selective marker gene for further culture (including passage, growth, culture, and division at optimal cell density). In some embodiments, the entire expression vector is integrated into the genome of the cell, and in some embodiments, only part of the expression vector is integrated into the genome of the cell. Cells that "stably express" recombinant polypeptides or RNA are stably transfected with a gene encoding the recombinant polypeptide or a polynucleotide sequence encoding the RNA. Therefore, the sequence encoding the recombinant polypeptide or ribonucleic acid is retained in the genome of the cell and its daughter cells.

[0081] In some embodiments, the recombinant vector system includes a nucleic acid sequence encoding a GPI anchor polypeptide. In some embodiments, the nucleic acid sequence encodes a portion of a GPI anchor protein. In some embodiments, the nucleic acid sequence encodes the N-terminal domain of a GPI anchor protein. In some embodiments, the nucleic acid sequence encodes the C-terminal domain of a GPI anchor protein. In some embodiments, the nucleic acid sequence encodes the N and C-terminal domains of a GPI anchor protein.

[0082] In some embodiments, the nucleic acid sequence encodes the N-terminal domain of any of the polypeptides listed in Table A. In some embodiments, the nucleic acid sequence encodes the C-terminal domain of any of the polypeptides listed in Table A. In some embodiments, the nucleic acid sequence encodes the N- and C-terminal domains of any of the polypeptides listed in Table A. In some embodiments, the nucleic acid sequence encodes the N-terminal domain of CD52. In some embodiments, the nucleic acid sequence encodes the C-terminal domain of CD52. In some embodiments, the nucleic acid sequence encodes the N- and C-terminal domains of CD52. In some embodiments, the nucleic acid sequence encodes the N-terminal domain of CD55. In some embodiments, the nucleic acid sequence encodes the C-terminal domain of CD55. In some embodiments, the nucleic acid sequence encodes the N- and C-terminal domains of CD55. In some embodiments, the nucleic acid sequence encodes the N-terminal domain of CD58. In some embodiments, the nucleic acid sequence encodes the C-terminal domain of CD58. In some embodiments, the nucleic acid sequence encodes the N- and C-terminal domains of CD58. In some embodiments, the nucleic acid sequence encodes the N-terminal domain of CD59. In some embodiments, the nucleic acid sequence encodes the C-terminal domain of CD59. In some embodiments, the nucleic acid sequence encodes the N- and C-terminal domains of CD59. In some embodiments, the nucleic acid sequence encodes the N-terminal domain of CD46. In some embodiments, the nucleic acid sequence encodes the C-terminal domain of CD46. In some embodiments, the nucleic acid sequence encodes the N- and C-terminal domains of CD46.

[0083] In some embodiments, the nucleic acid sequence encodes an amino acid sequence selected from Table 1.

[0084] Table 1 Amino acid sequence

[0085] In some embodiments, the recombinant vector system includes an expression control sequence operably linked to the nucleic acid sequence. In some embodiments, the expression control sequence is a promoter. For example, a nucleic acid sequence corresponding to a polypeptide can be inserted into a recombinant vector containing a promoter sequence compatible with a specific RNA polymerase. For example, an exemplary vector can include T3 and T7 promoter sequences compatible with T3 and T7 RNA polymerases, respectively. Examples of other promoter sequences (e.g., for expression in mammalian cells) are promoters and / or enhancers derived from (CMV) (e.g., CMV Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters (e.g., natural immunoglobulin and actin promoters). In some embodiments, the expression control sequence is a polyadenylation signal, such as BGHpolyA, SV40 late or early polyA; or the 3′UTR of an immunoglobulin gene. In one In some embodiments, the nucleic acid sequence of the polypeptide can be fused to at least one active domain at the N-terminus and / or C-terminus, and the active domain can be selected from: nucleases (such as endonucleases or exonucleases), polymerases, kinases, phosphatases, methylases, demethylases, acetylases, deacetylases, topoisomerases, integrases, transposases, ligases, helicases, recombinases, transcription activators (such as VP64, VP16), transcription inhibitors (such as KRAB), DNA end-processing enzymes (such as Trex2, Tdt) and reporter molecules (such as fluorescent proteins, lacZ, luciferases).

[0086] In some embodiments, the recombinant vector system includes a selection marker. In some embodiments, the selection marker is ampicillin, chloramphenicol, kanamycin, tetracycline, blasticidin S, neomycin, hygromycin B, or any combination thereof.

[0087] In some embodiments, the nucleic acid sequence includes a sequence corresponding to at least one fluorescent marker. In some embodiments, the fluorescent marker is a green fluorescent protein (e.g., GFP, EGFP, AmCyan, etc.), a red fluorescent protein (e.g., mCherry, DsRed, td to ma to, mStrowberry, etc.), an orange and yellow fluorescent protein (e.g., mOrange, mBanana, ZsYellow, etc.), a far red fluorescent protein (e.g., E-Crimson, HcRed, mRasberry, mPlum, etc.), or any combination thereof. In some embodiments, the fluorescent marker is mCherry.

[0088] In some embodiments, the identity or quantity of the producing cells or exosomes can be assessed by in vitro detection. For example, the identity or quantity of the producing cells or exosomes is assessed by counting the number of cells or complexes in the population, such as by microscopy, flow cytometry, or hemocytometer. Alternatively, or in addition, the identity or quantity of the producing cells or exosomes is assessed by analyzing the protein content of the cells or complexes, such as by flow cytometry, immunoblotting, immunoprecipitation, fluorescence spectroscopy, chemiluminescence, mass spectrometry, or absorption spectroscopy. In some embodiments, the protein content measured is a surface protein, such as a differentiation marker, a receptor, a co-receptor, a transporter, a glycoprotein. In some embodiments, the identity or quantity of the producing cells or exosomes is assessed by analyzing the receptor content of the cells or complexes, such as by flow cytometry, immunoblotting, immunoprecipitation, fluorescence spectroscopy, chemiluminescence, mass spectrometry, or absorption spectroscopy. For example, the identity or quantity of the producing cells or exosomes can be assessed by the mRNA content of the cells or complexes, such as by RT-PCR, flow cytometry, or Northern blotting. The identity or quantity of the producer cells can be assessed by the content of nuclear material, for example by flow cytometry, microscopy or Northern blotting, using, for example, nuclear stains or nucleic acid probes. Alternatively or additionally, the identity or quantity of the producer cells or exosomes can be assessed by the lipid content of the cells or complexes, for example by flow cytometry, liquid chromatography or mass spectrometry.

[0089] In another aspect, the present disclosure provides a method for preparing a stable cell line for producing EVs. In some cases, the method comprises the following steps: a) transfecting EV-producing cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence of at least one polypeptide and a selection marker, wherein at least one peptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group; b) screening and selecting transfected cells; c) culturing the selected cells.

[0090] In some cases, the polypeptide is selected from LFA-3, NCAM, PH-20, CD9, CD14, CD16b, CD40, CD46, CD47, CD52, CD55 (DAF), CD58, CD59, CD63, CD81, CD133, Thy-1, Qa-2, carcinoembryonic antigen (CEA), scrapie prion protein, folate binding protein, any one of the polypeptides listed in Table A, and any combination thereof. In some cases, the polypeptide is derived from LFA-3, NCAM, PH-20, CD9, CD14, CD16b, CD40, CD46, CD47, CD52, CD55 (DAF), CD58, CD59, CD63, CD81, CD133, Thy-1, Qa-2, carcinoembryonic antigen (CEA), scrapie prion protein, folate binding protein, any one of the polypeptides listed in Table A, and any combination thereof. In some embodiments, the polypeptide is derived from CD52, CD55, CD58 or CD59. In some embodiments, the polypeptide is derived from CD59. In some embodiments, the polypeptide is derived from CD55. In some embodiments, the polypeptide is CD59. In some embodiments, the polypeptide is CD55.

[0091] In certain embodiments, the expression vector comprises an expression control sequence operably connected to the nucleic acid sequence. In certain embodiments, the expression control sequence is a promoter. In certain embodiments, the nucleic acid sequence comprises at least one fluorescent marker. In certain embodiments, the selective marker is a group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance and histidinol resistance.

