Methods for producing and using extracellular vesicles

Abstract

A method for enhancing extracellular vesicle production is disclosed herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to Chinese Patent Application No. PCT / CN2022 / 102338, filed on June 29, 2022, the content of each of which is incorporated herein by reference.

[0002] The present disclosure relates to methods for manufacturing and using extracellular vesicles. In particular, the present disclosure provides methods for the production of extracellular vesicles in cells and for enhancing programmable engineered extracellular vesicles.

Background Art

[0003] Extracellular vesicles (EVs) are naturally derived and secreted by all cells, both prokaryotic and eukaryotic. When secreted in both normal and pathophysiological states, EVs act as cargo assemblies for transporting essential cellular components (i.e., soluble proteins, or active enzymes, lipids, and nucleic acids such as mRNA, microRNA, long non - coding RNA, and metabolites), thereby facilitating the cell - to - cell communication process.

[0004] Such transport capabilities of EVs have prompted the exploration of the use of EVs in the delivery of drugs (e.g., therapeutic agents) to target cells or into target cells. Compared to other well - known synthetic drug delivery vehicles (e.g., liposomes, lipid nanoparticles, or viral vectors, etc.), EVs offer many advantages as drug carriers due to their characteristics as natural secretomes from cells for short - term or long - term cell - to - cell communication, having directionality to specific organs or cells through binding to specific surface receptors, excellent cargo transport efficiency through multiple cell uptake pathways that may include endocytosis, and the ability to avoid immunological clearance due to phagocytosis, micropinocytosis, or direct fusion with the recipient cell membrane and the inherent properties of the carrier.

[0005] EVs are classified broadly into exosomes and exosome-like vesicles. Exosomes typically have an average diameter range of about 40 to about 160 nm, which is smaller than red blood cells. Exosomes are also highly effective in crossing the blood-brain barrier, and such ability further attracts their use in drug delivery for various types of brain diseases. However, the production of exosomes in large scale is difficult because the amount of exosomes naturally produced in cells is limited.

[0006] Therefore, there is a need in the art for enhancing EV manufacturing methods, particularly for enhancing exosome production in mammalian cells. The present disclosure addresses these needs. SUMMARY OF THE INVENTION

[0007] Methods for producing extracellular vesicles (EVs) are disclosed herein. In particular, the present disclosure provides methods for enhancing the exosome manufacturing process.

[0008] In one aspect, the present disclosure provides a method for enhancing the production of extracellular vesicles (EVs) comprising: a) genetically engineering a production cell to overexpress at least one or more polypeptides; and b) harvesting a plurality of EVs from the production cell. In some cases, the polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) moiety. In some cases, the polypeptide is derived from any one of the polypeptides in Table A. In some cases, the polypeptide is derived from a protein selected from the group consisting of CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, and GPC6. In some cases, the polypeptide is derived from CD59. In some cases, the polypeptide is derived from CD55. In some cases, the polypeptide is selected from the group consisting of CD52, CD55, CD58, and CD59. In some cases, the polypeptide is CD59. In some cases, the polypeptide is CD55. In some cases, the EVs are exosomes, ectosomes, microvesicles, apoptotic bodies, or any combination thereof. In some cases, the EVs are exosomes.

[0009] In some cases, the production cell is genetically engineered by transfection with a recombinant vector system. In some cases, the recombinant vector system comprises a nucleic acid sequence encoding the polypeptide. In some cases, the recombinant vector system comprises an expression control sequence operably linked to the nucleic acid sequence. In some cases, the nucleic acid sequence comprises at least one fluorescent marker. In some cases, the expression control sequence is a promoter. In some cases, the recombinant vector system comprises a selectable marker. In some cases, the production cell is a genetically engineered stable cell line. In some cases, the plurality of EVs are harvested by dialysis or ultracentrifugation. In some cases, the plurality of EVs are harvested by ultracentrifugation.

[0010] In another aspect, the present disclosure provides a method for generating EVs that produce stable cell lines. In some cases, the method comprises: 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, and the polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group; b) screening and selecting the transfected cells; and c) culturing the selected cells. In some cases, the polypeptide is a protein selected from the group consisting of CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, and GPC6. In some cases, the polypeptide is CD59. In some cases, the polypeptide is CD55. In some cases, the polypeptide is derived from a protein selected from the group consisting of CD52, CD55, CD58, and CD59. In some cases, the polypeptide is derived from CD59. In some cases, the polypeptide is derived from CD55. In some cases, the expression vector comprises an expression control sequence operably linked to the nucleic acid sequence. In some cases, the expression control sequence is a promoter. In some cases, the nucleic acid sequence comprises at least one fluorescent marker. In some cases, 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.

[0011] In some cases, the amount of the concentration of EVs collected from the producing cells is at least 2-fold higher than the concentration from the control cells. In some cases, the amount of the concentration of EVs collected from the producing cells is 2-fold to 40-fold higher than the concentration from the control cells. In some cases, the producing cells are mammalian cells. In some cases, the producing cells are stem cells, mesenchymal stem cells (MSCs), HEK 293F cells, HEK 293T cells, or any combination thereof.

[0012] In some cases, EVs have cargo molecules incorporated therein. In some cases, the cargo molecules include a pharmaceutical active ingredient (API). In some cases, the API includes small molecule therapeutics. In some cases, the cargo molecules include polypeptides, proteins, lipids, nucleic acids, carbohydrates, lipids, metabolites, or any combination thereof. In some cases, the nucleic acids include DNA. In some cases, the nucleic acids include peptide nucleic acid (PNA). In some cases, the nucleic acids include RNA. In some cases, the RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), antisense RNA, microRNA (mi-RNA), and long non-coding RNA (lncRNA). In some cases, the proteins include antibodies or enzymes. In some cases, the cargo molecules include antisense oligonucleotides. In some cases, the cargo molecules include morpholino oligomers. In some cases, the cargo molecules include one or more components of a gene editing system. In some cases, the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nucleases, transcription activator-like effector nucleases (TALENs).

[0013] In yet another aspect, the disclosure provides a cell line produced according to any one of the methods described herein. The disclosure also provides a kit for enhancing EV production, comprising any one of the producer cells or stable cell lines described herein. The disclosure also provides a composition comprising a plurality of EVs produced according to any one of the EV production methods described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0014] In some embodiments, the present disclosure provides a composition comprising extracellular vesicle (EV)-producing cells, wherein the EV-producing cells are genetically engineered to overexpress at least one or more polypeptides, and the polypeptides are linked to glycosyl-phosphatidyl-inositol (GPI) groups.

[0015] The novel features of the invention are described in detail in the appended claims. A better understanding of the features and advantages of the invention can be obtained by reference to the following detailed description, which describes exemplary embodiments in which the principles of the invention are utilized, and the accompanying drawings.

Brief Description of the Drawings

[0016]

FIG. 1A

FIG. 1B

FIG. 2

FIG. 3A

FIG. 3B

FIG. 4A

FIG. 4B

FIG. 4C

FIG. 5

FIG. 6

FIG. 7

FIG. 8A

FIG. 8B

FIG. 8C

FIG. 9A

FIG. 9B

FIG. 9C

FIG. 10A

FIG. 10B

FIG. 10C

FIG. 11A

FIG. 11B

FIG. 11C

FIG. 12A

FIG. 12B

FIG. 12C

FIG. 12D

FIG. 13A

FIG. 13B

FIG. 13C

FIG. 13D

FIG. 14A

FIG. 14B

FIG. 14C

FIG. 14D

FIG. 14E

FIG. 15A

FIG. 15B

FIG. 16A

FIG. 16B

FIG. 16C

FIG. 16D

FIG. 16E

FIG. 16F

FIG. 17

FIG. 18A

FIG. 18B

FIG. 18C

FIG. 18D

FIG. 18E

FIG. 19A

FIG. 19B

FIG. 20A

FIG. 20B

FIG. 21

FIG. 22A

FIG. 22B

FIG. 22C

FIG. 23

FIG. 24A

FIG. 24B

FIG. 24C

FIG. 24D

FIG. 24E

FIG. 24F

FIG. 25A

FIG. 25B

Modes for Carrying Out the Invention

[0017] Definitions

[0018] As used herein, the term "about" and its grammatical equivalents with respect to reference numerical values, and its grammatical equivalents, can include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% etc. from that value. For example, the quantity "about 10" includes quantities from 9 to 11. Unless otherwise indicated, some embodiments herein contemplate numerical ranges. When a numerical range is provided, unless otherwise indicated, the range includes the range endpoints. Unless otherwise indicated, numerical ranges include all values and sub - ranges therein as if explicitly recited.

[0019] The singular forms "a", "an", and "the" include the plural unless it is clearly specified otherwise in the context. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by this application.

[0020] Unless otherwise indicated, open terms, such as "contain", "containing", "include", "including", and the like are meant to include.

[0021] The terms "agent", "active pharmaceutical ingredient (API)", "treatment method", "therapeutic agent", and "drug" are used interchangeably herein and include agents having pharmacological effects that induce biological or medical responses in animal, or human tissue, or cell lines desired by researchers, veterinarians, general practitioners, or other physicians, including changing biological systems at the molecular level (e.g., acting as inhibitors, activators, or regulators of proteins), including alleviation of symptoms, diseases, or disorders to be treated, and the agent can be a chemical compound, a biological molecule having therapeutic activity (e.g., siRNA, miRNA, anti-miRNA, shRNA, etc., antibodies, antibody fragments that recognize specific epitopes), an antitumor drug, or a radiation therapy drug.

[0022] The term "cargo molecule" refers to any molecule, or compound that can be incorporated, encapsulated, fused, or injected into molecular transport cargo (e.g., vesicles, exosomes, etc.) and can be a chemical molecule, or biological molecule, with or without therapeutic activity.

