Composition for ameliorating functional prognosis of cerebral infarction

Abstract

The present invention addresses the problem of providing a novel technique for ameliorating the function prognosis of cerebral infarction. A transcript of a gene encoding miR-451, miR-4739, or miR-3541 or a processed product of the transcript is used as an active ingredient.

Description

Composition for improving functional prognosis after cerebral infarction

[0001] The present invention relates to a composition for improving functional prognosis of cerebral infarction.

[0002] The number of stroke patients is 1.74 million, and annual medical expenses amount to 1,808.5 billion yen (see Non-Patent Document 1). Stroke is also the fourth leading cause of death, the second leading cause of nursing care, and the leading cause of severe nursing care, making it an urgent need to develop new treatments for the chronic stage of cerebral infarction.

[0003] Japanese Patent Application Laid-Open No. 2023-011580 Special Publication No. 2019-513414

[0004] Ministry of Health, Labour and Welfare, 2016 (Heisei 28), "Overview of the National Survey of Living Conditions" Xu, Y., Lai, Y., Cao, L., Li, Y., Chen, G., Chen, L., Weng, H., Chen, T., Wang, L., & Ye, Y. (2020). Human umbilical cord mesenchymal stem cells-derived exosomal microRNA-451a represses epithelial-mesenchymal transition of hepatocellular carcinoma cells by inhibiting ADAM10. RNA Biology, 18, 1408-1423. Liu, J. , Xing, F. , Fu, Q. ,He,B. , Jia, Z. , Du, J. , Li, Y. , Zhang, X. , &Chen, X. (2022). hUC-MSCs exosomal miR-451 alleviated acute lung injury by modulating macrophage M2 polarization via regulating MIF-PI3K-AKT signaling pathway. Environmental Toxicology, 37, 2819-2831. Zhang Y, Bi J, Huang J, Tang Y, Du S, Li P. Exosome: A Review of Its Classification, Isolation Techniques, Storage, Diagnostic and Targeted Therapy Applications. Int J Nanomedicine. 2020;15:6917-6934.

[0005] Cerebral infarction causes neurological deficits, such as motor paralysis (e.g., paralysis of the limbs) and sensory impairment, but rehabilitation can only achieve limited recovery. Therefore, there is a need for the development of therapeutic drugs that can improve the functional prognosis of cerebral infarction. Therefore, an objective of the present invention is to provide a novel technology for improving the functional prognosis of cerebral infarction.

[0006] The present invention, which solves the above-mentioned problems, is as follows: [1] A composition for improving the functional prognosis of cerebral infarction, comprising any one of the following (1) to (8) as an active ingredient: (1) A transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, or a processed product thereof. (2) A pre-miRNA that has a substitution, addition, and / or deletion of 1 to 10 bases relative to a pre-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, and that can form a stem-loop hairpin structure. (3) A pre-miRNA that has 70% or more sequence homology to a pre-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, and that can form a stem-loop hairpin structure. (4) A mature-miRNA having 1 to 5 base substitutions, additions, and / or deletions relative to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. (5) A mature-miRNA that has 80% or more sequence homology to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. (6) A silencing molecule for the MIF gene and / or CCND1 gene. (7) An expression vector encoding one or more types of RNA corresponding to any of (1) to (6) above. (8) Extracellular vesicles derived from placenta-derived adherent cells, extracellular vesicles derived from umbilical cord blood mononuclear cells, or extracellular vesicles derived from mesenchymal stem cells.

[0007] [2] The miRNA described in (2) to (5) above comprises at least a sequence identical to 5'-aaccguu-3' (SEQ ID NO: 5) or 5'-cccuccc-3' (SEQ ID NO: 6) or a sequence having a substitution, addition, and / or deletion of 1 to 2 bases. The composition described in [1].

[0008] [3] The composition according to [1] or [2], comprising a vesicle made of a lipid bilayer membrane encapsulating the RNA molecule according to any one of (1) to (6).

[0009] [4] The composition according to [3], wherein the vesicle comprising the lipid bilayer membrane is selected from extracellular vesicles, liposomes, and lipid nanoparticles.

[0010] [5] The composition according to [4], wherein the vesicle made of a lipid bilayer membrane is an exosome.

[0011] [6] The composition according to any one of [1] to [5], wherein the mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451 is miR-451-5p.

[0012] [7] The composition according to any one of [1] to [6], which is used for reducing cerebral infarction lesions.

[0013] [8] The composition according to any one of [1] to [7], which is used for promoting the expression of S100A10-positive astrocytes and / or suppressing the expression of C3d-positive astrocytes.

[0014] [9] The composition according to any one of [1] to [8], which is used for inhibiting glial scar formation.

[0015]

[10] The composition according to any one of [1] to [9], which is used for suppressing the expression of the MIF gene in neurons and / or the expression of the CCND1 gene in astrocytes.

[0016]

[11] The composition according to any one of [1] to

[10] , which is used for improving functional prognosis in the chronic phase of cerebral infarction.

[0017]

[12] The composition according to any one of [1] to

[11] , which is administered during the acute to subacute phase of cerebral infarction.

[0018]

[13] The composition according to any one of [1] to

[12] , which is used in combination with one or more therapeutic agents selected from t-PA, free radical scavengers, antiplatelet agents, antithrombin agents, anticoagulants, antiplatelet agents, dextran 40 preparations, and concentrated glycerin-fructose preparations.

[0019]

[14] A cell into which an expression vector encoding the RNA molecule of any one of (1) to (5) above has been introduced, and which expresses the RNA molecule.

[0020]

[15] A cell composition comprising cells expressing any one of the RNA molecules (1) to (5), the cells according to

[14] , placenta-derived adherent cells, umbilical cord blood mononuclear cells, or mesenchymal stem cells, for use in improving functional prognosis following cerebral infarction, or as a source of extracellular vesicles for use in improving functional prognosis following cerebral infarction.

[0021]

[16] A composition for improving the functional prognosis of cerebral infarction, comprising as an active ingredient a culture supernatant of the cell composition described in

[15] or extracellular vesicles separated from the culture supernatant.

[0022] Further, examples of the present invention that solve the above problems include the following: [1] A method for improving functional prognosis of cerebral infarction, which comprises administering to a subject a composition containing any one of the above (1) to (8) as an active ingredient.

[0023] [2] The method according to [1], wherein the miRNA described in (2) to (5) above comprises at least a sequence identical to 5'-aaccguu-3' (SEQ ID NO: 5) or 5'-cccuccc-3' (SEQ ID NO: 6) or a sequence having a substitution, addition, and / or deletion of 1 to 2 bases.

[0024] [3] The method according to [1] or [2], comprising administering to a subject a composition comprising a vesicle made of a lipid bilayer membrane encapsulating the RNA molecule according to any one of (1) to (6).

[0025] [4] The method according to [3], wherein the vesicle comprising a lipid bilayer membrane is selected from extracellular vesicles, liposomes, and lipid nanoparticles.

[0026] [5] The method according to [4], wherein the vesicles made of lipid bilayer membranes are exosomes.

[0027] [6] The method according to any one of [1] to [5], wherein the mature-miRNA, which is a processed product of a transcription product of a gene encoding miR-451, is miR-451-5p.

[0028] [7] The method according to any one of [1] to [6], which comprises reducing a cerebral infarction lesion.

[0029] [8] The method according to any one of [1] to [7], comprising enhancing the expression of S100A10-positive astrocytes and / or suppressing the expression of C3d-positive astrocytes.

[0030] [9] The method according to any one of [1] to [8], which comprises inhibiting the formation of glial scars.

[0031]

[10] The method according to any one of [1] to [9], comprising suppressing expression of the MIF gene in neurons and / or suppressing expression of the CCND1 gene in astrocytes.

[0032]

[11] The method according to any one of [1] to

[10] , which is a method for improving functional prognosis in the chronic phase of cerebral infarction.

[0033]

[12] The method according to any one of [1] to

[11] , comprising administering the composition to the subject in the acute to subacute phase of cerebral infarction.

[0034]

[13] The method according to any one of [1] to

[12] , comprising administering to the subject the composition in combination with one or more therapeutic agents selected from t-PA, a free radical scavenger, an antiplatelet agent, an antithrombin agent, an anticoagulant, an antiplatelet agent, a dextran 40 preparation, and a concentrated glycerin-fructose preparation.

[0035]

[14] A cell into which an expression vector encoding the RNA molecule of any one of (1) to (5) above has been introduced, and which expresses the RNA molecule.

[0036]

[15] A method for improving functional prognosis of cerebral infarction, comprising administering to a subject cells expressing any of the RNA molecules of (1) to (5), the cells described in

[14] , placenta-derived adhesive cells, umbilical cord blood mononuclear cells, or mesenchymal stem cells; or A method for improving functional prognosis of cerebral infarction, comprising administering to a subject cells expressing any of the RNA molecules of (1) to (5), the cells described in

[14] , placenta-derived adhesive cells, umbilical cord blood mononuclear cells, or extracellular vesicles secreted from mesenchymal stem cells.