[0092] In some embodiments, EVs are collected by dialysis or ultracentrifugation. In some embodiments, EVs are collected by ultracentrifugation. In some embodiments, any cell including EVs disclosed herein can be filtered through a filter (e.g., a nylon mesh cell filter) with a suitable mesh or pore size. The pore size of the filter may be 50nm to 100mM. In some embodiments, the pore size of the filter is 80nm to 90mM. In some embodiments, the pore size of the filter may be 100nm to 80mM. In some embodiments, the pore size of the filter is about 200nm to about 70mM, about 400nm to about 60mM, about 600nm to about 50mM, about 800nm ​​to about 40mM, or about 1nm to about 20mM.

[0093] In some embodiments, the concentration of EVs obtained from the production cells is 2 to 300 times, 250 times, 200 times, 150 times or 100 times higher than the concentration of EVs obtained from the control cells (i.e., wild-type cells or vector-transfected cells), or any value or range therebetween. In some embodiments, the concentration of EVs obtained from the production cells is 2 to 90 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the production cells is 2 to 80 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the production cells is 2 to 70 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the production cells is 2 to 60 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the production cells is 2 to 50 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the production cells is 2 to 40 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is 2 to 30 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is 2 to 20 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is 2 to 10 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is 2 to 8 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is 2 to 4 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is 2 to 2 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 2 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 4 times higher than the concentration in the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 6 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 8 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 10 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 15 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 20 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 25 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 30 times higher than the concentration of EVs obtained from the control cells.In some embodiments, the concentration of EVs obtained from the producer cells is at least 35 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 40 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 45 times higher than the concentration of EVs obtained from the control cells. In some embodiments, the concentration of EVs obtained from the producer cells is at least 50 times higher than the concentration of EVs obtained from the control cells.

[0094] Extracellular vesicles (EVs)

[0095] The present invention provides a method for enhancing EV production in mammalian cells.

[0096] In some embodiments, the EVs of the present disclosure are exosomes. In some embodiments, the exosomes include a membrane that forms particles with a diameter of 30-100nm, 30-200nm, or 30-500nm. In some embodiments, the exosomes include a membrane that forms particles with a diameter of 10-100nm, 20-100nm, 30-100nm, 40-100nm, 50-100nm, 60-100nm, 70-100nm, 80-100nm, 90-100nm, 100-200nm, 100-150nm, 150-200nm, 100-250nm, 250-500nm, or 10-1000nm. In some embodiments, the average diameter length of EVs is at least about 180nm. In some cases, the average diameter length of EVs is at least about 80nm to about 180nm, about 85nm to about 175nm, about 90nm to about 160nm, about 92nm to about 150nm, about 96nm to about 140nm, about 98nm to about 130nm, about 100nm to about 120nm, about 102nm to about 112nm, or about 105nm to about 110nm. The size of EVs may vary with the loading of cargo molecules. In some embodiments, the size of EVs may remain unchanged after loading. In some cases, exosomes have an average diameter length of at least about 80nm. In some embodiments, the membrane is composed of lipids and fatty acids. In some embodiments, the membrane includes one or more of phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. In some embodiments, the membrane may include one or more polypeptides and one or more polysaccharides, such as polysaccharides. In some embodiments, the specific density of the produced exosomes can be between 0.5-2.0, 0.6-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.4-1.5, 1.0-1.5, 1.5-2.0 and 1.0-2.0 kg / m3.

[0097] In some embodiments, as described in Example 1, multiple EVs can be made, purified or isolated from cells, cell culture media or tissues. In some embodiments, multiple EVs can be purified or isolated before contacting a detergent or cargo molecule. In some embodiments, multiple EVs can be purified or isolated after contacting a detergent remover. In some embodiments, EVs can be purified or isolated into multiple cargo-loaded EVs.

[0098] In some embodiments, EVs are loaded with cargo molecules. In some cases, the cargo molecules include active pharmaceutical ingredients (APIs). In some embodiments, APIs include small molecule therapeutics. In some embodiments, the cargo molecules include polypeptides, proteins, lipids, nucleic acids, carbohydrates, lipids, metabolites, or any combination thereof. In some embodiments, nucleic acids include DNA. In some embodiments, nucleic acids include peptide nucleic acids (PNAs). In some embodiments, the nucleic acids include RNA. In some cases, wherein the RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, microRNA (mi RNA), and long non-coding RNA (lncRNA). In some embodiments, proteins include antibodies or enzymes. In some embodiments, cargo molecules include antisense oligonucleotides. In some embodiments, cargo molecules include morpholino oligomers. In some embodiments, cargo molecules include one or more components of a gene editing system. In some embodiments, the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs).

[0099] In another aspect, the present disclosure provides a cell line manufactured according to any of the methods described herein. The present disclosure also provides a kit for enhancing EV production, comprising any of the production cells or cell lines described herein. The present disclosure also provides a composition comprising a plurality of EVs according to any of the EVs described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0100] Disclosed herein are EVs comprising at least one cargo molecule. Also disclosed herein is a method for making EVs for loading a sufficient amount of one or more cargo molecules so as to deliver an appropriate amount of one or more cargo molecules to or into a target cell or tissue of interest.

[0101] In some embodiments, the final concentration of the one or more cargo molecules is about 0.1 μM to 300 μM. In some embodiments, the final concentration of the one or more cargo molecules is at least about 0.1 μM to about 1 μM, about 0.1 μM to about 10 μM, about 0.1 μM to about 20 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 100 μM, about 0.1 μM to about 150 μM, about 0.1 μM to about 200 μM, about 0.1 μM to about 300 μM, about 1 μM to about 10 μM, about 1 μM to about 20 μM, about 1 μM to about 50 μM, about 1 μM to about 100 μM, about 1 μM to about 150 μM, about 1 μM to about 200 μM, about 1 μM to about 300 μM, about 1 μM to about 2 In the present invention, the present invention relates to an aqueous solution of at least about 1% to about 5% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, wherein the aqueous solution is at least about 1% iodine, In some embodiments, the total concentration of one or more cargo molecules is at least about 0.1 μM, about 1 μM, about 10 μM, about 20 μM, about 50 μM, about 100 μM, about 150 μM, about 200 μM, or about 300 μM.

[0102] In some embodiments, transfection or overexpression of the polypeptide produces low or no toxicity in any of the cells or kits disclosed herein. In some embodiments, the cells, EVs, exosomes or kits disclosed herein may include a pharmaceutically acceptable detergent. In some embodiments, the detergent is a nonionic detergent. In some embodiments, the detergent is polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether. In some embodiments, the detergent is octaethylene glycol monododecyl ether (OEG). In some cases, EVs include polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether at a final concentration of about 0.03mM to about 4mM, about 0.04mM to about 3mM, about 0.05mM to about 2.5mM, about 0.06mM to about 2.2mM, about 0.08mM to about 2mM, about 0.1mM to about 1.8mM, about 0.2mM to about 1.5mM, or any concentration therebetween.

[0103] In some cases, the EVs of the present disclosure can form a detergent mixture solution having a final detergent concentration of about 0.005% v / v to about 10% v / v, about 0.01% v / v to about 9.8% v / v, about 0.02% v / v to about 9.6% v / v, about 0.04% v / v to about 9.4% v / v, about 0.06% v / v to about 9.2% v / v, about 0.08% v / v to about 9.0%, From about 0.1% v / v to about 8.0% v / v, from about 0.1% v / v to about 7.0% v / v, from about 0.1% v / v to about 6.0% v / v, from about 0.1% v / v to about 5.0% v / v, from about 0.2% v / v to about 4.0% v / v, from about 0.4% v / v, from about 0.5% v / v, from about 0.6% v / v, from about 0.8% v / v, from about 1.0% v / v, or any concentration therebetween.

[0104] In some embodiments, the EVs may be contacted with the cargo molecule and the detergent by adding the cargo molecule and the detergent simultaneously. In some embodiments, the biological sample comprising extracellular vesicles may be contacted with the cargo molecule and the detergent by adding the cargo molecule and the detergent sequentially. In some cases, the method includes the step of contacting the biological sample comprising a plurality of EVs with the cargo molecule and the detergent by adding the cargo molecule prior to adding the detergent.