[0023] The term "extracellular vesicle" should be understood in the meaning generally known in the art and refers to vesicles containing membrane-coated cytoplasmic parts that are released from cells in the microenvironment. These vesicles represent a heterogeneous population that includes multiple types of vesicles, such as "exosomes", and vesicles, or apoptotic bodies, which can be classified separately based on size, antigen composition, and secretion mode. The terms "therapeutic agent delivery vesicle" and "therapeutic agent cargo" refer to, for example, vesicles that can be obtained from cells, such as microvesicles (any vesicles shed from the cell membrane of cells), exosomes (any vesicles derived from the endosomal pathway), apoptotic bodies (derived from apoptotic cells), microparticles (e.g., those that can be derived from platelets), exosomes (e.g., those that can be derived from neutrophils and monocytes in serum), prostasomes (obtainable from prostate cancer cells), cardiosomes (derived from heart cells), etc., of any type. Further, the terms "cargo molecule delivery vesicle" and "delivery vesicle" should also be understood to be potentially related to lipoprotein particles such as LDL, VLDL, HDL, and chylomicrons, as well as liposomes, lipid-like particles, lipidoids, etc. Essentially, the present disclosure can be related to any type of lipid-based structure (vesicles, or any other suitable form) that can act as a delivery or transport vehicle for cargo molecules.

[0024] As used herein, the terms "fusion", or "fusion polypeptide" refer to a recombinant protein of two or more polypeptides. A fusion protein can be produced, for example, by binding a nucleic acid sequence encoding one polypeptide to a nucleic acid encoding another polypeptide, or protein domain, such that, in a cell, they are translated into a single polypeptide carrying all the intended proteins and constitute a single open reading frame. The order of polypeptide arrangement can vary. A fusion polypeptide can include an epitope tag, or a half-life extender. Epitope tags include biotin, FLAG tag, c-myc, hemagglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tag, GST, β-galactosidase, AU1, AU5, and avidin. Half-life extenders include the Fc domain, and serum albumin.

[0025] The terms "linked", "anchored", or "associated" are understood in the present disclosure as any interaction between two groups, such as the interaction between a polypeptide and a GPI moiety, or the interaction between a GPI-anchored polypeptide and a membrane. This includes enzyme interactions, ionic bonds, covalent bonds, non-covalent bonds, hydrogen bonds, London forces, van der Waals forces, hydrophobic interactions, lipophilic interactions, magnetic interactions, electrostatic interactions, and the like.

[0026] The term "incorporation", or "incorporating extracellular vesicles" is understood in the present disclosure as an activity, or state, that results in a vesicle containing one or more target molecules that are not normally present inside, within, and / or on the membrane surface of the vesicle. In some embodiments, the cargo molecule is incorporated into the lumen of the extracellular vesicle. In some embodiments, the cargo molecule is incorporated onto the outer surface of the extracellular vesicle. In some embodiments, the cargo molecule is incorporated into the membrane of the extracellular vesicle.

[0027] The term "nucleic acid molecule" refers to a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases. This includes chromosomal DNA, which may be recombinant and can express exogenous polypeptides when introduced into cells, as well as self-replicating plasmids, vectors, mRNA, tRNA, siRNA, etc.

[0028] The term "polypeptide" or "peptide" is understood in the present disclosure as a sequence of amino acids composed of amino acids linked by peptide bonds. This term can be used interchangeably with "protein" in its broadest sense to refer to a molecule of two or more amino acids, amino acid analogs, or peptidomimetics. In some cases, the amino acids are linked by peptide bonds. In some cases, the amino acids are linked by other types of bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to any natural and / or non-natural or synthetic amino acid, including glycine, as well as both D- or L-optical isomers, and amino acid analogs, and peptidomimetics.

[0029] In some cases, the peptides or polypeptides of the present disclosure contain at least two amino acid residues and have a length of less than about 50 amino acids (e.g., 40 amino acids, 30 amino acids, 20 amino acids, or any number thereof). In some cases, the peptides or polypeptides of the present disclosure contain at least 50 amino acids, 100 amino acids, 150 amino acids, or more. In some cases, counterions are provided to the peptides or polypeptides. In some embodiments, the peptides or polypeptides include N-terminal and / or C-terminal modifications, such as blocking modifications that reduce degradation or have a GPI group linked post-translationally.

[0030] The terms "purified," "isolated," and "obtained" are used interchangeably and are intended to mean removed from its natural environment. The terms purified or isolated do not require absolute purity or isolation, but rather are intended as relative terms.

[0031] The term "vector" is a nucleic acid molecule, preferably self-replicating, that transfers and / or replicates an inserted nucleic acid molecule, such as a transgene or exogenous nucleic acid, within and / or between host cells. This includes plasmids or viral chromosomes into which a fragment of recombinant DNA is inserted and used to introduce the recombinant DNA or transgene into the polypeptides of the present disclosure.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the preferred methods and materials are described herein. Unless otherwise stated, the techniques employed herein are standard methodologies. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0033] Summary

[0034] Disclosed herein are methods for enhancing the production of extracellular vesicles (EVs) derived from or secreted by genetically engineered mammalian EV-producing cells. The EVs of the present disclosure can be incorporated with a sufficient amount of one or more therapeutic agents or any target molecule to deliver an effective amount of the therapeutic agent or any target molecule to a target site.

[0035] Previously, various manufacturing strategies have been contemplated to increase the production of EVs in cells. Some of the strategies include hypoxia induction, overexpression of tetraspanin proteins, and overexpression of hypoxia-inducible factor-1α. Genetic modification of genes (e.g., Nad B, SCD4, STEAP3) in some exosome-producing cells has been previously contemplated for EV production in cells, but the overall effect of overexpressing these genes has not been impressive. Here, the present disclosure provides a very efficient and excellent method for manufacturing EVs that enhances the production of EVs in exosome-producing cells.

[0036] Method for enhancing extracellular vesicle (EV) production

[0037] In one aspect, the present disclosure provides a method for enhancing extracellular vesicle (EV) production, comprising: a) genetically engineering a production cell to overexpress at least one or more polypeptides; and b) collecting a plurality of EVs from the production cell.

[0038] In some cases, the EVs are ectosomes, exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some cases, the EVs are exosomes.

[0039] In some cases, the producer cells are genetically modified to contain one or more polypeptides. In some cases, the producer cells naturally contain one or more polypeptides, and exosomes derived therefrom also contain polypeptides. The level of any desired polypeptide can be directly modified on the exosome (e.g., by contacting the complex with a recombinantly produced polypeptide to effect insertion into the membrane of the complex, or conjugation). Alternatively, or in addition, the level of any desired polypeptide can be directly modified on the producer cell (e.g., by contacting the complex with a recombinantly produced polypeptide to effect insertion into the membrane of the cell, or conjugation). Alternatively, the producer cells can be modified by transfecting the producer cells with exogenous nucleic acid to express the desired polypeptide. The polypeptide can already naturally exist on the producer cell, in which case the exogenous construct can result in overexpression of the polypeptide and an increase in the concentration of the polypeptide within or on the producer cell. Alternatively, the naturally expressed protein can be removed from the producer cell (e.g., by inducing gene silencing in the producer cell). The polypeptide can confer different functions on the exosome (e.g., specific targeting ability, delivery function (e.g., fusion molecule), enzymatic function, increase or decrease of half-life in vivo, etc.).

[0040] In some cases, the polypeptide includes, but is not limited to, any one of 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, cadherin, carcinoembryonic antigen (CEA), scrapie prion protein, folate binding protein, or any polypeptide listed in Table A, or any combination thereof.

[0041] In some cases, the EVs of the present disclosure are exosomes that contain on their surface one or more polypeptides selected from any one of 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 the polypeptides listed in Table A, or any combination thereof. In some cases, the exosomes are modified to contain one or more polypeptides.

[0042] In some cases, the producing cells are mammalian cells. In some cases, the producing cells are selected from human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080 cells, human embryonic retina PER.C6 cells, kidney / B cell hybrid HKB-11 cells, primary human amnion CAP cells, human mesenchymal stem cells (MSC), or hepatoma HuH-7 human cells. In some cases, the producing cells are HEK293-H, HEK293-T, HEK293-EBNA1, or HEK293-F. In some cases, the producing cells are genetically engineered to provide transient overexpression of the polypeptide. In some cases, the producing cells are genetically engineered stable cell lines that constitutively overexpress the polypeptide.

[0043] In some cases, the polypeptide comprises a glycosyl-phosphatidyl-inositol (GPI) moiety. In some cases, the GPI moiety is added post-translationally at the C-terminus of the polypeptide. GIP is a lipid moiety that includes a phosphoethanolamine linker, a glycan core, and a phospholipid tail. In some cases, the GPI moiety is covalently attached to the polypeptide as a post-translational modification marker, enabling lipid raft partitioning, signal transduction, cell-cell communication, or apical membrane targeting. In some cases, GPI moiety addition enables the modified polypeptide to anchor in the outer leaflet of the membrane region. In some cases, GPI-anchored polypeptides are classified as exosomes. In some cases, GPI-anchored polypeptides are exposed on the surface of exosomes.

[0044] In some cases, the GPI-anchored polypeptide is any one of the polypeptides listed in Table A. In some embodiments, the GPI-anchored polypeptide is any one or more polypeptides selected from Table A.

[0045] In some cases, the producing cell is genetically engineered to overexpress any one of the polypeptides listed in Table A, or a functional polypeptide fragment thereof, in an amount or copy number sufficient to be present during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more cycles. In some embodiments, the producing cell is genetically engineered to overexpress the polypeptide of Table A, or a functional polypeptide fragment thereof, in an amount, copy number, and / or ratio sufficient to be present during 15, 21, 30, 45, 60, 100, 120 days or more cycles.

[0046] In some cases, the GPI-anchored polypeptide is selected from the group consisting of CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, and GPC6.

