Extracellular vesicle secretion promoter that promotes the secretion of extracellular vesicles, and use thereof

Abstract

Relating to the secretion of extracellular vesicles from cells, provided are a novel secretion booster and a method for boosting the secretion.　This extracellular vesicle secretion booster is characterized by containing an ABCA1 inhibitor. This method for boosting extracellular vesicle secretion is characterized by comprising a boosting step for boosting the extracellular vesicle secretion by allowing a subject having cells to coexist with the aforesaid extracellular vesicle secretion booster of the present invention.

Description

[Technical Field]

[0001] The present invention relates to an extracellular vesicle secretion-promoting agent that promotes the secretion of extracellular vesicles from cells, and uses thereof. [Background technology]

[0002] Various functions of extracellular vesicles such as exosomes secreted from cells have been reported, and their applications have attracted attention. These extracellular vesicles encapsulate, for example, nucleic acids such as microRNA (miRNA) and proteins. These encapsulated substances are transmitted from the cell that secreted the extracellular vesicles to the recipient cell via the extracellular vesicles, and are therefore thought to function as a communication tool between cells. Due to this same function, exosomes have also attracted attention for their use as carriers in drug delivery systems (DDS). Summary of the Invention [Problem to be solved by the invention]

[0003] However, the mechanisms underlying the regulation of extracellular vesicle secretion from cells remain unclear. Therefore, efficient production of extracellular vesicles, which can be used as carriers for drugs or DDS for treating diseases, is difficult. Furthermore, no effective methods have been established to promote the secretion of endogenous EVs in vivo to achieve therapeutic effects.

[0004] Therefore, an object of the present invention is to provide a new secretion-promoting agent and a new method for promoting the secretion of extracellular vesicles from cells. [Means for solving the problem]

[0005] To achieve the above object, the extracellular vesicle secretion promoter of the present invention is characterized by containing an ABCA1 inhibitor.

[0006] The method for promoting secretion of the present invention is characterized by comprising a promoting step of promoting the secretion of extracellular vesicles by bringing a subject having cells into contact with the extracellular vesicle secretion promoter of the present invention.

[0007] The method for producing extracellular vesicles of the present invention is characterized by comprising a promotion step of promoting the secretion of extracellular vesicles by coexisting cells with the extracellular vesicle secretion promoter of the present invention, and a recovery step of recovering the extracellular vesicles secreted from the cells. [Effects of the Invention]

[0008] The extracellular vesicle secretion promoter of the present invention can promote the secretion of extracellular vesicles from cells. Therefore, for example, by secreting extracellular vesicles from cells, it is possible to efficiently prepare extracellular vesicles as materials or secrete endogenous extracellular vesicles in living organisms. Therefore, the present invention can be said to be a very useful technology in various fields, including, for example, medicine. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 is a graph showing the amount of EVs secreted from miR-3202-transfected HCT116 cells. [Figure 2] Figure 2 is a graph showing the amount of EVs secreted from miR-3202-transfected A549 cells. [Figure 3] Figure 3 is a graph showing the amount of EVs secreted from multiple cells transfected with miR-3202. [Figure 4] Figure 4 is a graph showing the size distribution of secreted EVs from miR-3202-transfected HCT116 cells. [Figure 5] Figure 5 is a graph showing the size distribution of secreted EVs from miR-3202-transfected A549 cells. [Figure 6] FIG. 6 is a graph showing caspase activity in cells transfected with miR-3202. [Figure 7] Figure 7 is a graph showing the amount of EVs secreted from miR-3202-transfected MSC cells. [Figure 8] Figure 8 is a graph showing the particle size distribution of EVs secreted from miR-3202-transfected MSC cells. [Figure 9] FIG. 9 is a graph showing the relative expression levels of ABCA1 mRNA in cells transfected with siRNA against the ABCA1 gene. [Figure 10] Figure 10 shows the results for cells transfected with siRNA against the ABCA1 gene. The upper graph is a graph of cell viability, and the lower graph is a graph of the relative amount of EV secretion. [Figure 11] Figure 11 shows the results for A549 cells treated with an ABCA1 inhibitor. The upper graph shows cell viability, and the lower graph shows the relative amount of EV secretion. [Figure 12] Figure 12 shows the results for HCT116 cells treated with an ABCA1 inhibitor. The upper graph shows cell viability, and the lower graph shows the relative amount of EV secretion. DETAILED DESCRIPTION OF THE INVENTION

[0010] In the present invention, extracellular vesicles (EVs) are also referred to hereinafter as EVs. The extracellular vesicle secretion promoter of the present invention is also referred to hereinafter as an EV secretion promoter.

[0011] As described above, the extracellular vesicle secretion promoter of the present invention is characterized by containing an ABCA1 inhibitor.

[0012] In the EV secretion promoter of the present invention, for example, the ABCA1 inhibitor is an expression inhibitor or function inhibitor of ABCA1 protein.

[0013] In the EV secretion promoter of the present invention, for example, the expression-inhibiting substance is at least one selected from the group consisting of a substance that inhibits transcription from the gene encoding the ABCA1 protein, a substance that degrades the transcribed transcript, and a substance that inhibits protein translation from the transcript.

[0014] In the EV secretion promoter of the present invention, for example, the expression-inhibiting substance is at least one nucleic acid substance selected from the group consisting of miRNA, siRNA, antisense, ribozyme, and precursors thereof.

[0015] In the EV secretion promoter of the present invention, for example, the expression-inhibiting substance is an expression vector that expresses the nucleic acid substance.

[0016] In the EV secretion-promoting agent of the present invention, for example, the expression-inhibiting substance is miR-3202 or a precursor thereof.

[0017] In the EV secretion promoter of the present invention, for example, the precursor is at least one selected from the group consisting of a nucleic acid encoding the miRNA, a primary miRNA transcript, a precursor miRNA, and an expression vector for the miRNA.

[0018] In the EV secretion promoter of the present invention, for example, the function-suppressing substance is a function-inhibiting or function-neutralizing substance against ABCA1 protein.

[0019] In the EV secretion-promoting agent of the present invention, for example, the function-neutralizing substance is an antibody or antigen-binding fragment against the ABCA1 protein.

[0020] The EV secretion promoter of the present invention is, for example, an expression vector in which the function-suppressing substance expresses the function-neutralizing substance.

[0021] The EV secretion promoter of the present invention is, for example, an extracellular vesicle secretion promoter that promotes the secretion of extracellular vesicles from cells, and the cells are mesenchymal stem cells.

[0022] As described above, the secretion promoting method of the present invention is characterized by including a promoting step of promoting the secretion of extracellular vesicles by coexisting a subject having cells with the extracellular vesicle secretion promoting agent of the present invention.

[0023] In the method for promoting EV secretion of the present invention, for example, in the promotion step, the extracellular vesicle secretion promoter is added to the subject in vivo, ex vivo, or in vitro.

[0024] In the method for promoting EV secretion of the present invention, for example, in the promotion step, the cells are transfected with the extracellular vesicle secretion promoter.

[0025] In the method for promoting EV secretion of the present invention, for example, the cells are mesenchymal stem cells.

[0026] In the method for promoting EV secretion of the present invention, for example, the cells are bone marrow-derived or adipose tissue-derived mesenchymal stem cells.

[0027] In the method for promoting EV secretion of the present invention, for example, the cells are cells collected from a living organism or cells of a cell line.

[0028] In the method for promoting EV secretion of the present invention, for example, in the promotion step, the cells and the extracellular vesicle secretion promoter are allowed to coexist in a medium.

[0029] In the method for promoting EV secretion of the present invention, the subject is, for example, a tissue, an organ, or a living organism.

