Method for repairing hair cycle-related genes using miR-520d-5p and method for treating hair cycle-related diseases

Abstract

It is an object of the present invention to provide a novel means for treating a hair cycle-related disease or condition, such as a hair cycle-related disease. Provided is a miR-520d-p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p, for use in reverting a mutation in a hair cycle-related gene to wild type in a cell, wherein the polynucleotide comprises (a1) the base sequence set forth in SEQ ID NO: 1, or (a2) the base sequence set forth in SEQ ID NO: 1 having a deletion, substitution, insertion or addition of 1 to 3 bases. SEQ ID NO:1 (mature single-stranded miRNA sequence) 5'-CUACAAAGGGAAGCCCUUUC-3'

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to Singapore patent application 10202114489Q filed on December 29, 2021, the contents of which are incorporated herein by reference.

[0002] The present invention relates to a miR-520d-5p polynucleotide and a compound for upregulating miR-520d-5p, in particular to their novel uses. The present invention also relates to a method and composition for treating a disease or condition (e.g., alopecia) related to the hair cycle. [Background technology]

[0003] In WO2012 / 008302A, the present inventors disclosed that by using miR-520d-5p, it is possible to induce pluripotent stem cells from somatic cells or malignant tumor cells (i.e., an induction method or induction agent), and that malignant tumors can also be treated (i.e., a treatment method or treatment agent).

[0004] In WO2017 / 073689A, the present inventors disclosed that by using miR-520d-5p, DNA damage caused by ultraviolet light can be improved (i.e., a composition for improving DNA damage or a composition for improving ultraviolet damage) and that a decrease in collagen production caused by ultraviolet light can be suppressed (i.e., a composition for promoting collagen production or a composition for suppressing a decrease in collagen production).

[0005] In WO2018 / 203553A, the present inventors disclosed that the quality of differentiated cells obtained from stem cells such as iPS cells can be improved by using miR-520d-5p (i.e., a method for improving the quality of differentiated cells).

[0006] In WO2017 / 073692A, the present inventors disclosed that malignant tumors can be treated, stem cells can be induced, and DNA damage can be improved by using HAT1 and / or KAT8 inhibitors (i.e., malignant tumor treatment drugs, stem cell inducers, DNA damage improvement drugs), etc. Examples of HAT1 and / or KAT8 inhibitors include chlorpropamide, vancomycin hydrochloride, betaxolol hydrochloride, cholesterol sulfate, bisoprolol fumarate, pinaverium bromide, oxprenolol hydrochloride, methylbenzethonium chloride, demepotassium bromide, celiprolol hydrochloride, amikacin hydrate, alprenolol hydrochloride, etc.

[0007] References: Ohyama, M. et al., J. Cell Sci. 125, 4114-4125 (2012); Ceruti, JM et al., Mol. Cell Endocrinol. 465, 122-133 (2018); and Lolli, Fr et al., Endocrine. 57, 9-17 (2017) each describe genes expressed in (and associated with the proliferation or function of) dermal papilla cells, and signaling pathways, etc. and the references Nowak, JA et al., Cell Stem Cell. 3, 33-43 (2008); Liu, Y. et al., J. Invest. Dermatol. 121, 963-968 (2003); Rhee, H. et al., Science. 312, 1946-1949 (2006); Horsley, V. et al., Cell. 132, 299-310 (2008); and Xu, Z. et al., Elife. 4, e10567 (2015) describe genes expressed in (and function as cellular markers of) bulge stem cells.

[0008] Diseases and conditions related to the hair cycle, such as alopecia, remain a problem. Summary of the Invention

[0009] Various means (treatment methods, treatment compositions, etc.) have been proposed for treating diseases or symptoms (e.g., alopecia) related to the hair cycle, but new means are still needed to meet the needs of patients for whom known means do not provide sufficient effects.

[0010] An objective of the present invention is to provide a novel means for treating diseases or symptoms associated with the hair cycle (such as hair cycle-related diseases).

[0011] The present inventors have found that by using miR-520d-5p, mutations in genes related to the hair cycle (hair cycle-related genes) can be reverted to wild type in cells related to the hair cycle (hair cycle-related cells). The present inventors have also found that some compounds have the effect of enhancing (upregulating) the expression of miR-520d-5p, and that mutations in hair cycle-related genes can also be reverted to wild type by using such compounds for upregulating miR-520d-5p. Furthermore, the present inventors have also found that miR-520d-5p is presumed to suppress or regulate the expression of some hair cycle-related genes or other genes in hair cycle-related cells. Thus, the present inventors have found that miR-520d-5p polynucleotides or polynucleotides encoding the same, or compounds for upregulating miR-520d-5p can be used as activity promoters for treating hair cycle-related diseases, etc., and have completed the present invention.

[0012] That is, the present invention provides at least the following:

[0013] [Section 1] In one embodiment, A miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p, is provided for use in reverting a mutation in a hair cycle-related gene in a cell to a wild type; Here, the polynucleotide is (a1) the nucleotide sequence of SEQ ID NO: 1, or (a2) A nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 1 Includes. SEQ ID NO: 1 (single-stranded miRNA sequence) 5'-CUACAAAGGGAAGCCCUUUC-3'

[0014] [Section 2] Item 2. The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to Item 1, wherein the cell is a hair papilla cell, a skin-derived progenitor cell, or a bulge stem cell, or a stem cell capable of differentiating into said cell.

[0015] [Section 3] Item 3. The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to Item 1 or 2, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 142 types of genes: ACT1, ALPL, ALX4, AQP3, AR, ASCL1, BCL2, BDNF, BMI1, BMP2, BMP4, BMP6, CCHCR1, CD271, CDKN1C, CDKN2A, Chr.20 1122 PAX1, CPE, CRHR2, CTGF, CTNNB1, CYP1B1, DDR2, DKK2, DKK4, DLL1, EDN3, EFNA, EGF, EGR1, EGR3, FBN2, FGF5, FGF7(KGF), FGF18, FN1, FOXE1, FOXN1, FOXO3, FZD1, FZD4, FZD8, GADD45G, GATA3, GLI2, GLI3, GRID1, GUCY1A3, HES1, HES5, HEY1, HEY2, HEYL, HGF, HR(HAIRLESS), HSD3B1, IGF1, IRF4, ITGB1, JAG1, JAG2, KAT2A, KAT2B, KCNJ8(KIR6), KDR, KRT5, KRT14, KRT15, ​​KRT19, LAMC3, LRIG1, LATS2, LEF1, LFNG, LGR5, LHX2, LMO1, LPL, LRP4, LRP5, MAPK1, NCAM1, NCOA3, NDP, NELL2, NFATC1, NFKB, NOG, NOS3, NOTCH2, NOTCH3, NOTCH4, NTRK, PDGFA, PTCH1, RBPJ, RGS2, RHOB, RUNX1, SDKN2A, SERPINE2, SHH, SIRT7, SLC25A37(MSCP), SMAD4, SMAD6, SMO, SMOC2, SMTN, SOSTDC1, SOX2, SOX9, SPON1, SPRY2, SP5, SRD5A2, STAT3, STS, SUR2(ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1(TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, w VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

[0016] [Section 4] The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to any one of items 1 to 3, which is used to revert a mutation in a hair cycle-related gene in a cell to a wild type and to suppress or regulate the expression of at least one gene selected from the group consisting of the following 18 types of genes: A.R., DIwO2, EDN3, FGF7 (KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTH LH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

[0017] [Section 5] The cells are hair papilla cells or stem cells capable of differentiating into said cells, and The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to any one of items 1 to 4, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 26 types of genes: ALX4, AR, BMP2, BMP4, CPE, EDN3, FGF7 (KGF), GUCY1A3, HEY1, HEY2, LAMC3, LEF1, LPL, LRP4, NDP, NOG, NTRK, SOSTDC1, SPON1, TFAP2A, TFAP2C, TRPS1, TWIST1, VAV3, VCAN and WNT5A.