[0047] In some cases, the GPI-anchor polypeptide is CD55, a 70 kDa membrane protein also known as complement decay-accelerating factor, or DAF. CD55 recognizes the complement system C4b and C3b fragments generated in C4 (classical complement pathway and lectin pathway), as well as C3 (alternative complement pathway). CD55 can block the formation of the membrane attack complex or prevent lysis by the complement cascade. In some cases, the producing cell is genetically engineered to overexpress the CD55 polypeptide, or a functional polypeptide fragment thereof, in an amount, or copy number, sufficient to be present during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more cycles. In some embodiments, the producing cell is genetically engineered to overexpress the CD55 polypeptide, or a functional polypeptide fragment thereof, in an amount, copy number, and / or ratio sufficient to be present during 15, 21, 30, 45, 60, 100, 120 days or more cycles.

[0048] In some cases, the GPI-anchor polypeptide is CD59, also known as membrane inhibitor of reactive lysis (MAC-IP), membrane inhibitor of reactive lysis (MIRL), protectin, or homologous restriction factor (HRF), a protein that binds to host cells via glycosylphosphatidylinositol (GPI) anchoring. In some cases, the producing cell is genetically engineered to overexpress the CD59 polypeptide, or a functional polypeptide fragment thereof, in an amount, or copy number, sufficient to be present during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more cycles. In some embodiments, the producing cell is genetically engineered to overexpress the CD59 polypeptide, or a functional polypeptide fragment thereof, in an amount, copy number, and / or ratio sufficient to be present during 15, 21, 30, 45, 60, 100, 120 days or more cycles.

[0049] In some cases, the GPI anchor polypeptide is CD52. CD52 is not present on granulocytes and bone marrow precursors, while being expressed at high levels on T lymphocytes and B lymphocytes, and at low levels on monocytes. In some cases, the producing cells are genetically engineered to overexpress the CD52 polypeptide, or a functional polypeptide fragment thereof, in an amount, or copy number, sufficient to be present during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more of circulation. In some embodiments, the producing cells are genetically engineered to overexpress the CD52 polypeptide, or a functional polypeptide fragment thereof, in an amount, copy number, and / or ratio sufficient to be present during 15, 21, 30, 45, 60, 100, 120 days or more of circulation.

[0050] In some cases, the GPI anchor polypeptide is selected from the group consisting of CD52, CD55, CD58, and CD59. In some cases, the polypeptide comprises a glycosyl-phosphatidyl-inositol (GPI) moiety, an extracellular domain, a transmembrane domain, a cytoplasmic domain, or combinations thereof.

[0051] In some cases, the producer cells are genetically engineered by transfection with a recombinant vector system to overexpress a polypeptide. As used herein, the terms "transfection" or "transfect" refer to a method of introducing exogenous genetic material into a host cell (e.g., mammalian cells via a lentivirus), and the host cell may be transiently transfected or stably transfected. The genetic material may be an expression vector containing a polynucleotide sequence encoding a gene of interest (e.g., a recombinant GPI-anchor polypeptide), or siRNA, or shRNA. Also, it may refer to introducing viral nucleic acid sequences in a manner that is natural for each virus. The viral nucleic acid sequences do not necessarily have to exist as naked nucleic acid sequences, but may be packaged in a viral protein envelope.

[0052] Transfection of eukaryotic host cells with polynucleotides, or expression vectors, resulting in genetically modified cells, or transgenic cells, can be carried out 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 co-precipitation, electroporation, nucleofection, nucleoporation, microporation, polycation (such as DEAE-dextran) - mediated transfection, protoplast fusion, viral infection, and microinjection. Transformation can result in transient or stable transformation of the host cell. In some cases, transfection is stable transfection. In some cases, transfection is transient transfection. Preferred are transfection methods that provide optimal transfection frequencies, as well as expression of heterologous genes in specific host cell lines and types. Appropriate methods can be determined by routine procedures. For stable transfectants, the construct is either integrated into the host cell genome or artificial chromosome / minichromosome, or episomally located so as to be stably maintained within the host cell. Thus, stably transfected sequences effectively remain in the genome of the cell and its daughter cells. Typically, this involves the use of a selectable marker gene, and the polynucleotide sequence encoding the gene or RNA of interest is integrated together with the selectable marker gene. Cells having such a selectable marker gene are screened and selected for further culture (including passage, growth, culturing, splitting at optimal cell density). In some cases, the entire expression vector is integrated into the cell genome, and in other cases, only a part of the expression vector is integrated into the cell genome.A cell that "stably expresses" a recombinant polypeptide or RNA is stably transfected with the gene encoding the recombinant polypeptide or the polynucleotide sequence encoding the RNA. Thus, the sequence encoding the recombinant polypeptide or RNA remains in the genome of the cell and its daughter cells.

[0053] In some cases, the recombinant vector system includes a nucleic acid sequence encoding a GPI-anchor polypeptide. In some cases, the nucleic acid sequence encodes a portion of the GPI-anchor protein. In some cases, the nucleic acid sequence encodes the N-terminal domain of the GPI-anchor protein. In some cases, the nucleic acid sequence encodes the C-terminal domain of the GPI-anchor protein. In some cases, the nucleic acid sequence encodes the N-terminal and C-terminal domains of the GPI-anchor protein.

[0054] In some cases, the nucleic acid sequence encodes the N-terminal domain of any one of the polypeptides listed in Table A. In some cases, the nucleic acid sequence encodes the C-terminal domain of any one of the polypeptides listed in Table A. In some cases, the nucleic acid sequence encodes the N-terminal and C-terminal domains of any one of the polypeptides listed in Table A. In some cases, the nucleic acid sequence encodes the N-terminal domain of CD52. In some cases, the nucleic acid sequence encodes the C-terminal domain of CD52. In some cases, the nucleic acid sequence encodes the N-terminal and C-terminal domains of CD52. In some cases, the nucleic acid sequence encodes the N-terminal domain of CD55. In some cases, the nucleic acid sequence encodes the C-terminal domain of CD55. In some cases, the nucleic acid sequence encodes the N-terminal and C-terminal domains of CD55. In some cases, the nucleic acid sequence encodes the N-terminal domain of CD58. In some cases, the nucleic acid sequence encodes the C-terminal domain of CD58. In some cases, the nucleic acid sequence encodes the N-terminal and C-terminal domains of CD58. In some cases, the nucleic acid sequence encodes the N-terminal domain of CD59. In some cases, the nucleic acid sequence encodes the C-terminal domain of CD59. In some cases, the nucleic acid sequence encodes the N-terminal and C-terminal domains of CD59. In some cases, the nucleic acid sequence encodes the N-terminal domain of CD46. In some cases, the nucleic acid sequence encodes the C-terminal domain of CD46. In some cases, the nucleic acid sequence encodes the N-terminal and C-terminal domains of CD46.

[0055] In some cases, the nucleic acid sequence encodes an amino acid sequence selected from Table 1.

[0056]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0057] Table 1. Amino acid sequence

[0058] In some cases, the recombinant vector system includes an expression control sequence operably linked to a nucleic acid sequence. In some cases, the expression control sequence is a promoter. For example, the 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, exemplary vectors may each contain T3 and T7 promoter sequences compatible with T3 and T7 RNA polymerases, respectively. Examples of other promoter sequences (exemplified for expression in mammalian cells) are promoters and / or enhancers derived from strong mammalian promoters such as (CMV) (cytomegalovirus), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyomavirus, as well as natural immunoglobulin promoters, and actin promoters. In some cases, the expression control sequence is a polyadenylation signal such as BGH polyA, SV40 late, or early polyA, or alternatively, the 3’UTR of an immunoglobulin gene. In some cases, the nucleic acid sequence of a polypeptide may be fused to at least one active domain at the N-terminus and / or C-terminus, and the active domain may be selected from the group consisting of nucleases (e.g., endonucleases, or exonucleases), polymerases, kinases, phosphatases, methylases, demethylases, acetylases, deacetylases, topoisomerases, integrases, transposases, ligases, helicases, recombinases, transcriptional activators (e.g., VP64, VP16), transcriptional inhibitors (e.g., KRAB), DNA end processing enzymes (e.g., Trex2, Tdt), and reporter molecules (e.g., fluorescent proteins, lacZ, luciferase).

[0059] In some cases, the recombinant vector system includes a selectable marker. In some cases, the selectable marker is ampicillin, chloramphenicol, kanamycin, tetracycline, blasticidin S, neomycin, hygromycin B, or any combination thereof.

[0060] In some cases, the nucleic acid sequence includes a sequence corresponding to at least one fluorescent marker. In some cases, the fluorescent marker is a green fluorescent protein (e.g., GFP, EGFP, AmCyan, etc.), a red fluorescent protein (e.g., mCherry, DsRed, tdTomato, mStrawberry, etc.), an orange-yellow, and a yellow fluorescent protein (e.g., mOrange, mvanana, ZsYellow, etc.), a far-red fluorescent protein (e.g., E-Crimson, HcRed, mRasberry, mPlum, etc.), or any combination thereof. In some cases, the fluorescent marker is mCherry.

[0061] In some cases, the identity or quantity of the producing cells or exosomes can be evaluated by in vitro assays. For example, the identity or quantity of the producing cells or exosomes is evaluated by counting the number of cells or complexes in the population, for example, by microscopy, flow cytometry, or hemocytometry. As another method, or additionally, the identity or quantity of the producing cells or exosomes is evaluated by analysis of the protein content of the cells or complexes, for example, by flow cytometry, Western blot, immunoprecipitation, fluorescence spectroscopy, chemiluminescence, mass spectrometry, or absorbance spectroscopy. In some cases, the protein content assayed is surface protein, such as a differentiation marker, receptor, co-receptor, transporter, glycoprotein. In some embodiments, the identity or quantity of the producing cells or exosomes is evaluated by analysis of the receptor content of the cells or complexes, for example, by flow cytometry, Western blot, immunoprecipitation, fluorescence spectroscopy, chemiluminescence, mass spectrometry, or absorbance spectroscopy. For example, the identity or quantity of the producing cells or exosomes can be evaluated by the mRNA content of the cells or complexes, for example, by RT-PCR, flow cytometry, or Northern blot. The identity or quantity of the producing cells can be evaluated by the nuclear material content, for example, by nuclear staining or using nucleic acid probes, for example, by flow cytometry, microscopy, or Southern blot. As another method, or additionally, the identity or quantity of the producing cells or exosomes is evaluated by the lipid content of the cells or complexes, for example, by flow cytometry, liquid chromatography, or mass spectrometry.