[0030] As described above, the method for producing extracellular vesicles of the present invention is characterized by comprising a promotion step of promoting the secretion of extracellular vesicles by causing cells to coexist with the extracellular vesicle secretion promoter of the present invention, and a recovery step of recovering the extracellular vesicles secreted from the cells.

[0031] The method for producing EVs of the present invention involves, for example, transfecting the cells with the extracellular vesicle secretion promoter.

[0032] The method for producing EVs of the present invention is, for example, such that the cells are mesenchymal stem cells.

[0033] The method for producing EVs of the present invention is, for example, such that the cells are mesenchymal stem cells derived from bone marrow or adipose tissue.

[0034] The method for producing EVs of the present invention is, for example, such that the cells are cells collected from a living body or cells of a cell line.

[0035] The method for producing EVs of the present invention is, for example, in the promoting step, the cells and the extracellular vesicle secretion promoter are allowed to coexist in a culture medium.

[0036] The method for producing EVs of the present invention is, for example, a method for producing the extracellular vesicles as a carrier of a drug delivery system.

[0037] The method for producing EVs of the present invention is, for example, a method for producing the extracellular vesicles as a therapeutic agent.

[0038] <EV secretion promoter> The EV secretion promoter of the present invention is characterized by containing an ABCA1 inhibitor as described above. The EV secretion promoter of the present invention is characterized by containing the ABCA1 inhibitor, and other configurations and conditions are not particularly limited. Further, the EV secretion promoter of the present invention can incorporate the descriptions of the method for promoting EV secretion of the present invention described below and the like.

[0039] The present invention is characterized in that it has been found that ABCA1 is involved in EV secretion and that the secretion of EVs from cells can be suppressed by suppressing ABCA1. Therefore, the type of substance for suppressing ABCA1 and the method of suppression are not limited in any way.

[0040] ABCA1 is an ATP-binding cassette transporter 1 (member 1 of the human transporter subfamily ABCA). The ABCA1 protein and the ABCA1 gene encoding it are registered in the Genetic Testing Registry (GTR) under Gene ID: 19.

[0041] In the present invention, an "ABCA1 inhibitor" may be any agent capable of suppressing ABCA1, and may, for example, suppress the expression of ABCA1 protein or the function of ABCA1 protein. In the former case, the ABCA1 inhibitor is, for example, a substance that suppresses the expression of ABCA1 protein, and in the latter case, the ABCA1 inhibitor is, for example, a substance that suppresses the function of ABCA1 protein. In the present invention, "suppression" may also mean inhibition.

[0042] The EV secretion promoter of the present invention may contain, for example, only the ABCA1 inhibitor as an active ingredient, or may contain, in addition to the ABCA1 inhibitor, other ingredients that promote EV secretion. For example, the EV secretion promoter of the present invention may contain, as the ABCA1 inhibitor, either the expression inhibitor or the function inhibitor, or both.

[0043] The type of the ABCA1 inhibitor is not particularly limited, and examples thereof include nucleic acid substances, low molecular weight compounds such as organic compounds, proteins such as antibodies, peptides such as antigen-binding fragments, and the like.

[0044] The expression-inhibiting substance is not particularly limited, and may suppress either the transcription or translation process in the expression of a protein (hereinafter also referred to as the target protein) from a gene encoding the ABCA1 protein (hereinafter also referred to as the ABCA1 gene or the target gene). Examples of transcription inhibition include inhibition of transcription from DNA to pre-mRNA, inhibition of RNA processing to form mature mRNA from pre-mRNA, and degradation of pre-mRNA or mature mRNA. Examples of translation inhibition include inhibition of translation from mature mRNA and inhibition of modification of translation products.

[0045] The expression-inhibiting substance is, for example, a nucleic acid substance (hereinafter also referred to as a nucleic acid-type inhibitor), and may be in a form that inhibits expression as it is (first form), or in a precursor form (second form) that becomes expression-inhibiting under an in vivo, in vitro, or ex vivo environment.

[0046] Examples of the first type of expression-inhibiting substance include antigens, antisense (antisense oligonucleotides), RNA interference (RNAi) substances, ribozymes, etc. Examples of RNAi substances include siRNA, miRNA, etc. Antigenes, for example, inhibit mRNA transcription, antisense and miRNA, for example, inhibit translation from mRNA, and siRNA and ribozymes, for example, degrade mRNA. These expression-inhibiting substances may target, for example, the entire region or a partial region of the target gene. As a specific example, antisense and miRNA can be designed to bind to, for example, the 3'UTR region of mRNA transcribed from the target gene, and siRNA and ribozymes can be designed to bind to, for example, a partial region of mRNA transcribed from the target gene in a completely complementary manner.

[0047] A specific example of the miRNA is miR-3202. The miRNA is registered, for example, in the NCBI DB (https: / / www.ncbi.nlm.nih.gov / nuccore) under the following accession number: miR-3202: No.NR_036175

[0048] The miRNA in the active ingredient may be, for example, any one of them, or a combination of two or more of them. miRNA (microRNA) is, for example, a single-stranded RNA molecule.

[0049] The first type of expression-inhibiting substance can be obtained, for example, by a screening method as described below, or can be designed from the sequence of the target gene.

[0050] The expression inhibitor of the first type may be, for example, single-stranded or double-stranded. The structural units of the expression inhibitor are not particularly limited and include, for example, a deoxyribonucleotide or ribonucleotide backbone containing a sugar, a base such as purine or pyrimidine, and phosphate. Other examples include non-nucleotide backbones containing a base such as pyrrolidine or piperidine. These backbones may be modified or unmodified. Furthermore, the structural units may be, for example, natural or artificially non-natural. The expression inhibitor may be formed, for example, from the same structural units, or from two or more types of structural units.

[0051] As described above, the expression-inhibiting substance of the second type is the precursor, and a specific example is a precursor that expresses the expression-inhibiting substance of the first type. When the precursor is administered to a subject, the expression-inhibiting substance of the first type can be expressed and made to function, for example, in vivo, in vitro, or ex vivo.

[0052] The precursor may be, for example, a form containing the expression-inhibiting substance of the first form and a linker. Specific examples of the precursor include a form in which both strands of siRNA are linked by the linker. For example, such a precursor can be cleaved in an in vivo, in vitro, or ex vivo environment, thereby removing the linker from the precursor and generating (expressing) a double-stranded siRNA. Specific examples of the precursor include shRNAs that generate siRNAs upon cleavage.

[0053] Specifically, the type of precursor of the miRNA is not particularly limited as long as it produces the miRNA in cells. Examples of the precursor include a nucleic acid encoding the miRNA, a primary miRNA transcript (pri-miRNA), and a precursor miRNA (pre-miRNA).

[0054] Furthermore, the precursor may be, for example, an expression vector into which the coding sequence of the expression inhibitor of the first type has been inserted. For example, the coding sequence of the precursor (for example, the above-mentioned shRNA, etc.) may be inserted into the expression vector. The expression vector allows the expression of the expression inhibitor of the first type, for example, in vivo, in vitro, or ex vivo. The type of expression vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector, and examples of the viral vector include an adenovirus vector and a Sendai virus vector.

[0055] Specific examples of the expression vector include an expression vector for the miRNA. The coding sequence in the expression vector includes, for example, a gene that generates a pri-miRNA by transcription. The expression vector includes, for example, an expression vector in which a miRNA coding sequence has been inserted into a miRNA cloning site in a vector. The miRNA coding sequence is, for example, a coding sequence for a single-stranded pri-miRNA or pre-miRNA having a stem-loop structure.

[0056] Examples of the function-inhibiting substance include a function-inhibiting substance (also called an activity-inhibiting substance) that inhibits the function of the ABCA1 protein, and a function-neutralizing substance (also called an activity-neutralizing substance) that neutralizes the function of the enzyme protein.