[0105] In some embodiments, the present invention provides a composition comprising any of the extracellular vesicle (EV) producing cells of the present invention, wherein the EVs producing cells are genetically engineered to overexpress at least one or more polypeptides, wherein the polypeptides are linked to a glycosyl-phosphatidyl-inositol (GPI) group. In some embodiments, the polypeptide is derived from any of the polypeptides listed in CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, GPC6, or Table A. In some cases, the EVs producing cells include a recombinant vector system comprising a nucleic acid sequence encoding a polypeptide coding sequence. In some embodiments, the recombinant vector system includes a polypeptide coding sequence selected from any of the sequences in Table 1. In some embodiments, the recombinant vector system includes a coding sequence for mCherry-CD46 (short), HA-CD46 short, or CD46 short HA polypeptides in Table 1.

[0106] In some cases, the production cell further comprises a release aid selected from the group consisting of protein G (G) of vesicular stoma virus (VSV), glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1), baculovirus fusion protein gp64, and gB of Epstein-Barr virus (EBV). In some embodiments, the release aid is vesicular stomatitis virus glycoprotein (VSVG).

[0107] How to use EVs

[0108] The present disclosure also provides methods, kits and reagents for treating diseases or conditions of subjects in need using EVs. For example, a method for using EVs to treat patients with chronic and recurrent diseases by administering an effective amount of EVs to the patient is provided herein. In some embodiments, chronic and recurrent diseases may be diabetes, infection, protein deficiency or immune system disease.

[0109] In some embodiments, the invention provides a method for delivering cargo molecules to target cells, wherein the cargo molecules are delivered by extracellular vesicles, wherein the extracellular vesicles comprising cargo molecules are produced by any genetic engineering production cell of the present invention. In some embodiments, the production cell overexpresses at least one or more polypeptides. In some embodiments, one or more polypeptides are connected to a glycosyl-phosphatidyl-inositol (GPI) group. In some embodiments, one or more polypeptides are connected to a cargo molecule. In some embodiments, one or more polypeptides are connected to a glycosyl-phosphatidyl-inositol (GPI) group and a cargo molecule. In some embodiments, polypeptides are derived from any of the polypeptides listed in Table A.

[0110] In some embodiments, the production cell is genetically engineered by transfection of a recombinant vector system. In some embodiments, the recombinant vector system includes a nucleic acid sequence encoding a glycosyl-phosphatidyl-inositol (GPI) group. In some embodiments, the recombinant vector system includes a nucleic acid sequence encoding a cargo molecule. In some embodiments, the recombinant vector system includes a nucleic acid sequence encoding a glycosyl-phosphatidyl-inositol (GPI) group and a cargo molecule. In some embodiments, the cargo molecule is a polypeptide, a protein, a lipid, a nucleic acid, a carbohydrate, a metabolite, or any combination thereof. In some embodiments, the nucleic acid includes RNA. In some embodiments, RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, microRNA (mi RNA) and long non-coding RNA (lncRNA). In some embodiments, the protein is an antibody or an enzyme.

[0111] In some embodiments, the extracellular vesicles produced by the production cells carry the cargo molecules on the outer surface of the extracellular vesicles. In some embodiments, the extracellular vesicles produced by the production cells carry the cargo molecules on the outer surface of the extracellular vesicles. In some embodiments, the extracellular vesicles produced by the production cells carry the cargo molecules in the lumen of the extracellular vesicles.

[0112] In the treatment methods disclosed herein, EVs can be administered to the patient via intravenous, intraarterial, intranasal or topical routes of administration. The effective dose can be empirically estimated by the attending physician or can be set by in vivo or in vitro evaluation for each pathology.

[0113] Example

[0114] Although preferred embodiments of the present invention have been shown and described herein, it will be appreciated by those skilled in the art that these embodiments are provided as examples only, as will be apparent. Without departing from the present invention, many changes, modifications and substitutions will now occur to those skilled in the art. It should be understood that various alternatives to the embodiments of the present invention described herein may be adopted in implementing the present invention. The following claims are intended to define the scope of the present invention and encompass methods and structures within the scope of these claims and their equivalents.

[0115] Example 1. Enhancing the production of extracellular vesicles (EVs) in mammalian cells

[0116] Cell culture

[0117] Human embryonic kidney 293 cells (HEK 293T) were placed in a DMEM cell culture medium containing 10% fetal bovine serum in a humidified incubator at 37°C, 5% CO2. Before suspension adaptation, cells were inoculated in a serum-free medium containing 5% serum (v / v) at 5x105 viable cells / mL and placed in a low binding 6-well plate on a shaking table at 120rpm. The cells were then cultured in an incubator at 37°C to reach a maximum cell density of approximately>85% cell viability, which is considered to be a sign that cells are fully adapted under experimental conditions. Adapted cells were passaged in a cell culture medium by sequential division, with serum concentrations reduced from 5% serum to 2%, 1%, 0.5%, 0.1% and 0% during each division.

[0118] For suspensions of 293F cells, cells were cultured in serum-free medium in a humidified incubator at 37°C, 5% CO2, with constant shaking at 120 rpm.

[0119] Plasmid construction and overexpression of target peptides

[0120] To generate stable cell lines overexpressing the polypeptide of interest, the PipyBac (PB) transposon system or the lentiviral system was used. Briefly, a nucleic acid fragment encoding the coding sequence (CDS) of the polypeptide of interest was synthesized and cloned into the BglII and XhoI restriction endonuclease sites of the PB transposon vector or into the XbaI and SalI restriction endonuclease sites of the pLenti vector. In this study, the CDS of CD46, CD55, and CD59 were synthesized and cloned into each vector for overexpression. Figure 1A As shown, an artificial GPI-anchored protein sequence was also synthesized and cloned into the vector. The artificial sequence in this example includes the mCherry sequence fused to the N-terminal and C-terminal domains of CD55.

[0121] According to the manufacturer's instructions, 2 μg of PB transposon vector carrying the polypeptide nucleic acid fragment was combined with 1 μg of PB transposase vector to transfect approximately 5×105 293T cells using Lipofectamine 3000. Alternatively, 2×105 293T cells were infected with lentivirus containing the target gene. The transfected or infected cells were then incubated and selected at 200 μg / mL hygromycin B for 7-8 days to obtain stable cell lines overexpressing the gene of interest.

[0122] Immunoblotting

[0123] Transfected cells were lysed in cell lysis buffer and the protein concentration of the lysates was measured by BCA protein assay to ensure equal loading. Samples were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were immunoblotted with anti-CD46 (sc-166159, Santa Cruz Biotechnology), anti-CD55 (sc-51733, Santa Cruz Biotechnology), anti-CD59 (sc-133170, Santa Cruz Biotechnology), and anti-GAPDH (2118S, CST). ECL-based immunoblotting detection was performed using updated HRP-linked secondary antibodies. Figure 1B As shown, overexpressed CD59, CD46 and CD55 protein levels were observed.

[0124] Example 2. Isolation and Quantification of Extracellular Vesicles

[0125] Isolation of small extracellular vesicles (sEVs)

[0126] Figure 2The isolation and purification process of extracellular vesicles (EVs), as well as their subsequent quantification, are summarized in . Briefly, cells were cultured in Condition cell culture medium. The conditioned medium was collected into a 50 mL centrifuge tube and immediately centrifuged at 500 x g for 10 min at 4 °C to remove susceptible cells. The supernatant was transferred to a new 50 mL centrifuge tube and centrifuged at 2000 x g for 20 min at 4 °C to remove dead cells. The supernatant was then filtered through a 0.2 μm membrane to remove cell debris and other large extracellular vesicles. The filtered sEVs were pelleted after ultracentrifugation at 100,000 x g for 85 min at 4 °C. The collected sEVs pellet was washed with PBS and further purified by a second ultracentrifugation at 100,000 x g for 85 min at 4 °C. The purified sEVs pellet was resuspended in an appropriate volume of PBS and then purified using a size exclusion column (SEC). The protein concentration of sEVs was quantified by BCA protein quantification assay. The particle concentration of sEVs was measured using an ApogeeMicro GxP flow cytometer, and the productivity (particles / L) was calculated as follows:

[0127] Nanoparticle tracking analysis (NTA) and nanoflow cytometry (nFCM) were used to measure the size and concentration of the acquired EVs. For NTA measurements, the NanoSight NS300 from Malvern Panalytical was used. For nFCM measurements, the Micro-GxP flow cytometer from Apogee flow System was used. In this disclosure, unless otherwise stated, the Micro-GxP system was used to measure the concentration of EVs particles in the acquired culture medium.

