Angiogenesis promoter, ischemia therapeutic agent, and method for promoting angiogenesis

Abstract

To provide a new system that allows introduction of a nucleic acid encoding a protein into a cell and enables enhancement of translation efficiency of the protein expressed from the introduced encoding nucleic acid.SOLUTION: An angiogenesis promoter of the present invention comprises a group of artificial microvesicles composed of multiple artificial microvesicles, the group of artificial microvesicles containing at least one microRNA selected from the group consisting of miR-126, miR-135, and miR-210. An ischemia therapeutic agent of the present invention comprises the angiogenesis promoter of the present invention.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an angiogenesis promoter, an agent for treating ischemia, and a method for promoting angiogenesis. [Background technology]

[0002] Limb ischemia is a condition in which arteriosclerosis prevents blood from reaching the lower limbs, resulting in poor circulation. Severe cases can lead to gangrene due to skin ulcers and infections, sometimes necessitating amputation. For this reason, clinical trials are underway to establish therapeutic methods that utilize angiogenesis. Currently, attempts are being made to administer known angiogenic proteins, such as VEGF and HGF, but the sustained effectiveness of these proteins in the body remains an issue, necessitating the development of new drugs. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, an object of the present invention is to provide a new drug that can promote angiogenesis. [Means for solving the problem]

[0004] In order to achieve the above object, the angiogenesis promoter of the present invention comprises: A group of artificial microvesicles consisting of a plurality of artificial microvesicles, The artificial microvesicles are characterized in that they contain at least one microRNA selected from the group consisting of miR-126, miR-135, and miR-210.

[0005] The therapeutic agent for ischemia of the present invention is characterized by containing the angiogenesis promoter of the present invention.

[0006] The method for promoting angiogenesis of the present invention is characterized by comprising a contacting step of contacting the angiogenic agent of the present invention. [Effects of the Invention]

[0007] The angiogenesis promoter of the present invention can promote angiogenesis, and is therefore effective in treating, for example, ischemic diseases. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a graph showing the number of lattice points of a luminal structure composed of HUVECs in Example 1. [Figure 2] FIG. 2 is a graph showing the number of lattice points of a luminal structure composed of HUVECs in Example 2. [Figure 3] FIG. 3 is a graph showing the number of junctions in the HUVEC tube in Example 3. [Figure 4] FIG. 4 is a graph showing the overall segment length of the HUVEC tube in Example 3. [Figure 5] FIG. 5 is a graph showing the number of pixels of the segment length of the HUVEC tube in Example 3. [Figure 6] FIG. 6 is a graph showing the results of macroscopic classification of the severity of neovascularized ischemic limbs in mice administered with exosome samples in Example 4. [Figure 7] FIG. 7 is a graph showing the flux values, which represent changes in blood flow, for mice administered with the exosome sample in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention includes, for example, the following aspects. [1] A group of artificial microvesicles comprising a plurality of artificial microvesicles; An angiogenesis promoter, characterized in that the group of artificial microvesicles contains at least one microRNA selected from the group consisting of miR-126, miR-135, and miR-210. [2] The angiogenesis promoter according to claim 1, wherein the group of artificial microvesicles contains, as the microRNA, miR-126 and miR-135, miR-126 and miR-210, or miR-135 and miR-210. [3] The angiogenesis promoter according to [1], wherein the group of artificial microvesicles contains miR-126, miR-135, and miR-210 as the microRNAs. [4] The angiogenesis promoter according to any one of [1] to [3], wherein the artificial microvesicles are artificial exosomes. [5] A therapeutic agent for ischemia, comprising the angiogenesis promoter according to any one of [1] to [4]. [6] A method for promoting angiogenesis, comprising a contact step of contacting target cells with an angiogenesis promoter described in any one of claims 1 to 4. [7] The method for promoting angiogenesis described in [6], wherein the contacting step is carried out in vivo, in vitro, or ex vivo. [8] The method for promoting angiogenesis according to [6] or [7], wherein the target cells are cells of a human or non-human animal.

[0010] Unless otherwise specified, terms used in this specification can be used in the sense commonly used in the art. [1] Angiogenesis promoters As described above, the angiogenesis promoter of the present invention comprises a group of artificial microvesicles consisting of a plurality of artificial microvesicles, and is characterized in that the group of artificial microvesicles comprises at least one microRNA selected from the group consisting of miR-126, miR-135, and miR-210.