[0057] The function inhibitor is not particularly limited, and examples thereof include low molecular weight compounds, etc. Specific examples include cyclosporin, probucol, valspodar, and analogs thereof.

[0058] Examples of the function-neutralizing substance include an antibody or antigen-binding fragment (antigen-binding peptide) against the ABCA1 protein (hereinafter, collectively referred to as an antibody-type inhibitor). The antibody-type inhibitor can inhibit the function of the ABCA1 protein by binding to the ABCA1 protein, and is therefore also referred to as a neutralizing antibody or neutralizing antigen-binding fragment. The antibody-type inhibitor can also be obtained, for example, by a screening method such as that described below.

[0059] The antibody may be, for example, a monoclonal antibody or a polyclonal antibody, and its isotype is not particularly limited and includes, for example, IgG, IgM, IgA, etc. When the antibody is to be administered to humans, for example, a fully human antibody, a humanized antibody, a chimeric antibody, etc. is preferred.

[0060] The antigen-binding fragment may be, for example, a fragment having the complementarity-determining region (CDR) of the antibody, as long as it can recognize and bind to the target site of the target protein. Specific examples of the antigen-binding fragment include Fab, Fab', F(ab') and the like.

[0061] The function-inhibiting substance may be, for example, a first form that inhibits the function of the ABCA1 protein as it is, or a second form of a precursor that inhibits the function of the ABCA1 protein under in vivo, in vitro, or ex vivo conditions. The first form of the function-inhibiting substance is, for example, an antibody-type inhibitor as described above. Furthermore, the second form of the precursor may be, for example, an expression vector into which a coding sequence for a protein (e.g., an antibody) or peptide (e.g., an antigen-binding fragment) that inhibits the function of the ABCA1 protein has been inserted. The type of expression vector is not particularly limited, and, as described above, examples include a plasmid vector, a viral vector, etc.

[0062] Furthermore, the function-inhibiting substance may be, for example, a substance that inhibits a state in which the ABCA1 protein retains its function but does not lose that function, but is still functional. That is, as a specific example, the function-inhibiting substance may be an inhibitor that reduces a substance (also called a substrate) necessary for the function of the ABCA1 protein, or that alters the substrate. The reduction of the substrate may be, for example, an inhibition of the production of the substrate, or may be a degradation of the substrate.

[0063] The EV secretion promoter of the present invention may contain, for example, only the active ingredient, or may further contain other additive ingredients. The EV secretion promoter of the present invention may contain, for example, only the ABCA1 inhibitor as the active ingredient, or may contain, in addition to the ABCA1 inhibitor, other active ingredients involved in promoting EV secretion. The additive ingredients are not particularly limited and include, for example, the following ingredients, preferably pharmacologically acceptable ingredients. The additive ingredients can be appropriately selected depending on, for example, the administration method, recipient, and dosage form of the EV secretion promoter.

[0064] The additive component may be, for example, an excipient. Examples of the excipient include liquid media such as aqueous solvents, alcoholic solvents, polyalcoholic solvents, oily solvents, and mixed solvents thereof (e.g., emulsifying solvents), as well as lactose and starch. Examples of the aqueous solvent include isotonic solutions such as water, physiological saline, and sodium chloride, and examples of the oily solvent include soybean oil. Other examples of the additive component include binders such as starch paste; disintegrants such as starch and carbonates; and lubricants such as talc and wax. The additive component may also include, for example, a DDS agent for delivering the active ingredient to the target site.

[0065] The additive component may be, for example, a transfection agent for transfecting the ABCA1 inhibitor of the present invention into cells, or a DDS agent for delivering the ABCA1 inhibitor of the present invention to target cells of the subject.

[0066] The additive component is preferably, for example, a pharmacologically acceptable component, and can be appropriately selected depending on, for example, the method for delivering the EV secretion promoter of the present invention to a subject, the type of subject, and the dosage form of the EV secretion promoter.

[0067] The EV secretion-promoting agent of the present invention can promote EV secretion from cells. The target cells of the present invention are not particularly limited and include various cells derived from humans or non-human animals. The cells may be, for example, normal cells or cells that are abnormal in the target item.

[0068] In the present invention, extracellular vesicles (EVs) include exosomes, microvesicles, apoptotic bodies, and the like secreted via the endocytic pathway, and particularly exosomes. Exosomes can generally be detected using marker molecules such as Alix, Tsg101, CD81, CD63, CD9, and flotillin.

[0069] The aforementioned EVs contain informational substances such as proteins and RNAs, and are known to have a function of mediating intercellular information transmission by moving to other cells when secreted from cells. Based on such a function, it has been reported that EVs secreted from cells exhibit a therapeutic effect on diseases at the cellular level. In addition, attention has also been paid to using the secreted EVs, for example, as carriers for drug delivery systems in drug delivery. Therefore, according to the EV secretion promoter of the present invention, for example, depending on the purpose, it can be supplied to target cells in vivo, ex vivo or in vitro to promote the secretion of EVs from cells. Regarding the use of the EV secretion promoter of the present invention, for example, it will be described in more detail in the EV secretion promotion method and the EV production method of the present invention described below, and these descriptions can be incorporated by reference.

[0070] <Method for Promoting Secretion of EVs> The method for promoting the secretion of EVs of the present invention, as described above, includes a promoting step of promoting the secretion of extracellular vesicles by coexisting a subject having cells with the EV secretion promoter of the present invention. The method for promoting the secretion of EVs of the present invention can promote the secretion of extracellular vesicles from cells in the subject, and thus can also be referred to as a method for producing EVs.

[0071] The subject is not particularly limited. In the method for promoting the secretion of EVs of the present invention, for example, the EV secretion promoter of the present invention may be supplied to a subject having cells from which the secretion of EVs is desired to be promoted. The type of the cells is not particularly limited, and specific examples include mesenchymal stem cells, epithelial cells, etc. The origin of the mesenchymal stem cells is not particularly limited, and examples include bone marrow, umbilical cord, dental pulp, adipose tissue, etc. The cells may be, for example, normal cells or abnormal cells.

[0072] The method for supplying the EV secretion promoter of the present invention is not particularly limited, and for example, it may be in vitro, ex vivo or in vivo.

[0073] In vitro, the subject to which the EV secretion promoter of the present invention is supplied is, for example, a cell. Examples of the cells include cells isolated from a living organism, cell line cells, and cultures thereof. Ex vivo, the subject to which the EV secretion promoter of the present invention is supplied is, for example, a tissue composed of cells or an organ composed of tissues, for example, isolated from a living organism. In vivo, the subject to which the EV secretion promoter of the present invention is supplied is, for example, a living organism. The origin or type of the cells, tissue, or organ is not particularly limited, and examples include adipose tissue, large intestine, lung, skin, breast, milk duct, mammary gland, pancreas, bone marrow, etc.

[0074] The cells, tissues, and organs may be derived from, for example, humans or non-human animals. The living organism may be, for example, humans or non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cattle, and camels. When the subject is derived from or is a non-human animal, the ABCA1 inhibitor is preferably, for example, an inhibitor that corresponds relatively specifically to the ABCA1 protein or ABCA1 gene derived from that specific non-human animal. When the subject is derived from or is a human, the ABCA1 inhibitor is preferably, for example, an inhibitor that corresponds relatively specifically to the ABCA1 protein or ABCA1 gene derived from humans.

[0075] The types of the cells, tissues, and organs are not particularly limited, and any target cells from which EVs can be secreted can be selected. Examples of the cells, tissues, and organs include those listed above.