[0128] Increased EV production in 293T cells overexpressing CD55 and CD59

[0129] refer to Figure 3A -D, compared with vector control (293T WT Compared with 293T cells overexpressing CD59 and CD55, an approximately 8- to 40-fold increase in EV production (particles / L or μg / mL) was observed in 293T cells overexpressing CD59 and CD55.

[0130] The obtained particles were further characterized to measure the particle size distribution (e.g., <200 nm were identified as small EVs) and to investigate the morphology of the obtained EVs. Figure 4A -C, the particle size was measured by using nano flow cytometry with a commercially available silica bead standard (nFCM silica nanosphere mixture 1).

[0131] The protein expression levels of CD59, CD46, and CD55 in overexpressed cells and EVs were determined by immunoblotting. Figure 5 As shown, higher concentrations of CD55 and CD59 were observed in EVs than in total cell lysates. In contrast, overexpression of CD46 resulted in relatively lower expression levels of CD46 in EVs than in cells.

[0132] Transmission Electron Microscopy (TEM)

[0133] The morphology and viability of the obtained EVs were observed under TEM. The resuspended purified exosome samples were further diluted to 0.1 μg / μL. An equal volume of 4% paraformaldehyde was added to the exosome samples and incubated for 2 hours. 3uL of the mixture was dropped onto the TEM grid and fixed with 2% paraformaldehyde for 20 minutes. The grid was washed with 3XPBS and fixed with 1% glutaraldehyde for 5 minutes. After 8 PBS washes (2 minutes each) and ddH2O washes (9 times), the grids were stained with uranyl oxalate for 5 minutes, and in 1% methylcellulose: 4% uranyl acetate (9:1) on ice for 10 minutes. Excess liquid was removed with filter paper and the grids were air-dried for 5 to 10 minutes. Exosomes were examined at 60 kV in a JEOL 1100 transmission electron microscope, and images were acquired with an ATM digital camera, as shown in Figure 2. Figure 6 shown.

[0134] LC-MS / MS

[0135] Proteins were extracted from cell and EV samples and quantified using the BCA method. Appropriate amounts of protein were enzymatically digested and then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS). The raw data were analyzed by MaxQuant to obtain quantitative results for LFQ and iBAQ.

[0136] LFQ is a method for relative protein quantification when comparing multiple samples, and can compare the expression of the same protein. iBAQ is a method that approximates absolute quantification. It can be used to compare the abundance of different proteins in the same sample.

[0137] Example 3. GPI-anchored proteins enhance the production of extracellular vesicles (EVs)

[0138] Table A lists small polypeptides and proteins known to have glycosyl-phosphatidyl-inositol (GPI) groups. Among them, CD55, CD59, GPC1, GPC3, GPC4, GPC6 and SMPDL3B were identified as 293T from Example 1. WT 、293T CD46 、293T CD59 and 293T CD55 GPI proteins are highly enriched and concentrated in cell-derived EVs ( Figure 7 All identified GPI-anchored proteins (i.e., CD55, CD59, GPC1, GPC3, GPC4, GPC6, and SMPDL3B) were enriched at higher concentrations in EVs than in the total cellular fraction, as shown by the relative protein enrichment quantification at EV / cell > 1 in Table 2.

[0139] Table A: GPI-anchored polypeptides *LFQ:EV / Cell WT CD46 CD55 CD59 CD55 194180001 530230001 18.3650627 172840001 CD59 89.0901689 470260001 196300001 8.28679451 GPC1 491270001 387390001 202620001 484780001 GPC3 91153001 565660001 196960001 1466200001 GPC4 838300001 2491800001 978250001 1799800001 GPC6 150560001 330800001 343950001 129630001 SMPDL3B 162910001 144220001 146630001 86740001

[0140] *LFQ: relative protein quantification. EV enrichment indicates proteins detected in >1 EV / cell when equal protein amounts were input.

[0141] Table 2: Protein enrichment in EVs

[0142] CD52 overexpression also enhanced EV production in cells ( Fig. 8A -B). From 293T CD52 The EVs obtained are in the form of Figure 8C In addition, a significant increase in EV production was observed after co-overexpression of GPI-anchored proteins (i.e., CD55 and CD59) compared to wild-type controls. However, only a slight increase in EV production was observed when CD55 and CD59 were overexpressed separately ( Fig. 9A -C).

[0143] In other cell lines, such as 293F cells ( Fig. 10A -C) and mesenchymal stem cells (MSC) ( Fig.11A -C), similar improvement effects of GPI anchor proteins on EV production were observed. In MSC cell-based experiments, human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in mesenchymal hematopoietic stem cell basal medium (MSCBM, Dakewe6114011) containing 5% EliteGro-Advance (EPA-050, Elite cells). MSCs were infected with CD55 lentivirus produced by the 3-plasmid system and selected with 50ug / mL hygromycin. CD55 The cells were stably expressed. The selected cells were then cultured in conditioned medium for 2 to 3 days. The culture medium was collected for EVs isolation.

[0144] For decades, mesenchymal stem cells (MSCs) have attracted great attention in regenerative medicine due to their differentiation capacity, strong immunomodulatory properties, and good culture and manipulation capabilities. Recent studies have shown that the pleiotropic effects of MSCs are independent of their differentiation capacity but are mediated by the secretion of soluble paracrine factors. Extracellular vesicles are one of these paracrine mediators. EVs transfer functional substances such as miRNA and mRNA molecules, peptides, proteins, cytokines, and lipids from MSCs to recipient cells. EVs participate in intercellular communication events and contribute to the healing of injured or diseased tissues and organs. Previous studies have reported that EVs are solely responsible for the therapeutic effects of MSCs in many experimental models. Based on this, MSC-derived EVs will be studied as a new cell-free therapy for the treatment of heart disease, kidney disease, liver disease, immune system diseases, and nervous system diseases, or to promote skin wound healing.

[0145] Example 4. Overexpression of the GPI domain improves the production of extracellular vesicles (EVs)

[0146] To identify GPI-anchored protein regions that provide enhanced EV production, full-length and truncated CD55 constructs were generated (e.g. Fig. 12A As shown) and transfected into 293F cells. Fig. 12B As shown in Figure 3-C, overexpression of both truncated CD55 (i.e., constructs 0-3) and full-length CD55 (i.e., construct 4) increased EV production. The morphology of the test EVs was observed using cryo-EM ( Fig.12D ). Briefly, to prepare cryo-EM samples, lace carbon EM grids were glow-discharged. A 2 μL EVs sample aqueous solution was applied to the carbon side of the EM grid, which was then blotted dry and frozen in pre-cooled liquid ethane. The samples were studied in a Talos Arctic cryo-EM microscope.

[0147] Example 5. Effect of GPI-anchored proteins on the production of extracellular vesicles (EVs)

[0148] like Fig.13A As shown, two CD55-based constructs encoding mCherry (short: mCherry-CD5-short, and long: MHerry-CD5-long) were transfected into 293T cells as described in Example 1. Immunoblotting was performed to determine the expression level of mCherry (ab213511, Abcam) protein in total cell lysates and purified EVs ( Fig. 13B ).like Fig. 13B-C, the CD55-based GPI anchor structure was used as a scaffold to load and carry the mCherry sequence onto EVs. Significantly higher levels of mCherry were detected in EVs derived from 293T cells transfected with the L structure compared to 293T cells transfected with the S structure. The uptake of mCherry-loaded EVs by target cells expressing H2B-GFP was observed using fluorescence microscopy ( Fig.13D ).

[0149] As another example, two CD46-based constructs were fused to the mCherry sequence and transfected into 293T cells for overexpression ( Fig.14A -B). Interestingly, the enrichment of mCherry protein in EVs purified from cells transfected with the S construct was much higher than that in EVs purified from cells transfected with the L construct ( Fig. 14C Considering that CD46 is a transmembrane protein, two forms of CD46 short constructs were generated, each encoding nanoluciferase (Nluc) at a specific position ( Fig.14D , O: Nluc outside, I: Nluc inside). Fig.14E As shown, Nluc loading inside or outside EVs was controlled by fusing Nluc sequences near the N-terminus (outside) or C-terminus (inside, EVs lumen).