[0037]

[16] A method for improving functional prognosis of cerebral infarction, comprising administering to a subject a composition containing, as an active ingredient, a culture supernatant of cells expressing any of the RNA molecules of (1) to (5), the cells according to

[14] , placenta-derived adherent cells, umbilical cord blood mononuclear cells, or mesenchymal stem cells, or extracellular vesicles isolated from the culture supernatant.

[0038] Further, the present invention for solving the above problems includes the following: [1] Use of a composition containing any one of the above (1) to (8) for the manufacture of an agent for improving the functional prognosis of cerebral infarction.

[0039] [2] The miRNA described in (2) to (5) above includes at least a sequence identical to 5'-aaccguu-3' (SEQ ID NO: 5) or 5'-cccuccc-3' (SEQ ID NO: 6) or a sequence having 1 to 2 base substitutions, additions, and / or deletions. [1] The application described in [1].

[0040] [3] The application according to [1] or [2], wherein the composition comprises a vesicle made of a lipid bilayer membrane encapsulating the RNA molecule according to any one of (1) to (6).

[0041] [4] The application according to [3], wherein the vesicles made of lipid bilayer membranes are selected from extracellular vesicles, liposomes, and lipid nanoparticles.

[0042] [5] The application according to [4], wherein the vesicle made of a lipid bilayer membrane is an exosome.

[0043] [6] The application according to any one of [1] to [5], wherein the mature-miRNA, which is a processed product of a transcription product of a gene encoding miR-451, is miR-451-5p.

[0044] [7] The application according to any one of [1] to [6], wherein the agent for improving functional prognosis of cerebral infarction is an agent for reducing cerebral infarction lesions.

[0045] [8] The application according to any one of [1] to [7], wherein the agent for improving functional prognosis of cerebral infarction is an agent for promoting the expression of S100A10-positive astrocytes and / or an agent for suppressing the expression of C3d-positive astrocytes.

[0046] [9] The application according to any one of [1] to [8], wherein the agent for improving functional prognosis of cerebral infarction is an agent for inhibiting glial scar formation.

[0047]

[10] The application according to any one of [1] to [9], wherein the agent for improving functional prognosis of cerebral infarction is an inhibitor of expression of the MIF gene in neurons and / or an inhibitor of expression of the CCND1 gene in astrocytes.

[0048]

[11] The application according to any one of [1] to

[10] , wherein the agent for improving functional prognosis of cerebral infarction is an agent for improving functional prognosis in the chronic phase of cerebral infarction.

[0049]

[12] The application according to any one of [1] to

[11] , wherein the agent for improving functional prognosis of cerebral infarction is administered during the acute to subacute phase of cerebral infarction.

[0050]

[13] The composition according to any one of [1] to

[12] , wherein the agent for improving functional prognosis of cerebral infarction is used in combination with one or more therapeutic agents selected from t-PA, free radical scavengers, antiplatelet agents, antithrombin agents, anticoagulants, antiplatelet agents, dextran 40 preparations, and concentrated glycerin-fructose preparations.

[0051]

[14] A cell into which an expression vector encoding the RNA molecule of any one of (1) to (5) above has been introduced, and which expresses the RNA molecule.

[0052]

[15] An application of cells or extracellular vesicles for the production of an agent for improving functional prognosis of cerebral infarction, wherein the cells are cells expressing any of the RNA molecules (1) to (5), the cells according to

[14] , placenta-derived adherent cells, umbilical cord blood mononuclear cells, or mesenchymal stem cells, and the extracellular vesicles are secreted from the cells expressing any of the RNA molecules (1) to (5), the cells according to

[14] , placenta-derived adherent cells, umbilical cord blood mononuclear cells, or mesenchymal stem cells.

[0053]

[16] Application of a culture supernatant or extracellular vesicles for the production of an agent for improving the functional prognosis of cerebral infarction, wherein the culture supernatant is a culture supernatant of the cell composition described in

[15] , and the extracellular vesicles are extracellular vesicles separated from the culture supernatant.

[0054] Further, the present invention for solving the above problems includes the following: [1] A composition used for improving functional prognosis of cerebral infarction, comprising any one of the above (1) to (8) as an active ingredient.

[0055] [2] The miRNA described in (2) to (5) above comprises at least a sequence identical to 5'-aaccguu-3' (SEQ ID NO: 5) or 5'-cccuccc-3' (SEQ ID NO: 6) or a sequence having a substitution, addition, and / or deletion of 1 to 2 bases. The composition described in [1].

[0056] [3] The composition according to [1] or [2], comprising a vesicle made of a lipid bilayer membrane encapsulating the RNA molecule according to any one of (1) to (6).

[0057] [4] The composition according to [3], wherein the vesicle comprising the lipid bilayer membrane is selected from extracellular vesicles, liposomes, and lipid nanoparticles.

[0058] [5] The composition according to [4], wherein the vesicle made of a lipid bilayer membrane is an exosome.

[0059] [6] The composition according to any one of [1] to [5], wherein the mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451 is miR-451-5p.

[0060] [7] The composition according to any one of [1] to [6], which is used for reducing cerebral infarction lesions.

[0061] [8] The composition according to any one of [1] to [7], which is used for promoting the expression of S100A10-positive astrocytes and / or suppressing the expression of C3d-positive astrocytes.

[0062] [9] The composition according to any one of [1] to [8], which is used for inhibiting glial scar formation.

[0063]

[10] The composition according to any one of [1] to [9], which is used for suppressing the expression of the MIF gene in neurons and / or the expression of the CCND1 gene in astrocytes.

[0064]

[11] The composition according to any one of [1] to

[10] , which is used for improving functional prognosis in the chronic phase of cerebral infarction.

[0065]

[12] The composition according to any one of [1] to

[11] , which is administered during the acute to subacute phase of cerebral infarction.

[0066]

[13] The composition according to any one of [1] to

[12] , which is used in combination with one or more therapeutic agents selected from t-PA, free radical scavengers, antiplatelet agents, antithrombin agents, anticoagulants, antiplatelet agents, dextran 40 preparations, and concentrated glycerin-fructose preparations.

[0067]

[14] A cell into which an expression vector encoding the RNA molecule of any one of (1) to (5) above has been introduced, and which expresses the RNA molecule.

[0068]

[15] A cell or extracellular vesicle used for improving functional prognosis of cerebral infarction, wherein the cell is a cell expressing any one of the RNA molecules (1) to (5), the cell according to

[14] , a placenta-derived adherent cell, a cord blood mononuclear cell, or a mesenchymal stem cell, and the extracellular vesicle is secreted from the cell expressing any one of the RNA molecules (1) to (5), the cell according to

[14] , a placenta-derived adherent cell, a cord blood mononuclear cell, or a mesenchymal stem cell.

[0069]

[16] A culture supernatant or extracellular vesicles used for improving functional prognosis of cerebral infarction, wherein the culture supernatant is a culture supernatant of the cell composition described in

[15] , and the extracellular vesicles are extracellular vesicles separated from the culture supernatant.

[0070] According to the present invention, the effect of improving the functional prognosis of cerebral infarction can be obtained.