[0076] The method of promoting EV secretion of the present invention may further include, for example, a step of recovering EVs secreted from the cells. The step of recovering EVs is also referred to as, for example, a step of isolating EVs secreted from the cells.

[0077] Specific examples of the EV secretion promoting method of the present invention are given below, but the present invention is not limited to these examples in any way.

[0078] As mentioned above, in recent years, it has been reported that EVs can be used to treat various diseases. For example, in the case of EVs derived from mesenchymal stem cells, the therapeutic effects on immune diseases, ischemic diseases, inflammatory diseases, neurological diseases, diabetes, and the like have been reported. Thus, since EVs derived from mesenchymal stem cells can serve as active ingredients in therapeutic drugs for these diseases, for example, by promoting the secretion of EVs from mesenchymal stem cells using the EV secretion promoter of the present invention, it is possible to efficiently prepare EVs that can be used as the active ingredient. The origin of the mesenchymal stem cells is not particularly limited, and examples include those mentioned above. Furthermore, when EVs are used as an active ingredient in the treatment of the diseases, the mesenchymal stem cells are preferably, for example, normal cells.

[0079] Other sources of EVs that can be used as active ingredients in therapeutic drugs include epithelial cells, specifically normal epithelial cells (Kadota et al., JEV, 2021), but the present invention is not limited to these.

[0080] Furthermore, EVs are known to encapsulate substances and mediate intercellular signaling, for example, as described above. Therefore, EVs can be used, for example, as carriers in drug delivery systems. Therefore, for example, by promoting the secretion of EVs from any cells using the EV secretion promoter of the present invention, it is possible to efficiently prepare EVs that can be used as carriers. The target cells for promoting the secretion of EVs that can serve as carriers are not particularly limited, and examples include mesenchymal stem cells, and the cells are preferably, for example, normal cells.

[0081] Furthermore, in the case of abnormal cells involved in various diseases, for example, the coexistence of the EV secretion promoter of the present invention can promote EV secretion and allow EV collection. Since EVs derived from the abnormal cells can be analyzed in research on the diseases and abnormal cells, analysis samples can be efficiently obtained by using the present invention.

[0082] As described above, the method for promoting EV secretion from cells in a subject using the EV secretion promoter of the present invention simply involves coexisting the subject with the EV secretion promoter. Specific examples are provided below, but the present invention is not limited to these examples. The following embodiments may be incorporated by reference unless otherwise specified.

[0083] (Embodiment 1) When the EV secretion promoter of the present invention is provided in vitro, it is preferable to coexist the cells with the EV secretion promoter in a medium, and to incubate the cells in the medium in the presence of the EV secretion promoter. This allows, for example, EVs to be secreted from the cells into the medium, allowing the secreted EVs to be easily recovered from the medium. Incubation of the cells in the medium may involve, for example, two-dimensional cell culture or three-dimensional cell culture using a scaffold or the like.

[0084] The cells to be coexisted with the EV secretion promoter of the present invention may be, for example, uncultured cells or cultured cells, preferably the latter. Specifically, for example, the cells are pre-cultured, and then cultured in the medium in the presence of the EV secretion promoter to promote EV secretion from the cells.

[0085] The EV secretion promoter of the present invention may be simply coexistent with the cells, for example, but it is preferable to transfect the cells with the ABCA1 inhibitor of the present invention. In this case, it is preferable that the EV secretion promoter of the present invention further coexists with a transfection agent (also called a gene transfer agent). The type of transfection agent is not particularly limited, and for example, commercially available reagents such as Lipofectamine (registered trademark) can be used. The conditions for adding the transfection agent are not particularly limited, and can be appropriately set depending on, for example, the type of cell, the number of cells, the type of medium, the amount of medium, etc.

[0086] The incubation conditions for the cells (e.g., medium, temperature, time, humidity, etc.) are not particularly limited and can be set appropriately depending on, for example, the type of cell, the type of medium, etc. As a specific example, when the cells are mesenchymal stem cells, the incubation conditions are, for example, MesenPRO RS medium, a culture time of 24 to 96 hours, and a culture temperature of 36-37° C. Similar conditions can also be used for other cells, for example.

[0087] The amount of the EV secretion promoter of the present invention added to the cells is not particularly limited. For example, when the EV secretion promoter is an expression-suppressing substance (e.g., an RNAi substance such as miRNA or siRNA), it is added to the medium to a final concentration of, for example, 25 to 100 nmol / L.

[0088] As a specific example, when the EV secretion promoter is a function-inhibiting substance (for example, a function-inhibiting substance such as the low-molecular-weight compound), it is added to the medium to a final concentration of, for example, 10 to 100 nmol / L.

[0089] The method for promoting EV secretion of this embodiment may further include, for example, a step of recovering EVs secreted from the cells. The step of recovering EVs is also referred to as, for example, a step of isolating EVs secreted from the cells.

[0090] The method for recovering EVs is not particularly limited. For example, the cells may be incubated in the medium in the presence of the EV secretion promoter, the cells may be removed from the medium, a liquid fraction such as a supernatant may be recovered, and EVs may be recovered from the liquid fraction. EVs can be recovered from the supernatant by, for example, filtration such as ultrafiltration or centrifugation such as ultracentrifugation.

[0091] (Embodiment 2) When the EV secretion promoter of the present invention is supplied ex vivo to the tissue or organ, the description of in vitro supply to the cells can be applied. Similarly, incubation conditions are not particularly limited and can be appropriately set depending on, for example, the type and size of the tissue or organ. For the amount of the EV secretion promoter of the present invention to be added, see, for example, the examples of the amount to be added to the cells.

[0092] The method for promoting EV secretion of this embodiment may further include, for example, a step of collecting EVs secreted from cells of the tissue or organ. The step of collecting EVs is also referred to as, for example, a step of isolating EVs secreted from the cells.

[0093] The method for recovering EVs is not particularly limited. For example, the tissue or organ may be incubated in the medium in the presence of the EV secretion promoter, and then the tissue or organ may be removed from the medium to recover a liquid fraction such as a supernatant. EVs may then be recovered from the liquid fraction. EVs can be recovered from the supernatant by, for example, filtration such as ultrafiltration or centrifugation such as ultracentrifugation.

[0094] (Embodiment 3) When the EV secretion promoter of the present invention is delivered in vivo, the subject is, for example, a living organism containing the cells. The living organism may be, for example, a human or a non-human animal. Examples of non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cattle, and camels.

[0095] As mentioned above, EVs have been reported to have therapeutic effects on various diseases. Therefore, for example, by administering the EV secretion-promoting agent of the present invention to a living body to promote EV secretion from cells, therapeutic effects can be obtained.

[0096] The method of administration of the EV secretion promoter of the present invention is not particularly limited, and examples include parenteral administration, oral administration, and intravenous administration. The conditions of administration are also not particularly limited, and can be determined appropriately depending on, for example, the type of living organism and the type of cells, tissues, or organs to be administered. The amount of the EV secretion promoter of the present invention to be added can be determined by reference to, for example, the examples of the amount to be added to the cells.

[0097] In the case of parenteral administration, the administration site may be, for example, a target tissue or organ (i.e., a tissue or organ containing cells whose EV secretion is desired to be promoted (also referred to as a target site)), or a site where the ABCA1 inhibitor of the present invention, the active ingredient of the EV secretion promoter, can be delivered to the target site. Specifically, for example, if the target cells are colon cells, the administration site may be the colon, or a site where the ABCA1 inhibitor of the present invention can be delivered to the colon. Furthermore, for example, if the target cells are lung cells, the administration site may be the lung, or a site where the ABCA1 inhibitor of the present invention can be delivered to the lung. The same applies to other organs. Examples of parenteral administration methods include intravenous injection, subcutaneous injection, intradermal injection, infusion injection, and transdermal administration. The form of the EV secretion promoter of the present invention is not particularly limited and can be appropriately determined depending on the administration method, as described above. The above descriptions are applicable.