[0150] Interestingly, unlike co-overexpression of CD55 and CD59, overexpression of CD55 with the CD46 short construct resulted in a greater increase in EV production in 293T cells ( Fig.15A To determine whether other transmembrane proteins would provide the same effect, mOrange-tagged transmembrane proteins, namely CD9, CD63, and CD81, were overexpressed in the presence of CD55. Fig. 15B The data in show that overexpression of CD9 or CD81 with CD55 resulted in a synergistic effect (up to a 3-fold increase) in increased EV production in 293F cells. Co-expression of CD55 with either CD9 or CD81 provided an additive effect (a 2-fold increase).

[0151] Example 6. Preparation of ExoBoost cells and extracellular vesicles (EVs)

[0152] To industrialize the production of ExoBoost cells (i.e., cells transfected with GPI-anchored proteins), a single clone (clone number 28) was obtained from 293TCD55 cells and cultured as 28F cells. The main findings related to the upstream and downstream manufacturing processes are summarized below.

[0153] Upstream: Cell culture

[0154] 1. As Fig.16AAs shown, a decrease in cell viability reduced the percentage of EV production in both 28F and 293F cells.

[0155] 2. The use of the applicant's optimized culture medium significantly increased the concentration of EVs in 293F and 28F cells ( Fig. 16B -C).

[0156] 3. Addition of anticoagulants reduced EV% ( Fig.16D ).

[0157] 4. Compared with batch or fed-batch processes (e.g., one-pot reactions), perfusion significantly increased cell survival and EVs yield (particles / L / day) ( Fig.16E ).

[0158] 5. During the perfusion phase, hollow fiber filters with a pore size of 2-5 μm provide a higher permeability / retention ratio than hollow filters with smaller pore sizes or capsule filters with a pore size of 5 μm ( Fig.16F ).

[0159] Downstream: Purification

[0160] Fig.17 The downstream purification process is summarized. 28F cells were cultured by perfusion. The permeate was clarified by the first and second filtration and then concentrated by TFF. The concentrated sample was treated with benzonuclease and then purified by TFF and SEC. The sample was sterilized by 0.2um filtration, diluted to 2ug / uL and aliquoted and stored at -80℃. All materials used were sterilized at high level or designed for single use. The quality control methods are detailed in Table 3.

[0161] Table 3. Quality Control

[0162] EVs obtained from 28F cells were labeled with TREXO, and product quality was confirmed by measuring size distribution and EV% and acquiring TEM, Cryo-EM, nFCM, and immunoblot images ( Fig.18A -E). For nFCM results, 5uL EV sample (2E8 particles / uL) was mixed with 5uL anti-CD55-APC (10101-R028-A, Sinobiology) or PBS and then incubated at room temperature for 1 hour. The sample was then diluted 500 times for nFCM detection.

[0163] The obtained EVs were stable for 6 months at 4°C with a concentration higher than 10e9 particles / μL ( Fig.19A), even longer when stored at -80°C. The stability of EV samples under freeze-dried conditions was also tested. Briefly, frozen EVs were thawed from -80°C, and the particle concentration of the thawed samples was measured by nFCM. Then, the EV samples were diluted to a final concentration of 1e7 particles / uL with PBS or 10% (w / w) trehalose (T0167, Sigma) in PBS. The EVs samples were freeze-dried in a freeze dryer (FD-1A-50LGJ-10, Songyuan, Beijing). The freeze-dried samples were re-dissolved in ultrapure water at the initial volume before freeze-drying. After re-dissolution, the EV concentration was tested by nFCM, and the samples freshly thawed at -80°C were used as controls. As Fig.19B As shown, aggregation and degradation of freeze-dried EVs were observed.

[0164] Example 7. Loading EVs with cargo molecules

[0165] The loading of extracellular vesicles with siRNA as cargo molecules was studied.

[0166] Cargo molecules loaded onto the outside of EVs

[0167] Cholesterol-modified siRNA (GenePharm Co., Ltd) was dissolved in DEPC water without further purification. The purified EVs were dispersed in PBS and the concentration of EV particles was determined by nFCM. The siRNA solution was added to the EVs suspension by repeated pipetting, and the EVs and siRNA were mixed and then incubated at room temperature for 30 minutes before downstream purification. Unloaded free siRNA was purified from the final sample using a pre-filled chromatographic column. Cy5-dye-modified cholesterol siRNA (GenePharm Co., Ltd) was used for quantification of loading efficiency and loading capacity. Loading efficiency was measured by nFCM in terms of EV loading percentage.

[0168] Loading cargo molecules into the lumen of EVs

[0169] EVs were electroporated by a Gene Pulser Xcell electroporation system (BioRad) with a 0.1 cm cuvette (#1652089, BioRad). An exponential mode with variable voltage, fixed capacitance of 125 uF, and resistance of ∞ was used. Electroporation buffer was prepared and used freshly for each electroporation experiment. The particle concentration of EVs was adjusted to a final concentration ranging from 1E7 particles / uL to 1E9 particles / uL. After electroporation, the samples were kept on ice for at least half an hour and then purified using pre-packed chromatography columns to remove free RNA. The loading efficiency and loading capacity were measured by nFCM. To determine whether the RNA was packaged within the lipid membrane of EVs (and thus protected) or on the outer surface of EVs, the chromatographically purified samples were treated with RNase (RNase A / T1 mixture, Thermo Fischer) or an equal volume of PBS (no RNase control) for 10 minutes at 37°C. The samples were then measured by nFCM to determine changes in RNA loading efficiency with / without RNase treatment.

[0170] like Fig. 20A As shown in Figure 2B, siRNA loaded onto the outside via hydrophobic modification provided >99% efficiency. When siRNA was loaded into EVs via electroporation, approximately 40-60% efficiency was observed. Fig.21 As shown in Figure 2, siRNA loading was further verified by RNAse treatment. The loading efficiency is affected by many factors, such as the concentration of EVs ( Fig.22A ), the concentration ratio of EVs and RNA ( Fig. 22B ) and voltage ( Fig. 22C ). In general, higher loading efficiencies in the lumen of EVs were observed when higher EV concentrations of cargo molecules and higher voltages were used.

[0171] To determine whether cargo molecules can be loaded on the outside or surface of EVs, the CD46 short scaffold construct of Example 4 was fused with an HA tag at the N-terminus (outside) or C-terminus (inside) of the construct. After overexpression in cells, EVs were harvested and external loading of HA was measured by nFCM with anti-HA-488 (2350, CST), as shown in Fig.23 shown.

[0172] Typically, when EVs are taken up by target cells, they translocate to endosomes where they fuse with the endosomal membrane to release the cargo. For cargo molecules loaded into the lumen of EVs, such as enzymes and nucleic acids, endosomal escape is required to function at the target site within the cytoplasm. To study the release of cargo molecules from the lumen of EVs, β-lactamase (BLAM) was used as a cargo molecule. CCF4-AM is a cell-permeable, non-fluorescent compound that can cross the cell membrane due to its lipophilicity. Once inside the cell, cellular esterases cleave the acetoxymethyl ester group from CCF4-AM, thereby forming a carboxyfluorescein molecule. CCF4 is unique in that it can undergo a FRET-based color change upon interaction with β-lactamase, an enzyme that hydrolyzes the β-lactam ring present in antibiotics. In its intact state, CCF4 emits green fluorescence due to FRET between the donor fluorophore (carboxyfluorescein) and the acceptor fluorophore (coumarin). However, when β-lactamase is active and present in the cellular environment, it cleaves the CCF4 molecule, resulting in a loss of FRET and a fluorescence shift from green to blue. The activity of β-lactamase in the cytoplasm of cells was assessed by introducing CCF4-AM into the cells and monitoring the fluorescence emission.

[0173] VSVG (vesicular stomatitis virus glycoprotein) is a viral protein that facilitates the escape of cargo from endosomes into the cytoplasm of host cells. We fused BLAM to the CD63 N-terminus (inside) or the extracellular loop (outside). In another sample, BLAM was fused to the CD46-short N-terminus (inside) or C-terminus (outside). EVs (carrying the fusion peptide inside or outside the vesicle) were collected and taken up by the target 293T cells. Fig.24A As shown in Figure 2B, BLAM fusion polypeptides (i.e., BLAM-CD63-N-terminus, BLAM-CD63-extracellular loop, BLAM-CD46-short-N-terminus, and CD46-short-BLAM-C-terminus) are designed to be expressed on the membrane of target cells or EVs. Fig.24C As shown in Figure 5-F, BLAM activity was observed only when BLAM was placed inside EVs (not on the outer surface). In addition, the results showed that the cytoplasmic activity of BLAM requires the overexpression of the cargo molecule release facilitator VSVG. LiveBLAzer using CCF4-AM (K1096, ThermoFisher) TM The FRET-B / G loading tool was used to test the activity of BLAM.