[0071] Figure 1 is a graph showing the change in survival rate of rats in the vehicle group and the EVs-administered group. Figure 2A is a graph showing the change in neurological signs of rats in the vehicle group and each EVs-administered group. Figure 2B is a graph showing the change in motor function of rats in the vehicle group and each EVs-administered group. Figure 3 shows the cerebral infarction volume in rats in the vehicle group and each EVs-administered group 28 days after MCAO. Figure 4 shows the densities of pNFH, npNFH, and MAP2 in rats in the vehicle group and each EVs-administered group 28 days after MCAO. Figure 5 shows the GFAP densities in rats in the vehicle group and each EVs-administered group 28 days after MCAO. Figure 6 shows the densities of C3d and GFAP / C3d in rats in the vehicle group and each EVs-administered group 28 days after MCAO. Figure 7 shows the densities of S100A10 and GFAP / S100A10 in rats in the vehicle group and the EV-treated groups 28 days after MCAO. Figure 8 shows changes in the profile of activated astrocytes in the peri-infarct area (PIA) in rats before surgery, the vehicle group, and the EV-treated groups 28 days after MCAO. This graph shows the particle size distribution of pre-EVs, 3D-EVs, and 28D-EVs. This graph shows the intensity of the ischemic area in sham-operated rats and rats treated with pre-EVs, 3D-EVs, and 28D-EVs. This image shows immunofluorescence images of EVs co-expressed with MAP2, pNFH, npNFH, and GFAP in the peri-infarct area. This image shows the densities of GFAP and C3d in non-OGD astrocytes, OGD-treated astrocytes, and astrocytes treated with each EV. The density of GFAP and S100A10 in non-OGD astrocytes, OGD astrocytes, and astrocytes treated with each EV is shown. The amount of GFAP, C3d, and S100A10 protein in non-OGD astrocytes, OGD astrocytes, and astrocytes treated with each EV is shown. This figure shows miR-451-5p contained in 28D-EVs. This is a volcano plot showing miRNAs differentially expressed in 28D-EVs compared to pre-EVs. Points corresponding to mir-3541 and mir-451 are shown. The 47 diseases or functions most important to miR-451-5p identified using pathway analysis software (IPA) are shown. This suggests that miR-451-5p may be of myeloid origin.Figure 1 shows the ratio of tetraspanins in each EV bound to CD9, CD63, and CD81. Representative Western blots showing protein levels of pNFH, npNFH, and MAP2 in OGD neurons treated with 28D-EV, 28D-mimic-EV, and 28D-inhibitor-EV. Representative Western blots showing protein levels of GFAP, C3d, and S100A10 in OGD astrocytes treated with 28D-EV, 28D-mimic-EV, and 28D-inhibitor-EV. Representative Western blots showing protein levels of pNFH, npNFH, and MAP2 in OGD neurons, 28D-EV, 293T-EV, and miR-451-5p-rich-293T-EVs. Representative Western blots showing the protein levels of GFAP, C3d, and S100A10 in OGD astrocytes, 28D-EVs, 293T-EVs, and OGD astrocytes treated with miR-451-5p-rich-293T-EVs. Representative Western blots showing the protein levels of MIF, BCL2, and CCND1 in neurons and astrocytes treated with 293T-EVs and miR-451-5p-rich-293T-EVs. Representative Western blots showing the protein levels of MIF, BCL2, and CCND1 in neurons and astrocytes treated with Pre-EVs and 28D-EVs. Figure 26 shows representative images of hematoxylin-eosin-stained coronal brain sections from rats administered 293T-EV and miR-451-5p-rich-293T-EVs 28 days after MCAO, along with a graph showing the volume of cerebral infarction. Figure 26A shows graphs showing the progression of neurological signs in rats administered 293T-EVs and miR-451-rich-293T-EVs. Figure 26B shows graphs showing the progression of motor function in rats administered 293T-EVs and miR-451-rich-293T-EVs.

[0072] The present invention will be described in detail below. In the embodiments of the present invention, A (numerical value) to B (numerical value) means A or more and B or less. The preferred and more preferred embodiments exemplified below can be used in appropriate combinations, regardless of expressions such as "for example," "preferred," and "more preferred." Numerical ranges are merely examples, and ranges obtained by appropriately combining the upper and lower limits of each range and the numerical values ​​of the examples can also be used (for example, when A to B or C to D is stated, the combinations A to D or C to B can be used). Furthermore, terms such as "contain" or "comprise" may be interpreted as "essentially consisting of" or "consisting only of."

[0073] 1. Composition Section 1 details the composition of the present invention, which contains any of the above (1) to (8).

[0074] 1.1 Active Ingredients of the Present Invention Section 1.1 describes each of (1) to (8) in detail. In one embodiment, the composition of the present invention contains any of the ingredients (1) to (8) as an active pharmaceutical ingredient.

[0075] (1) Transcription products of genes encoding miR-451, miR-4739, or miR-3541, or their processing products. Typical miRNAs are biosynthesized through a series of processes. The primary transcription product of a gene encoding a miRNA is called a primary miRNA transcript (pri-miRNA), which generally has a stem-loop hairpin structure. The pri-miRNA is cleaved by the microprocessor complex and converted to a hairpin shape by the RNase III enzyme Drosha, producing precursor miRNA (pre-miRNA), an intermediate precursor of approximately 70 bases. The pre-miRNA is then transported from the nucleus to the cytoplasm. In the cytoplasm, it is further cleaved by another RNase III enzyme, Dicer, to produce double-stranded mature miRNA. Generally, the double-stranded miRNAs expressed from the 5'-end of the precursor are designated by "-5p" and those expressed from the 3'-end by "-3p," and are thus designated as "hsa-miR-21-5p" and "hsa-miR-21-3p." Publicly known miRNAs are, in principle, registered in miRBase (http: / / www.mirbase.org / ).

[0076] Only one strand of the mature-miRNA may exert the desired effect, both strands may exert the desired effect, or the desired effect may be exerted in a double-stranded state.Furthermore, the desired effect may be exerted in the form of pri-miRNA or pre-miRNA.

[0077] The nucleic acid contained in the composition of the present invention may be a transcription product or a processing product of a gene encoding one or two miRNAs selected from miR-451 and miR-4739 or miR-3541.

[0078] Specifically, the active ingredient in the present invention may be pri-miR-451, pre-miR-451, double-stranded mature-miR-451 or single-stranded mature-miR-451, pri-miR-4739, pre-miR-4739, double-stranded mature-miR-4739 or single-stranded mature-miR-4739, or pri-miR-3541, pre-miR-3541, double-stranded mature-miR-3541 or single-stranded mature-miR-3541.

[0079] miR-451 is a miRNA that is highly conserved between species. The gene encoding miR-451 is found not only in mammals such as humans, mice, and rats, but also in fish such as zebrafish, birds such as chickens, and amphibians such as Xenopus frogs. The sequence of the processed product of the transcription product of the gene encoding miR-451 used in one embodiment of the present invention is as follows. Note that the following sequence is common to both humans and rats.

[0080]

[0081] When miR-451-5p is used as the composition of the present invention, it may be in the form of a single-stranded mature-miRNA, or may be in the form of a double-stranded mature-miRNA consisting of a combination of miR-451-5p and a perfect or imperfect complementary strand.

[0082] The animal species from which the transcription product of the gene encoding miR-451 or its processing product used in the present invention is derived is not particularly limited, and may be human, mouse, or rat.

[0083] miR-4739 is encoded by the human MIR4739 gene. The sequence of the processed product of the transcription product of the gene encoding miR-4739, which is used in one embodiment of the present invention, is as follows:

[0084]

[0085] When miR-4739-5p is used as the composition of the present invention, it may be in the form of a single-stranded mature-miRNA, or may be in the form of a double-stranded mature-miRNA consisting of a combination of miR-4739-5p and a perfect or imperfect complementary strand.

[0086] miR-3541 is encoded by the Mir3541 gene in rats. The sequences of the pre-miRNA and mature-miRNA of miR-3541 are identical to those of miR-4739 shown in Table 2.

[0087] Because their sequences are identical, miR-4739 and miR-3541 may be collectively referred to herein as "miR-4739 / 3541."

[0088] When miR-3541-5p is used as the composition of the present invention, it may be in the form of a single-stranded mature-miRNA, or may be in the form of a double-stranded mature-miRNA consisting of a combination of miR-3541-5p and a perfect or imperfect complementary strand.

[0089] The miRNA described herein as an active ingredient may be chemically modified as known in the art for the purpose of improving RNA stability, specificity, etc. Chemical modifications that can be used in the present invention include, for example, LNA (Locked Nucleic Acid), BNA (Bridged Nucleic Acid), ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids), 2'-OMe modification, phosphorothioate (S-modification), S-TuD (Synthetic Tough Decoy), morpholino modification, peptide addition, sugar chain addition, aptamer addition, addition of hydrophobic molecules such as cholesterol, addition of polymers such as PEG, or addition of unmodified DNA. These chemical modifications can be carried out by known means in the art.

[0090] (2) A pre-miRNA that has a substitution, addition, and / or deletion of 1 to 10 bases relative to a pre-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541, and that can form a stem-loop hairpin structure. The present invention may be embodied as including a modified miRNA defined in (2). In this embodiment, the explanations for miR-451, miR-4739 / 3541, pre-miRNA, and chemical modifications are the same as those in (1) above.

[0091] The modified miRNA in this embodiment has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base substitutions, additions, and / or deletions relative to the wild-type pre-miRNA (SEQ ID NO: 2 or 4) described above, and may have a stem-loop hairpin structure. For ease of explanation, "substitutions, additions, and / or deletions" may be referred to as "substitutions, etc." in this specification.

[0092] As shown below, pre-miR-451 forms a hairpin structure by forming 31 pairs of hydrogen bonds in the stem portion. The substitution of 1 to 10 bases introduced into pre-miR-451 is not limited as long as this hairpin structure is maintained. This is because it can be understood that at least by forming a hairpin structure, it can be processed by Dicer, an RNase III enzyme.

[0093] More specifically, of the 31 pairs of hydrogen bonds, substitution of bases or the like may be permitted as long as preferably 20 or more, more preferably 22 or more, even more preferably 24 or more, even more preferably 26 or more, even more preferably 28 or more, and even more preferably 30 or more hydrogen bonds are maintained.