[0062] In another aspect, the present disclosure provides a method of generating EVs that produce stable cell lines. In some cases, the method comprises: a) transfecting EV-producing cells with an expression vector, the expression vector comprising nucleic acid sequences of at least one polypeptide and a selection marker, and at least one polypeptide being linked to a glycosyl-phosphatidyl-inositol (GPI) group; b) screening and selecting the transfected cells; and c) culturing the selected cells.

[0063] In some cases, the polypeptide is selected from the group consisting of 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 cases, the polypeptide is derived from CD52, CD55, CD58, or CD59. In some cases, the polypeptide is derived from CD59. In some cases, the polypeptide is derived from CD55. In some cases, the polypeptide is CD59. In some cases, the polypeptide is CD55.

[0064] In some cases, the expression vector includes an expression control sequence operably linked to a nucleic acid sequence. In some cases, the expression control sequence is a promoter. In some cases, the nucleic acid sequence includes at least one fluorescent marker. In some cases, 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.

[0065] In some cases, the EVs are harvested by dialysis or ultracentrifugation. In some cases, the EVs are harvested by ultracentrifugation. In some cases, any one of the cells of the present disclosure containing EVs can be filtered through a filter having a suitable mesh or pore size (e.g., a nylon mesh cell strainer). The filter may have a pore size of 50 nm to 100 mM. In some cases, the filter may have a pore size of 80 nm to 90 mM. In some cases, the filter may have a pore size of 100 nm to 80 mM. In some cases, the filter may have a pore size of about 200 nm to about 70 mM, about 400 nm to about 60 mM, about 600 nm to about 50 mM, about 800 nm to about 40 mM, or about 1 mM to about 20 mM.

[0066] In some cases, the concentration of EVs collected from the producing cells is 2 to 300 times, 250 times, 200 times, 150 times, or 100 times higher than that from the control cells (i.e., wild-type cells or vehicle-transfected cells), or any value or range in between. In some cases, the concentration of EVs collected from the producing cells is 2 to 90 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 80 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 70 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 60 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 50 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 40 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 30 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 20 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 10 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 8 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 4 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is 2 to 2 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is at least 2 times higher than the concentration from the control cells. In some cases, the concentration of EVs collected from the producing cells is at least 4 times higher than the concentration from the control cells. In some cases, the concentration of EVs collected from the producing cells is at least 6 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is at least 8 times higher than that from the control cells. In some cases, the concentration of EVs collected from the producing cells is at least 10 times higher than the concentration from the control cells.In some cases, the concentration of EVs harvested from the producer cells is at least 15 times higher than that from the control cells. In some cases, the concentration of EVs harvested from the producer cells is at least 20 times higher than the concentration from the control cells. In some cases, the concentration of EVs harvested from the producer cells is at least 25 times higher than that from the control cells. In some cases, the concentration of EVs harvested from the producer cells is at least 30 times higher than the concentration from the control cells. In some cases, the concentration of EVs harvested from the producer cells is at least 35 times higher than that from the control cells. In some cases, the concentration of EVs harvested from the producer cells is at least 40 times higher than that from the control cells. In some cases, the concentration of EVs harvested from the producer cells is at least 45 times higher than that from the control cells. In some cases, the concentration of EVs harvested from the producer cells is at least 50 times higher than that from the control cells.

[0067] Extracellular vesicles (EVs)

[0068] The present disclosure provides a method for enhancing EV production in mammalian cells.

[0069] In some cases, the EVs of the present disclosure are exosomes. In some cases, the exosome comprises a membrane that forms particles having a diameter of 30 to 100 nm, 30 to 200 nm, or 30 to 500 nm. In some cases, the exosome comprises a membrane that forms particles having a diameter of 10 to 100 nm, 20 to 100 nm, 30 to 100 nm, 40 to 100 nm, 50 to 100 nm, 60 to 100 nm, 70 to 100 nm, 80 to 100 nm, 90 to 100 nm, 100 to 200 nm, 100 to 150 nm, 150 to 200 nm, 100 to 250 nm, 250 to 500 nm, or 10 to 1000 nm. In some cases, the EV has an average diameter length of at least about 180 nm. In some cases, the EV has an average diameter length of at least about 80 nm to about 180 nm, about 85 nm to about 175 nm, about 90 nm to about 160 nm, about 92 nm to about 150 nm, about 96 nm to about 140 nm, about 98 nm to about 130 nm, about 100 nm to about 120 nm, about 102 nm to about 112 nm, or about 105 nm to about 110 nm. The size of the EV can change after the uptake of cargo molecules. In other cases, the size of the EV may remain the same after uptake. In some cases, the exosome has an average diameter length of at least about 80 nm. In some cases, the membrane comprises lipids and fatty acids. In some embodiments, the exosome membrane comprises one or more of phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. Further, the membrane can comprise one or more polypeptides and one or more polysaccharides, such as glycans, etc. In some cases, the produced exosomes can have a specific gravity between 0.5 to 2.0, 0.6 to 1.0, 0.7 to 1.0, 0.8 to 1.0, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.4 to 1.5, 1.0 to 1.5, 1.5 to 2.0, and 1.0 to 2.0 kg / m 3 and may have a specific gravity therebetween.

[0070] In some cases, multiple EVs may be manufactured, purified, or isolated from cells, cell culture media, or tissues as described in Example 1. In some cases, multiple EVs may be purified or isolated before contacting with a surfactant or cargo molecule. In some cases, multiple EVs may be purified or isolated after contacting with a surfactant removing agent. In some cases, EVs may be purified or isolated as multiple cargo-loaded EVs.

[0071] In some cases, cargo molecules are incorporated into EVs. In some cases, cargo molecules include pharmaceutical active ingredients (APIs). In some cases, APIs include small molecule therapeutics. In some cases, cargo molecules include polypeptides, proteins, lipids, nucleic acids, carbohydrates, lipids, metabolites, or any combination thereof. In some cases, nucleic acids include DNA. In some cases, nucleic acids include peptide nucleic acids (PNAs). In some cases, nucleic acids include RNA. In some cases, RNA is selected from the group consisting of mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), antisense RNA, microRNA (mi-RNA), and long non-coding RNA (lncRNA). In some cases, proteins include antibodies or enzymes. In some cases, cargo molecules include antisense oligonucleotides. In some cases, cargo molecules include morpholino oligomers. In some cases, cargo molecules include one or more components of a gene editing system. In some cases, the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nucleases, transcription, and transcription activator-like effector nucleases (TALEN).

[0072] In yet another aspect, the present disclosure provides a cell line produced according to any one of the methods described herein. The present disclosure also provides a kit for enhancing EV production, comprising any one of the producer cells or cell lines described herein. The present disclosure also provides a composition comprising a plurality of EVs by any one of the EVs described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0073] Disclosed herein are EVs comprising at least one cargo molecule. Also disclosed herein is a method of producing EVs for incorporating a sufficient amount of one or more cargo molecules such that an appropriate amount of the one or more cargo molecules is delivered to, or into, a target cell or tissue of interest.

[0074] In some cases, one or more cargo molecules have a final concentration of from about 0.1 μM to about 300 μM. In some cases, one or more cargo molecules have a final concentration of 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 10 μM to about 20 μM, about 10 μM to about 50 μM, about 10 μM to about 100 μM, about 10 μM to about 150 μM, about 10 μM to about 200 μM, about 10 μM to about 300 μM, about 20 μM to about 50 μM, about 20 μM to about 100 μM, about 20 μM to about 150 μM, about 20 μM to about 200 μM, about 20 μM to about 300 μM, about 50 μM to about 100 μM, about 50 μM to about 150 μM, about 50 μM to about 200 μM, about 50 μM to about 300 μM, about 100 μM to about 150 μM, about 100 μM to about 200 μM, about 100 μM to about 300 μM, about 150 μM to about 200 μM, about 150 μM to about 300 μM, or about 200 μM to about 300 μM. In some cases, one or more cargo molecules have a total concentration of 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.

[0075] In some cases, transfection or overexpression of a polypeptide is either low-toxicity or non-toxic in any one of the cells or kits of the present disclosure. In some cases, the cells, EVs, exosomes, or kits of the present disclosure may contain a pharmaceutically acceptable surfactant. In these cases, the surfactant is a non-ionic surfactant. In such cases, the surfactant is polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X-100®). In some cases, the surfactant is octaethylene glycol monododecyl ether (OEG). In some cases, the EVs contain polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether at a concentration of about 0.03 mM to about 4 mM, about 0.04 mM to about 3 mM, about 0.05 mM to about 2.5 mM, about 0.06 mM to about 2.2 mM, about 0.08 mM to about 2 mM, about 0.1 mM to about 1.8 mM, about 0.2 mM to about 1.5 mM, or any concentration therebetween.

[0076] In some cases, the EVs of the present disclosure may form a surfactant mixture solution having a final surfactant 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%, about 0.1% v / v to about 8.0% v / v, about 0.1% v / v to about 7.0% v / v, about 0.1% v / v to about 6.0% v / v, about 0.1% v / v to about 5.0% v / v, about 0.2% v / v to about 4.0% v / v, about 0.4% v / v, about 0.5% v / v, about 0.6% v / v, about 0.8% v / v, about 1.0%, or any concentration therebetween.

[0077] In some cases, the EVs may contact cargo molecules and detergents by adding the cargo molecules and detergents simultaneously. In some cases, a biological sample containing extracellular vesicles may contact cargo molecules and surfactants by continuously adding the cargo molecules and surfactants. In some cases, the method includes contacting a biological sample containing a plurality of EVs with cargo molecules and surfactants by adding the cargo molecules before adding the surfactant.