[0011] In the present invention, the artificial microvesicles may contain at least one of miR-126, miR-135, and miR-210 as microRNA. The artificial microvesicles may contain, for example, any two of miR-126, miR-135, and miR-210 as microRNA, or all three. In the present invention, the artificial microvesicles preferably contain miR-126 and miR-135, or may contain three of miR-126, miR-135, and miR-210. In the present invention, miR-126, miR-135, and miR-210 are hereinafter also referred to as effective microRNAs (effective miRNAs) in the present invention.

[0012] In the angiogenesis promoter of the present invention, when the group of artificial microvesicles contains two or more types of effective microRNA, for example, each of the multiple artificial microvesicles constituting the group of artificial microvesicles may contain two or more types of effective microRNA, or each may contain any one of the effective microRNAs. In other words, the angiogenesis promoter of the present invention is sufficient as long as the multiple artificial microvesicles as a whole contain two or more types of effective microRNA, and there is no particular limitation on whether each individual artificial microvesicle contains two or more types of effective microRNA.

[0013] The sequence information of the effective microRNA of the present invention is registered in databases (FANTOM5, RIKEN). miR-126: Accession number 3p: MIMAT0000445 miR-135: Accession number b-5p: MIMAT0000758 miR-210: Accession number 3p: MIMAT0000267

[0014] The artificial microvesicles of the present invention can be obtained, for example, as extracellular vesicles released from cells. The extracellular vesicles are, for example, membrane vesicles (microvesicles) secreted from cells and enclosed in a lipid bilayer membrane. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles.

[0015] For example, artificial microvesicles containing the effective microRNA can be obtained by artificially processing cells or cell lines (hereinafter also referred to as raw cells) collected from a living organism, causing the processed raw cells to secrete microvesicles, and then recovering the microvesicles. The artificial processing is, for example, so-called genetic processing. Specifically, for example, the raw cells are genetically processed to strongly express the effective microRNA, and then cultured, thereby obtaining artificial microvesicles containing the effective microRNA from the genetically processed raw cells.

[0016] The method for preparing the artificial microvesicles containing the effective microRNA is not particularly limited. As an example, a method for preparing exosomes encapsulating the effective microRNA is described below.

[0017] First, a gene encoding the effective microRNA is introduced into a host cell. The type of cell is not particularly limited, and for example, MSCs can be used. A vector such as a viral vector can be used to introduce the gene. The viral vector is not particularly limited, and for example, a lentiviral vector is preferred in terms of infection efficiency. For example, the sequence of the effective microRNA may be inserted into the viral vector. For example, a gene transfer reagent such as TransDux (System Biosciences) may be used in combination to introduce (infect) the viral vector into the cell, for example, in terms of improving infection.

[0018] Next, the cells into which the vector has been introduced are cultured, and exosomes are collected. Since exosomes are released extracellularly, for example, a culture supernatant containing exosomes is collected from the cell culture. The culture supernatant can be collected, for example, by centrifugation or filtration. When exosomes are further separated from the culture supernatant, for example, the culture supernatant can be subjected to ultracentrifugation, and a precipitate fraction containing exosomes can be collected.

[0019] The method for evaluating exosomes is not particularly limited. The amount of exosomes in the collected fractions can be converted into, for example, protein amount or can be counted as particle number. Protein quantification can be performed, for example, using a fluorometer. The number and particle size of exosomes can also be analyzed, for example, using a nanoparticle analyzer.

[0020] Artificial microvesicles obtained from the genetically-treated raw cells (hereinafter also referred to as "treated raw cells") have the following characteristics compared to, for example, microvesicles (natural microvesicles) obtained from raw cells that have not been genetically-treated (hereinafter also referred to as "untreated raw cells"). Here, "not genetically-treated" means that they have not been subjected to a treatment to strongly express the effective microRNA. As a specific example, when the treated raw cells and the untreated raw cells are cultured under the same conditions and microvesicles of the same size are collected, the artificial microvesicles will encapsulate more of the effective microRNA than, for example, the natural microvesicles.

[0021] When the angiogenesis promoter of the present invention is used to treat a living organism, for example, as described below, the source cells may be cells collected from the subject (autologous cells) or cells or cell lines collected from a living organism other than the subject (allogeneic cells). The type of source cells is not particularly limited, and mesenchymal stem cells (MSCs) are preferred. The origin of MSCs is not particularly limited, and examples include bone marrow. If such source cells are used, the resulting artificial microvesicles can be, for example, HLA-2-free, HLA-G-expressing, and CD47-expressing on the exosome surface. This can result in, for example, tolerance immunity, reduced antigenicity to the living organism, and avoidance of immune rejection.