[0094] Furthermore, it is preferable that the modified form of pre-miR-451 into which substitutions of 1 to 10 bases or the like have been introduced is in a form that exhibits the function of silencing the expression of the MIF gene and / or CCND1 gene when processed into mature-miRNA.

[0095] The following sequence contained in miR-451-5p is complementary to the 3'UTR in the MIF gene transcript: 5'-aaccguu-3' (SEQ ID NO: 5) Analysis using miRNA target sequence prediction tools (miRDB, TargetScan, miRTarBase, etc.) strongly predicted that miR-451 interacts with the 3'UTR in the MIF gene transcript at the sequence specified by SEQ ID NO: 5 and inhibits its expression. Therefore, it is preferable that a variant of pre-miR-451 into which substitutions of 1 to 10 bases or the like have been introduced contains the same sequence as the sequence specified by SEQ ID NO: 5 or a sequence having substitutions, additions, and / or deletions of 1 or 2 bases.

[0096] As shown below, pre-miR-4739 / 3541 forms a hairpin structure by forming 37 pairs of hydrogen bonds in the stem portion. The substitution of 1 to 10 bases to be introduced into pre-miR-4739 / 3541 is not limited as long as this hairpin structure is maintained. This is because it can be understood that at least by forming a hairpin structure, it can be processed by Dicer, an RNase III enzyme.

[0097] More specifically, of the 37 pairs of hydrogen bonds, substitution of bases, etc. may be permitted as long as 26 or more, more preferably 28 or more, even more preferably 30 or more, even more preferably 32 or more, even more preferably 34 or more, and even more preferably 36 or more hydrogen bonds are maintained.

[0098] Furthermore, it is preferable that the modified form of pre-miR-4739 / 3541 into which substitutions of 1 to 10 bases or the like have been introduced is in a form that exhibits the function of silencing the expression of the MIF gene and / or CCND1 gene when processed into mature-miRNA.

[0099] The following sequence contained in miR-4739 / 3541-5p is complementary to the 3'UTR in the transcript of the CCND1 gene: 5'-cccuccc-3' (SEQ ID NO: 6) Analysis using miRNA target sequence prediction tools (miRDB, TargetScan, miRTarBase, etc.) strongly predicted that miR-4739 / 3541 interacts with the 3'UTR in the transcript of the CCND1 gene in the sequence specified by SEQ ID NO: 6 and inhibits its expression. Therefore, it is preferable that variants of pre-miR-4739 / 3541 into which substitutions of 1 to 10 bases or the like have been introduced contain a sequence identical to the sequence specified by SEQ ID NO: 6 or a sequence having substitutions, additions, and / or deletions of 1 or 2 bases.

[0100] (3) A pre-miRNA that has 70% or more sequence homology to a pre-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541, and that can form a stem-loop hairpin structure. The present invention may be embodied as including a modified miRNA defined in (3). In this embodiment, the explanations for miR-451, miR-4739 / 3541, pre-miRNA, and chemical modifications are the same as those in (1) above.

[0101] The modified miRNA in this embodiment has sequence homology of preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and even more preferably 99% or more to the wild-type pre-miRNA (SEQ ID NO: 2 or 4) described above, and can have a stem-loop hairpin structure.

[0102] For the same reason as explained in (2) above, the form of the modified miRNA in this embodiment is not limited as long as it can maintain a hairpin structure. The explanation in (2) above also applies to the number of hydrogen bonds that are preferably maintained in the stem portion of the modified miRNA in this embodiment.

[0103] Furthermore, the modified miRNA in this embodiment is preferably in a form that exhibits the function of silencing the expression of the MIF gene and / or CCND1 gene when processed into mature-miRNA.

[0104] As explained in (2) above, it is preferable that the modified miRNA in this embodiment also contains a sequence identical to the sequence specified by SEQ ID NO: 5 or SEQ ID NO: 6, or a sequence having a substitution, addition, and / or deletion of one or two bases.

[0105] (4) Mature-miRNA having 1 to 5 base substitutions, additions, and / or deletions relative to mature-miRNA, which is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. The present invention may be embodied as including a modified miRNA defined in (4). In this embodiment, the explanations for miR-451, miR-4739 / 3541, pre-miRNA, and chemical modifications are the same as those in (1) above.

[0106] The modified miRNA in this embodiment has 1, 2, 3, 4 or 5 base substitutions, additions, and / or deletions relative to the wild-type mature-miRNA (SEQ ID NO: 1 or 3) described above.

[0107] The modified miRNA in this embodiment is preferably in a form that exhibits the function of silencing the expression of the MIF gene and / or CCND1 gene.

[0108] As explained in (2) above, it is preferable that the modified miRNA in this embodiment also contains a sequence identical to the sequence specified by SEQ ID NO: 5 or SEQ ID NO: 6, or a sequence having a substitution, addition, and / or deletion of one or two bases.

[0109] (5) A mature-miRNA that has 80% or more sequence homology to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. The present invention may be embodied in an embodiment that includes a modified miRNA defined in (5). In this embodiment, the explanations for miR-451, miR-4739 / 3541, pre-miRNA, and chemical modifications are the same as those in (1) above.

[0110] The variant of miRNA in this embodiment has a sequence homology of preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and even more preferably 99% or more to the wild-type mature-miRNA (SEQ ID NO: 1 or 3) described above.

[0111] Furthermore, the modified miRNA in this embodiment is preferably in a form that exhibits the function of silencing the expression of the MIF gene and / or CCND1 gene.

[0112] As explained in (2) above, it is preferable that the modified miRNA in this embodiment also contains a sequence identical to the sequence specified by SEQ ID NO: 5 or SEQ ID NO: 6, or a sequence having a substitution, addition, and / or deletion of one or two bases.

[0113] (6) Silencing molecule for MIF gene and / or CCND1 gene The present invention may be in an embodiment including a silencing molecule defined in (6). The silencing molecule may be siRNA (small interfering RNA), shRNA (short hairpin RNA), antisense nucleic acid, or miRNA against transcripts of the MIF gene and / or CCND1 gene.

[0114] siRNA is a 21-25 bp dsRNA with a dinucleotide 3' overhang, which is formed by cleaving a long dsRNA with Dicer in the RNA interference pathway. RNAi is induced by introducing synthetic siRNA into mammalian cells. siRNAs targeting transcripts of the MIF gene and / or CCND1 gene can be easily designed using known software, and can be synthesized by conventional methods.

[0115] shRNAs are hairpin-shaped RNA molecules used for gene silencing by RNA interference. The shRNA is cleaved by Dicer, and either strand of the double-stranded molecule is loaded into RISC, where it degrades the complementary mRNA target. shRNAs targeting transcripts of the MIF gene and / or CCND1 gene can be easily designed using known software. They can also be synthesized by conventional methods.

[0116] Antisense nucleic acids are nucleic acids that form hybrids with target RNA (such as mRNA). DNA or chemically modified versions thereof can also be used as antisense nucleic acids. In this case, RNase H recognizes the hybrid (DNA-RNA) between the target RNA and the antisense nucleic acid, degrading the target RNA and inhibiting its translation. Various chemical modifications (such as phosphorothioation) are performed to confer resistance to nucleases. RNA can also be used as antisense nucleic acids. In this case, translation is inhibited by forming a hybrid (RNA-RNA) between the nucleotide-resistant antisense RNA and the target RNA.

[0117] Antisense oligos against transcripts of the MIF gene and / or CCND1 gene can be easily designed using known software, etc. They can also be synthesized by conventional methods.

[0118] (7) An expression vector encoding one or more RNAs corresponding to any one of (1) to (6) above. The present invention may be in an embodiment including the expression vector defined in (7). Specifically, the embodiment may include, as an active ingredient, one or more RNA molecules selected from the miRNAs defined in (1) to (5) above and the silencing molecules defined in (6) above.

[0119] The expression vector is not particularly limited as long as it is a vector capable of expressing the above-mentioned RNA molecule. Typical examples include those having a structure in which an expression cassette for the above-mentioned RNA is inserted downstream of a promoter.

[0120] The vector used to prepare the expression vector can be any known vector, including commercially available products, but is preferably a viral vector when introduced into a mammal. Specific examples of viral vectors include murine leukemia retroviral vectors (Microbiology and Immunology (1992) 158, 1-23), adeno-associated viral vectors (Muzyczka N. Curr. Top. Microbiol. Immunol. (1992) 97-129), and adenoviral vectors (Rosenfeld M, et al. Science (1991) 252, 431-434).