[0018] [Section 6] Item 6. The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to any one of Items 1 to 5, which is used to revert a mutation in a hair cycle-related gene in a cell to the wild type and to suppress or regulate the expression of at least one gene selected from the group consisting of VAV3, NTRK (SLITRK2, 4, 6), HEY2, RORA, VCAN, HEY1, AR, TRPS1, TWIST1, LEF1, EDN3, SOSTDC1, FGF7 (KGF), DIO2, and RNF144A.

[0019] [Section 7] The cells are bulge stem cells or stem cells capable of differentiating into such cells; and The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to any one of items 1 to 6, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 18 types of genes: ASCL1, BMP2, DKK4, GADD45G, KRT15, LHX2, LMO1, LRP5, NFATC1, SHH, SMOC2, SOSTDC1, SOX2, SOX9, SP5, SRD5A2, STAT3, STS, SUR2(ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1(TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

[0020] [Section 8] Item 8. The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to any one of Items 1 to 7, which is used to revert a mutation in a hair cycle-related gene in a cell to the wild type and to suppress or regulate the expression of at least one gene selected from the group consisting of PTHLH, SOX9, and SMOC2.

[0021] [Section 9] The cells are hair papilla cells or stem cells capable of differentiating into said cells, and bulge stem cells or stem cells capable of differentiating into said cells, The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to any one of items 1 to 8, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following two types of genes: BMP2 and SOSTDC1.

[0022] [Section 10] The miR-520d-5p polynucleotide is (b1) the nucleotide sequence of SEQ ID NO: 2, or (b2) a polynucleotide having a base sequence in which 1 to 3 bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2; and (c1) the nucleotide sequence of SEQ ID NO: 3, or (c2) a double-stranded polynucleotide having a base sequence in which 1 to 3 bases have been deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 3; or (d1) the nucleotide sequence of SEQ ID NO: 4, or (d2) A polynucleotide having a base sequence in which 1 to 6 bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 4. Item 1, a miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p. SEQ ID NO:2 (Guide strand sequence) 5'-UCUACAAAGGGAAGCCCUUUCUG-3' SEQ ID NO:3 (Passenger strand sequence) 5'-AAAGUGCUUCUCUUUGGUGGGU-3' SEQ ID NO: 4 (Pre-miRNA sequence) 5'-UCUCAAGCUGUGAGUCUACAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCUCUUUGGUGGGUUACGGUUUGAGA-3'

[0023] [Section 11] The miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p according to any one of items 1 to 10, wherein the compound for upregulating miR-520d-5p is selected from the group consisting of the following 13 types of compounds: Vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacrynic acid, bisoprolol, candesartan, alexidine, bumetanide and glycopyrrolate.

[0024] [Section 12] In another aspect (1) Culturing cells using the miR-520d-5p polynucleotide according to any one of items 1 to 11, or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p; and (2) confirming whether the mutation in the hair cycle-related gene in the cell reverts to the wild type; A method for producing a cell is provided, comprising:

[0025] [Section 13] In another aspect (I) The miR-520d-5p polynucleotide according to any one of items 1 to 11, or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p, or (II) Cells produced by the method according to claim 12. The present invention provides a composition for use in the treatment of a hair cycle related disease or condition, comprising:

[0026] [Section 14] Item 14. The composition according to item 13, which is used for the therapeutic treatment of alopecia.

[0027] [Section 15] The composition according to item 13 or 14, wherein each of the compounds for upregulating miR-520d-5p is administered to achieve the following maximum effective blood concentration or applied externally at the following concentrations: Vancomycin: Administration, 30 μg / ml or less Metoprolol: administered, 41.8 ng / ml or less Benzethonium: For external use, 0.2% or less Chlorpropamide: Administration, 30 μg / ml or less Betaxolol: administered, 41.8 ng / ml or less Colistin: Administration, 4.4 μg / ml or less Oxprenolol: Administration, 30.18 μg / ml or less Ethacrynic acid: Dosage, 100 μg / ml or less Bisoprolol: Administration, 100 ng / ml or less Candesartan: Administration, 75 ng / ml or less Alexidine: Bumetanide: Administration, 0.4 μg / ml or less Glycopyrrolate: Administer, below 800 pg / ml.

[0028] It should be noted that a person skilled in the art can change the category of the above invention or other inventions included in this specification to make it into a different invention. For example, "miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for upregulating miR-520d-5p for use in reverting a mutation in a hair cycle-related gene to a wild type in a cell" may be changed to the invention of "a method for reverting a mutation in a hair cycle-related gene in a cell to a wild type using a miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for upregulating miR-520d-5p" and "miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for upregulating miR-520d-5p, or a cell produced by the method of the present invention" may be changed to the invention of "a method for treating a hair cycle-related disease or condition using (e.g., administering) a miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for upregulating miR-520d-5p, or a cell produced by the method of the present invention." A person skilled in the art would be able to replace the matters relating to the invention before the modification described in this specification with the matters relating to the invention after the modification. Effect of the Invention

[0029] Hair growth and hair loss are closely related to the aging of hair cycle-related cells. Not only aged hair cycle-related cells present in living organisms such as humans, but also iPS cells produced from somatic cells and mesenchymal stem cells derived from iPS cells are accompanied by many mutations and epigenetic modifications. By using miR-520d-5p, hair cycle-related genes in those cells can be restored to wild type, and the function of hair cycle-related cells can be normalized. Furthermore, miR-520d-5p not only has the effect of repairing the above-mentioned mutations, but also has the effect of suppressing or regulating the expression of mRNA having a complementary base sequence, for example, the mRNA of hair cycle-related genes or other genes that contribute to hair cycle-related diseases. Therefore, by using the miR-520d-5p polynucleotide or the polynucleotide encoding it or the compound for upregulating miR-520d-5p according to the present invention, any of the above-mentioned actions, preferably the synergistic action of both, can be used to show favorable effects in the treatment of hair cycle-related diseases (e.g., alopecia). [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows an overview of 178 hair cycle-related genes that are expressed during the anagen, catagen, telogen, and shedding phases of the hair cycle. [Diagram 2] FIG. 2 shows an overview of genes expressed in dermal papilla cells (DP cells) in Example 1. [Diagram 3] FIG. 3 shows an overview of genes expressed in bulge stem cells in Example 2. [Figure 4] FIG. 4 is a graph showing the expression level by compounds for upregulating miR-520d-5p in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] In various embodiments, a miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p (which may be collectively referred to herein as "miR-520d-5p-related substances") is used to revert a mutation in a hair cycle-related gene in a cell to the wild type.