[0098] For parenteral administration, the dosage form of the EV secretion promoter of the present invention is not particularly limited and can be appropriately determined depending on the administration method. For example, it can be a liquid, cream, gel, or the like, and can be prepared by mixing a vehicle with the ABCA1 inhibitor of the present invention. Among the vehicles, the aqueous solvent can be, for example, physiological saline or an isotonic solution, the oily solvent can be, for example, soybean oil, and the emulsifying solvent can be, for example, a mixture thereof. The parenteral administration agent can further contain, for example, alcohol, polyalcohol, surfactant, or the like. Furthermore, for parenteral administration, the EV secretion promoter of the present invention can contain, for example, a DDS agent for effectively delivering the ABCA1 inhibitor of the present invention from a site other than the target site to the target site. Furthermore, when effectively delivering the ABCA1 inhibitor of the present invention to, for example, specific cells (e.g., cancer cells) within the tissue of the target site, the EV secretion promoter of the present invention can contain, for example, a DDS agent that specifically recognizes the specific cells.

[0099] In the case of oral administration, the dosage form of the EV secretion promoter of the present invention is not particularly limited and may be, for example, tablets, pills, granules, powders, capsules, syrups, etc. The EV secretion promoter of the present invention may contain, for example, a diluent, excipient, carrier, etc. The EV secretion promoter of the present invention may also contain, for example, a DDS agent for effectively delivering the ABCA1 inhibitor of the present invention to the target site. Furthermore, when the ABCA1 inhibitor of the present invention is to be effectively delivered to, for example, specific cells (e.g., cancer cells) within the tissue of the target site, the EV secretion promoter of the present invention may also contain, for example, a DDS agent that specifically recognizes the specific cells.

[0100] When administering the EV secretion promoter of the present invention to a living body, the administration conditions can be appropriately determined depending on, for example, the age, body weight, type of tissue or organ to be administered, sex, etc.

[0101] As described above, regarding EVs, therapeutic effects on various diseases are known, and according to the EV secretion promoter of the present invention, the secretion of EVs having such effects can be promoted by administration to a living body. Therefore, the EV secretion promoter of the present invention itself can also be said to be, for example, a pharmaceutical composition for treating the above-mentioned diseases indirectly.

[0102] Also, as described above, EVs are known to contain, for example, information substances within a certain cell and mediate intercellular information transmission by moving. Therefore, the EV secretion promoter of the present invention can also be said to be, for example, an intercellular information transmission promoter, and the EV secretion promotion method of the present invention can also be said to be, for example, an intercellular information transmission promotion method.

[0103] <Method for producing EV> The method for producing EV of the present invention includes, as described above, a promotion step of promoting the secretion of extracellular vesicles by coexisting cells and the EV secretion promoter of the present invention, and a recovery step of recovering the extracellular vesicles secreted from the cells. In the method for producing EV of the present invention, the EV secretion promoter of the present invention used and the above-mentioned promotion step can be applied by referring to the description of the EV secretion promoter of the present invention and the EV secretion promotion method of the present invention.

[0104] The above-mentioned promotion step is the same as the in vitro or ex vivo form in the EV secretion promotion method of the present invention. And in particular, it is preferable to coexist the cells and the EV secretion promoter in a medium in the above-mentioned promotion step. According to this form, as described above, EVs can be secreted from the cells into the above-mentioned medium. Therefore, in the subsequent recovery step, that is, the step of recovering the EVs secreted from the cells, the EVs can be easily recovered from the medium.

[0105] The method for collecting EVs is not particularly limited, and any conventionally known method can be used. Examples of the collection method include filtration, centrifugation such as ultracentrifugation, and precipitation. Alternatively, for example, the EVs can be collected using a device on which antibodies against the aforementioned EV marker molecules are immobilized, utilizing binding and dissociation of the antibodies.

[0106] <Application> The present invention relates to an ABCA1 inhibitor for use in promoting the secretion of extracellular vesicles from cells. The present invention also relates to an ABCA1 inhibitor for use in producing a promoter for the secretion of extracellular vesicles from cells. The same description as for the EV secretion promoter of the present invention can be applied to the ABCA1 inhibitor. [Example]

[0107] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified.

[0108] [material and method] (cell) We used human colon adenocarcinoma cell line HCT116 (ATCC CCL-247) for colorectal cancer cells, human alveolar basal adenocarcinoma cell line A549 (ATCC CCL-185) for lung cancer cells, human prostate cancer cell line PC3M-Luc-C6 (PerkinElmer CVCL_D577), human melanoma cell line A375 (ATCC CRL-1619), human embryonic kidney cell line HEK293 (CRL-1573), human pancreatic adenocarcinoma cell line PANC-1 (ATCC CRL-1469), human breast cancer cell line MM231 (ATCC HTB-26), human normal prostate epithelial cell line PNT-2 (formerly DS Pharma, now KAC, EC95012613), and human preneoplastic breast epithelial cell line MCF10A (ATCC CRL-10317) derived from an adult female. The mesenchymal stem cells used were human bone marrow-derived mesenchymal stem cells BM-MSC (ATCC PCS-500-012™) and human subcutaneous adipose tissue-derived mesenchymal stem cells AT-MSC (ATCC PCS-500-011).

[0109] (miRNA) Example: miRNA miR-3202 (product number 4464066, Ambion) Negative control miRNA (hereafter referred to as NC1) miRNA Mimic Negative Control #1 (Product No. 4464058, Ambion)

[0110] (cell culture) HCT116, A549, PANC-1, A375, and HEK293 cells were cultured in DMEM (serum-supplemented) and advanced DMEM (serum-free). PNT2, MM231, and PC3M cells were cultured in RPMI 1640 (serum-supplemented) and advanced RPMI 1640 (serum-free). BM-MSCs and AT-MSCs were cultured in MesenPRO RS (serum-free). Culture conditions were 37°C, 5% carbon dioxide, and 95% relative humidity (RH). MCF10A cells were cultured in MEGM (Kit Catalog No. CC-3150, LONZA, serum-free).

[0111] (Transfection of miRNA and collection of secreted EVs) In the Examples, secreted exosomes were collected and used as a secreted EV sample.

[0112] To prepare secreted EV samples for the ExoScreen method, culture and supernatant collection were carried out as follows: First, 5 × 10 cells were cultured in serum-supplemented medium (the medium appropriate for the cell type) in a 96-well plate. 3 Cells were seeded at a ratio of 0.1 mL of cells / medium per well (Day 0). After 24 hours of incubation (Day 1), 10 fmol of the miRNA was added, and the miRNA was transfected into the cells using a transfection agent (Dhermafect1, product name, Dhermacon, hereinafter the same) according to the manufacturer's instructions. After a further 24 hours of incubation (Day 2), the culture supernatant was removed and replaced with fresh serum-free medium (the medium appropriate for the cell type). After 48 hours of incubation (Day 4), the supernatant was collected from the well. 10 μL of the collected supernatant was used as a secreted EV sample, and the following ExoScreen method was performed.