[0121] (8) Extracellular vesicles derived from placenta-derived adhesive cells, extracellular vesicles derived from umbilical cord blood mononuclear cells, or extracellular vesicles derived from mesenchymal stem cells Extracellular vesicles secreted by placenta-derived adhesive cells, umbilical cord blood mononuclear cells, and mesenchymal stem cells are known to contain miR-451 (Patent Documents 1 and 2, Non-Patent Documents 2 and 3). Therefore, the present invention may be embodied as comprising extracellular vesicles derived from placenta-derived adhesive cells, extracellular vesicles derived from umbilical cord blood mononuclear cells, or extracellular vesicles derived from mesenchymal stem cells, as defined in (8).

[0122] Here, "extracellular vesicles" refer to vesicles secreted from any cell. In this specification, "extracellular vesicles" may also be abbreviated as "EVs." The term "extracellular vesicles" encompasses, for example, membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles, exosome-like vesicles, exosomes, ectosome-like vesicles, ectosomes, exovesicles, apoptotic bodies, and the like.

[0123] Examples of extracellular vesicles according to this embodiment include microvesicles with a particle diameter of 50 nm to 1 μm, exosomes with a particle diameter of 30 to 150 nm, and apoptotic bodies with a particle diameter of 50 nm to 5 μm. A preferred example of a particle diameter of extracellular vesicles is 79.5 nm to 369.5 nm. Extracellular vesicles can also be distinguished based on their intracellular origin, density in sucrose, shape, sedimentation rate, lipid composition, marker proteins, and secretion pattern. Furthermore, extracellular vesicles contain any of phosphatidylserine, phosphatidylcholine, cholesterol, sphingomyelin, and ceramide as their constituent lipids.

[0124] In one embodiment, the extracellular vesicles defined in (8) are exosomes.

[0125] The extracellular vesicles defined in (8) can be isolated from the culture supernatant of placenta-derived adherent cells, umbilical cord blood mononuclear cells, and mesenchymal stem cells. The method for isolating extracellular vesicles from the culture supernatant is not particularly limited. Examples include ultracentrifugation, poly-ethylene glycol (PEG)-based precipitation, ultrafiltration, immunoaffinity capture, microfluidics, and size exclusion chromatography (SEC).

[0126] The method described in Patent Document 1 can be used to obtain extracellular vesicles derived from placenta-derived adherent cells. The method described in Patent Document 2 can be used to obtain extracellular vesicles derived from umbilical cord blood mononuclear cells.

[0127] Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. The present invention may use extracellular vesicles secreted by mesenchymal stem cells derived from any of these tissues.

[0128] As shown in the Examples below, it has been suggested that extracellular vesicles encapsulating miR-451, which are effective in improving functional prognosis after cerebral infarction, originate from bone marrow. Therefore, in one embodiment, extracellular vesicles secreted by bone marrow-derived mesenchymal stem cells are used.

[0129] In one embodiment, extracellular vesicles derived from umbilical cord mesenchymal stem cells are used. As a method for obtaining extracellular vesicles derived from umbilical cord mesenchymal stem cells, the methods described in Non-Patent Documents 2 and 3 can be used.

[0130] 1.2 Uses Section 1.2 describes uses of the compositions of the present invention.

[0131] In one embodiment, the composition of the present invention is used to improve functional prognosis of cerebral infarction. "Improving functional prognosis of cerebral infarction" refers to improving neurological symptoms caused by cerebral infarction (stroke), such as motor paralysis, sensory disturbance, ataxia, higher brain function, and aphasia, and includes restoring lost motor function or sensory disturbance, as well as alleviating, mitigating, improving, relieving, or curing the sensory disturbance.

[0132] Prognosis refers to the predicted future course and outcome of an illness or disability, and involves making a forecast based on all information such as the state of the illness, patient factors, functional impairment, treatment progress, and level of activities of daily living (ADL), and involves deriving outcomes, survival rates, length of hospital stay, rehabilitation approaches, etc.

[0133] The pathological process caused by vascular disorders after cerebral infarction (after cerebral artery occlusion) is divided into acute, subacute, and chronic phases. In this specification, the acute phase refers to the period from the onset of symptoms to within 7 days, the subacute phase refers to the period from 7 days to 27 days, and the chronic phase refers to the period from 28 days onwards.

[0134] In one embodiment, the composition of the present invention is administered from the acute phase to the subacute phase, which can suppress the formation of glial scars that can form from the subacute phase to the chronic phase.

[0135] As described above, the composition of the present invention can inhibit the formation of glial scars, which can form from the subacute phase to the chronic phase. Thus, in one embodiment, the composition of the present invention is used to inhibit glial scar formation.

[0136] Inhibition of glial scar formation, which can occur from the subacute to chronic phases, leads to improved effects on improving functional prognosis in the chronic phase. Thus, in one embodiment, the composition of the present invention can be used to improve functional prognosis in the chronic phase of cerebral infarction.

[0137] In one embodiment, the composition of the present invention is used to reduce cerebral infarction.

[0138] There are at least two types of GFAP-positive activated astrocytes that form scars after cerebral infarction: astrocytes that provide neuroprotection or promote the regeneration of neural circuits (S100A10-positive astrocytes), and astrocytes that have neuropathic effects (C3d-positive astrocytes). In one embodiment, the composition of the present invention is used to promote the expression of S100A10-positive astrocytes. In another embodiment, the composition of the present invention is used to suppress the expression of C3d-positive astrocytes.

[0139] In one embodiment, the composition of the present invention is used to suppress the expression of the MIF gene in neurons. In one embodiment, the composition of the present invention is used to suppress the expression of the CCND1 gene in astrocytes. In one embodiment, the composition of the present invention is used to regenerate and / or protect neurons after cerebral infarction. In one embodiment, the composition of the present invention is used to promote the extension of axons and dendrites of neurons after cerebral infarction. In one embodiment, the composition of the present invention is used to suppress cell death after cerebral infarction.

[0140] Examples of therapeutic agents for cerebral infarction include t-PA (tissue plasminogen activator), free radical scavengers, antiplatelet agents, antithrombin agents, anticoagulants, antiplatelet agents, dextran 40 preparations, and concentrated glycerin / fructose preparations. These therapeutic agents alone are insufficient in improving functional prognosis. A high therapeutic effect can be achieved by using these therapeutic agents in combination with the composition of the present invention. Therefore, in one embodiment of the present invention, the composition of the present invention is used in combination with one or more therapeutic agents selected from t-PA, free radical scavengers, antiplatelet agents, antithrombin agents, anticoagulants, antiplatelet agents, dextran 40 preparations, and concentrated glycerin / fructose preparations.

[0141] The "subject" to which the composition of the present invention is applied is a mammal (for example, a human, a mouse, a rat, a hamster, a rabbit, a cat, a dog, a cow, a sheep, a monkey, etc.), preferably a human.

[0142] 1.3 Vesicles Made of Lipid Bilayer Membrane The transcription products, processing products, or modified forms of genes encoding miRNAs described in Section 1.1 (1) to (5), or the silencing molecules described in (6) that are composed of RNA (e.g., siRNA, shRNA, antisense RNA), can be encapsulated in vesicles made of lipid bilayer membranes. By encapsulating them in vesicles made of lipid bilayer membranes, the active ingredient, RNA, can be protected from degradation by various nucleases, resulting in high efficacy.

[0143] Suitable examples of vesicles made of lipid bilayer membranes include extracellular vesicles, liposomes, and lipid nanoparticles.

[0144] The extracellular vesicles encapsulating the RNA molecules that are the active ingredients of the present invention include exosomes. Methods for obtaining exosomes loaded with desired RNA can be broadly classified into two types: indirect methods and direct methods.

[0145] The indirect method involves producing exosomes encapsulating the desired miRNA from cells carrying endogenous or exogenous genes encoding the miRNA. The miRNA expressed from these genes is naturally incorporated into exosomes and secreted extracellularly. By introducing an expression vector expressing an exogenous gene with a strong promoter upstream into cells and transiently or stably overexpressing the miRNA, exosomes enriched in the miRNA can be obtained.

[0146] The direct method is a method in which RNA is directly introduced into exosomes obtained from cultured cells or the like by chemical or physical means such as lipofection, electroporation, sonication, or extrusion.

[0147] Liposomes can be mentioned as vesicles encapsulating the RNA molecules, which are the active ingredients of the present invention. Liposomes are spherical vesicles having at least one lipid bilayer. Liposomes can be prepared by disrupting biological membranes (by methods such as sonication). Liposomes may contain phospholipids, particularly phosphatidylcholine, but may also contain other lipids such as egg yolk phosphatidylethanolamine. Major types of liposomes include multilamellar vesicles (MLVs containing several lamellar phase lipid bilayers), small unilamellar vesicles (SUVs containing one lipid bilayer), large unilamellar vesicles (LUVs), cochleate vesicles, and multivesicular liposomes (one vesicle containing one or more smaller vesicles).

[0148] RNA can be incorporated into liposomes by conventional methods. For example, liposomes encapsulating RNA molecules can be produced by incubating lipids and RNA at a certain ratio. RNA can also be introduced into liposomes by means of electroporation, extrusion, or the like.