[0174] Example 8: Extracellular vesicles (EVs) loaded with siRNA or shRNA

[0175] To determine whether EVs can deliver siRNA cargo molecules into cells and provide sufficient knockdown efficiency, cholesterol-modified siGFP labeled with Cy5 was loaded on the outer surface of EVs (loading efficiency determined by nFCM >90%) and introduced into cells in the absence of VSVG. RNAiMAX direct transfection was used as a positive control. No knockdown of GFP was observed when siRNA targeting GFP was loaded onto the outside of EVs. However, when shGFP was loaded into EVs in the presence or absence of VSVG in ExoBoost cells, GFP expression levels in cells were reduced (approximately 10% knockdown efficiency) after expressing shGFP-loaded EVs with VSVG, even when the loading efficiency was as low as <10%. The results are shown in Figure 2 Fig.25A -B.

[0176] Incorporated by Reference

[0177] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0178] Numbered embodiments of the present disclosure

[0179] Other subject matter encompassed by the present disclosure is listed in the following numbered embodiments:

[0180] 1. A method for enhancing the production of extracellular vesicles (EVs), comprising: obtaining a plurality of EVs from a production cell, wherein the production cell is genetically engineered to overexpress at least one polypeptide, and wherein the at least one polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group.

[0181] 2. The method of embodiment 1, wherein the polypeptide is derived from any one of the polypeptides in Table A.

[0182] 3. The method according to embodiment 1 or 2, wherein the polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4 or GPC6.

[0183] 4. The method of any one of embodiments 1-3, wherein the polypeptide is derived from CD59.

[0184] 5. The method of any one of embodiments 1-3, wherein the polypeptide is derived from CD55.

[0185] 6. The method according to any one of embodiments 1-5, wherein the production cell is genetically engineered by transfection of a recombinant vector system.

[0186] 7. The method according to Example 6, wherein the recombinant vector system comprises a nucleic acid sequence encoding the polypeptide coding sequence.

[0187] 8. According to the method described in Example 7, the recombinant vector system includes an expression control sequence operably linked to the nucleic acid sequence.

[0188] 9. The method according to Example 8, wherein the nucleic acid sequence includes at least one fluorescent marker.

[0189] 10. The method of embodiment 8, wherein the expression control sequence is a promoter.

[0190] 11. The method of embodiment 6, wherein the recombinant vector system comprises a selection marker.

[0191] 12. The method of embodiment 11, wherein the selection marker is selected from the group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance and histidinol resistance.

[0192] 13. A method according to embodiment 1, wherein the production cell further includes a release aid, and the release aid is selected from the group consisting of vesicular stomatitis virus glycoprotein (VSVG), glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1), baculovirus fusion protein gp64 and gB of Epstein-Barr virus (EBV).

[0193] 14. The method of any one of embodiments 1-13, wherein the production cell is a stable cell line that has been genetically engineered.

[0194] 15. The method according to any one of embodiments 1-14, wherein the plurality of EVs are obtained by dialysis or ultracentrifugation.

[0195] 16. The method according to Example 15, wherein the multiple EVs are obtained by ultracentrifugation.

[0196] 17. A method according to any one of embodiments 1-16, wherein the concentration of EVs obtained from the production cells is at least 2 times higher than the concentration of EVs obtained from wild-type cells.

[0197] 18. The method according to any one of embodiments 1-17, wherein the concentration of the EVs obtained from the production cells is 2 to 250 times higher than the concentration of the EVs obtained from wild-type cells.

[0198] 19. The method of any one of embodiments 1-18, wherein the production cell is a mammalian cell.

[0199] 20. The method according to embodiment 19, wherein the production cells are HEK 293F cells, HEK293T cells, mesenchymal stem cells (MSC) or any combination thereof.

[0200] 21. The method according to any one of embodiments 1-20, wherein the EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies or any combination thereof.

[0201] 22. The method of embodiment 21, wherein the EVs are exosomes.

[0202] 23. The method of embodiment 1, wherein the EVs are loaded with cargo molecules, wherein the cargo molecules include active pharmaceutical ingredients (API).

[0203] 24. The method of embodiment 23, wherein the API comprises a small molecule therapeutic agent.

[0204] 25. The method of embodiment 24, wherein the cargo molecule comprises a polypeptide, a protein, a lipid, a nucleic acid, a carbohydrate, a metabolite or any combination thereof.

[0205] 26. The method of embodiment 25, wherein the nucleic acid comprises DNA.

[0206] 27. The method of embodiment 25, wherein the nucleic acids comprise peptide nucleic acids (PNAs).

[0207] 28. The method of embodiment 25, wherein the nucleic acid comprises RNA.

[0208] 29. The method of embodiment 28, wherein the RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, micro RNA (mi-RNA) and long non-coding RNA (lncRNA).

[0209] 30. The method of embodiment 25, wherein the protein comprises an antibody or an enzyme.

[0210] 31. The method of embodiment 25, wherein the cargo molecule comprises an antisense oligonucleotide.

[0211] 32. The method of embodiment 25, wherein the cargo molecule comprises a morpholino oligomer.

[0212] 33. The method of embodiment 25, wherein the cargo molecule comprises one or more components of a gene editing system.

[0213] 34. A method according to embodiment 33, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, and transcription activator-like effector nuclease (TALEN).

[0214] 35. The method of any one of embodiments 1-34, wherein the cargo molecule is located on the inner surface or outer surface of the plurality of EVs.

[0215] 36. The method of embodiment 35, wherein the cargo molecule comprises a polypeptide fused to a polypeptide derived from CD46 or CD63.

[0216] 37. The method of embodiment 35, wherein the cargo molecule is located on the inner surface of the plurality of EVs, and wherein the cargo molecule has a higher efficacy in the presence of a release aid than in the absence of the release agent.

[0217] 38. A method for preparing a stable cell line for producing extracellular vesicles (EVs), comprising:

[0218] a) transfecting an EV production cell with an expression vector, wherein the expression vector comprises a nucleic acid sequence of one or more polypeptides and a selection marker, wherein the polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group;

[0219] b) screening and selecting transfected cells; and

[0220] c) culturing the selected cells.

[0221] 39. The method of embodiment 38, wherein the polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4 or GPC6.

[0222] 40. The method of embodiment 39, wherein the polypeptide is derived from CD59.

[0223] 41. The method of embodiment 39, wherein the polypeptide is derived from CD55.

[0224] 42. According to the method described in Example 38, the expression vector includes an expression control sequence operably linked to the nucleic acid sequence.

[0225] 43. The method of embodiment 42, wherein the expression control sequence is a promoter.

[0226] 44. The method of embodiment 42, wherein the nucleic acid sequence comprises at least one fluorescent marker.

[0227] 45. The method of embodiment 38, wherein the selectable marker is selected from the group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance, and histidinol resistance.

[0228] 46. ​​A method according to any one of embodiments 38-45, wherein the concentration of EVs obtained from the production cells is at least 2 times higher than the concentration of EVs obtained from wild-type cells.

[0229] 47. A method according to any one of embodiments 38-46, wherein the concentration of the EVs obtained from the production cells is 2 to 250 times higher than the concentration of the EVs obtained from wild-type cells.

[0230] 48. The method of any one of embodiments 38-47, wherein the production cell is a mammalian cell.

[0231] 49. The method of embodiment 48, wherein the production cells are HEK 293F cells, HEK 293T cells, mesenchymal stem cells (MSCs), or any combination thereof.

[0232] 50. The method of any one of embodiments 38-49, wherein the EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies, or any combination thereof.

[0233] 51. The method of embodiment 50, wherein the EVs are exosomes.

[0234] 52. The method of any one of embodiments 38-51, wherein the EVs are loaded with cargo molecules.

[0235] 53. The method of embodiment 52, wherein the cargo molecule comprises an active pharmaceutical ingredient (API).

[0236] 54. The method of embodiment 53, wherein the API comprises a small molecule therapeutic agent.

[0237] 55. The method of embodiment 52, wherein the cargo molecule comprises a polypeptide, a protein, a lipid, a nucleic acid, a carbohydrate, a metabolite or any combination thereof.