[0022] The artificial microvesicles of the present invention may contain, for example, microRNAs other than the effective microRNAs. Specifically, the artificial microvesicles may further contain, for example, microRNAs originally contained in the extracellular vesicles derived from the raw material cells used in their preparation.

[0023] The angiogenesis-promoting agent of the present invention may contain, as an active ingredient, the artificial cell population containing the effective microRNA. The angiogenesis-promoting agent of the present invention may contain, as the active ingredient, only the artificial cell population, or may contain the artificial cell population and other components involved in angiogenesis.

[0024] The angiogenesis-promoting agent of the present invention may contain only the active ingredient, or may contain the active ingredient and other additive ingredients. Examples of the additive ingredients include pharmaceutically acceptable ingredients. Specific examples of the additive ingredients are not particularly limited and include excipients, carriers (base materials), and the like. Examples of the excipients and carriers include aqueous solvents such as water, physiological saline, and buffer solutions; oils and fats such as soybean oil; petrolatum; alcohols such as glycerol; sugars such as maltose, dextrose, and dextrin; sugar alcohols such as xylitol; phospholipids; and liposomes. Other examples of the additive ingredients include binders, disintegrants, surfactants, emulsifiers, antioxidants, lubricants, humectants, thickeners, stabilizers, UV filters, antiseptics, preservatives, vitamins, minerals, and colorants.

[0025] In the angiogenesis promoter of the present invention, the amount of the artificial microvesicles containing the effective microRNA is not particularly limited, and it is preferable that the content be such that an effective amount is administered depending on the purpose, for example.

[0026] In the angiogenesis promoter of the present invention, the concentration of the artificial microvesicles containing the effective microRNA is not particularly limited. In the angiogenesis promoter of the present invention, the concentration of the effective microRNA contained in the artificial microvesicles is not particularly limited.

[0027] When the angiogenesis promoter of the present invention contains two or more types of effective microRNAs, the ratio (molar ratio) between them is not particularly limited.

[0028] The angiogenesis promoter of the present invention may be used, for example, at the living body level (in vivo), at the cellular level (in vitro), or at the tissue or organ level (ex vivo).

[0029] When the angiogenesis promoter of the present invention is used in vivo, the type of living organism to be treated is not particularly limited, and examples thereof include humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, dogs, cats, monkeys, rabbits, cows, goats, camels, and horses.

[0030] When the angiogenesis promoter of the present invention is used in vivo, the method of administration is not particularly limited and may be, for example, oral administration or parenteral administration. Examples of parenteral administration include topical, transdermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intraintestinal, intracerebral, and nasal administration. The administration method can be appropriately determined depending on, for example, the purpose of administration, the presence or absence of a disease, the severity of the disease, the type of living organism, age, sex, etc.

[0031] When the angiogenesis promoter of the present invention is administered to an adult human, the following conditions can be exemplified. Number of doses per day 1 to 3 times per location Administration method Intramuscular administration

[0032] The angiogenesis-promoting agent of the present invention can promote angiogenesis. Therefore, the angiogenesis-promoting agent of the present invention can be used, for example, for diseases that can be treated by angiogenesis. The disease is not particularly limited, and examples thereof include ischemia, more specifically, lower limb ischemia, myocardial ischemia, intestinal ischemia, cerebral ischemia, and the like, which are caused by ischemia. The administration site of the angiogenesis-promoting agent of the present invention is not particularly limited, and examples thereof include the affected area, muscles surrounding the affected area, etc.

[0033] When the angiogenesis promoter of the present invention is used in vitro or ex vivo, the origin of the target cells, tissues, or organs is not particularly limited, and may be human or the non-human animals. The type of the target cells is not particularly limited, and the type of the target tissues and organs is not particularly limited.

[0034] When the angiogenesis-promoting agent of the present invention is used in vitro or ex vivo, it is preferable to add the angiogenesis-promoting agent to a culture medium and then incubate the cells, tissues, or organs. The culture medium and incubation conditions are not particularly limited and can be appropriately set depending on the type of cells, tissues, or organs, for example.

[0035] [2] Ischemia treatment As described above, the therapeutic agent for ischemia of the present invention is characterized by containing the angiogenesis promoter of the present invention.

[0036] The therapeutic agent for ischemia of the present invention is characterized by containing the angiogenesis promoter of the present invention, and other configurations, conditions, etc. are not limited in any way. Unless otherwise specified, the description of the angiogenesis promoter of the present invention can be applied to the therapeutic agent for ischemia of the present invention.