[0149] Lipid nanoparticles (LNPs) can be mentioned as vesicles encapsulating RNA molecules, which are the active ingredients of the present invention. LNPs are nanoparticles containing lipids. Lipid nanoparticles may be spherical. The average diameter of LNPs is about 10 to 1000 nm. Lipid nanoparticles may contain a solid lipid core matrix that may be stabilized by an emulsifier.

[0150] RNA-containing LNPs can be prepared by conventional methods. For example, lipids in ethanol and RNA in a low pH buffer are rapidly mixed in a microfluidic mixer. The ionized lipids become protonated and begin to bind to and encapsulate the RNA. The pH is gradually increased to 7.4, and the ethanol is removed to form LNPs encapsulating the RNA.

[0151] 1.4 Dosage Form, Dosage and Administration The composition of the present invention may be formulated into any dosage form, including, for example, tablets, capsules, powders, granules, liquids, suspensions, emulsions, inhalants, and injections.

[0152] The composition of the present invention may contain, in addition to any of the above-mentioned active ingredients (1) to (8), a pharmaceutically acceptable carrier and / or additive. Pharmaceutically acceptable carriers include lactose, mannitol, corn starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carmellose calcium, carmellose sodium, sterilized water, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, etc. Additives include disintegrants, stabilizers, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, lubricants, etc.

[0153] The compositions of the present invention may be administered to a subject by any method, including systemic or local administration, and may be administered orally or parenterally (e.g., intracerebral, intraarterial, intramedullary, subcutaneous, intradermal, intravenous, intramuscular, etc.).

[0154] The composition of the present invention is administered to a subject in an amount capable of exerting the desired effect (referred to herein as an "effective amount"). The dosage is determined appropriately depending on the age, weight, health condition, etc. of the subject. For example, when administered to a human, the amount of the active ingredient may be 0.00001 g / kg to 10 g / kg per day. Here, the "amount of active ingredient" refers to the amount of the nucleic acid component described in (1) to (7) above, the amount of the structure encapsulating the nucleic acid (extracellular vesicles, vesicles composed of lipid bilayer membranes such as liposomes and lipid nanoparticles), or the amount of the extracellular vesicles described in (8) above.

[0155] The ameliorating composition may be administered once a day, or in divided doses (e.g., 2, 3, or 4 times), or may be administered continuously by infusion or the like. The ameliorating composition may be administered once, daily, or at regular intervals, for example, 1 to several days (e.g., 1, 2, 3, 4, 5, or 6 days), 1 to several weeks (e.g., 1, 2, 3, 4, 5, or 6 weeks), or 1 to several months (e.g., 1, 2, 3, 4, 5, or 6 months). The administration period may be acute, subacute, or chronic, and is not particularly limited, and may be 1 to several days (e.g., 1, 2, 3, 4, 5, or 6 days), 1 to several weeks (e.g., 1, 2, 3, 4, 5, or 6 weeks), 1 to several months (e.g., 1, 2, 3, 4, 5, or 6 months), or longer.

[0156] 1.5 Cells The present invention also relates to cells that have been introduced with an expression vector encoding any one of the RNA molecules (1) to (5) above and that express the RNA molecule. The cells of the present invention secrete exosomes encapsulating miRNA or modified miRNAs derived from an exogenous gene encoding any one of the RNA molecules (1) to (5).

[0157] The structure of the expression vector is not limited as long as it expresses any one of the RNA molecules (1) to (5) when introduced into a cell. The type of promoter provided upstream of the sequence encoding the RNA molecule in the expression vector is also not limited. The promoter is preferably one that has transcription activity for downstream genes in mammalian cells. Examples of promoters that can be used include the EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter. Of these, the EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, and SRα promoter are preferred, with the CAG promoter being more preferred. Furthermore, the expression cassette may contain, in addition to the basal promoter sequence, an enhancer sequence (for example, a CMV immediate early enhancer) for enhancing expression.

[0158] There is no limitation on the cells into which an expression vector encoding any of the above-mentioned RNA molecules (1) to (5) is introduced. Cells obtained from a subject to which the composition of the present invention is to be administered may be used, or an established cell line may be used.

[0159] The cells of the present invention may be those that transiently express any of the RNA molecules (1) to (5) above, or may be constructed so as to stably express the RNA molecule by integrating an expression vector into a chromosome.

[0160] 1.6 Cell Composition The cell composition of the present invention is a composition containing cells that express any of the above-mentioned RNA molecules (1) to (5). Examples of the cells include cells that endogenously express any of the above-mentioned RNA molecules (1) to (5), such as placenta-derived adherent cells, umbilical cord blood mononuclear cells, or mesenchymal stem cells, and cells that express the exogenous RNA molecules described in Section 1.5.

[0161] The cell composition of the present invention can be used as a cell preparation itself, or can be used as a source of exosomes encapsulating any of the RNA molecules (1) to (5) described above.

[0162] When the cell composition of the present invention is administered as a cell preparation, exosomes encapsulating any of the RNA molecules (1) to (5) described above are secreted in the body of the subject. These exosomes exert an effect of improving the functional prognosis of cerebral infarction. In other words, when the cell composition of the present invention is used as a cell preparation, it can be used to improve the functional prognosis of cerebral infarction. Furthermore, the cell composition of the present invention can be used for any of the applications described in Section 1.2.

[0163] Exosomes secreted from the cell composition of the present invention encapsulate any of the RNA molecules (1) to (5) described above, and therefore exhibit the effect of improving the functional prognosis of cerebral infarction. In other words, the cell composition of the present invention can be used as a source of extracellular vesicles used to improve the functional prognosis of cerebral infarction. Furthermore, the cell composition of the present invention can be used as a source of extracellular vesicles used for any of the applications described in Section 1.2.

[0164] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations can also be adopted.

[0165] 2. Treatment Methods Section 2 describes the method of the present invention. The method of the present invention comprises administering to a subject a composition containing one or more compounds selected from (1) to (8) described in Section 1.

[0166] The subjects to which one or more selected from (1) to (8) above are administered are mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.), preferably humans.

[0167] In one embodiment, a composition containing one or more compounds selected from (1) to (8) above is administered to a patient after cerebral infarction.

[0168] In one embodiment, the method of the present invention is a method for improving functional prognosis after cerebral infarction.

[0169] In one embodiment, the present invention is a method for improving neurological deficits such as motor paralysis, sensory impairment, ataxia, higher brain function, and aphasia caused by cerebral infarction (stroke), restoring lost motor function and sensory impairment, and alleviating, mitigating, improving, relieving, or curing such sensory impairment.

[0170] In one embodiment, a composition containing one or more compounds selected from (1) to (8) above is administered to a patient in the acute to subacute phase.

[0171] Inhibition of glial scar formation, which can occur from the subacute to chronic phases, leads to improved effects on improving functional prognosis in the chronic phase. Thus, in one embodiment, a composition containing one or more compounds selected from (1) to (8) is administered to a patient in a state before or in the process of forming a glial scar.

[0172] In one embodiment, the present invention comprises administering a composition containing one or more selected from (1) to (8) to a patient in need of inhibition of glial scar formation.

[0173] In one embodiment, the present invention involves inhibiting the formation of glial scars by administering to a subject a composition containing one or more of the compounds selected from (1) to (8).

[0174] In one embodiment, the present invention includes reducing cerebral infarction by administering to a subject a composition containing one or more compounds selected from (1) to (8).

[0175] In one embodiment, the present invention comprises administering a composition containing one or more compounds selected from (1) to (8) to a patient in need of shrinkage of cerebral infarction.

[0176] In one embodiment, the present invention involves increasing the expression of S100A10-positive astrocytes by administering to a subject a composition containing one or more of the compounds selected from (1) to (8).

[0177] In one embodiment, the present invention comprises administering a composition containing one or more selected from (1) to (8) to a patient with reduced expression of S100A10-positive astrocytes.

[0178] In one embodiment, the present invention includes suppressing the expression of C3d-positive astrocytes by administering to a subject a composition containing one or more compounds selected from (1) to (8).

[0179] In one embodiment, the present invention comprises administering a composition comprising one or more selected from (1) to (8) to a patient with increased expression of C3d-positive astrocytes.

[0180] In one embodiment, the present invention includes suppressing the expression of the MIF gene in nerve cells by administering to a subject a composition containing one or more compounds selected from (1) to (8).

[0181] In one embodiment, the present invention comprises administering a composition containing one or more selected from (1) to (8) to a patient with increased expression of the MIF gene in nerve cells.

[0182] In one embodiment, the present invention involves suppressing expression of the CCND1 gene in astrocytes by administering to a subject a composition containing one or more compounds selected from (1) to (8).

[0183] In one embodiment, the present invention involves administering a composition containing one or more selected from (1) to (8) to a patient with increased expression of the CCND1 gene in astrocytes.