[0032] The use of miR-520d-5p-related substances means that the expression level of miR-520d-5p in cells is increased more than usual (when no miR-520d-5p-related substances are used). By using miR-520d-5p-related substances that can increase the expression level of miR-520d-5p to a desired level depending on the amount of miR-520d-5p-related substances introduced into cells, the expression level of miR-520d-5p can be, for example, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 2 times (200%) or more, 3 times (300%) or more, 4 times (400%) or more, 5 times (500%) or more, 10 times (1000%) or more, or 20 times (2000%). As mentioned above, the increase can be 30 times (3000%) or more, 40 times (4000%) or more, or 50 times (5000%) or more compared to normal levels.

[0033] In the present invention, the term "hair cycle-related gene" refers to a gene related to the hair cycle that is expressed in cells related to the hair cycle (hair cycle-related cells). The hair cycle includes an anagen phase (a period in which hair matrix cells divide actively and hair grows rapidly, usually 2 to 6 years), a catagen phase (a period in which the function of hair papilla cells that support the division of hair matrix cells decreases and hair papilla cells move away from hair matrix cells, slowing hair growth, usually 2 to 3 weeks), a telogen phase (a period in which hair papilla cells shrink and hair does not grow, usually 3 to 4 months), and an effluvium phase (a period in which hair falls out, usually 2 to 5 months). The hair cycle-related gene is not particularly limited as long as it has the effect of returning a mutation to the wild type by a miR-520d-5p-related substance.

[0034] In various embodiments, the hair cycle-related cells include, for example, dermal papilla cells (DP cells), skin-derived progenitor cells (SKP cells) and bulge stem cells, as well as stem cells that can differentiate into these cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Dermal papilla cells are classified as mesenchymal stem cells and interact with skin-derived progenitor cells. Bulge stem cells originate from the peripheral ring of the placode basement membrane and are essential for hair growth. Such stem cells also maintain the potential to differentiate into other cell lineages that are important for promoting hair growth, such as blood cells, fibroblasts, adipocytes, and nerve cells. The hair cycle-related cells may be any one type of cell, or may be two or more types of cells.

[0035] The target cells (hair cycle-related cells) for reverting the mutation in the hair cycle-related gene to the wild type may be those existing in a living body (in vivo) or those existing outside a living body (ex vivo, in vitro). For example, when carrying out the treatment method of the present invention (details are described separately), the target cells are those existing in a living body, and when carrying out the cell production method of the present invention (details are described separately), the target cells are those existing outside a living body.

[0036] In various embodiments, the hair cycle-related gene whose mutation is repaired is at least one gene selected from the group consisting of the following 142 genes: ACT1, ALPP, ALX4, AQP3, AR, ASCL1, BCL2, BDNF, BMI1, BMP2, BMP4, BMP6, CCHCR1, CD271, CDKN1C, CDKN2A, Chr.20 1122 PAX1, CPE, CRHR2, CTGF, CTNNB1, CYP1B1, DDR2, DKK2, DKK4, DLL1, EDN3, EFNA, EGF, EGR1, EGR3, FBN2, FGF5, FGF7(KGF), FGF18, FN1, FOXE1, FOXN1, FOXO3, FZD1, FZD4, FZD8, GADD45G, GATA3, GLI2, GLI3, GRID1, GUCY1A3, HES1, HES5, HEY1, HEY2, HEYL, HGF, HR(HAIRLESS), HSD3B1, IGF1, IRF4, ITGB1, JAG1, JAG2, KAT2A, KAT2B, KCNJ8(KIR6), KDR, KRT5, KRT14, KRT15, ​​KRT19, LAMC3, LRIG1, LATS2, LEF1, LFNG, LGR5, LHX2, LMO1, LPL, LRP4, LRP5, MAPK1, NCAM1, NCOA3, NDP, NELL2, NFATC1, NFKB, NOG, NOS3, NOTCH2, NOTCH2, NOTCH4, NTRK, PDGFA, PTCH1, RBPJ, RGS2, RHOB, RUNX1, SDKN2A, SERPINE2, SHH, SIRT7, SLC25A37(MSCP), SMAD4, SMAD6, SMO, SMOC2, SMTN, SOSTDC1, SOX2, SOX9, SPON1, SPRY2, SP5, SRD5A2, STAT3, STS, SUR2(ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1(TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

[0037] Examples of hair cycle-related genes include 178 types of genes, as shown in Table 2 in Example 1 below. In another embodiment, genes including the above 178 types of genes but not including the above 142 types of genes, i.e., at least one type of gene selected from the following 36 types of genes (genes with a gray background in Table 2), may be subject to mutation repair in some cases. ANGPT, APOE, ASCL4, BAX, CD200, CD34, CDH1, CDH3, CLCA2, CSTA, DIO2, DKK1, EDA2R, EDAR, ENG(CD105), FGF2, GLI1, GPRC5B, IFITM3, IL6, LAMA5 MKI67(Ki67) NANOGNB(HOXC14), NCOA4 PRSS53, PTGDS, PTHLH, RNF144A, SHISA2, SPECC2, SPP1, SPRY1, SRD5A1, STAR, STAT2 and WIF1.

[0038] In various embodiments, the miR-520d-5p-related substance can repair the mutation of the hair cycle-related gene and suppress or regulate the expression of the gene (mRNA) containing a base sequence having a certain degree of complementarity with miR-520d-5p among the genes expressed in cells, preferably hair cycle-related cells. The gene suppressed or regulated by miR-520d-5p can be estimated using a known miRNA target search database or website (e.g., miRBase, EumiR, miRanda, PicTar, TargetScanHuman 7.0). For example, genes with a target score of 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more may also be targeted. The gene whose expression is suppressed or regulated may be a hair cycle-related gene or another gene, and may be, for example, at least one gene selected from the group consisting of the following 18 types of genes: A.R., DIO2, EDN3, FGF7 (KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTH LH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

[0039] The "suppression" of gene expression by miR-520d-5p (related substance) means that the expression level of the target gene in the cell is reduced compared to when the miR-520d-5p related substance is not used. Depending on the amount of the miR-520d-5p related substance introduced into the cell, the expression level of the target gene can be suppressed to a desired level, for example, compared to when the miR-520d-5p related substance is not used, it can be 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, and 0.01% or less. On the other hand, the "regulation" of gene expression by miR-520d-5p (related substance) means that the expression level of the target gene in the cell is kept within a certain range from the expression level in normal cells. Depending on the amount of miR-520d-5p related substance introduced into cells, the expression level of the target gene can be adjusted to a desired level, for example, to ±100% or less, ±50% or less, ±20% or less, ±10% or less, or ±5% or less compared to the expression level in normal cells. An embodiment in which miR-520d-5p (related substance) acts on cells in which a target gene is overexpressed to "suppress" (reduce) gene expression and thereby keep the intracellular expression level within a certain range also corresponds to "suppressing or regulating" gene expression by miR-520d-5p (related substance).