[0078] In some embodiments, the present disclosure provides a composition comprising any one of the extracellular vesicle (EV)-producing cells of the present disclosure, wherein the EV-producing cells are genetically engineered to overexpress at least one or more polypeptides, and the polypeptides are linked to glycosyl-phosphatidyl-inositol (GPI) groups. In some cases, the polypeptide is derived from any one of CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, GPC6, or the polypeptides listed in Table A. In some cases, the EV-producing cells comprise a recombinant vector system comprising a nucleic acid sequence encoding the coding sequence of the polypeptide. In some cases, the recombinant vector system comprises a coding sequence of a polypeptide selected from any one of the sequences in Table 1. In some cases, the recombinant vector system comprises a coding sequence of the polypeptide of mCherry-CD46(Short), HA-CD46Short, or CD46Short-HA in Table 1.

[0079] In some cases, the producing cells further comprise a release helper selected from the group consisting of the protein G (G) of vesicular stomatitis virus (VSV), the glycoprotein B (gB) of herpes simplex virus 1 (HSV-1), the baculovirus fusion protein gp64, and the gB derived from Epstein-Barr virus (EBV). In some cases, the release helper is vesicular stomatitis virus glycoprotein (VSVG).

[0080] Methods of using EVs

[0081] The present disclosure also provides methods, kits, and reagents for using EVs to treat a disease or disorder in a subject in need thereof. For example, methods for using EVs to treat patients suffering from chronic and recurrent diseases are provided herein by administering to the patient an effective amount of EVs. In some cases, the chronic and recurrent diseases can be diabetes, an infectious disease, protein deficiency, or an immunological disorder.

[0082] In some embodiments, the present disclosure provides a method for delivering a cargo molecule to a target cell, wherein the cargo molecule is delivered by an extracellular vesicle, and the extracellular vesicle containing the cargo molecule is produced by any one of the genetically engineered producer cells of the present disclosure. In some embodiments, the producer cell overexpresses at least one or more polypeptides. In some embodiments, the one or more polypeptides are linked to a glycosyl-phosphatidyl-inositol (GPI) group. In some embodiments, the one or more polypeptides are linked to the cargo molecule. In some embodiments, the one or more polypeptides are linked to a glycosyl-phosphatidyl-inositol (GPI) group and the cargo molecule. In some embodiments, the polypeptide is derived from any one of the polypeptides listed in Table A.

[0083] In some embodiments, the producer cells are genetically engineered by transfecting a recombinant vector system. In some embodiments, the recombinant vector system comprises a nucleic acid sequence encoding a glycosyl-phosphatidyl-inositol (GPI) moiety. In some embodiments, the recombinant vector system comprises a nucleic acid sequence encoding a cargo molecule. In some embodiments, the recombinant vector system comprises a nucleic acid sequence encoding a glycosyl-phosphatidyl-inositol (GPI) moiety and a nucleic acid sequence encoding a cargo molecule. In some embodiments, the cargo molecule is a polypeptide, protein, lipid, nucleic acid, carbohydrate, metabolite, or any combination thereof. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), antisense RNA, microRNA (mi-RNA), and long non-coding RNA (lncRNA). In some embodiments, the protein is an antibody or an enzyme.

[0084] In some embodiments, the producer cells produce extracellular vesicles that carry cargo molecules on the outer surface of the extracellular vesicles. In some embodiments, the producer cells produce extracellular vesicles that carry cargo molecules on the inner surface of the extracellular vesicles. In some embodiments, the producer cells produce extracellular vesicles that carry cargo molecules in the lumen of the extracellular vesicles.

[0085] In the therapeutic methods of the present disclosure, the EVs may be administered to a patient via intravenous, intraarterial, intranasal, or local administration routes. The effective dose may be evaluated by the attending physician on an empirical basis or may be set by in vivo or in vitro evaluation for each pathology.

[0086] Examples

[0087] Preferred embodiments of the present disclosure have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely by way of example. Numerous variations, modifications, and substitutions will be envisioned by those skilled in the art without departing from the present disclosure hereinafter. Of course, various alternatives to the embodiments of the present disclosure described herein may be used in the practice of the present disclosure. In the claims of the present application, it is intended that the scope of the present disclosure, and the methods and structures within these claims, and their equivalents, be encompassed thereby.

[0088] Example 1. Enhancement of extracellular vesicle (EV) production in mammalian cells

[0089] Cell culture

[0090] Human embryonic kidney 293 cells (HEK 293T) were cultured in DMEM cell medium supplemented with 10% fetal bovine serum in a 5% CO2, 37 °C, humidified incubator. Prior to suspension adaptation, the cells were seeded at 5 × 10 5 viable cells / mL in serum-free medium containing 5% serum (v / v) in a low-binding 6-well plate placed on a shaker at 120 rpm. The cells were then incubated in an incubator at 37 °C to achieve a maximum cell density with a cell viability of approximately >85%, which was considered an appropriate indicator of cell adaptation under the experimental conditions. The adapted cells were subcultured by successively dividing them in the cell culture medium with the serum concentration decreased from 5% to 2%, 1%, 0.5%, 0.1%, and 0% of the serum in the medium in each passage.

[0091] For the suspension of 293F cells, the cells were cultured in serum-free medium placed in a 5% CO 2、 37 °C, humidified incubator with constant shaking at 120 rpm.

[0092] Plasmid construction and overexpression of the polypeptide of interest

[0093] To generate stable cell lines that overexpress the target polypeptide, the PippyBac (PB) transposon system or the lentiviral system was used. Briefly, a nucleic acid fragment encoding the coding sequence (CDS) of the target polypeptide was synthesized and cloned into the PB transposon vector at the BglII and XhoI restriction enzyme digestion sites, or into the pLenti vector at the XbaI and SalI restriction enzyme digestion sites. In this study, the CDSs of CDCD46, CD55, and CD59 were synthesized and cloned into each vector for overexpression. As shown in Figure 1A, an artificial GPI anchor protein sequence was also synthesized and cloned into the vector. The artificial sequence of this example contains an mCherry sequence fused to the N-terminal and C-terminal domains of CD55.

[0094] According to the manufacturer's instructions, approximately 5×10 of 293T cells 5 were transfected with a combination of 2 μg of the PB transposon vector carrying the nucleic acid fragment of the polypeptide and 1 μg of the PB transposase vector by using Lipofectamine 3000. Alternatively, 2×10 of 293T cells 5 were infected with lentivirus containing the target gene. Then, the transfected cells or the infected cells were incubated under 200 μg / mL of hygromycin B for 7 - 8 days and selected to obtain stable cell lines that overexpress the target gene.

[0095] Western blot

[0096] The transfected cells were lysed in cell lysis buffer, and the protein concentration of the lysate was measured by the BCA protein assay to ensure equal uptake. The samples were resolved by SDS-PAGE and subsequently transferred to a PVDF membrane. The membrane was 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). An updated HRP-conjugated secondary antibody was used for ECL-based Western blot detection. As shown in Figure 1B, overexpressed CD59, CD46, and CD55 protein levels were observed.

[0097] Example 2. Isolation and Quantification of Extracellular Vesicles (EVs)

[0098] Isolation of Small Extracellular Vesicles (sEVs)

[0099] The isolation, purification process, and subsequent quantification of extracellular exosomes (EVs) are summarized in Figure 2. Briefly, cells were cultured in conditioned cell culture medium. The conditioned culture medium was collected in 50 mL centrifuge tubes and immediately centrifuged at 500×g for 10 minutes at 4°C to remove the adherent cells. The supernatant was transferred to a new 50 mL centrifuge tube and centrifuged at 2000×g for 20 minutes 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 ultracentrifuged at 100,000×g for 85 minutes at 4°C to pellet them. The collected sEV pellet was washed with PBS and further purified by a second ultracentrifugation at 100,000×g for 85 minutes at 4°C. The purified sEV pellet was resuspended in an appropriate amount of PBS and then purified by size exclusion chromatography (SEC). The protein concentration of sEVs was quantified by the BCA protein quantification assay. The particle concentration of sEVs was measured with an Apogee Micro-GxP flow cytometer, and the productivity (particles / L) was calculated as follows.

[0100] [Number]

[0101] The size and concentration of the collected EVs were measured using nanoparticle tracking analysis (NTA) and nanoflow cytometry (nFCM). For NTA measurements, a NanoSight NS300 from Malvern Panalytical was used. For nFCM measurements, a Micro-GxP flow cytometer from Apogee Flow System was used. In the present disclosure, unless otherwise specified, the Micro-GxP system was used to measure the EV particle concentration in the collected culture medium.

[0102] The increase in EV production in CD55 and CD59 was overexpressed in 293T cells

[0103] Referring to FIGS. 3A - 3D, an approximately 8 - 40-fold increase in EV production (particles / L or μg / mL) was observed in CD59 and CD55 overexpressed in 293T cells compared to the vehicle control (293T WT ), and / or CD46 overexpressing 293T cells.

[0104] The recovered particles were further characterized to measure the particle size distribution (e.g., identified as small EVs <200 nm), and the morphology of the recovered EVs was studied. As shown in FIGS. 4A - C, the particle size was measured using a nanoflow cytometer with a commercially available silicon bead standard (nFCM silica nanosphere cocktail 1).

[0105] The protein expression levels of CD59, CD46, and CD55 in cells and EVs after overexpression were measured by Western blotting. As shown in Figure 5, higher concentrations of CD55 and CD59 were observed in EVs than in total cell lysates. In contrast, overexpression of CD46 resulted in relatively lower levels of CD46 expression in EVs than in cells.

[0106] Transmission electron microscopy (TEM)

[0107] The morphology and viability of the collected EVs were observed under TEM. The resuspended purified exosome sample was further diluted to 0.1 μg / μL. An equal volume of 4% paraformaldehyde was added to the exosome sample and incubated for 2 hours. 3 μL of the mixture was dropped onto a TEM grid and fixed with 2% paraformaldehyde for 20 minutes. The grid was washed with 3X PBS and fixed with 1% glutaraldehyde for 5 minutes. After 8 washes with PBS (2 minutes each), followed by 9 washes with ddH2O, the grid was 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 grid was air-dried for 5 - 10 minutes. As shown in Figure 6, exosomes were examined with a JEOL 1100 transmission electron microscope at 60 kV and images were acquired using an ATM digital camera.