[0184] In one embodiment, the present invention includes promoting regeneration and / or protection of nerve cells after cerebral infarction by administering to a subject a composition containing one or more selected from (1) to (8).

[0185] In one embodiment, the present invention includes promoting the extension of axons and dendrites of neurons after cerebral infarction by administering to a subject a composition containing one or more compounds selected from (1) to (8).

[0186] In one embodiment, the present invention includes suppressing cell death after cerebral infarction by administering to a subject a composition containing one or more compounds selected from (1) to (8).

[0187] In one embodiment, the present invention includes administering to a subject a composition containing one or more therapeutic agents selected from (1) to (8) above in combination with one or more therapeutic agents selected from t-PA, free radical scavengers, antiplatelet agents, antithrombin agents, anticoagulants, antiplatelet agents, dextran 40 preparations, and concentrated glycerin-fructose preparations.

[0188] In the method of the present invention, the method of administering the composition containing one or more compounds selected from (1) to (8) above is not particularly limited. The administration may be systemic administration or local administration, and may be oral administration or parenteral administration (e.g., intracerebral administration, intraarterial administration, intramedullary administration, subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, etc.).

[0189] A composition containing one or more compounds selected from (1) to (8) is administered to a subject in an effective amount. The effective amount is determined appropriately depending on the age, weight, health condition, etc. of the subject. For example, when administered to a human, the amount of the active ingredient may be 0.00001 g / kg to 10 g / kg per day. The composition may be administered once a day, or in divided doses (e.g., 2, 3, or 4 times a day), or may be administered continuously by infusion or the like. The composition may be administered once, daily, or at regular intervals, for example, 1 to several days (e.g., 1, 2, 3, 4, 5, or 6 days), 1 to several weeks (e.g., 1, 2, 3, 4, 5, or 6 weeks), or 1 to several months (e.g., 1, 2, 3, 4, 5, or 6 months). The administration period may be acute, subacute, or chronic, and is not particularly limited, and may be 1 to several days (e.g., 1, 2, 3, 4, 5, or 6 days), 1 to several weeks (e.g., 1, 2, 3, 4, 5, or 6 weeks), 1 to several months (e.g., 1, 2, 3, 4, 5, or 6 months), or longer.

[0190] In addition, the matters described in Section 1 can be applied to specific embodiments of the method of the present invention.

[0191] 3. Application of the composition for the production of a medicine Section 3 describes the invention of an application for the production of a medicine. The present invention relates to one or more applications selected from the above (1) to (8) for the production of a medicine.

[0192] In one embodiment, the medicament is an agent for improving functional prognosis after cerebral infarction.

[0193] In one embodiment, the pharmaceutical is a pharmaceutical for improving neurological deficit symptoms such as motor paralysis, sensory disturbance, ataxia, higher brain function, and aphasia caused by cerebral infarction (stroke), for recovering lost motor function and sensory disturbance, and for alleviating, mitigating, improving, relieving, or curing the sensory disturbance.

[0194] In one embodiment, the medicament is a therapeutic agent for patients in the acute to subacute phase of cerebral infarction.

[0195] In one embodiment, the medicament is an inhibitor of glial scar formation.

[0196] In one embodiment, the medicament is an agent for reducing cerebral infarction.

[0197] In one embodiment, the medicament is an agent that enhances the expression of S100A10-positive astrocytes.

[0198] In one embodiment, the medicament is an agent that suppresses the expression of C3d-positive astrocytes.

[0199] In one embodiment, the pharmaceutical agent is an agent for suppressing the expression of the MIF gene in nerve cells.

[0200] In one embodiment, the pharmaceutical agent is an agent for suppressing the expression of the CCND1 gene in astrocytes.

[0201] In one embodiment, the medicament is a regenerating and / or protecting agent for nerve cells after cerebral infarction.

[0202] In one embodiment, the medicament is an agent for promoting the outgrowth of axons and dendrites of nerve cells after cerebral infarction.

[0203] In one embodiment, the medicament is an agent for inhibiting cell death after cerebral infarction.

[0204] The pharmaceutical agent is an agent for oral administration or a agent for parenteral administration.

[0205] In addition, the matters described in Sections 1 and 2 can be applied to specific embodiments of the invention of application for the manufacture of medicines.

[0206] In vivo EV administration study in rats. Pre-EVs, 3D-EVs, and 28D-EVs were prepared from serum isolated from whole blood (7 ml) collected from the hearts of rats before surgery, model rats 3 days after MCAO (intracavitary middle cerebral artery occlusion), and model rats 28 days after MCAO. The method for purifying extracellular vesicles from serum was employed, as described in Non-Patent Document 4. All collected EVs were combined with PBS to make a 200 μl volume and administered via the tail vein of rats from the acute to subacute phase 7 days after MCAO. Neurological signs (mNSS) and motor function (rotarod) were assessed every 7 days after administration until 28 days after administration, which corresponds to the chronic phase. The survival rate tended to be better in the EV-administered group compared to the untreated group (vehicle) (Figure 1). Neurological symptoms and motor function were significantly improved in the 3D-EVs and 28D-EVs groups compared to the vehicle group (Figure 2).

[0207] Brains were removed from rats that had received these EVs, and the volume of the infarct lesion and the neurons and astrocytes in the PIA (cerebral cortex within 300 μm of the infarct) were compared using histological techniques or immunohistochemical staining using antibodies against cell-specific markers.

[0208] H&E staining showed that the infarct size was significantly smaller in the EVs-administered group compared to the vehicle (Figure 3). Furthermore, immunohistochemical staining showed that in PIA, axons (pNFH) and dendrites (npNFH) were significantly increased in the 3D-EVs and 28D-EVs-administered groups compared to the vehicle. There was no change in neurons (MAP2) (Figure 4).

[0209] GFAP-positive astrocytes (activated astrocytes) were significantly decreased in the EVs-administered group compared to the vehicle group (Figure 5). C3d-positive astrocytes (cytotoxic astrocytes) were also significantly decreased (Figure 6). S100A10-positive astrocytes (neuroprotective astrocytes) were significantly increased in the EVs-administered group compared to the vehicle group (Figure 7).

[0210] The pia mater (PIA) was removed, and GFAP-positive activated astrocytes were sorted by flow cytometry. From these, C3d- and S100A10-positive astrocytes were further sorted. The results showed that the number of S100A10-positive astrocytes was significantly increased in the 28D-EVs-treated group compared to the vehicle group (Figure 8).

[0211] The above results indicate that 28D-EVs have the effect of improving the functional prognosis of cerebral infarction (improving neurological symptoms and motor function); more specifically, they have the effect of reducing infarct size, promoting axonal-dendritic extension, inhibiting glial scar formation, promoting the expression of S100A10-positive astrocytes (neuroprotective astrocytes), and inhibiting the expression of C3d-positive astrocytes (cytotoxic astrocytes).

[0212] <Analysis of EV Characteristics> Quantitative analysis was performed using Nanosight to examine changes in circulating EVs induced by MCAO. Results showed significant differences in EVs within the 79.5-369.5 nm range, with significantly fewer 3D-EVs and significantly more 28D-EVs in total (Figure 9). These results suggest that EVs decrease during the acute phase of cerebral infarction and become more abundant during the chronic phase.

[0213] Fluorescently labeled Pre-EVs, 3D-EVs, and 28D-EVs were injected intravenously via the tail vein. The brains were removed and used for immunohistochemical studies. These EVs accumulated in the cerebral infarct area, with significantly greater accumulation in the EV-administered group compared to the sham (no cerebral infarction) group (Figure 10). There was no significant difference in the number of EVs accumulated in neurons, axons, dendrites, or GFAP-positive activated astrocytes in the infarcted area (Figure 11). The results confirmed that EVs accumulate in the infarcted area.

[0214] Next, the accumulation of circulating EVs throughout the body was observed. 28D-EVs accumulated significantly more in the lungs, liver, and bones than sham controls. 3D-EVs accumulated more in the muscles. These results indicate that circulating EVs after MCAO accumulate in different organs over time.

[0215] Next, primary cultured neurons and cultured astrocytes from rat brain were exposed to hypoxia-low glucose conditions (ODG) and then administered pre-EVs, 3D-EVs, and 28D-EVs. Immunostaining (Figures 12 and 13) and Western blotting (Figure 14) were performed. Cultured neurons showed significantly greater axon extension in the EVs-treated group compared to the OGD group. pNFH and npNFH levels were significantly increased in the 3D-EVs-treated and 28D-EVs-treated groups compared to the OGD group. MAP2 levels remained unchanged. Cultured astrocytes also showed significantly decreased GFAP and C3d levels in the EVs-treated group compared to the OGD group. S100A10 levels were significantly increased in the 3D-EVs-treated and 28D-EVs-treated groups compared to the OGD group. As described above, the changes observed in in vivo studies were also confirmed in in vitro studies.