[0040] In various embodiments, the hair cycle-related gene whose mutation is repaired is at least one gene expressed in dermal papilla cells or stem cells capable of differentiating into said cells, and is selected from the group consisting of the following 26 genes: ALX4, AR, BMP2, BMP4, CPE, EDN3, FGF7 (KGF), GUCY1A3, HEY1, HEY2, LAMC3, LEF1, LPL, LRP4, NDP, NOG, NTRK, SOSTDC1, SPON1, TFAP2A, TFAP2C, TRPS1, TWIST1, VAV3, VCAN and WNT5A.

[0041] In addition to the above 26 types of genes, there are seven types of hair cycle-related genes expressed in hair papilla cells (APOE, CLCA2, CSTA, DIO2, GPRC5B, RNF144A, WIF1). In another embodiment, for example, when cells are cultured under different conditions from the examples disclosed herein and have different mutations, at least one of these seven types of genes may be the target for mutation repair in some cases.

[0042] Furthermore, in the above embodiment in which hair papilla cells are the target cells, in addition to the above 26 types of genes (or other 7 types of genes), at least one type of gene among 6 types of genes (ALPL, CCND2, CTSH, FRZB, PTGS2, RORA) that are expressed in hair papilla cells but do not fall under the hair cycle-related genes may be targeted for mutation repair.

[0043] Furthermore, in the above embodiment in which the target cells are hair papilla cells, miR-520d-5p can be used to repair the mutation of a specific gene and simultaneously suppress or regulate the expression of a specific gene (mRNA) among genes expressed in hair papilla cells. Examples of genes expressed in hair papilla cells that can be the target of such expression suppression or regulation include at least one gene selected from the group consisting of VAV3, NTRK (SLITRK2, 4, 6), HEY2, RORA, VCAN, HEY1, AR, TRPS1, TWIST1, LEF1, EDN3, SOSTDC1, FGF7 (KGF), DIO2 and RNF144A. In particular, VAV3, NTRK, HEY2, VCAN, HEY1, AR, TRPS1, TWIST1, LEF1, EDN3, SOSTDC1 and FGF7 (KGF) may be subjected to both mutation repair and expression suppression or regulation by miR-520d-5p. Among these, AR is an important hair cycle-related gene expressed in hair papilla cells, as it is a gene that is overexpressed in androgenetic alopecia (AGA) and female androgenetic alopecia (FAGA).

[0044] In various embodiments, the hair cycle-related gene whose mutation is repaired is at least one gene expressed in bulge stem cells or stem cells capable of differentiating into said cells, selected from the group consisting of the following 18 genes: ASCL1, BMP2, DKK4, GADD45G, KRT15, LHX2, LMO1, LRP5, NFATC1, SHH, SMOC2, SOSTDC1, SOX2, SOX9, SP5, SRD5A2, STAT3, STS, SUR2(ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1(TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

[0045] In addition to the above 18 genes, there are 5 hair cycle-related genes expressed in bulge stem cells (CDH3, EDAR, IFITM3, PTHLH, WIF1). In another embodiment, for example, when cells are cultured under different conditions and have different mutations from those in the examples disclosed herein, at least one of these 5 genes may be targeted for mutation repair.

[0046] Furthermore, in the above embodiment in which the target cells are bulge stem cells, miR-520d-5p can be used to repair the mutation of a specific gene and simultaneously suppress or regulate the expression of a specific gene (mRNA) among genes expressed in hair papilla cells. Examples of genes expressed in bulge stem cells that can be the target of such expression suppression include at least one gene selected from the group consisting of PTHLH, SOX9, and SMOC2. In particular, SOX9 is important as a hair cycle-related gene expressed in bulge stem cells because it can be both repaired by miR-520d-5p and suppressed or regulated by miR-520d-5p.

[0047] In various embodiments, the hair cycle-related gene whose mutation is repaired is a gene expressed in both dermal papilla cells or stem cells capable of differentiating into said cells and bulge stem cells or stem cells capable of differentiating into said cells, and is at least one gene selected from the group consisting of the following two types of genes: BMP2 and SOSTDC1.

[0048] In this specification, "converting a mutation in a hair cycle-related gene to a wild type" means repairing a base sequence of a hair cycle-related gene that is different from the wild type (i.e., mutated) in a cell so that it is the same as the wild type base sequence. The number of mutated bases may be a range (which may or may not include the upper limit and / or lower limit) determined by any lower limit and / or upper limit selected from the group consisting of, for example, 1 base, 5 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 60 bases, 70 bases, 80 bases, 90 bases, 95 bases, and 100 bases, for example, 1 base or more and 100 bases or less. The converted base may be all or a part of the mutated base, and for example, the conversion rate (the ratio of the number of converted bases to the number of mutated bases) may be, for example, a range determined by any lower limit and / or upper limit selected from the group consisting of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% and 100% (the upper limit and the lower limit may be included or not included), for example, 1% or more and 100% or less. Such an action effect may be observed in one cell (for example, a hair cycle-related cell), or may be observed in a certain proportion (preferably 50% or more) of cells in a cell population containing a certain number of cells.

[0049] -miR-520d-5p polynucleotide / polynucleotide encoding same- As used herein, "miR-520d-5p polynucleotide" may refer to (a1) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, or (a2) a nucleotide sequence in which several nucleotides have been deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 1. The "several nucleotides" in the nucleotide sequence (a2) is preferably 1 to 3 nucleotides, more preferably 1 to 2 nucleotides, and even more preferably 1 nucleotide.

[0050] SEQ ID NO: 1 (single-stranded miRNA sequence) 5'-CUACAAAGGGAAGCCCUUUC-3'

[0051] In the present invention, the "polynucleotide encoding miR-520d-5p polynucleotide" refers to a polynucleotide that expresses a polynucleotide having the base sequence (a1) or (a2), i.e., that can produce a polynucleotide having the base sequence (a1) or (a2) by transcription, typically an expression vector (expression plasmid, viral vector, etc.). The expression vector may be double-stranded or single-stranded, and may be DNA or RNA. The expression vector may exist temporarily or persistently in the nucleus or cytoplasm while being replicated, or may exist permanently by being integrated into genomic DNA.

[0052] In various embodiments, a "polynucleotide" may be composed of nucleotides constituting natural RNA or DNA molecules, i.e., adenine (A), guanine (G), cytosine (C), uracil (U) and thymine (T), or may be composed of nucleotides, nucleotide analogs, nucleotide derivatives and other nucleotide equivalents. For example, a polynucleotide may contain a nucleotide analog or a nucleotide derivative for the purpose of improving the nuclease resistance of the polynucleotide, stabilizing the polynucleotide, increasing the affinity with a complementary strand nucleic acid, avoiding off-target effects, increasing the efficiency of delivery to a target cell or cell permeability, or for visualization.