[0238] 56. The method of embodiment 55, wherein the nucleic acid comprises DNA.

[0239] 57. The method of embodiment 55, wherein the nucleic acids comprise peptide nucleic acids (PNAs).

[0240] 58. The method of embodiment 55, wherein the nucleic acid comprises RNA.

[0241] 59. A method according to embodiment 58, wherein the RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, micro RNA (mi-RNA) and long non-coding RNA (lncRNA).

[0242] 60. The method of embodiment 55, wherein the protein comprises an antibody or an enzyme.

[0243] 61. The method of embodiment 55, wherein the cargo molecule comprises an antisense oligonucleotide.

[0244] 62. The method of embodiment 55, wherein the cargo molecule comprises a morpholino oligomer.

[0245] 63. The method of embodiment 38, wherein the cargo molecule comprises one or more components of a gene editing system.

[0246] 64. A method according to embodiment 63, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, and transcription activator-like effector nuclease (TALEN).

[0247] 65. A cell line manufactured according to any one of embodiments 38-64.

[0248] 66. A kit for enhancing the production of EVs, comprising a production cell according to any one of embodiments 1-37 or a cell line according to embodiment 65.

[0249] 67. A composition comprising a plurality of EVs according to any one of embodiments 1-66.

[0250] 68. The composition of embodiment 67, further comprising a pharmaceutically acceptable excipient.

[0251] 69. A composition comprising extracellular vesicle (EVs) producing cells, wherein the EV producing cells are genetically engineered to overexpress at least one polypeptide, wherein the at least one polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group.

[0252] 70. The composition of embodiment 69, wherein the polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4 or GPC6.

[0253] 71. A composition according to embodiment 69 or 70, wherein the production cell is genetically engineered by a transfection recombinant vector system.

[0254] 72. A composition according to embodiment 71, wherein the recombinant vector system comprises a nucleic acid sequence encoding the polypeptide coding sequence.

[0255] 73. According to the composition of embodiment 71, the recombinant vector system includes an expression control sequence operably linked to the nucleic acid sequence.

[0256] 74. A composition according to embodiment 72, wherein the nucleic acid sequence includes at least one fluorescent label.

[0257] 75. The composition of embodiment 73, wherein the expression control sequence is a promoter.

[0258] 76. The composition of embodiment 71, wherein said recombinant vector system comprises a selectable marker.

[0259] 77. A composition according to any one of embodiments 69-76, wherein the production cell is a stable cell line that has been genetically engineered.

[0260] 78. The composition of embodiment 72, wherein the polypeptide is derived from any one of the polypeptides in Table 1.

[0261] 79. A composition according to Example 78, wherein the polypeptide comprises the sequence of mCherry-CD46(Short), HA-CD46Short or CD46Short HA in Table 1.

[0262] 80. The composition of embodiment 76, wherein the selectable marker is selected from the group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance, and histidinol resistance.

[0263] 81. A composition according to any one of embodiments 69-80, wherein the concentration of EVs obtained from the production cells is at least 2 times higher than the concentration of EVs obtained from wild-type cells.

[0264] 82. A composition according to any one of embodiments 69-81, wherein the concentration of the EVs obtained from the production cells is 2 to 250 times higher than the concentration of the EVs obtained from wild-type cells.

[0265] 83. A composition according to any one of embodiments 69-82, wherein the production cell is a mammalian cell.

[0266] 84. The composition of embodiment 83, wherein the production cells are HEK 293F cells, HEK 293T cells, mesenchymal stem cells (MSCs), or any combination thereof.

[0267] 85. A composition according to any one of embodiments 69-84, wherein the EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies or any combination thereof.

[0268] 86. The composition of embodiment 85, wherein the EVs are exosomes.

[0269] 87. A composition according to any one of embodiments 69-86, wherein the EVs are loaded with cargo molecules.

[0270] 88. The composition of embodiment 87, wherein the cargo molecule comprises an active pharmaceutical ingredient (API).

[0271] 89. The composition of embodiment 88, wherein the API comprises a small molecule therapeutic.

[0272] 90. The composition of embodiment 88, wherein the cargo molecule comprises a polypeptide, a protein, a lipid, a nucleic acid, a carbohydrate, a metabolite, or any combination thereof.

[0273] 91. The composition of embodiment 90, wherein the nucleic acid comprises DNA.

[0274] 92. The composition of embodiment 90, wherein the nucleic acids comprise peptide nucleic acids (PNAs).

[0275] 93. The composition of embodiment 90, wherein the nucleic acid comprises RNA.

[0276] 94. The composition of embodiment 93, wherein the RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, micro RNA (mi-RNA) and long non-coding RNA (lncRNA).

[0277] 95. The composition of embodiment 90, wherein the protein comprises an antibody or an enzyme.

[0278] 96. The composition of embodiment 90, wherein the cargo molecule comprises an antisense oligonucleotide.

[0279] 97. The composition of embodiment 90, wherein the cargo molecule comprises a morpholino oligomer.

[0280] 98. The composition of embodiment 87, wherein the cargo molecule comprises one or more components of a gene editing system.

[0281] 99. A composition according to embodiment 98, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, and transcription activator-like effector nuclease (TALEN).

[0282] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature mentioned in this specification, as well as those listed in the application data sheet, are incorporated into this specification by reference in their entirety. As needed, certain aspects of the embodiments can be modified to apply the concepts in various patents, applications, and documents to provide more embodiments.

Claims

1. A method for enhancing the production of extracellular vesicles (EVs), comprising: A plurality of EVs is obtained from a producer cell, wherein the producer cell is genetically engineered to overexpress at least one polypeptide, and wherein the at least one polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group.

2. The method according to claim 1, characterized in that The polypeptide is derived from any one of the polypeptides in Table A.

3. The method according to claim 1 or 2, characterized in that: The polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4 or GPC6.

4. The method according to any one of claims 1 to 3, characterized in that The polypeptide is derived from CD59.

5. The method according to any one of claims 1 to 3, characterized in that: The polypeptide is derived from CD55.

6. The method according to any one of claims 1 to 5, characterized in that The production cells are genetically engineered by transfecting a recombinant vector system.

7. The method according to claim 6, characterized in that The recombinant vector system includes a nucleic acid sequence encoding the polypeptide coding sequence.

8. The method according to claim 7, wherein the recombinant vector system comprises an expression control sequence operably linked to the nucleic acid sequence.

9. The method according to claim 8, characterized in that: The nucleic acid sequence includes at least one fluorescent label.

10. The method according to claim 8, characterized in that The expression control sequence is a promoter.

11. The method according to claim 6, characterized in that The recombinant vector system includes a selectable marker.

12. The method according to claim 11, characterized in that: The selection marker is selected from the group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance and histidinol resistance.

13. The method according to claim 1, characterized in that The production cell further comprises a release aid selected from the group consisting of vesicular stomatitis virus glycoprotein (VSVG), glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1), baculovirus fusion protein gp64, and gB of Epstein-Barr virus (EBV).

14. The method according to any one of claims 1 to 13, characterized in that The production cell is a stable cell line that has been genetically engineered.

15. The method according to any one of claims 1 to 14, characterized in that The multiple EVs are obtained by dialysis or ultracentrifugation.

16. The method according to claim 15, characterized in that: The multiple EVs are obtained by ultracentrifugation.

17. The method according to any one of claims 1 to 16, characterized in that The concentration of EVs obtained from the production cells is at least 2 times higher than the concentration of EVs obtained from wild-type cells.

18. The method according to any one of claims 1 to 17, characterized in that The concentration of the EVs obtained from the production cells is 2 to 250 times higher than that of the EVs obtained from wild-type cells.

19. The method according to any one of claims 1 to 18, characterized in that The producer cells are mammalian cells.

20. The method according to claim 19, characterized in that The production cells are HEK 293F cells, HEK293T cells, mesenchymal stem cells (MSCs) or any combination thereof.

21. The method according to any one of claims 1 to 20, characterized in that The EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies or any combination thereof.

22. The method according to claim 21, characterized in that: The EVs are exosomes.

23. The method according to claim 1, characterized in that: The EVs are loaded with cargo molecules, wherein the cargo molecules include active pharmaceutical ingredients (API).

24. The method according to claim 23, characterized in that The API includes a small molecule therapeutic.

25. The method according to claim 23, characterized in that The cargo molecule includes polypeptides, proteins, lipids, nucleic acids, carbohydrates, metabolites or any combination thereof.