[0216] The results so far suggest that the cargo carried by circulating EVs may be an effective ingredient in improving the prognosis of cerebral infarction.

[0217] <miRNA analysis> To elucidate the molecular mechanism of the effect of 28D-EVs on functional recovery after MCAO, microarray analysis was used to identify miRNAs significantly higher in 28D-EVs than in pre-EVs. As a result, miR-451-5p was identified as the miRNA most influential in the chronic phase after cerebral infarction (Figure 15). Furthermore, a volcano plot revealed that miR-3541 was significantly higher in 28D-EVs (Figure 16).

[0218] To explore the origin of miR-451-5p, we performed a Function and Disease core analysis using pathway analysis software, which suggested that miR-451-5p may have a bone marrow origin (Figure 17).

[0219] To explore the possibility that beneficial EVs, which contribute to improved prognosis in the chronic phase after stroke, selectively accumulate around the infarct area, we focused on tetraspanins on the surface of EVs and examined the distribution of tetraspanins on pre-EVs, 3D-EVs, and 28D-EVs by counting the number of EVs bound to CD9, CD81, and CD63 capture antibodies using ExoView (Figure 18). As a result, we confirmed that CD9+ 28D-EVs adhered significantly more strongly than pre-EVs and 3D-EVs in the presence of anti-CD9 antibodies.

[0220] <Confirmation of functional control by miR-451> The results of the analysis so far indicate that miR-451-5p is likely to contribute to improving functional prognosis after cerebral infarction. Therefore, to explore the role of miR-451-5p itself, we prepared 28D-EVs transfected with mirVana™ hsa-miR-451 mimic (28D-mimic-EVs) and 28D-EVs transfected with mirVana™ hsa-miR-451 inhibitor (28D-inhibitor-EVs), and administered them to cultured neurons and astrocytes after OGD. The 28D-EVs-administered group, the 28D-mimic-EVs-administered group, and the 28D-inhibitor-EVs-administered group were collected and subjected to Western blotting. In both neurons and astrocytes, the 28D-mimic-EVs-administered group showed the same improvement effects as seen in previous studies (increases in pNFH, npNFH, and S100A10, and decreases in C3d and GFAP) (Figures 19 and 20).

[0221] A plasmid encoding miR-451-5p (Origene #SC403409) was transfected into 293T cells to obtain cells secreting exosomes rich in miR-451-5p (miR-451-5p-rich-293T). EVs rich in miR-451-5p (miR-451-5p-rich-293T-EVs) were isolated from the culture supernatant of these cells. 293T-EVs and miR-451-5p-rich-293T-EVs were administered to cultured neurons and astrocytes after OGD, and Western blotting was performed. In both neurons and astrocytes, the miR-451-5p-rich-293T-EVs administration group showed the same improvement effects as those observed in previous studies (increases in pNFH, npNFH, and S100A10, and decreases in C3d and GFAP) (Figures 21 and 22).

[0222] To elucidate the molecular mechanism of miR-451-5p, we performed proteomics on cultured neurons and astrocytes treated with miR-451-5p-rich 293T-EVs and 293T-EVs. The results showed that miR-451-5p suppressed MIF and BCL2 in neurons and CCND1 in astrocytes. WB analysis confirmed this, revealing a decrease in MIF in neurons and CCND1 in astrocytes (Figures 23 and 24). Similar results were obtained when comparing the Pre-EVs group with the 28D-EVs group. These results indicate that miR-451-5p suppresses MIF and CCND1 in neurons and astrocytes, respectively.

[0223] Using the same procedures as in the in vivo study described above, 293T-EVs and miR-451-5p-rich-293T-EVs were administered to rats after MCAO. As a result, infarct size was reduced and neurological symptoms and motor function improved in the miR-451-5p-rich-293T-EVs-administered group.

[0224] The composition of the present invention has the effect of improving the functional prognosis of cerebral infarction, and is useful for treating sequelae of cerebral infarction.

Claims

1. A composition for improving the functional prognosis of cerebral infarction, comprising any one of the following (1) to (8) as an active ingredient: (1) a transcription product or a processed product of a gene encoding miR-451, or miR-4739, or miR-3541; (2) a pre-miRNA that has a substitution, addition, and / or deletion of 1 to 10 bases relative to a pre-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, and that can form a stem-loop hairpin structure; (3) a pre-miRNA that has 70% or more sequence homology to a pre-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, and that can form a stem-loop hairpin structure. (4) A mature-miRNA having 1 to 5 base substitutions, additions, and / or deletions relative to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. (5) A mature-miRNA that has 80% or more sequence homology to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. (6) A silencing molecule for the MIF gene and / or CCND1 gene. (7) An expression vector encoding one or more types of RNA falling under any of (1) to (6) above. (8) Extracellular vesicles derived from placenta-derived adherent cells, extracellular vesicles derived from umbilical cord blood mononuclear cells, or extracellular vesicles derived from mesenchymal stem cells.

2. The composition according to claim 1, wherein the miRNA described in (2) to (5) above comprises at least a sequence identical to 5'-aaccguu-3' (SEQ ID NO: 5) or 5'-cccuccc-3' (SEQ ID NO: 6) or a sequence having a substitution, addition, and / or deletion of 1 to 2 bases.

3. The composition according to claim 1, comprising a vesicle made of a lipid bilayer membrane encapsulating an RNA molecule according to any one of (1) to (6).

4. The composition according to claim 3, wherein the vesicles made of lipid bilayer membranes are selected from extracellular vesicles, liposomes, and lipid nanoparticles.

5. The composition according to claim 4, wherein the vesicles made of lipid bilayer membranes are exosomes.

6. The composition according to claim 1, wherein the mature-miRNA, which is a processed product of the transcription product of the gene encoding miR-451, is miR-451-5p.

7. A composition according to any one of claims 1 to 6, which is used for reducing cerebral infarction lesions.

8. A composition described in any one of claims 1 to 6, which is used for promoting the expression of S100A10-positive astrocytes and / or suppressing the expression of C3d-positive astrocytes.

9. A composition described in any one of claims 1 to 6, which is used for inhibiting glial scar formation.

10. A composition described in any one of claims 1 to 6, which is used for suppressing the expression of the MIF gene in neurons and / or the expression of the CCND1 gene in astrocytes.

11. A composition described in any one of claims 1 to 6, used to improve functional prognosis in the chronic phase of cerebral infarction.

12. The composition according to any one of claims 1 to 6, which is administered during the acute to subacute phase of cerebral infarction.

13. The composition according to any one of claims 1 to 6, which is used in combination with one or more therapeutic agents selected from t-PA, free radical scavengers, antiplatelet agents, antithrombin agents, anticoagulants, antiplatelet agents, dextran 40 preparations, and concentrated glycerin / fructose preparations.

14. A cell into which an expression vector encoding any one of the following RNA molecules (1) to (5) has been introduced, and which expresses said RNA molecule: (1) A transcription product or a processing product of a gene encoding miR-451, or miR-4739, or miR-3541. (2) A pre-miRNA that has a substitution, addition, and / or deletion of 1 to 10 bases relative to a pre-miRNA that is a processing product of a transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, and that can form a stem-loop hairpin structure. (3) A pre-miRNA that has 70% or more sequence homology to a pre-miRNA that is a processing product of a transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, and that can form a stem-loop hairpin structure. (4) A mature-miRNA having a substitution, addition, and / or deletion of 1 to 5 bases relative to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. (5) A mature-miRNA that has 80% or more sequence homology to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541.

15. A cell composition for use in improving the functional prognosis of cerebral infarction, or as a source of extracellular vesicles to be used in improving the functional prognosis of cerebral infarction, comprising cells expressing any of the RNA molecules (1) to (5) below, the cells according to claim 14, placenta-derived adherent cells, umbilical cord blood mononuclear cells, or mesenchymal stem cells: (1) a transcription product or a processing product thereof of a gene encoding miR-451, or miR-4739, or miR-3541; (2) a pre-miRNA that has 1 to 10 base substitutions, additions, and / or deletions relative to a pre-miRNA that is a processing product of a transcription product of a gene encoding miR-451, or miR-4739, or miR-3541, and that can form a stem-loop hairpin structure. (3) A pre-miRNA that has 70% or more sequence homology to a pre-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541, and that can form a stem-loop hairpin structure. (4) A mature-miRNA that has 1 to 5 base substitutions, additions, and / or deletions to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541. (5) A mature-miRNA that has 80% or more sequence homology to a mature-miRNA that is a processed product of a transcription product of a gene encoding miR-451, or miR-4739 or miR-3541.

16. A composition for improving the functional prognosis of cerebral infarction, comprising as an active ingredient the culture supernatant of the cell composition described in claim 15 or extracellular vesicles isolated from the culture supernatant.