[0037] As used herein, the term "treatment" refers to, for example, treatment in a broad sense, and includes prevention in addition to treatment in the narrow sense. Treatment in the narrow sense includes, for example, curing a disease, alleviating a disease, or inhibiting the progression of a disease, and prevention of a disease includes, for example, preventing contraction of a disease, preventing the onset of a disease, preventing the recurrence of a disease, etc. Treatment or prevention of a disease can also be referred to as, for example, treatment or prevention of a symptom of a disease.

[0038] In this specification, the subject to be treated is, for example, a living organism, and may be a human or a non-human animal, and may be a living organism that has developed a disease or a living organism that has not developed a disease (also referred to as a healthy organism). When the purpose is treatment in the narrow sense, the subject may be, for example, a patient (human) or a veterinary patient (non-human animal), and when the purpose is prevention, the subject may be, for example, a healthy individual (human) or a healthy non-human animal.

[0039] [3] Method for promoting angiogenesis As described above, the method for promoting angiogenesis of the present invention is characterized by comprising a contacting step of bringing the angiogenesis-promoting agent of the present invention into contact with target cells.

[0040] The method for promoting angiogenesis of the present invention is characterized by using the angiogenesis promoter of the present invention, and other steps, conditions, etc. are not limited in any way. Unless otherwise specified, the description of the angiogenesis promoter of the present invention can be applied to the method for promoting angiogenesis of the present invention.

[0041] In the angiogenesis-promoting method of the present invention, for example, the contacting step may be carried out in vivo, in vitro, or ex vivo.

[0042] In the angiogenesis-promoting method of the present invention, when the contacting step is performed in vivo, for example, the angiogenesis-promoting agent of the present invention may be administered to a living body that is a subject. The administration of the angiogenesis-promoting agent of the present invention to a living body is as described above.

[0043] In the angiogenesis-promoting method of the present invention, when the contacting step is performed in vitro or ex vivo, for example, the angiogenesis-promoting agent of the present invention may be contacted with cells, tissues, or organs of a subject. The conditions for contacting the angiogenesis-promoting agent of the present invention with the subject are as described above.

[0044] [4] Treatment of ischemia The method for treating ischemia of the present invention is characterized by comprising the step of administering the angiogenesis-promoting agent of the present invention or the therapeutic agent for ischemia of the present invention to a living subject.

[0045] The method for treating ischemia of the present invention is characterized by using the angiogenesis-promoting agent of the present invention or the therapeutic agent for ischemia of the present invention, and other steps, conditions, etc. are not limited in any way. Unless otherwise specified, the description of the angiogenesis-promoting agent of the present invention and the method for promoting ischemia of the present invention can be cited for the method for treating ischemia of the present invention.

[0046] [5]Usage The present invention relates to the use of the artificial microvesicles containing the effective microRNA for use in promoting angiogenesis or treating ischemia, and also to the use of the artificial microvesicles containing the effective microRNA for producing an agent for promoting angiogenesis or treating ischemia.

[0047] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. [Example]

[0048] [Example 1] The effectiveness of effective microRNAs on angiogenesis was confirmed.

[0049] Commercially available mimic RNAs (Thermo Fisher) of miR-126, miR-135, and miR-210 were used as effective microRNAs. Human umbilical vein endothelial cells (HUVEC, LONZA) were used as cells. EGM-2 medium was used.

[0050] 200 μL of EGM-2 medium, 1 nM (1 × 10 -12 A transfection mixture was prepared by mixing 11 μL of mimicRNA (100 mol / ml) and 4 μL of transfection reagent (product name INTERFERin, Polyplus Transfection). The effective miRNAs in each transfection mixture were as follows: Example 1-1: Combination of miR-126 + miR-135 + miR-210 (mixing ratio 1:1) Example 1-2: miR-126 alone Example 1-3: miR-135 alone Example 1-4: miR-210 alone Control: mirVana miRNA Mimic, Negative Control #1 (Invitrogen)

[0051] Confluent HUVECs were cultured at 1.6 × 10 5 The cells were seeded onto a 6-well plate at 2 mL / well and incubated at 37°C and 5% CO for 24 hours. The medium was then removed from the 6-well plate, and fresh medium was added at 2 mL / well. 215 μL of the transfection mixture was then added to each well, and the cells were incubated at 37°C and 5% CO for 48 hours. The medium was then removed from each well of the 6-well plate and washed with PBS(-). The cells in the wells were then detached with trypsin, collected, and the number of HUVEC cells was counted.