[0053] Nucleotide analogs can be naturally occurring or non-naturally occurring molecules and examples include nucleoside modified nucleotide analogs, sugar moiety modified nucleotide analogs, and phosphodiester linkage modified nucleotide analogs.

[0054] Specific examples of nucleoside-modified nucleotide analogues include nucleotide derivatives in which the nucleoside moiety (purine ring, pyrimidine ring) is substituted with a group derived from an aromatic compound such as a benzene derivative or a pyridine derivative.

[0055] Specific examples of sugar-modified nucleotide analogues include nucleotide analogues in which the sugar moiety is substituted with 2'-O-methylribose, 2'-O-propylribose, 2'-methoxyethoxyribose, 2'-O-methoxyethylribose, 2'-O-[2-(guanidinium)ethyl]ribose or 2'-fluororibose; nucleotide analogues in which the sugar moiety is substituted with a morpholino ring; bridged nucleotide analogues (BNAs) in which a bridged structure has been introduced into the sugar moiety, such as locked nucleic acid (LNA) having a structure in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged with a methylene group, and ethylene-bridged artificial nucleic acid (ENA) having a structure in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged with an ethylene group.

[0056] Specific examples of phosphodiester bond modified nucleotide analogs include those in which the phosphodiester bond is replaced with a phosphorothioate bond or an N3'-P5' phosphoamidate bond.

[0057] Specific examples of nucleotide derivatives include molecules obtained by adding another chemical substance to a nucleotide, such as DNA-added derivatives, 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0058] In various embodiments, the miR-520d-5p polynucleotide may be a polynucleotide consisting of nucleotides corresponding to the base sequence (a1) or (a2), or may be a polynucleotide containing nucleotides corresponding to the base sequence (a1) or (a2) and nucleotides having another base sequence. For example, when the miR-520d-5p polynucleotide is in the form of a mature miRNA or Pre-miRNA described below, the miR-520d-5p may contain a portion consisting of nucleotides corresponding to the base sequence (a1) or (a2) and a portion consisting of nucleotides corresponding to another base sequence contained in the mature miRNA or Pre-miRNA.

[0059] A polynucleotide consisting of nucleotides corresponding to the base sequence (a1) or (a2) is incorporated into the RISC complex as a single-stranded miRNA and degrades an mRNA having a base sequence 100% complementary to the single-stranded miRNA. Alternatively, the polynucleotide inhibits the translation of an mRNA having a base sequence incompletely complementary to the single-stranded miRNA. Based on online algorithmic analysis (miRBase - http: / / www.mirbase.org / ; RNAcentral - https: / / rnacentral.org / , TargetScanHuman - http: / / www.targetscan.org / vert_72 / ), the polynucleotide having the sequence of SEQ ID NO: 1 (natural miR-520d-5p) is predicted to bind to more than 9000 mRNAs.

[0060] In various embodiments, the "miR-520d-5p polynucleotide" may be a double-stranded polynucleotide (mature miRNA) composed of (b1) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2 (guide strand sequence), or (b2) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2 with several bases deleted, substituted, inserted or added, and (c1) a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 (passenger strand sequence), or (c2) a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 with several bases deleted, substituted, inserted or added. The "several" in (b2) and (c2) is preferably 1 to 3, more preferably 1 to 2, and even more preferably 1, respectively.

[0061] SEQ ID NO:2 (Guide strand sequence) 5'-UCUACAAAGGGAAGCCCUUUCUG-3' SEQ ID NO:3 (Passenger strand sequence) 5'-AAAGUGCUUCUCUUUGGUGGGU-3'

[0062] In various embodiments, the "miR-520d-5p polynucleotide" is (d1) a polynucleotide having the nucleotide sequence of SEQ ID NO: 4 (Pre-miRNA sequence), or (d2) a nucleotide sequence in which several nucleotides have been deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 4. The "several nucleotides" in the nucleotide sequence (d2) is preferably 1 to 6 nucleotides, more preferably 1 to 4 nucleotides, even more preferably 1 to 2 nucleotides, and particularly preferably 1 nucleotide.

[0063] SEQ ID NO: 4 (Pre-miRNA sequence) 5'-UCUCAAGCUGUGAGUCUACAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCUCUUUGGUGGGUUACGGUUUGAGA-3'

[0064] The base length of miR-520d-5p polynucleotide is not particularly limited, and can be adjusted within the range that exerts the action effect. For example, when miR-520d-5p polynucleotide is composed of nucleotides corresponding to the nucleotide sequence (a1), (b1), (c1), or (d1), the polynucleotide may have a base length of 20, 23, 22, or 87 as per the respective nucleotide sequences, and when composed of nucleotides corresponding to the nucleotide sequence (a2), (b2), (c2), or (d2), the polynucleotide may have a base length of 20, 23, 22, or 87 with a number of deletions, substitutions, insertions, or additions.

[0065] --Compounds for upregulating miR-520d-5p-- In this specification, the term "miR-520d-5p upregulating compound" refers to a compound that has the effect of enhancing (upregulating) the expression of miR-520d-5p polynucleotide in cells. The compound having such an effect is not particularly limited, and can be selected from various compounds (preferably compounds that have already been used as medicamentously active compounds for other indications and are considered to be relatively safe) and used in the present invention. The compound for upregulating miR-520d-5p may be used alone or in combination of two or more.

[0066] Specific examples of compounds for upregulating miR-520d-5p include the following 13 compounds: vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacrynic acid, bisoprolol, candesartan, alexidine, bumetanide, and glycopyrrolate. Any one of these 13 compounds may be used, or two or more may be used in combination.

[0067] The compound for upregulating miR-520d-5p may form a pharma- ceutically acceptable salt. The pharma- ceutically acceptable salt may be selected from, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, halides, etc., taking into consideration the chemical structure of the compound for upregulating miR-520d-5p. Examples of metal salts include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), and aluminum salts. Examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine. Examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, mesylic acid, tosylic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid. Examples of salts with basic amino acids include salts with arginine, lysine, or ornithine, and examples of salts with acidic amino acids include salts with aspartic acid or glutamic acid. Examples of halides include chloride, bromide, and iodide. A preferred salt of vancomycin is, for example, the hydrochloride. A preferred salt of metoprolol is, for example, the tartrate. A preferred salt of benzethonium is, for example, the chloride. A preferred salt of betaxolol is, for example, the hydrochloride. A preferred salt of colistin is, for example, the sulfate. A preferred salt of oxprenolol is, for example, the hydrochloride. A preferred salt of bisoprolol is, for example, the fumarate salt.

[0068] The compound for upregulating miR-520d-5p or its pharma- ceutically acceptable salt may be a solvate. The solvate may vary depending on the solvent in which the compound is dissolved or the environment in which the compound is placed (e.g., in air with a certain degree of humidity), and examples of the solvate include hydrates (monohydrate, dihydrate, trihydrate, etc.), ethanol solvates, and acetic acid solvates.

[0069] -Cell production method- In various embodiments, the method for producing cells includes (1) a step of culturing cells using a miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p (a miR-520d-5p-related substance) (hereinafter referred to as the "culturing step"), and (2) a step of confirming whether a mutation in a hair cycle-related gene in the cells has reverted to the wild type (hereinafter referred to as the "confirmation step").