[0108] LC-MS / MS Proteins were extracted from cell and EV samples and quantified by the BCA method. An appropriate amount of protein was subjected to enzymatic digestion and then to liquid chromatography mass spectrometry (LC-MS / MS) analysis. The raw data were analyzed by MaxQuant to obtain the quantification results of LFQ and iBAQ.

[0109] 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 of approximate absolute quantification that can be used to compare the abundance of different proteins in the same sample.

[0110] Example 3. GPI-anchored proteins that enhance extracellular vesicle (EV) production Table A lists small polypeptides and proteins known to have a glycosyl-phosphatidyl-inositol (GPI) moiety. Among these, CD55, CD59, GPC1, GPC3, GPC4, GPC6, and SMPDL3B were highly enriched and identified as enriched GPI proteins from 293T WT 293T CD46 293T CD59 and 293T CD55 cells. All of the identified GPI-anchored proteins (i.e., CD55, CD59, GPC1, GPC3, GPC4, GPC6, and SMPDL3B) were enriched in EVs at a higher concentration than in the total cell fraction, as represented by the relative protein concentration quantification of EV / cell > 1 in Table 2.

[0111] [Table 2]

[0112] [Table 3]

[0113] [Table 4]

[0114] *LFQ: Relative protein quantification EV enrichment is represented as proteins detected at EV / Cell > 1 while inputting equal amounts of protein.

[0115] Table 2: Protein enrichment in EVs

[0116] CD52 overexpression also enhanced EV production in cells (Figs. 8A - B). EVs collected from 293T CD52 are shown in Fig. 8C. Furthermore, a significant increase in EV production was observed after co-expression of GPI-anchor proteins (i.e., CD55 and CD59) compared to WT controls. However, only a slight increase in EV production was observed compared to when CD55 and CD59 were overexpressed separately (Figs. 9A - C).

[0117] A similar effect of improving EV production by GPI-anchor proteins was observed in other cell lines such as 293F cells (Figs. 10A - C) and mesenchymal stem cells (MSCs) (Figs. 11A - C). In MSC cell-based experiments, human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in mesenchymal stem cell basal medium (MSCBM, Dakewe 6114011) supplemented with 5% EliteGro-Advance (EPA-050, EliteCell). MSC were infected with CD55 lentivirus generated by a 3-plasmid system and selected using 50 μg / mL hygromycin for stable expression. CD55 The selected cells were then cultured in conditioned medium for 2 - 3 days. Culture medium was collected for EV isolation.

[0118] Mesenchymal stem cells (MSCs) have been highly regarded in regenerative medicine for decades due to their differentiation ability, strong immunomodulatory properties, and their ability to be cultured and manipulated favorably. Recent investigations have suggested that the multifaceted effects of MSCs are not related to their differentiation ability but rather are mediated by the secretion of soluble paracrine factors. Extracellular vesicles are one of these paracrine mediators. EVs transfer functional cargos such as miRNA molecules, mRNA molecules, peptides, proteins, cytokines, and lipids from MSCs to recipient cells. EVs are involved in cell-to-cell communication events and contribute to the healing of damaged tissues, or diseased tissues, and organs. In past studies, it has been reported that EVs alone are involved in the therapeutic effects of MSCs in numerous experimental models. Based on this, MSC-derived EVs should be studied as a novel cell-free therapeutic agent for treating heart disease, kidney disease, liver disease, immune disorders, and neurological diseases, or for promoting skin wound healing.

[0119] Example 4. Overexpression of the glycosyl-phosphatidyl-inositol (GPI) region improves extracellular vesicle (EV) production

[0120] To identify the region of the GPI-anchor protein that provides enhanced EV production, full-length and truncated CD55 constructs (as shown in Figure 12A) were generated and transfected into 293F cells. As shown in Figures 12B - C, both truncated CD55 (i.e., construct numbers 0 - 3), and full-length CD55 (i.e., construct number 4) increased EV production. The morphology of the tested EVs was observed using cryo-EM (Figure 12D). Briefly, to prepare samples for cryo-EM, lacey carbon EM grids were glow discharged. An aliquot (2 μL) of the aqueous solution of the EV sample was applied to the carbon side of the EM grid and then blotted and plunge frozen into pre-cooled liquid ethane. The samples were studied with a cryo-electron microscope Taros Arctica.

[0121] Example 5. Effect of GPI-anchor proteins on extracellular vesicle (EV) production

[0122] As shown in Fig. 13A, CD55-based constructs encoded with mCherry (Short: mCherry-CD55-Short, and Long: mCherry-CD55-Long) were transfected into 293T cells as described in Example 1. Western blotting was performed to determine the mCherry (ab213511, Abcam) protein expression levels in total cell lysates and purified EVs (Fig. 13B). As shown in Figs. 13B - C, the CD55-based GPI anchor constructs functioned as scaffolds for incorporating and carrying the mCherry sequence onto EVs. Significantly higher levels of mCherry were detected in EVs derived from 293T cells transfected with the L construct compared to those from 293T cells transfected with the S construct. The uptake of mCherry-incorporated EVs by H2B-GFP-expressing target cells was observed by fluorescence microscopy (Fig. 13D).

[0123] As another example, two CD46-based constructs were fused with the mCherry sequence and transfected into 293T cells for overexpression (Figs. 14A - B). Interestingly, the enrichment of the mCherry protein was much higher in EVs purified from S construct-transfected cells than in EVs from L construct-transfected cells (Fig. 14C). Considering that CD46 is a transmembrane protein, two forms of the CD46-short chain constructs were generated, each encoding nanoluciferase (Nluc) at specific positions (Fig. 14D, O: Nluc outside, and I: Nluc inside). As shown in Fig. 14E, the incorporation of Nluc inside or outside the EVs was controlled by fusing the Nluc sequence near the N-terminus (outside) or the C-terminus (inside, the lumen of the EV).

[0124] Interestingly, unlike the co-overexpression of CD55 and CD59, overexpression of CD55 with the CD46-short construct led to a further increase in EV production in 293T cells (Figure 15A). To determine whether other transmembrane proteins would have the same effect, transmembrane proteins tagged with mOrange, namely CD9, CD63, and CD81, were overexpressed in the presence of CD55. In Figure 15B, the data show that overexpression of CD9 or CD81 with CD55 had a synergistic effect on increasing EV production in 293F cells (up to a 3-fold increase). Co-expression of CD55 and CD9 or CD81 resulted in an additive effect (a 2-fold increase).

[0125] Example 6. Preparation of ExoBoost cells and extracellular vesicles (EVs)

[0126] For the industrial production of ExoBoost cells (i.e., cells transfected with GPI-anchored proteins), a single colony (colony number 28) was obtained from 293TCD CD55 cells and grown as 28F cells. The main findings related to the upstream and downstream manufacturing processes are summarized below.

[0127] Upstream: Cell culture

[0128] 1. As shown in Figure 16A, a decrease in cell viability decreased the rate of EV production in 28F and 293F cells.

[0129] 2. The use of the applicant's optimized culture medium significantly increased the concentration of EVs in both 293F and 28F cells (Figures 16B - C).

[0130] 3. The addition of anti-clumping agents decreased the EV%. (Figure 16D).

[0131] 4. Compared with the batch process or the fed-batch process (e.g., one-pot reaction), the perfusion method significantly increased cell viability and EV production (particles / L / day) (Figure 16E).

[0132] 5. In the perfusion stage, hollow fiber filters with a pore size of 2 - 5 μm provided a higher permeate / retentate ratio than hollow filters with smaller pore sizes or capsule filters with a pore size of 5 μm (Figure 16F).

[0133] Downstream: Purification The downstream purification process is summarized in Figure 17. 28F cells were cultured by the perfusion method. The permeate was purified by the first and second filtrations and then concentrated by TFF. The concentrated sample was treated with benzonase and then purified by TFF and SEC. The sample was sterilized by 0.2 um filtration, diluted to 2 ug / uL, aliquoted, and then stored at -80°C. All materials used were designed for high-level sterilization or single-use. The details of the quality control method are summarized in Table 3.

[0134]

Table 5

[0135] Table 3: Quality Control

[0136] EVs collected from 28F cells were labeled as TREXO, and the product quality was confirmed by measuring the size distribution and EV%, and obtaining TEM, cryo-EM, nFCM, and immunoblot images (Figure 18A - E). For the nFCM results, 5 uL of the EV sample (2E8 particles / uL) was mixed with 5 uL of anti-CD55-APC (10101-R028-A Sinobiology) or PBS and then incubated at room temperature for 1 hour. The sample was then diluted 500-fold for nFCM detection.

[0137] The EVs taken were stable at concentrations higher than 10e9 particles / μL for 6 months at 4 °C (Figure 19A), and when stored at -80 °C, they were stored at even lower temperatures for longer. The stability of the EV samples was also tested under lyophilization conditions. Briefly, frozen EVs were thawed from -80 °C, and the particle concentration of the thawed samples was measured by nFCM. The EV samples were then diluted to a final concentration of 1e7 particles / μL with either PBS alone or 10% (w / w) trehalose (T0167, Sigma) in PBS. The EV samples were lyophilized in a lyophilizer (FD-1A-50 LGJ-10, Beijing SongYuan). The lyophilized samples were redissolved in ultrapure water with the initial volume before lyophilization. The EV concentration was tested by nFCM after redissolving from the -80 °C samples as a control and freshly thawing. Aggregation and degradation of the lyophilized EVs were observed as shown in Figure 19B.

[0138] Example 7. Incorporating Cargo Molecules into EVs

[0139] The uptake of siRNA as a cargo molecule by extracellular exosomes was tested.

[0140] Incorporating Cargo Molecules into the Outer Portion of EVs 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. EV and siRNA were mixed by adding the siRNA solution to the EV suspension using repetitive pipetting and then maintained at room temperature for 30 minutes before downstream purification. A pre-packed chromatography column was used to purify the free siRNA that was not incorporated from the final sample. Cy5-dye-modified cholesterol siRNA (GenePharm Co., Ltd) was used for quantification of the uptake efficiency and uptake capacity. The uptake efficiency was measured by nFCM in % EV uptake.