[0052] 280 μL to 300 μL of Matrigel basement membrane matrix (trade name: Corning® Matrigel basement membrane matrix, Corning®) was added to the entire 24-well plate. The collected HUVECs were added to a 24-well plate at a concentration of 3.5 × 10 4 The cells were seeded at 300 μL per well in medium.

[0053] After further culturing for 24 hours, HUVEC proliferation on the matrix, i.e., angiogenic potential, was evaluated (n = 3). Specifically, angiogenic potential was evaluated by analyzing the luminal structure (luminal area and HUVEC elongation) formed by HUVECs using the Angiogenesis Analyzer in Image J, and the number of grid points was calculated.

[0054] A higher grid point number indicates more angiogenesis, while a lower number indicates less angiogenesis. These results are shown in Figure 1. In Figure 1, the bold line indicates the mean value. Points above and below the mean indicate mean + SE and mean - SE, respectively. Points above and below the mean indicate mean + SD and mean - SD, respectively. All represents a system that combines miR-126, miR-135, and miR-210; 126 represents a system that uses miR-126 alone; 135 represents a system that uses miR-135 alone; and 210 represents a system that uses miR-210 alone.

[0055] Figure 1 is a graph showing the number of lattice points of luminal structures. As shown in Figure 1, compared to the control, the number of lattice points increased in all systems in which microRNA was added to the medium.

[0056] [Example 2] Effective microRNAs were confirmed.

[0057] (cell) Human bone marrow-derived mesenchymal stem cells (MSCs: Lonza, Lot No. 19TL28109) were cultured using MesenPro (Gibco), GlutaMax (Thermo Fisher Scientific), and StemPro (Gibco). Human umbilical vein endothelial cells (HUVECs) were cultured using EBM-2 (Lonza). Cell culture was performed in a chamber controlled at 37°C and 5% CO2. Matrigel basement membrane matrix (Corning) for 3D culture was used to evaluate angiogenic potential.

[0058] (lentiviral vector) The lentiviral vectors used were miRNA-expressing lentiviral particles (BiOSETTIA) expressing RFP (Red Fluorescent Protein)-labeled active miRNAs (hsa-mir-126, hsa-mir-135b, hsa-mir-210) or miRNA-expressing lentiviral particles (BiOSETTIA) expressing a control miRNA (hsa-mir-ctrl). The lentiviral particles contained a puromycin selection marker. Therefore, when miRNAs were expressed in cells infected with the lentiviral vector, they also acquired puromycin resistance. To increase the infection efficiency of the lentiviral vector, a transfection reagent (trade name TransDux, System Biosciences) was used during infection.

[0059] (1) Infection of MSCs with lentivirus The MSCs were thawed and cultured in the medium (MesenPro+GlutaMax). 2.0 × 10 5 The MSCs were seeded at a density of 3000 μL of medium per well, and a transfection reagent (TransDux) and the lentiviral vector were added to the wells at 1 / 200 the volume (15 μL) of the medium. The MSCs in the experimental group (6 wells) of the well plate were added with the lentiviral vectors hsa-mir-126, hsa-mir-135b, and hsa-mir-210 at an MOI of 1, while the MSCs in the control group (6 wells) were added with the lentiviral vector hsa-mir-ctrl at an MOI of 1. 24 hours after the addition of the lentiviral vector, the medium was replaced, and puromycin was added at 2 μg / mL. MSCs exhibiting puromycin resistance were selected over a 3-day period.

[0060] The fluorescence of labeled RFP was confirmed for the resistant MSCs. As a result, the fluorescence of RFP was confirmed, indicating that the MSCs were infected with the lentiviral vector.

[0061] (2) Exosome collection and evaluation The infected MSCs were cultured and passaged in a 10 cm dish. 7 When the cells proliferated to approximately 1000 cells, the medium was replaced with StemPro. After culturing for an additional 24 hours, the culture in the wells was filtered through a 0.22 μm filter, and the culture supernatant was collected. The collected culture supernatant was ultracentrifuged at 35,000 rpm for 70 min at 4°C to precipitate exosomes. The supernatant fraction was removed, and the precipitate fraction containing exosomes was washed with PBS(-) and ultracentrifuged again to collect the precipitate fraction. The collected precipitate fraction was used as the exosome fraction, and exosomes were evaluated. The exosome fraction was subjected to protein quantification using a fluorometer and particle size analysis using a nanoparticle analyzer to evaluate its properties. Hereinafter, the weight of the precipitate fraction was referred to as the weight of exosomes.