[0113] To prepare secreted EV samples for the NTA method, we cultured, collected the supernatant, and purified the samples as follows. First, we placed 1 × 10 5Cells were seeded in a 4 mL / well ratio (Day 0). Three wells of the 6-well dish were used for miR-3202, and the remaining three wells were used for NC1. After 24 hours of incubation (Day 1), the miRNA was added, and the cells were transfected with the miRNA using the transfection agent according to the manufacturer's instructions. After another 24 hours of incubation (Day 2), the culture supernatant was removed and replaced with fresh serum-free medium (the medium appropriate for the cell type). After 96 hours of incubation (Day 6), the supernatant was collected from each well and mixed with the supernatant from three wells containing the same miRNA to obtain approximately 12 mL of mixed supernatant. After collecting the supernatant, the number of cells in each well was counted. The collected mixed supernatant was then centrifuged at 2000 × g for 10 minutes to remove cell debris, and then filtered through a 0.22 μm filter (Millipore), and the filtrate was collected. EV (exosomes) were further purified using 11 mL of the filtrate by ultracentrifugation. Specifically, the filtrate was centrifuged at 110,000 × g for 70 minutes to obtain a pellet enriched in secreted EVs. The pellet was washed with 11 mL of PBS and further ultracentrifuged at 110,000 × g for 70 minutes, and then re-collected. This was used as the secreted EV sample and subjected to the NTA method described below.

[0114] (ExoScreen method) EV detection was performed using the ExoScreen method, utilizing the exosome markers CD9 or C63. EV detection was performed using AlphaLISA reagents (Perkin Elmer) consisting of AlphaScreen streptavidin-coated donor beads (6760002), AlphaLISA non-binding acceptor beads (6062011), and AlphaLISA universal buffer (AL001F), a 96-well half-area white plate (6005560, Perkin Elmer), and an EnSpire Alpha 2300 Multilabel plate reader (Perkin Elmer). Specifically, 10 μL of the secreted EV sample for the ExoScreen method, 10 μL of 5 nmol / L biotinylated antibody prepared in the buffer, and 10 μL of 50 μg / mL AlphaLISA acceptor bead-bound antibody were added to each well of the plate. Biotinylated anti-human CD9 antibody and AlphaLISA acceptor bead-bound anti-human CD9 antibody were used to detect CD9 / CD9 double-positive EVs, while biotinylated anti-human CD63 antibody and AlphaLISA acceptor bead-bound anti-human CD63 antibody were used to detect CD63 / CD63 double-positive EVs. After incubating the plate at 37°C for 1 hour, 25 μL of 80 μg / mL AlphaScreen streptavidin-coated donor beads were added and incubated for an additional 30 minutes at 37°C in the dark. The detector was then set to an excitation wavelength of 680 nm and an emission detection wavelength of 615 nm, and the luminescence in the plate wells was measured. Commercially available antibodies were mouse monoclonal anti-human CD9 (clone 12A12) and CD63 (clone 8A12) antibodies (both from Cosmo Bio).

[0115] (Analysis of EVs by Nanoparticle Tracking Analysis (NTA)) The secreted EV samples for the NTA assay were suspended in PBS, and further dilutions were prepared in PBS. These samples were analyzed using NanoSight particle tracking analysis (LM10, software version 2.03). The particle tracking assay acquired at least five 60-second videos from each sample at camera level 14. Analysis settings were optimized and maintained constant across samples. EV concentration was calculated as particles / cell in the culture medium, and the net EV secretion rate was obtained. The EV measurement results using the ExoScreen assay correlated with those using the NTA assay, confirming that the ExoScreen assay can measure secreted EV levels.

[0116] Unless otherwise specified, data in the examples are presented as mean ± standard error. Statistical significance was determined by Student's t-test. In dot plots, bars indicate the median and interquartile range, and statistical significance was determined by Student's t-test. P < 0.05 was considered statistically significant. *P<0.05, **P<0.01.

[0117] [Example A1] We confirmed that miR-3202 promotes EV secretion.

[0118] (1) Promotion of EV secretion from HCT and A549 cells Using the method described above, HCT116 and A549 were transfected with miR-3202, and secreted EV samples were prepared from the collected culture supernatant. The amount of secreted EVs was measured using the ExoScreen and NTA methods. The amount of secreted EVs was measured in the same manner as for the negative control (NC), except that the negative control miRNA (NC1) was used. The amount of secreted EVs for the NC was set at 1, and the relative values ​​(n = 3) for the secreted EVs in the examples were calculated.

[0119] These results are shown in Figures 1 and 2. Figure 1 shows the secreted EV yield of transfected HCT116 cells, and Figure 2 shows the secreted EV yield of transfected A549 cells. In Figures 1 and 2, the upper left graph shows the results for CD9 / CD9 double-positive EVs by ExoScreen, the upper right graph shows the results for CD63 / CD63 double-positive EVs by ExoScreen, and the lower graph shows the results by NTA. As shown in Figures 1 and 2, miR-3202 transfection significantly enhanced EV secretion, increasing the amount of secreted EVs compared to NC cells in both HCT116 and A549 cells. Furthermore, immunofluorescence staining confirmed increased levels of the EV marker CD63 in the cytoplasm of cells cultured in the presence of miR-3202.

[0120] (2) Promotion of EV secretion from other cells As shown in (1) above, miR-3202 was found to promote EV secretion from cells in both HCT116 and A549. Therefore, we further confirmed that miR-3202 can promote EV secretion in a broad range of cell types, regardless of cell type. The amount of secreted EVs was measured in the same manner as in (1) above, except that the cells shown in Figure 3 were used. These results are shown in Figure 3. Figure 3 shows the results of CD63-positive EVs measured by ExoScreen, and the signal intensity is shown relative to the measured value for NC.

[0121] As shown in Figure 3, in all cell types, the relative value exceeded 1. These results confirmed that miR-3202 can promote EV secretion from cells, regardless of the cell type.

[0122] (3) EVs obtained by secretion stimulation A comparison was made between an EV sample recovered from cells cultured in the presence of miR-3202 (miR-3202) and an EV sample recovered from cells cultured in the absence of miR-3202 (C).

[0123] When the EV sample (miR-3202) and the EV sample (C) were examined by Western blotting for EV markers (CD9, CD63, CD81, TSG101) and endosomal markers (RAB5, EEA1, LAMP1, RAB7), all markers were detected in both samples, with no significant differences between them.

[0124] We also compared the functions of EV samples collected from cells cultured in the presence of miR-3202 with those collected from cells cultured in the absence of miR-3202. Specifically, EV sample (miR-3202) was collected from BM-MSCs cultured in the presence of miR-3202, and EV sample (C) was collected from BM-MSCs cultured in the absence of miR-3202. HEL293 cells were then cultured in the absence of the EV sample, the presence of the EV sample (miR-3202), or the presence of the EV sample (C), and cell proliferation was compared by cell count. Compared to cells cultured in the absence of the EV sample, both the EV sample (miR-3202) and the EV sample (C) significantly increased cell proliferation, but there was no significant difference between the presence of the EV sample (miR-3202) and the EV sample (C). This indicates that even when EV secretion is promoted in target cells using miR-3202, EVs similar to those obtained in the target cells that have not been contacted with miR-3202 are obtained.

[0125] (3) Analysis of secreted EVs The size of the EVs contained in the secreted EV sample obtained in (1) was confirmed. The secreted EV sample was subjected to a nanoparticle analysis system (trade name NanoSight, Malvern) to measure the particle size distribution of the particles (EVs) contained in the secreted EV sample.

[0126] The particle size distribution results are shown in Figures 4 and 5. Figure 4 shows the results for secreted EV samples from HCT116 cells. The upper graph shows the results for secreted EV samples from miR-3202-transfected cells, and the lower graph shows the results for secreted EV samples from NC (negative control). Figure 5 shows the results for secreted EV samples from A549 cells. The upper graph shows the results for secreted EV samples from miR-3202-transfected cells, and the lower graph shows the results for secreted EV samples from NC (negative control). In Figures 4 and 5, the vertical axis represents particle concentration (particles / mL), the horizontal axis represents particle diameter (nm), the thin line represents the average concentration of triplicate measurements for each particle diameter, and the thick line represents each concentration (concentration range) of triplicate measurements for each particle diameter. In HCT116 (Figure 4), the peak particle size (97 nm) of the secreted EV sample from miR-3202-transfected cells was comparable to that of the NC-transfected EV sample (94 nm). Similarly, in A549 (Figure 5), the peak particle size (111 nm) of the secreted EV sample from miR-3202-transfected cells was comparable to that of the NC-transfected EV sample (102 nm). These results confirmed that the EVs released from miR-3202-transfected cells were the same as those from the control.