[0141] Loading cargo molecules into the lumen of EVs

[0142] Electroporation of EVs was performed using a Gene Pulser Xcell Electroporation System (BioRad) (#1652089, BioRad) with a 0.1 cm cuvette. The exponential mode was selected with a varying voltage, a fixed capacitance of 125 uF, and an ∞ resistance. Electroporation buffer was prepared and freshly used in each electroporation experiment. The particle concentration of EVs was adjusted to a final concentration in the range of 1E7 particles / μL to 1E9 particles / μL. After electroporation, the samples were placed on ice for at least 30 hours and then purified using a pre-packed chromatography column to remove free RNA. Uptake efficiency and uptake capacity were measured by nFCM. To determine whether the RNA was loaded inside the lipid membrane of the EVs (and thus protected) or on the external surface of the EVs, the samples after chromatographic purification were treated with RNase (RNase A / T1 mixture, Thermo Fischer), or an equal volume of PBS (no RNase control) at 37 °C for 10 minutes. The samples were then measured by nFCM to determine the change in RNA uptake efficiency with / without RNase treatment.

[0143] As shown in FIGS. 20A - B, siRNA taken up externally by hydrophobic modification resulted in an efficiency of >99%. When siRNA was taken up into the interior of EVs by electroporation, an efficiency of approximately 40 - 60% was observed. Uptake of siRNA was further confirmed by RNase treatment as shown in FIG. 21. The uptake efficiency was affected by multiple factors such as the concentration of EVs (FIG. 22A), the ratio between the concentration of EVs and RNA (FIG. 22B), and the voltage (FIG. 22C). Generally, higher uptake efficiency was observed in the lumen of EVs when the EV concentration was higher and the voltage was higher than that of the cargo molecules.

[0144] To determine whether cargo molecules can be incorporated into the outer portion, or surface, of the EV, the CD46 Short scaffold construct of Example 4 was fused with an HA tag at the N-terminus (outer) or C-terminus (inner) of the construct. After overexpression in cells, EVs were harvested and the outer incorporation of HA was measured by nFCM using anti-HA-488 (2350, CST) as shown in Figure 23.

[0145] Generally, when EVs are taken up by target cells, the EVs translocate to the endosome where they fuse with the endosomal membrane and release the cargo. For cargo molecules incorporated into the lumen of EVs, such as enzymes and nucleic acids, endosomal escape is required for them to function at their target sites in 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, resulting in the formation of carboxyfluorescein molecules. A unique feature of CCF4 is that it can undergo an FRET-based color change upon interaction with β-lactamase, an enzyme that hydrolyzes the beta-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 the loss of FRET and a shift in fluorescence from green to blue. The activity of β-lactamase in the cytoplasm of cells was evaluated by introducing CCF4-AM into the cells and monitoring the emission of fluorescence.

[0146] VSVG (vesicular stomatitis virus glycoprotein) is a viral protein that can promote the escape of cargo from endosomes into the cytoplasm of host cells. The applicants fused BLAM to the CD63 N-terminus (internal) or extracellular loop (outer). In another sample, BLAM was fused to the CD46-Short N-terminus (internal) or C-terminus (outer). EVs (carrying the fusion polypeptide either inside or outside the vesicles) were harvested and taken up by target 293T cells. The BLAM fusion polypeptides (i.e., BLAM-CD63-N-terminal, BLAM-CD63-extracellular loop, BLAM-CD46-Short-N-terminal, and CD46-Short-BLAM-C-terminal) were designed to be expressed on target cells or EV membranes as shown in FIGS. 24A - B. In FIGS. 24C - F, BLAM activity was observed only when BLAM was placed inside (not outside) the EVs. Also, the results showed that overexpression of VSVG, a cargo molecule release helper, is required for the cytoplasmic activity of BLAM. The activity of BLAM was tested with the LiveBLAzer® FRET-B / G uptake kit containing CCF4-AM (K1096, ThermoFisher).

[0147] Example 8: Extracellular vesicles (EVs) incorporating siRNA or shRNA

[0148] To determine whether EVs can deliver siRNA cargo molecules into cells and result in sufficient knockdown efficiency, cholesterol-modified siGFP labeled with Cy5 was incorporated onto the outer surface of EVs (>90% incorporation efficiency, determined by nFCM) and introduced into cells in the absence of VSVG. Direct transfection with RNAiMAX was used as a positive control. When siRNA targeting GFP was incorporated outside the EVs, no knockdown of GFP was observed. However, even with a low incorporation efficiency <10%, a decrease in GFP expression levels in cells was observed after the expression of shGFP-incorporated EVs using VSVG, in the presence or absence of VSVG in ExoBoost cells (approx. 10% knockdown efficiency). The results are shown in FIGS. 25A - B.

[0149] Incorporation by reference

[0150] All publications, patent applications, and issued patents cited herein are hereby incorporated by reference into this specification as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