[0062] As a result, the exosome fraction derived from MSCs in the experimental group had a protein concentration of 2300 μg / ml and a particle density of 1.44 × 10 11 The most common detected particle size was 108.0 nm. The MSC-derived exosome fraction in the control group had a protein concentration of 2470 μg / ml and a particle density of 3.22 × 10 11 The most common particle size was 116.5 nm.

[0063] Furthermore, miRNA was extracted from the exosome fraction, and amplification of the introduced effective miRNA was confirmed by RT-PCR. miRNA was extracted using the phenol-chloroform extraction method with a reagent (product name QIAzol Lysis Reagent, QIAGEN). Similarly, miRNA was extracted from the exosome fraction and RT-PCR was performed on MSCs not infected with the lentiviral vector.

[0064] As a result, the exosome fraction from the example group infected with the lentiviral vector showed increased, i.e., higher expression, of the effective miRNAs compared to the exosome fraction from uninfected MSCs. Specifically, the expression of miR-126 was 4-fold, miR-135 was 20-fold, and miR-210 was 4-fold.

[0065] (3) Evaluation of angiogenic potential The HUVECs were thawed and cultured in the medium (EBM-2). 1.5 × 10 cells were placed in a 24-well plate containing the Matrigel basement membrane matrix. 4 The HUVECs were seeded at a density of 300 μL of cells / well. The exosome fraction was then added to the well at a predetermined concentration (1.0 μg / mL, 5.0 μg / mL) and cultured. After 24 hours, the proliferation of HUVECs on the matrix, i.e., their angiogenic potential, was assessed. Specifically, angiogenic potential was assessed by analyzing the luminal structure (luminal area and HUVEC elongation) formed by HUVECs using an Image J Angiogenesis Analyzer, and the number of grid points was obtained. Furthermore, HUVECs were cultured in the same manner, except that the exosome fraction was not added to the culture medium, and their luminal structure was analyzed.

[0066] These results are shown in Figure 2, which is a graph showing the number of lattice points in the luminal structures. As shown in Figure 3, compared to the luminal structures of HUVECs produced using the exosome fraction from the control group, the luminal structures of HUVECs produced using the exosome fraction from the experimental group, which highly expressed three types of effective miRNAs, showed significant differences in luminal area and HUVEC elongation.

[0067] These examples demonstrate that by highly expressing at least one of miR-126, miR-135, and miR-210 as an effective miRNA and encapsulating it in exosomes, the exosomes can effectively function as an angiogenesis promoter.

[0068] [Example 3] HUVEC tube formation assay was performed using exosomes from cells that overexpressed effective microRNAs.

[0069] (1) Preparation of exosome samples Human bone marrow mesenchymal stem cells (BMMSCs) were transfected with the lentiviral vectors containing the nucleotide sequences of effective miRNAs to generate MSCs with high miRNA expression. The lentiviral vectors used were those containing the nucleotide sequences of miRNA126, miRNA135b, and miRNA210. The lentiviral vectors were transfected at a multiplicity of infection (MOI) of 1, and transfection efficiency was enhanced using a transfection reagent (TransDux™, System Bioscience). After transfection, puromycin was added to the medium to a final concentration of 2 μg / ml, and infected MSCs were selected.

[0070] In addition, native MSCs without the lentiviral vector and control MSCs with a control vector (a lentiviral vector with a random base sequence inserted instead of the miRNA that does not express specific RNA) were also prepared in the same manner. MSCs were cultured at 37°C and 5% CO 2 It was decided.

[0071] Each MSC was cultured in a commercially available MSC medium (StemPro™, Thermo Fisher Scientific), and exosomes were collected from the culture supernatant by ultracentrifugation. The quality of the collected exosomes was assessed using a commercially available reagent (Qubit, Thermo Fisher Scientific) and a nanoparticle analyzer (Nanosight, Malvern Panalytical).

[0072] The following exosome samples were prepared using exosome solutions derived from each MSC. (Example: Exosomes) miRNA126-transfected MSC-derived exosomes (126) miRNA135b-transfected MSC-derived exosomes (135b) miRNA210-transfected MSC-derived exosomes (210) A 1:1 mixture of exosomes derived from MSCs transfected with miRNA126 and exosomes derived from MSCs transfected with miRNA135b (126 + 135b). A 1:1 mixture of miRNA126-transfected MSC-derived exosomes and miRNA210-transfected MSC-derived exosomes (126+210). A 1:1 mixture of miRNA135b-transfected MSC-derived exosomes and miRNA210-transfected MSC-derived exosomes (135b + 210).