[0127] (4) Confirmation that the increase in EV amount is due to the promotion of secretion When cell death occurs due to apoptosis, apoptotic bodies may be released. Therefore, in this example, we confirmed that the EVs secreted from cells were not apoptotic bodies.

[0128] Caspase activity is known to be a marker of apoptosis. Therefore, caspase activity was measured for cell samples obtained by culturing various cells transfected with the miRNAs (1) and (2). The cell samples were also measured for cell viability using the MTS assay. The caspase activity of the cell samples was then normalized by the respective cell viability, and the normalized caspase activity of the miR-3202-transfected cells was divided by the normalized caspase activity of the NC1-transfected cells to obtain the relative caspase activity (miR-3202 / NC1).

[0129] These results are shown in Figure 6. Figure 6 is a graph showing the caspase activity of cells, with the vertical axis representing the relative value of caspase activity (miR-3202 / NC1). If the relative value of caspase activity is approximately 1 or less than 1, it can be determined that caspase activity is not increased by miRNA transfection. As shown in Figure 6, the relative value was approximately 1 or less than 1 for all cells, indicating that caspase activity was not significantly increased in cells transfected with miR-3202 compared to cells transfected with the control NC1. Therefore, it was found that the EV secretion promoter of the present invention can maintain cell survival and promote EV secretion, rather than promoting cell death by apoptosis, for example. Therefore, the EVs in the present invention can be said to be EVs derived from viable cells.

[0130] [Example A2] We confirmed that miR-3202 promotes EV secretion from mesenchymal stem cells.

[0131] In Example A1, it was confirmed that the miRNA of the present invention can promote EV secretion from cells with broad applicability. We further confirmed that it promotes EV secretion from bone marrow-derived mesenchymal stem cells.

[0132] (1) Promotion of EV secretion from bone marrow-derived MSCs BM-MSCs were transfected with miR-3202 in the same manner as in Example A1, except that BM-MSCs were used as the cells and MesenPRO RS (serum-free) was used as the medium. Secreted EV samples were prepared from the collected culture supernatant, and the amount of secreted EVs was measured using the NTA method. The amount of secreted EVs was measured in the same manner as in Example A1, except that the negative control miRNA (NC1) was used. The amount of secreted EVs in the NC was set to 1, and the relative values ​​(n = 3) of the secreted EVs in the examples were calculated. The secreted EV samples were also subjected to the nanoparticle analysis system (trade name NanoSight, Malvern) to measure the particles (EVs) contained in the secreted EV samples.

[0133] These results are shown in Figure 7. In Figure 7, the graph on the left shows the amount of EVs secreted by transfected MSCs as measured using the nanoparticle analysis system, the graph on the right shows the amount of EVs secreted by transfected MSCs as measured using the NTA method, and the table below shows the results of counting the number of miRNA-transfected cells in each well (n = 3).

[0134] The amount of secreted EVs measured by the NTA method is normalized by the number of viable cells. Therefore, assuming that apoptosis occurs due to miR-3202 transfection, the amount of secreted EVs measured by the nanoparticle analysis system, which is not normalized by the number of viable cells, is likely to differ from the amount of secreted EVs measured by the NTA method. However, as shown in the graph in Figure 7, the amount of secreted EVs measured by the nanoparticle analysis system and the NTA method were comparable. Furthermore, as shown in the table in Figure 7, when comparing the cell counts of BM-MSCs transfected with NC1 miRNA and those transfected with miR-3202, no significant decrease in cell count was observed due to miR-3202 transfection. These results suggest that miR-3202 transfection did not induce apoptosis in BM-MSCs, and the significant increase in the amount of secreted EVs in miR-3202-transfected BM-MSCs compared to NC is likely due to the promotion of EV secretion by miR-3202.

[0135] (2) Analysis of secreted EVs The size of the EVs contained in the secreted EV sample obtained in (1) above was confirmed in the same manner as in Example A1(3) above.

[0136] The particle size distribution results are shown in Figure 8. Figure 8 shows the results for the secreted EV samples from BM-MSCs. The upper graph shows the results for the secreted EV samples from miR-3202-transfected cells, and the lower graph shows the results for the secreted EV samples from NC (negative control). In Figure 8, the vertical axis represents particle concentration (particles / mL), the horizontal axis represents particle diameter (nm), the thin line represents the average concentration of triplicate measurements, and the thick line represents the concentration range of triplicate measurements. As shown in Figure 8, the particle diameter peak (106 nm) of the secreted EV sample from miR-3202-transfected cells was similar to that of the secreted EV sample from NC (112 nm). These results confirmed that the EVs released from miR-3202-transfected BM-MSCs were the same as those from the control.

[0137] [Example A3] We confirmed that miR-3202 suppresses the expression of the ABCA1 gene.

[0138] As in Example A2, A549, HCT116, and Panc6 were transfected with miR-3202 and cultured, and the expression levels of ABCA1 mRNA were measured for the resulting cultured cells. ABCA1 mRNA was measured by RT-PCR. As a negative control, culture and expression level measurement were performed in the same manner, except that miR-3202 was not added. The expression level of the negative control was set as a relative value of 1, and the relative expression level of the miR-3202-transfected cells was calculated.

[0139] The relative expression levels in A549, HCT116, and Panc1 were 0.61, 0.63, and 0.63, respectively. In all cell types, the relative values ​​were less than 1, confirming that ABCA1 gene expression was suppressed compared to NC. These results confirmed that the target gene of miR-3202 is the ABCA1 gene. Furthermore, since the aforementioned examples confirmed that miR-3202 can promote EV secretion, it can be concluded that EV secretion can be promoted not only by miR-3202, but also by suppressing the expression of the ABCA1 gene, a target gene of miR-3202, or by suppressing the function of the ABCA1 protein encoded by the target gene.

[0140] [Example B] In Example A, it was confirmed that miR-3202, which suppresses ABCA1 expression, promotes EV expression. Therefore, it was confirmed that ABCA1 inhibitors other than miR-3202 can also promote EV secretion.

[0141] [Example B1] We confirmed that knockdown of the ABCA1 gene promoted EV secretion.

[0142] (1) Silencing of ABCA1 gene expression by siRNA The following two types of siRNA were used as siRNAs against the ABCA1 gene. siABCA1_1: Product name: siGENOME siRNA Human ABCA1 (D-004128-01-0002), Dharmacon siABCA1_2: Product name: siGENOME siRNA Human ABCA1 (D-004128-03-0002), Dharmacon

[0143] A549 and HCT116 were transfected with the siRNA and cultured, and the resulting cultured cells were measured for ABCA1 mRNA expression levels. Cell culture was performed according to the Materials and Methods section above, and siRNA transfection was performed in the same manner as for miRNA introduction described in Materials and Methods. ABCA1 mRNA was measured by RT-PCR. As a negative control, culture and expression levels were measured in the same manner except that the siRNA was not added. The expression level of the negative control was set as a relative value of 1, and the relative expression level of the siRNA-transfected cells was calculated. The results are shown in Figure 9.