[0151] The numbered embodiments of the present disclosure Other subject matter contemplated by the present disclosure is described in the following numbered embodiments. 1. A method for enhancing extracellular vesicle (EV) production, comprising harvesting a plurality of EVs from producer cells, wherein the producer cells are genetically engineered to overexpress at least one polypeptide, and the at least one polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group. 2. The method according to embodiment 1, wherein the polypeptide is derived from any one of the polypeptides in Table A. 3. The method according to embodiment 1 or 2, wherein the polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, or GPC6. 4. The method according to any one of embodiments 1 to 3, wherein the polypeptide is derived from CD59. 5. The method according to any one of embodiments 1 to 3, wherein the polypeptide is derived from CD55. 6. The method according to any one of embodiments 1 to 5, wherein the producing cell is genetically engineered by transfection with a recombinant vector system. 7. The method according to embodiment 6, wherein the recombinant vector system comprises a nucleic acid sequence encoding the coding sequence of the polypeptide. 8. The method according to embodiment 7, wherein the recombinant vector system comprises an expression control sequence operably linked to the nucleic acid sequence. 9. The method according to embodiment 8, wherein the nucleic acid sequence comprises at least one fluorescent marker. 10. The method according to embodiment 8, wherein the expression control sequence is a promoter. 11. The method according to embodiment 6, wherein the recombinant vector system comprises a selectable marker. 12. The method according to embodiment 11, 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. 13. The method according to embodiment 1, wherein the producing cell further comprises a release helper selected from the group consisting of vesicular stomatitis virus glycoprotein (VSVG), glycoprotein B (gB), baculovirus fusion protein gp64, and gB derived from Epstein-Barr virus (EBV)-derived herpes simplex virus 1 (HSV-1). 14. The method according to any one of embodiments 1 to 13, wherein the producing cell is a genetically engineered stable cell line. 15. The method according to any one of embodiments 1 to 14, wherein the plurality of EVs are collected by dialysis or ultracentrifugation. 16. The method according to embodiment 15, wherein the plurality of EVs are collected by ultracentrifugation. 17. The method according to any one of embodiments 1 to 16, wherein the concentration of EVs collected from the producer cells is at least twice as high as the concentration from wild-type cells. 18. The method according to any one of embodiments 1 to 17, wherein the concentration of EVs collected from the producer cells is 2 to 250 times higher than the concentration from wild-type cells. 19. The method according to any one of embodiments 1 to 18, wherein the producer cells are mammalian cells. 20. The method according to embodiment 19, wherein the producer cells are HEK 293F cells, HEK 293T cells, mesenchymal stem cells (MSC), or any combination thereof. 21. The method according to any one of embodiments 1 to 20, wherein the EVs are exosomes, ectosomes, microvesicles, apoptotic bodies, or any combination thereof. 22. The method according to embodiment 21, wherein the EVs are exosomes. 23. The method according to embodiment 1, wherein the cargo molecule comprises a pharmaceutical active ingredient (API). 24. The method according to embodiment 23, wherein the API comprises a small molecule therapeutic agent. 25. The method according to embodiment 24, wherein the cargo molecule comprises a polypeptide, protein, lipid, nucleic acid, carbohydrate, metabolite, or any combination thereof. 26. The method according to embodiment 25, wherein the nucleic acid comprises DNA. 27. The method according to embodiment 25, wherein the nucleic acid comprises peptide nucleic acid (PNA). 28. The method according to embodiment 25, wherein the nucleic acid comprises RNA. 29. The method according to 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 (snoRNA), antisense RNA, microRNA (mi-RNA), and long non-coding RNA (lncRNA). 30. The method according to embodiment 25, wherein the protein comprises an antibody or an enzyme. 31. The method according to embodiment 25, wherein the cargo molecule comprises an antisense oligonucleotide. 32. The method according to embodiment 25, wherein the cargo molecule comprises a morpholino oligomer. 33. The method according to embodiment 25, wherein the cargo molecule comprises one or more components of a gene editing system. 34. The method according to embodiment 33, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, transcription, and transcription activator-like effector nuclease (TALEN). 35. The method according to any one of embodiments 1 to 34, wherein the cargo molecule is located on the inner surface or the outer surface of the plurality of EVs. 36. The method according to embodiment 35, wherein the cargo molecule is a polypeptide fused with a polypeptide derived from CD46 or CD63. 37. The method according to embodiment 35, wherein the cargo molecule is located on the inner surface of the plurality of EVs, and the cargo molecule has higher efficacy in the absence of the release in the presence of the release helper. 38. A method for producing extracellular vesicles (EVs) that produce a stable cell line, comprising: a) Transfecting EV-producing cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence of one or more polypeptides and a selection marker, and the polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group; b) Screening and selecting the transfected cells; and c) Culturing the selected cells. 39. The method according to embodiment 38, wherein the polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, or GPC6. 40. The method according to embodiment 39, wherein the polypeptide is derived from CD59. 41. The method according to embodiment 39, wherein the polypeptide is derived from CD55. 42. The method according to embodiment 38, wherein the expression vector comprises an expression control sequence operably linked to the nucleic acid sequence. 43. The method according to embodiment 42, wherein the expression control sequence is a promoter. 44. The method according to embodiment 42, wherein the nucleic acid sequence comprises at least one fluorescent marker. 45. The method according to embodiment 38, 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. 46. The method according to any one of embodiments 38 to 45, wherein the concentration of EVs collected from the producer cells is at least 2-fold higher than the concentration from wild-type cells. 47. The method according to any one of embodiments 38 to 46, wherein the concentration of EVs collected from the producer cells is 2-fold to 250-fold higher than the concentration from wild-type cells. 48. The method according to any one of embodiments 38 to 47, wherein the producer cells are mammalian cells. 49. The method according to embodiment 48, wherein the producer cells are HEK 293F cells, HEK 293T cells, mesenchymal stem cells (MSCs), or any combination thereof. 50. The method according to any one of embodiments 38 to 49, wherein the EVs are exosomes, ectosomes, microvesicles, apoptotic bodies, or any combination thereof. 51. The method according to embodiment 50, wherein the EVs are exosomes. 52. The method according to any one of embodiments 38 to 51, wherein the EVs take up cargo molecules. 53. The method according to embodiment 52, wherein the cargo molecules comprise a pharmaceutical active ingredient (API). 54. The method according to embodiment 53, wherein the API comprises a small molecule therapeutic agent. 55. The method according to embodiment 52, wherein the cargo molecule comprises a polypeptide, a protein, a lipid, a nucleic acid, a carbohydrate, a metabolite, or any combination thereof. 56. The method according to embodiment 55, wherein the nucleic acid comprises DNA. 57. The method according to embodiment 55, wherein the nucleic acid comprises a peptide nucleic acid (PNA). 58. The method according to embodiment 55, wherein the nucleic acid comprises RNA. 59. The 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 (snoRNA), antisense RNA, microRNA (mi-RNA), and long non-coding RNA (lncRNA). 60. The method according to embodiment 55, wherein the protein comprises an antibody or an enzyme. 61. The method according to embodiment 55, wherein the cargo molecule comprises an antisense oligonucleotide. 62. The method according to embodiment 55, wherein the cargo molecule comprises a morpholino oligomer. 63. The method according to embodiment 38, wherein the cargo molecule comprises one or more components of a gene editing system. 64. The method according to embodiment 63, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, transcription activator-like effector nuclease (TALEN). 65. The produced cell line according to any one of embodiments 38 to 64. 66. A kit for enhancing EV production, comprising the production cell according to any one of embodiments 1 to 37 or the cell line according to embodiment 65. 67. A composition comprising a plurality of EVs according to any one of embodiments 1 to 66. 68. The composition according to embodiment 67, further comprising a pharmaceutically acceptable carrier. 69. A composition comprising extracellular vesicle (EV)-producing cells, wherein the EV-producing cells are genetically engineered to overexpress at least one polypeptide, and the at least one polypeptide is linked to a glycosyl-phosphatidyl-inositol (GPI) group. 70. The composition according to embodiment 69, wherein the polypeptide is derived from CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, or GPC6. 71. The composition according to embodiment 69 or 70, wherein the producing cells are genetically engineered by transfecting a recombinant vector system. 72. The composition according to embodiment 71, wherein the recombinant vector system comprises a nucleic acid sequence encoding the coding sequence of the polypeptide. 73. The composition according to embodiment 71, wherein the recombinant vector system comprises an expression control sequence operably linked to the nucleic acid sequence. 74. The composition according to embodiment 72, wherein the nucleic acid sequence comprises at least one fluorescent marker. 75. The composition according to embodiment 73, wherein the expression control sequence is a promoter. 76. The composition according to embodiment 71, wherein the recombinant vector system comprises a selectable marker. 77. The composition according to any one of embodiments 69 to 76, wherein the producing cells are a genetically engineered stable cell line. 78. The composition according to embodiment 72, wherein the coding sequence of the polypeptide is selected from any one of the sequences in Table 1. 79. The composition according to embodiment 78, wherein the coding sequence of the polypeptide is mCherry-CD46(Short), HA-CD46Short, or CD46Short-HA in Table 1. 80. The composition according to 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. 81. The composition according to any one of embodiments 69 to 80, wherein the concentration of EV collected from the producer cells is at least 2-fold higher than the concentration from wild-type cells. 82. The composition according to any one of embodiments 69 to 81, wherein the concentration of EV collected from the producer cells is 2-fold to 250-fold higher than the concentration from wild-type cells. 83. The composition according to any one of embodiments 69 to 82, wherein the producer cells are mammalian cells. 84. The composition according to embodiment 83, wherein the producer cells are HEK 293F cells, HEK 293T cells, mesenchymal stem cells (MSC), or any combination thereof. 85. The composition according to any one of embodiments 69 to 84, wherein the EV is an exosome, an ectosome, a microvesicle, an apoptotic body, or any combination thereof. 86. The composition according to embodiment 85, wherein the EV is an exosome. 87. The composition according to any one of embodiments 69 to 86, wherein the EV takes up cargo molecules. 88. The method according to embodiment 87, wherein the cargo molecule comprises a pharmaceutical active ingredient (API). 89. The method according to embodiment 88, wherein the API comprises a small molecule therapeutic agent. 90. The method according to embodiment 88, wherein the cargo molecule comprises a polypeptide, a protein, a lipid, a nucleic acid, a carbohydrate, a metabolite, or any combination thereof. 91. The method according to embodiment 90, wherein the nucleic acid comprises DNA. 92. The method according to embodiment 90, wherein the nucleic acid comprises a peptide nucleic acid (PNA). 93. The method according to embodiment 90, wherein the nucleic acid comprises RNA. 94. The method according to embodiment 93, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), antisense RNA, microRNA (mi-RNA), and long non-coding RNA (lncRNA). 95. The method according to embodiment 90, wherein the protein comprises an antibody or an enzyme. 96. The method according to embodiment 90, wherein the cargo molecule comprises an antisense oligonucleotide. 97. The method according to embodiment 90, wherein the cargo molecule comprises a morpholino oligomer. 98. The method according to embodiment 87, wherein the cargo molecule comprises one or more components of a gene editing system. 99. The method according to embodiment 98, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, zinc finger nuclease, transcription activator-like effector nuclease (TALEN).

[0152] Additional embodiments can be provided by combining the various embodiments described above. All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to adopt concepts from various patents, applications, and publications to provide additional embodiments.

Claims

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

2. The method according to claim 1, wherein the at least one polypeptide is derived from one of the polypeptides in Table A: CD52, CD55, CD58, CD59, CD109, GPC1, GPC4, or GPC6.

3. The method according to any one of claims 1 or 2, wherein the producing cells are genetically engineered by transfecting them with a recombinant vector system.

4. The method according to claim 3, wherein the recombinant vector system comprises a nucleic acid sequence encoding the at least one polypeptide, and comprises an expression control sequence operably linked to the nucleic acid sequence.

5. The method according to claim 4, wherein the nucleic acid sequence comprises at least one fluorescent marker, promoter, selection marker, neomycin resistance, puromycin resistance, hygromycin resistance, DHFR resistance, GPT resistance, zeosin resistance, G418 resistance, phreomycin resistance, blastosidine resistance, and histidinol resistance, or a combination thereof.

6. The method according to claim 3, wherein the producing cells further include a release helper selected from the group consisting of vesicular stomatitis virus glycoprotein (VSVG), glycoprotein B (gB), baculovirus fusion protein gp64, and gB of herpes simplex virus 1 (HSV-1) derived from Epstein-Barr virus (EBV).

7. The method according to claim 3, wherein the producing cells are a genetically modified stable cell line selected from mammalian cells, HEK 293F cells, HEK 293T cells, mesenchymal stem cells (MSCs), or a combination thereof.

8. The method according to any one of claims 1 to 2 or 4 to 7, wherein the plurality of EVs are EVs collected by dialysis or ultracentrifugation.

9. The method according to any one of claims 1 to 2 or 4 to 7, wherein the concentration of EV collected from the aforementioned producing cells is 2 to 250 times higher than the concentration from wild-type cells.

10. The method according to claim 9, wherein the EV is an ectosome, exosome, microvesicle, apoptotic body, or any combination thereof.

11. The method according to claim 10, wherein the EV is an exosome, and the EV is taken up by a cargo molecule comprising a pharmaceutical active ingredient (API), a small molecule therapeutic agent, a polypeptide, a polypeptide fused with a polypeptide derived from CD46 or CD63, a protein, a lipid, a nucleic acid, a carbohydrate, a metabolite, DNA, peptide nucleic acid (PNA), RNA, mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA), nucleolar small RNA (snoRNA), antisense RNA, microRNA (mi-RNA), long non-coding RNA (lncRNA), an antibody, an enzyme, an antisense oligonucleotide, a morpholino oligomer, one or more components of a gene editing system, CRISPR / Cas, a zinc finger nuclease, a transcription agent, and an activator-like effector nuclease (TALEN), or a combination thereof.

12. The method according to claim 11, wherein the cargo molecules are located on the inner surfaces of the plurality of EVs, and the cargo molecules have a higher effectiveness in the presence of the release helper than in the absence of the release helper.

13. A method for producing extracellular vesicles (EVs) that produce stable cell lines, a) Transfecting EV-producing cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding one or more polypeptides and a selection marker, and the polypeptides are linked to a glycosyl-phosphatidyl-inositol (GPI) group. b) Screening and selecting the transfected cells, c) A method comprising culturing the selected cells.

14. A manufactured cell line according to claim 13.

15. A kit for enhancing EV production, comprising the cell line described in claim 14.

16. A composition comprising a plurality of EVs manufactured according to any one of claims 1 to 2, 4 to 7, or 10 to 12, or a cell line according to claim 14.

17. The composition according to claim 16, further comprising a pharmaceutically acceptable carrier.

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

19. The composition according to claim 18, wherein the polypeptide is derived from any one of the polypeptides in Table 1.

Images