[0073] (2) HUVEC tube formation A non-contact co-culture model was created to confirm the effect of exosome samples on HUVEC tube formation. First, the creation of the non-contact co-culture model, HUVEC tube formation, and evaluation methods are described below.

[0074] First, 1.5 × 10 cells were placed in a 24-well multiwell plate coated with 150 μg of Matrigel (registered trademark, Corning). 4 Human umbilical vein endothelial cells (HUVECs) were seeded at a density of 1.0 × 10 cells / well, and 300 μl / well of medium (EBM™-2, LONZA) was added. An insert membrane (Transwell™, Corning) was then placed on top of the multiwell plate on which the HUVECs had been seeded, and 1.0 × 10 MSCs were added to the insert membrane. 4 The non-contact co-culture model was cultured under the conditions of 37°C, 5% CO 2After 24 hours of culture, the tube formation status of HUVECs was observed using a fluorescence microscope (BZ-X800, KEYENCE) and images were taken. The images were then analyzed using an analyzer (Image J, Angiogenesis Analyzer) to measure the number of tube (lumen) junctions, total length of segments, and total mesh area, which are indicators of angiogenesis.

[0075] In the example systems, the example exosome sample was added together with the medium when HUVECs were seeded on the multiwell plate. In the native system (native), the native exosomes were added instead of the example exosome sample. In the control system (control), the control vector exosome sample was added instead of the example exosome sample. In each system, the amount of exosome sample added was adjusted so that the concentration in EBM medium was 1.0 μg / ml. In the no-addition system (coculture), the exosome sample was not added when HUVECs were seeded on the multiwell plate.

[0076] These results are shown in Figures 3, 4, and 5. Figure 3 is a graph showing the number of branches in HUVEC tubes, with the vertical axis representing the number of branches (junctions) measured by image analysis. Figure 4 is a graph showing the total length of HUVEC tube segments, with the vertical axis representing the number of pixels in the segment length measured by image analysis. Figure 5 is a graph showing the total mesh area, with the vertical axis representing the number of pixels in the area of ​​the mesh area measured by image analysis. In each graph, square plots represent the average value. In each graph, "coculture" represents the results for a system without the exosome sample added, "native" represents the results for a system with the native exosome added, "control" represents the results for a system with the control vector exosome added, and the remaining figures represent the results for systems with the exosomes added from various examples.

[0077] A comprehensive evaluation of the three angiogenesis evaluation items, the number of branches (Figure 3), the segment length (Figure 4), and the mesh area (Figure 5), showed that the system to which the exosomes of the Example were added showed better results than the system to which the exosomes of the Example were not added (coculture), the system to which the native exosomes were added (native), and the system to which the control vector exosomes were added (control). In other words, it was found that angiogenesis can be enhanced by adding the exosomes of the Example containing various overexpressed proteins.

[0078] [Example 4] Exosomes from cells that highly express effective microRNAs were administered to mice, and angiogenesis in vivo was confirmed.

[0079] (1) Exosome sample Nine types of exosome samples were prepared using the exosome solutions derived from each MSC from Example 3. The protein amount of each sample was 4 μg / 50 μl.

[0080] Native MSC-derived exosomes (native exosomes) Control vector-transfected MSC-derived exosomes (control vector exosomes) (Example: Exosomes) miRNA126-transfected MSC-derived exosomes (126) miRNA135b-transfected MSC-derived exosomes (135b) miRNA210-transfected MSC-derived exosomes (210) A 1:1 mixture of exosomes derived from MSCs transfected with miRNA126 and exosomes derived from MSCs transfected with miRNA135b (126 + 135b). A 1:1 mixture of miRNA126-transfected MSC-derived exosomes and miRNA210-transfected MSC-derived exosomes (126+210). A 1:1 mixture of miRNA135b-transfected MSC-derived exosomes and miRNA210-transfected MSC-derived exosomes (135b + 210). A 1:1:1 mixture of exosomes derived from miRNA126-transfected MSCs, exosomes derived from miRNA135b-transfected MSCs, and exosomes derived from miRNA210-transfected MSCs (ALL, 126+135b+210)

[0081] (2) Creation of a mouse model of hindlimb ischemia Ten-week-old male BALB / c mice were used. The mice were anesthetized intraperitoneally. After sufficient sedation, the hair on the abdomen and lower limbs of the mice was removed with hair removal cream. The mice were placed on their backs (supine position), with both lower limbs extended and fixed in an abducted position, and the skin was wiped clean with alcohol wipes. An oblique incision was made in the left groin of the mice, the subcutaneous fat was removed, and the femoral artery, vein, and femoral nerve were identified and dissected. The common femoral artery was ligated in two locations and transected. 50 μl of the exosome sample from the example derived from MSCs overexpressing miRNA was administered to two locations, the quadriceps and adductor muscles, visible through the oblique incision (example exosome group). The incised skin was then closed.