26. The method according to claim 25, characterized in that The nucleic acid includes DNA.

27. The method according to claim 25, characterized in that The nucleic acids include peptide nucleic acids (PNAs).

28. The method according to claim 25, characterized in that The nucleic acid includes RNA.

29. The method according to claim 28, characterized in that The RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, micro RNA (mi-RNA) and long non-coding RNA (lncRNA).

30. The method according to claim 25, characterized in that The protein includes an antibody or an enzyme.

31. The method according to claim 25, characterized in that The cargo molecule comprises an antisense oligonucleotide.

32. The method according to claim 25, characterized in that The cargo molecule comprises a morpholino oligomer.

33. The method according to claim 25, characterized in that The cargo molecule includes one or more components of the gene editing system.

34. The method according to claim 33, characterized in that The gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, and transcription activator-like effector nuclease (TALEN).

35. The method according to any one of claims 1 to 34, characterized in that The cargo molecules are located on the inner surface or the outer surface of the plurality of EVs.

36. The method according to claim 35, characterized in that The cargo molecule includes a polypeptide fused to a polypeptide derived from CD46 or CD63.

37. The method according to claim 35, characterized in that The cargo molecule is located on the inner surface of the plurality of EVs, and wherein the cargo molecule has a higher efficacy in the presence of a release assisting agent than in the absence of the release assisting agent.

38. A method for preparing a stable cell line for producing extracellular vesicles (EVs), comprising: a) transfecting an EV production cell with an expression vector, wherein the expression vector comprises a nucleic acid sequence of one or more polypeptides and a selection marker, wherein the polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group; b) screening and selecting transfected cells; and c) culturing the selected cells.

39. The method according to claim 38, characterized in that The polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4 or GPC6.

40. The method according to claim 39, characterized in that The polypeptide is derived from CD59.

41. The method according to claim 39, characterized in that The polypeptide is derived from CD55.

42. The method of claim 38, wherein the expression vector comprises an expression control sequence operably linked to the nucleic acid sequence.

43. The method of claim 42, wherein the expression control sequence is a promoter.

44. The method according to claim 42, characterized in that The nucleic acid sequence includes at least one fluorescent label.

45. The method according to claim 38, characterized in that The selection marker is selected from the group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance and histidinol resistance.

46. ​​The method according to any one of claims 38 to 45, characterized in that The concentration of EVs obtained from the production cells is at least 2 times higher than the concentration of EVs obtained from wild-type cells.

47. The method according to any one of claims 38 to 46, characterized in that The concentration of the EVs obtained from the production cells is 2 to 250 times higher than that of the EVs obtained from wild-type cells.

48. The method according to any one of claims 38 to 47, characterized in that The producer cells are mammalian cells.

49. The method according to claim 48, characterized in that The production cells are HEK 293F cells, HEK293T cells, mesenchymal stem cells (MSCs) or any combination thereof.

50. The method according to any one of claims 38 to 49, characterized in that The EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies or any combination thereof.

51. The method according to claim 50, characterized in that The EVs are exosomes.

52. The method according to any one of claims 38 to 51, characterized in that The EVs are loaded with cargo molecules.

53. The method according to claim 52, characterized in that The cargo molecule comprises an active pharmaceutical ingredient (API).

54. The method according to claim 53, characterized in that The API includes a small molecule therapeutic.

55. The method according to claim 52, characterized in that The cargo molecule includes polypeptides, proteins, lipids, nucleic acids, carbohydrates, metabolites or any combination thereof.

56. The method according to claim 55, characterized in that The nucleic acid includes DNA.

57. The method according to claim 55, characterized in that The nucleic acids include peptide nucleic acids (PNAs).

58. The method according to claim 55, characterized in that The nucleic acid includes RNA.

59. The method according to claim 58, characterized in that The RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, micro RNA (mi-RNA) and long non-coding RNA (lncRNA).

60. The method according to claim 55, characterized in that The protein includes an antibody or an enzyme.

61. The method according to claim 55, characterized in that The cargo molecule comprises an antisense oligonucleotide.

62. The method according to claim 55, characterized in that The cargo molecule comprises a morpholino oligomer.

63. The method according to claim 38, characterized in that The cargo molecule includes one or more components of the gene editing system.

64. The method according to claim 63, characterized in that The gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, and transcription activator-like effector nuclease (TALEN).

65. A cell line manufactured according to any one of claims 38-64.

66. A kit for enhancing the production of EVs, comprising a production cell according to any one of claims 1-37 or a cell line according to claim 65.

67. A composition comprising a plurality of EVs according to any one of claims 1-66.

68. The composition of claim 67, further comprising a pharmaceutically acceptable excipient.

69. A composition comprising extracellular vesicle (EVs) producing cells, characterized in that The EV producing cells are genetically engineered to overexpress at least one polypeptide, wherein the at least one polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group.

70. The composition according to claim 69, characterized in that The polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4 or GPC6.

71. The composition according to claim 69 or 70, characterized in that The production cells are genetically engineered by transfecting a recombinant vector system.

72. The composition according to claim 71, characterized in that The recombinant vector system includes a nucleic acid sequence encoding the polypeptide coding sequence.

73. The composition of claim 71, wherein the recombinant vector system comprises an expression control sequence operably linked to the nucleic acid sequence.

74. The composition according to claim 72, characterized in that The nucleic acid sequence includes at least one fluorescent label.

75. The composition of claim 73, wherein the expression control sequence is a promoter.

76. The composition of claim 71, wherein the recombinant vector system comprises a selectable marker.

77. A composition according to any one of claims 69 to 76, characterized in that The production cell is a stable cell line that has been genetically engineered.

78. The composition according to claim 72, characterized in that The polypeptide is derived from any one of the polypeptides in Table 1.

79. The composition according to claim 78, characterized in that The polypeptide includes the sequence of mCherry-CD46 (Short), HA-CD46Short or CD46Short HA in Table 1.

80. The composition according to claim 76, characterized in that The selection marker is selected from the group consisting of neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeocin resistance, G418 resistance, phleomycin resistance, blasticidin resistance and histidinol resistance.

81. A composition according to any one of claims 69-80, characterized in that The concentration of EVs obtained from the production cells is at least 2 times higher than the concentration of EVs obtained from wild-type cells.

82. The composition according to any one of claims 69-81, characterized in that The concentration of the EVs obtained from the production cells is 2 to 250 times higher than that of the EVs obtained from wild-type cells.

83. A composition according to any one of claims 69-82, characterized in that The producer cells are mammalian cells.

84. The composition according to claim 83, characterized in that The production cells are HEK 293F cells, HEK293T cells, mesenchymal stem cells (MSCs) or any combination thereof.

85. The composition according to any one of claims 69-84, characterized in that The EVs are extranuclear particles, exosomes, microvesicles, apoptotic bodies or any combination thereof.

86. The composition according to claim 85, characterized in that The EVs are exosomes.

87. A composition according to any one of claims 69-86, characterized in that The EVs are loaded with cargo molecules.

88. The composition according to claim 87, characterized in that The cargo molecule comprises an active pharmaceutical ingredient (API).

89. The composition according to claim 88, characterized in that The API includes a small molecule therapeutic.

90. The composition according to claim 88, characterized in that The cargo molecule includes polypeptides, proteins, lipids, nucleic acids, carbohydrates, metabolites or any combination thereof.

91. The composition according to claim 90, characterized in that The nucleic acid includes DNA.

92. The composition according to claim 90, characterized in that The nucleic acids include peptide nucleic acids (PNAs).

93. The composition according to claim 90, characterized in that The nucleic acid includes RNA.

94. The composition according to claim 93, characterized in that The RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi interacting RNA (piRNA), small nucleolar RNA (snoRNAs), antisense RNA, micro RNA (mi-RNA) and long non-coding RNA (lncRNA).

95. The composition according to claim 90, characterized in that The protein includes an antibody or an enzyme.

96. The composition according to claim 90, characterized in that The cargo molecule comprises an antisense oligonucleotide.

97. The composition according to claim 90, characterized in that The cargo molecule comprises a morpholino oligomer.

98. The composition according to claim 87, characterized in that The cargo molecule includes one or more components of the gene editing system.

99. The composition according to claim 98, characterized in that The gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, and transcription activator-like effector nuclease (TALEN).

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