[0070] The culture step can be performed by culturing target cells (hair cycle-related cells) in a medium containing a miR-520d-5p-related substance. The duration of the culture step can be adjusted within a range that provides the effect of returning the target cells to the wild type or partially returning them to the wild type, depending on the type of the target cells, and is not particularly limited. The duration of the culture step may be within a range defined by any lower limit and / or any upper limit selected from the group consisting of, for example, 1 day, 2 days, 4 days, 6 days, 7 days (1 week), 8 days, 10 days, 12 days, 14 days (2 weeks), 16 days, 18 days, 20 days, 21 days (3 weeks), 22 days, 24 days, 26 days, 28 days (4 weeks) and 30 days (which may or may not include the upper limit and / or the lower limit itself), for example, 6 days or 7 days (1 week) or more and 21 days (3 weeks) or less. The medium supplemented with the miR-520d-5p related substance may be used throughout the entire culture process, or the medium supplemented with the miR-520d-5p related substance may be used during part of the culture process, and the medium not supplemented with the miR-520d-5p related substance may be used during the remaining period of the culture process.

[0071] The concentration of the miR-520d-5p related substance in the culture medium during the culture process can be adjusted within a range that has the effect of restoring the miR-520d-5p to the wild type or partially restoring the miR-520d-5p related substance to the wild type, depending on the type of the miR-520d-5p related substance used, and is not particularly limited.

[0072] The type of medium, i.e., the basal medium, and the types and concentrations of components other than the miR-520d-5p-related substance added thereto can be appropriately adjusted depending on the type of target cells, etc., and are not particularly limited.

[0073] In the culture step, known means may be used to improve the efficiency of introduction of miR-520d-5p-related substances into target cells. For example, miR-520d-5p-related substances, particularly miR-520d-5p polynucleotides or polynucleotides encoding the same, can be introduced into target cells in the form of a plasmid vector by electroporation, microinjection, lipofection, or other transfection methods (e.g., by adding a lipofection reagent to the medium), or in the form of a viral vector by infecting cells.

[0074] The confirmation step can be carried out by determining the base sequence (target base sequence) of the hair cycle-related gene in the cell, comparing the target base sequence with the wild-type base sequence, and judging that the target base sequence has returned to the wild-type when the two base sequences match. The base sequence of the hair cycle-related gene can be determined using a known means. The wild-type base sequence can be referred to those registered in a database.

[0075] The hair cycle-related genes to be confirmed may be one type or two or more types depending on the purpose of the confirmation step and / or the cell production method, but may be all or a part of the hair cycle-related genes in the above-mentioned specific embodiment depending on the type of cell. In various embodiments, the confirmation step determines the proportion of hair cycle-related genes that have reverted to wild type among the preset hair cycle-related genes for the target cells obtained in the culture step, and confirms whether the proportion has reached a certain level.

[0076] Depending on the purpose of the confirmation step and / or the cell production method, the number of target cells may be one or more. In various embodiments, in the confirmation step, a part of the cell population obtained in the culture step is used as a sample, and the proportion of cells in which the mutation of the hair cycle-related gene has reverted to the wild type among the target cells contained therein (or the proportion of cells in which the proportion of hair cycle-related genes that have reverted to the wild type among the preset hair cycle-related genes has reached a certain level) is determined, and it is confirmed whether the proportion has reached a certain level.

[0077] The cell production method may include a step other than the culturing step and the recovery step, if necessary. An example of such an optional step is a step of recovering the cells in which the target nucleotide sequence has been restored to the wild-type nucleotide sequence (hereinafter referred to as the "recovery step").

[0078] In the recovery step, cells in which the target base sequence has reverted to the wild-type base sequence can be recovered using known means. Alternatively, the entire cell population in which the proportion of cells in which the mutation of the intracellular hair cycle-related gene has reverted to the wild-type has reached a certain level (which can be considered as such by measuring using a sample) may be recovered.

[0079] In one aspect, a method for restoring a hair cycle-related gene is provided, comprising culturing cells with a miR-520d-5p polynucleotide or a polynucleotide encoding the same, or a compound for upregulating miR-520d-5p (a miR-520d-5p-related substance) to revert a mutation in the hair cycle-related gene to a wild type.

[0080] -Treatment Methods / Compositions- In various embodiments, a method for treating a hair cycle-related disease or condition comprises administering (I) a miR-520d-5p polynucleotide or a polynucleotide encoding same, or a compound for upregulating miR-520d-5p (a miR-520d-5p related substance), or (II) a cell obtained by the method.

[0081] In various embodiments, the composition is used to treat a hair cycle-related disease or condition (i.e., to practice the treatment methods described hereinabove), and contains (I) a miR-520d-5p polynucleotide or a polynucleotide encoding same, or a compound for upregulating miR-520d-5p (a miR-520d-5p related substance), or (II) a cell obtained by the method described hereinabove.

[0082] By administering the miR-520d-5p-related substance (I), the action and effect of the miR-520d-5p-related substance to restore or partially restore the wild type in the hair cycle-related cells in vivo can be exerted, and hair cycle-related diseases or symptoms can be treated. In addition, hair cycle-related diseases or symptoms can also be treated by administering the cells (II) obtained by the method described above in this specification, that is, the cells that have already shown the effect of restoring the mutation of the cycle-related gene to the wild type. In the treatment method and composition of the present invention, as the miR-520d-5p-related substance (I), either one of the miR-520d-5p polynucleotide or the polynucleotide encoding it, and the compound for upregulating miR-520d-5p may be used, or both may be used (sequentially or simultaneously). Furthermore, either the miR-520d-5p-related substance (I) or the cells (II) obtained by the method described above in this specification may be used, or both may be used (sequentially or simultaneously).

[0083] "Hair cycle-related disease" refers to a disease related to the anagen, catagen, telogen or shedding phase of the hair cycle, particularly a disease related to the expression (inhibition or enhancement) of hair cycle-related genes in hair papilla cells, skin-derived precursor cells or bulge stem cells, or stem cells that can differentiate into said cells. The hair cycle-related disease that is the subject of symptom treatment by the present composition is not particularly limited, but a representative example is alopecia. Alopecia is generally a condition in which the anagen phase is shortened (for example, several months to a year) and the catagen and telogen phase are relatively long, resulting in hair loss, soft hair (thick and long hair does not regenerate, and most hair becomes thin and short), and hair fragility, and includes, for example, male pattern baldness, female pattern baldness, alopecia areata, trichotillomania, telogen baldness, hair loss due to endocrine abnormality, hair loss due to nutritional disorder, hair loss due to skin infection, hair loss due to skin tumor, cicatricial alopecia, hair loss due to drugs or chemicals, and various hair deformities.

[0084] As used herein, the term "treatment" refers to fully or partially therapeutically treating a hair cycle-related disease, fully or partially curing (alleviating or ameliorating) symptoms associated with a hair cycle-related disease, slowing the progression, delaying the onset, preventing, or reducing the risk of a hair cycle-related disease or a symptom thereof, or a combination thereof.