[0082] The PBS group (PBS) was treated in the same manner as the exosome group of the example, except that 50 μl of PBS was administered instead of 50 μl of the exosome sample of the example. The native exosome group (EV native) was treated in the same manner as the exosome group of the example, except that 50 μl of the native exosome sample was administered instead of 50 μl of the exosome sample of the example. The control exosome group (EV control) was treated in the same manner as the exosome group of the example, except that 50 μl of the control vector exosome sample was administered instead of 50 μl of the exosome sample of the example. Each group consisted of four individuals.

[0083] (3) Evaluation of angiogenesis (3-1) Macroscopic classification The ischemic hindlimb of each group of model mice was evaluated after femoral artery transection. Evaluation was performed according to a previously published macroscopic classification of mouse hindlimb ischemia (Goto T, Fukuyama N, Aki A, Kanabuchi K, Kimura K, Taira H, Tanaka E, Wakana N, Mori H, Inoue H. Search for appropriate experimental methods to create stable hindlimb ischemia in mice. Tokai J Exp Clin Med. 2006 Sep 20;31(3):128-32.). Specifically, the severity of ischemia was classified into the following four stages: 0: No necrosis 1:Finger tip necrosis 2: Foot necrosis 3: Knee necrosis

[0084] (3-2) Hind limb blood flow Using a laser speckle blood flow imaging system (Moor FLPI-2, Moor Instruments), we monitored hindlimb blood flow in mice before and after treatment on day 0 and on day 7. To compare changes in blood flow, we set a region of interest (ROI) around the ankle joint and analyzed the ratio of flux values ​​between the affected and unaffected sides.

[0085] Figure 6 shows the results of macroscopic classification of limb ischemia severity. The first row shows the PBS group (no exosome administration), the second row shows the results for the native exosome group, the third row shows the control exosome group, and the fourth row and beyond show the results for the exosome group of the example. In Figure 6, four mice (n = 4) in each group are set as 100%, and the percentage (%) of mice that fit into each classification is represented by color. As a result, compared to the PBS group, the native exosome group, and the control exosome group, the example group administered with exosomes derived from MSCs that strongly expressed miRNA showed a reduced level of severity in all cases.

[0086] The ratio of flux values ​​between the affected and healthy sides is shown in Figure 7 (p<0.05). As shown in Figure 7, compared to the PBS group, the control vector group, and the native group, enhanced angiogenesis was confirmed in the example group administered with example exosomes derived from MSCs that strongly expressed miRNA.

[0087] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments. 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.

[0088] This application claims priority based on Japanese Patent Application No. 2024-46258, filed March 22, 2024, the disclosure of which is incorporated herein in its entirety. [Industrial Applicability]

[0089] The angiogenesis promoter of the present invention can promote angiogenesis, and is therefore effective in treating, for example, ischemic diseases.

Claims

1. A group of artificial microvesicles consisting of a plurality of artificial microvesicles, An angiogenesis promoter, characterized in that the group of artificial microvesicles contains at least one microRNA selected from the group consisting of miR-126, miR-135 and miR-210.

2. The artificial microvesicles contain miR-126 and miR-135 as the microRNAs, The angiogenesis promoter according to claim 1, comprising miR-126 and miR-210, or miR-135 and miR-210.

3. The angiogenesis promoter according to claim 1, wherein the group of artificial microvesicles comprises miR-126, miR-135, and miR-210 as the microRNAs.

4. The angiogenesis promoter according to any one of claims 1 to 3, wherein the artificial microvesicles are artificial exosomes.

5. A therapeutic agent for ischemia, comprising the angiogenesis promoter according to any one of claims 1 to 4.

6. A method for promoting angiogenesis, comprising a contacting step of contacting a target cell with the angiogenesis-promoting agent according to claim 1 .

7. The method for promoting angiogenesis according to claim 6 , wherein the contacting step is carried out in vivo, in vitro, or ex vivo.

8. The method for promoting angiogenesis according to claim 6 or 7, wherein the target cells are cells of a human or non-human animal.

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