[0144] Figure 9 is a graph showing the relative expression levels of ABCA1 mRNA in cells transfected with siRNA against the ABCA1 gene. The upper graph shows the results for A549 cells, and the lower graph shows the results for HCT116 cells. As shown in Figure 9, when either siABCA1_1 or siABCA1_2 was used, the expression levels of ABCA1 mRNA in A549 and HCT116 were significantly lower than those of the negative control. These results confirmed that the ABCA1 gene was knocked out by ABCA1 siRNA.

[0145] (2) Promotion of EV secretion by suppressing ABCA1 gene expression A549 and HCT116 were transfected with the siRNA and cultured in the same manner as described in (1) above. After confirming the cell count after culture, secreted EV samples were prepared from the collected culture supernatant according to the Materials and Methods section above, and the amount of secreted EVs was measured by the NTA method. The amount of secreted EVs was also measured for a negative control (NC) in the same manner as described in (1) above. The amount of secreted EVs from the NC was set as a relative value of 1, and the relative value of the secreted EVs from the siRNA-transfected cells was calculated. The number of NC cells was set as a relative value of 1, and the relative value of the number of siRNA-transfected cells was calculated as cell viability.

[0146] These results are shown in Figure 10. Figure 10 shows the results for cells transfected with ABCA1 siRNA. The top graph shows cell viability, and the bottom graph shows the relative amount of EV secretion. The left column shows the results for A549 cells, and the right column shows the results for HCT116 cells. First, as shown in the top panel of Figure 10, siRNA transfection did not decrease viability in either A549 or HCT116 cells compared to NC. On the other hand, as shown in the bottom panel of Figure 10, siRNA transfection increased EV secretion in both A549 and HCT116 cells compared to NC. In particular, transfection with siABCA1_2 significantly increased EV secretion. These results confirm that knockout of the ABCA1 gene can promote EV secretion without affecting cell viability.

[0147] [Example B2] We confirmed that EV secretion was promoted by an inhibitor of the ABCA1 protein.

[0148] Cyclosporine, probucol, and valspodar were used as inhibitors. A549 and HCT116 cells were cultured as described in the Materials and Methods section (miRNA transfection and secreted EV recovery). After 24 hours of incubation (Day 1), cell counts and secreted EV levels were measured in the same manner, except that the inhibitors were added instead of the miRNA. The inhibitors were added at 10 nmol / L or 100 nmol / L per well. As a negative control, the culture, cell counts, and secreted EV levels were measured in the same manner, except that the inhibitors were not added. The amount of secreted EVs from the NC cells was set as a relative value of 1, and the relative value of the secreted EVs from the inhibitor-treated cells was calculated. Cell viability was calculated based on the relative value of the number of cells from the NC cells set as a relative value of 1.

[0149] These results are shown in Figures 11 and 12. Figure 11 shows the results for A549 cells treated with the inhibitors. The upper graph shows cell viability, and the lower graph shows the relative amount of EV secretion. Figure 12 shows the results for HCT116 cells treated with the inhibitors. The upper graph shows cell viability, and the lower graph shows the relative amount of EV secretion. First, as shown in the upper panels of Figures 11 and 12, the addition of the inhibitors did not significantly reduce viability in either A549 or HCT116 cells compared to NC. On the other hand, as shown in the lower panels of Figures 11 and 12, the addition of the inhibitors increased EV secretion in both A549 and HCT116 cells compared to NC. Furthermore, EV secretion was promoted in a concentration-dependent manner with both inhibitors. Among the inhibitors, probucol and valspodar were particularly effective in promoting EV secretion.

[0150] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0151] This application claims priority based on Japanese Patent Application No. 2021-166634, filed on October 11, 2021, the disclosure of which is incorporated herein in its entirety. [Industrial Applicability]

[0152] According to the present invention, the secretion of extracellular vesicles from cells can be promoted. Therefore, for example, by secreting extracellular vesicles from cells, it is possible to efficiently prepare extracellular vesicles as materials or secrete endogenous extracellular vesicles in living organisms. Therefore, the present invention can be said to be a very useful technology, for example, in the medical field.

Claims

1. The ABCA1 inhibitor includes an expression inhibitor that suppresses the expression of a gene encoding the ABCA1 protein or a function inhibitor of the ABCA1 protein; the expression-inhibiting substance is at least one nucleic acid substance selected from the group consisting of an RNA interference substance, an antisense, an antigene, and an expression vector that expresses any of these; the function-suppressing substance is a function-inhibiting substance or a function-neutralizing substance against the ABCA1 protein, the function inhibitor is at least one selected from the group consisting of cyclosporine, probucol, and valspodar; The function-neutralizing substance is an antibody against ABCA1 protein, an antigen-binding fragment thereof, or an expression vector expressing either of these. An extracellular vesicle secretion promoter characterized by:

2. the expression-inhibiting substance is the RNA interference substance, and the RNA interference substance is at least one nucleic acid substance selected from the group consisting of miRNA, siRNA, and precursors thereof; The extracellular vesicle secretion promoter according to claim 1, wherein the precursor is at least one selected from the group consisting of shRNA, a nucleic acid encoding the miRNA, a primary miRNA transcript, a precursor miRNA, and an expression vector for the miRNA.

3. The extracellular vesicle secretion promoter according to claim 2, wherein the miRNA is miR-3202.

4. an extracellular vesicle secretion promoter that promotes the secretion of extracellular vesicles from cells; The extracellular vesicle secretion promoter according to claim 1 or 2, wherein the cells are mesenchymal stem cells.

5. A method for promoting the secretion of extracellular vesicles, comprising a step of promoting the secretion of extracellular vesicles by coexisting a subject having cells in vitro or ex vivo with the extracellular vesicle secretion promoter described in claim 1 or 2.

6. The method for promoting secretion according to claim 5 , wherein the extracellular vesicle secretion promoter is added to the subject in vivo or ex vivo in the promoting step.

7. The method for promoting secretion according to claim 5 , wherein the promoting step involves transfecting the cells with the extracellular vesicle secretion promoter.

8. The method for promoting secretion according to claim 5 , wherein the cells are mesenchymal stem cells.

9. The method for promoting secretion according to claim 8 , wherein the cells are bone marrow-derived or adipose tissue-derived mesenchymal stem cells.

10. The method for promoting secretion according to claim 5 , wherein the cells are cells collected from a living organism or cells of a cell line.

11. The secretion promoting method according to claim 10 , wherein the cells and the extracellular vesicle secretion promoting agent are allowed to coexist in a medium in the promoting step.

12. The method for promoting secretion according to claim 5 , wherein the subject is a tissue or an organ.

13. A method for promoting secretion of extracellular vesicles, comprising administering the extracellular vesicle secretion promoter described in claim 1 or 2 to a living non-human animal in vivo.

14. A step of promoting secretion of extracellular vesicles from cells by coexisting a subject having cells with the extracellular vesicle secretion promoter according to claim 1 or 2 in vitro or ex vivo; A method for producing extracellular vesicles, comprising a recovery step of recovering extracellular vesicles secreted from the cells.

15. The method of claim 14, wherein the cells are transfected with the extracellular vesicle secretion promoter.

16. The method of claim 14 , wherein the cells are mesenchymal stem cells.

17. The method of claim 16, wherein the cells are bone marrow-derived or adipose tissue-derived mesenchymal stem cells.

18. The method of claim 14 , wherein the cells are cells collected from a living organism or cells of a cell line.

19. The method according to claim 14 , wherein the cells and the extracellular vesicle secretion promoter are allowed to coexist in a medium in the promoting step.

20. The method of claim 14, which is a method for producing the extracellular vesicles as carriers for a drug delivery system.

21. The method of claim 14, which is a method for producing the extracellular vesicles as a therapeutic agent.

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