[0085] The subject of treatment may be a human or a non-human animal, and sex and age are not particularly limited. Examples of human subjects include patients with hair cycle-related diseases or patients who wish to prevent hair cycle-related diseases. Examples of non-human animals include non-human mammals such as mice, rats, rabbits, cows, monkeys, chimpanzees, pigs, horses, sheep, goats, dogs, cats, guinea pigs, and hamsters, and preferably mammals that can be models of hair cycle-related diseases. The subject of treatment may be a subject of non-clinical trials or clinical trials.

[0086] The dosage of the miR-520d-5p related substance or the cells obtained by the method described above in this specification can be adjusted within a range that exerts the action and effect of restoring the wild type or partially restoring the wild type, and is not particularly limited.

[0087] In various embodiments, the compound for upregulating miR-520d-5p can be administered in an amount that provides the maximum effective blood concentration shown in Table 1, or can be used as an external preparation having the concentration shown in Table 1. The maximum effective blood concentration or the concentration in the external preparation when the compound for upregulating miR-520d-5p is administered can be the same or lower (e.g., 100% or less, less than 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, 0.01% or less) than the maximum effective blood concentration or the concentration in the external preparation when the same compound is used as an active ingredient for other known indications.

[0088] [Table 1] JPEG2025501641000002.jpg9165

[0089] The amount (concentration) of the active ingredient contained in the composition, i.e., the miR-520d-5p-related substance or the cells obtained by the method described above in this specification, can be adjusted within a range in which the action and effect of returning to the wild type or partially returning to the wild type can be exerted, taking into consideration, for example, the administration form, administration route, number of administrations (frequency), etc., and is not particularly limited. The appropriate usage, dosage, etc. of the miR-520d-5p-related substance or the cells obtained by the method described above in this specification may be determined by non-clinical tests (cell tests, animal tests) and clinical tests.

[0090] The administration form and route of the composition can be appropriately selected. For example, topical preparations that are administered (applied) directly and transdermally or transcutaneously to the affected area (scalp, etc.), or injections that are administered locally, are suitable. By preparing the composition as topical preparations for transdermal administration, injections for local administration, etc., the active ingredient can be effectively applied to the affected area, and the effect of treatment can be exerted at a relatively low concentration, which is advantageous in terms of cost and avoids side effects that are a concern in the case of systemic administration forms containing high concentrations of active ingredients. On the other hand, if side effects are not substantially problematic, the composition may be administered in a systemic administration form such as by intravenous injection, or in an oral administration form.

[0091] Examples of dosage forms of topical skin preparations or topical mucosal preparations include liquids, sprays, ointments, creams, gels, patches, etc. When the topical skin preparation is a (medicinal) cosmetic, examples include hair care products such as shampoos, conditioners, and hair styling products (hair tonics, hair conditioners, hair treatments, hair lotions, hair creams, hair oils, hair gels, and hair sprays).

[0092] The injection may be intravenous, intraarterial, intramuscular, intradermal, subcutaneous, etc. When administering cells obtained by the methods described herein above, the composition may be administered as a cell preparation by infusion or injection.

[0093] Examples of dosage forms for oral preparations include tablets, capsules, granules, powders, fine granules, syrup, pills, and liquids, and the release may be controlled, such as sustained release.

[0094] Ingredients other than the active ingredient contained in the composition may include additives that are commonly used in the production of medicines, quasi-drugs, cosmetics, etc., preferably additives used in the production of external preparations, injections, oral preparations, etc., such as stabilizers, preservatives, buffers, pH adjusters, excipients, bases, etc. The content of such additives can be appropriately adjusted by those skilled in the art.

[0095] In addition to the treatment methods and compositions described above in this specification, it is also possible to apply known technical matters (preferred embodiments, etc.) regarding pharmaceuticals using nucleic acids such as miRNA, so-called oligonucleotide pharmaceuticals, etc.

[0096] For example, in the present composition, the miR-520d-5p-related substance is preferably in a state to which a technology related to a drug delivery system (DDS) used for injections, external preparations, etc. is applied. One specific example of DDS is nanoparticle formation, that is, encapsulation of a miR-520d-5p-related substance in nano-sized particles made of a specific compound to improve delivery efficiency to target cells. The nanoparticle may contain only a miR-520d-5p polynucleotide or a polynucleotide encoding the same, or only a compound for upregulating miR-520d-5p (e.g., benzethonium, etc.) as the miR-520d-5p-related substance, or may contain both a polynucleotide encoding a miR-520d-5p polynucleotide or a polynucleotide encoding the same and a compound for upregulating miR-520d-5p. Alternatively, the nanoparticle formulation may contain multiple types of nanoparticles, for example, nanoparticles containing miR-520d-5p polynucleotides or polynucleotides encoding the same, and nanoparticles containing compounds for upregulating miR-520d-5p. Various compounds constituting nanoparticles are known, and are not particularly limited, and examples thereof include biocompatible lipids (e.g., triglycerides, diglycerides, monoglycerides, fatty acids, steroids), surfactants (e.g., phospholipids), polymers (e.g., polylactic acid: PLA, polylactic acid-glycolic acid copolymer: PLGA, polyethylene glycol: PEG, etc.). By considering the type, solubility (water-soluble / liposoluble) and other properties of the miR-520d-5p-related substance, and using appropriate raw materials and manufacturing methods, nanoparticles having an appropriate encapsulation rate and an appropriate size (particle size distribution) can be manufactured.

[0097] In the present composition, the miR-520d-5p-related substance may be formulated in collagen, i.e., may be contained in a base (filler) containing collagen. In other words, the present composition may take the form of a collagen preparation containing a base (filler) containing a miR-520d-5p-related substance and collagen. The collagen preparation is gradually absorbed into the body over a certain period of time after intradermal or subcutaneous injection, so that the miR-520d-5p-related substance contained in the collagen can be gradually released. The collagen preparation may contain only a miR-520d-5p polynucleotide or a polynucleotide encoding the same, or only a compound for upregulating miR-520d-5p, or both a polynucleotide encoding a miR-520d-5p polynucleotide or a polynucleotide encoding the same and a compound for upregulating miR-520d-5p, as the miR-520d-5p-related substance (I). Furthermore, the collagen preparation may contain cells (II) obtained by the method described above in this specification. The base of the collagen preparation contains collagen such as type I collagen and type III collagen derived from humans or non-human animals (preferably human-derived when administered to humans), and optionally contains other components such as hyaluronic acid, gelatin (denatured collagen), etc.

[0098] The composition can be manufactured in various compositions according to classifications such as drugs, cosmetics, or quasi-drugs (medicated cosmetics), which are intermediate between the two, in accordance with the legal systems and guidelines of each country, and can claim various effects and efficacies.

[0099] In this specification, the description can be interpreted such that singular terms may be replaced with plural terms and vice versa, unless otherwise stated or obvious to one of ordinary skill in the art that such a substitution is not possible.

[0100] In this specification, "consisting of" means a so-called closed definition that does not include other elements, and "comprise," "contain," "include," etc. mean a so-called open definition that may include other elements. EXAMPLES

[0101] [Example 1] We targeted 178 hair cycle-related genes involved in the hair cycle, including the anagen, catagen, telogen, and shedding phases, and investigated whether mutations contained in genes of mesenchymal stem cells (MSCs) differentiated from iPSCs could be converted to wild type by introducing miR-520d-5p into iPSCs in vitro, i.e., whether the state of the genomic sequence could be improved, according to the following procedure. The 178 hair cycle-related genes were confirmed by NGS analysis (see Figure 1).

[0102] (1) Materials and methods Cell preparation Three cell lines and lentiviral vectors were used for DNA extraction. hiPSCs (HPS0002:253G1) and MSCs (hMSCs) were provided by the Riken BioResource Center Cell Bank (Nakagawa, M. et al., Generation of induced pluripotent stem cells without Myc from mouse 312 and human fibroblasts. Nat. Biotechnol. 26, 101-106 (2008)) and Takara Bio Development Laboratories, Biomedical Research Cell Resource Center (Kusatsu, Shiga, Japan), respectively. hiPSCs (253G1) and hMSCs were cultured in ReproStem medium (ReproCell, Tokyo, Japan) containing 10 ng / ml bFGF-2 and mesenchymal stem cell growth medium (Takara Bio, Kusatsu, Shiga, Japan), respectively. In addition, human mesangial cell line 293FT (Invitrogen Japan KK, Tokyo, Japan) was used to generate 520d-5p-expressing lentiviral particles for transfecting hMSCs. 293FT cells were cultured in DMEM supplemented with 10% FBS, 0.1 mM MEM non-essential amino acids, 2 mM L-glutamine, and 1% penicillin / streptomycin. hMSCs were also derived from iPSCs using the STEMdiff Mesenchymal Progenitor kit (STEMCELL Technologies, Seattle, WA, USA). hMSCs transfected with miR-520d-5p were defined as 520d / hMSCs.

[0103] Lentiviral vector constructs 293FT cells (5 x 10 6The effect of overexpression of miR-520d-5p was examined by transfecting 1000 cells / 10 cm culture dish with 20 μg of pMIRNA1-miR-520d-5p / GFP (System Biosciences, Mountain View, CA, USA) or mock vector pCDH. hMSCs with miR-520d-5p expression were investigated by centrifuging the cells at 170,000 × g for 120 min at 4 °C and recovering the viral particles produced in the culture medium of 293FT cells. The viral pellet was collected and incubated for 1 h at 37 °C for 24 h. TM Viral copy numbers were measured using a qRT-PCR titration kit (Clontech, Mountain View, CA, USA). Meanwhile, one million lentiviral copies per 10 cm culture dish were used for infection of 293FT cells. 293FT cells were transfected with 50 nM of synthetic oligonucleotides using FuGENE HD transfection reagent (Roche Diagnostics, Basel, Switzerland). Cells treated with pCDH / lenti / GFP were used as controls. Finally, 520d-5p-transfected hMSCs were generated by transfecting the hMSCs with 520d-5p lentivirus after inducing differentiation of iPSCs into hMSCs using System and Reagents for hiPSC-derived MSC Differentiation (Veritas Corporation, Tokyo, Japan).

[0104] Next-generation sequencing (NGS) analysis iPSCs (253G1), 520d-transfected 253G1-derived progenitor hMSCs (520d / hMSCs), and hMSCs were used for NGS analysis. DNA extraction was performed using the Qiagen DNeasy kit (QIAGEN, Tokyo, Japan) according to the manufacturer's instructions. Exome sequencing was performed using a next-generation sequencer (Illumina, Inc., San Diego, USA). Three replicates were analyzed per group (n = 3). Genomic DNA was processed using SureSelectXT Human All Exon v5 + UTR (Agilent Technologies, Inc.) and sequenced on an Illumina HiSeq2500 platform with 101bp paired-end reads (HSS, Sapporo, Japan). Paired-end reads in FastQ format were mapped to the reference genome HG19 using BWA-0.7.10. Mapping files in Sequence Alignment / Map (SAM) format were converted to BAM (binary version of SAM) format and sorted with SAMtools-1.2. Local realignment around known indels was performed on the sorted BAM files with GATK-Lite-2.3.0. PCR duplicates were removed with Picardtools-1.133 (Nielsen, R., Paul, JS, Albrechtsen, A., and Song, YS Genotype and SNP calling from next-generation sequencing data. Nat. Rev. Genet. 12, 443-451 (2011). Wysoker, A., Tibbetts, K., and Fennell, T. picardTools 1.5, vol. 3 (2011). Available from: http: / / sourceforge.net / projects / picard / files / picard-tools / .) Finally, a recalibration of base quality scores was performed using GATK. RNA sequencing was performed using a next-generation sequencer (Illumina).Total RNA was processed using the TruSeq Stranded mRNA Library Prep Kit (Illumina) and sequenced on an Illumina HiSeq2500 platform with 101 bp paired-end reads. Finally, the FastQ-formatted paired-end reads were mapped to the reference genome HG19 using TopHat.

[0105] Mutation analysis Samtools mpileup [options: -d 10000 -L 10000 -B -t DP,DV,SP,DP4,DPR] was piped through bcftools call [options: -A -v -m -f GQ] to generate variant calling format (VCF) files. VCF files were further filtered to select variants with at least two supported reads, a minimum allele frequency of 0.8, and a maximum read depth of 35 at the called site. For high throughput, deep sequencing of selected cDNA was performed by Sanger sequencing to screen for nonsynonymous mutations (Falk, MJ et al., Mitochondrial disease genetic diagnostics: optimized whole-exome analysis for all MitoCarta nuclear genes and the mitochondrial genome, Discov. Med. 14, 389-399(2012). Kawazu, M. et al., Transforming mutations of RAC guanosine triphosphatases in human cancers, Proc. Natl. Acad. Sci. US A. 110, 3029-3034(2013). Takahashi, Y., Mori, J. and Kami, M. BRAF mutations in hairy-cell leukemia, N. Engl. J. Med. 365, 960-961(2011).).

[0106] (2) Results and Discussion The results regarding the effect of converting the base sequence using miR-520d-5p targeting hair cycle-related genes are shown in Table 2.

[0107] For 142 of the 178 hair cycle-related genes, genomic conversion was observed, in which mutated bases (at least some of them) were restored to wild type by treating cells with miR-520d-5p. There were 26 genes in which all mutated bases were restored to wild type, i.e., the conversion rate was 100%. As an example, for the AR gene, 71.4% (5 out of 7 bases) of the bases mutated in iPSCs were reverted to wild type bases in 520d / hMSCs, but the repeat sequences (22 CAG repeats and 17 GGC repeats) contained in the AR gene were identical in the base sequences in 520d / hMSCs to those in iPSCs. In addition, conversion of consecutive 2, 3, or 4 bases was confirmed in 33, 8, and 1 genes, respectively (2bc, 3bc, or 4bc in Table 2).

[0108] On the other hand, for 36 of the 178 hair cycle-related genes (gray background in Table 2), no genomic conversion was observed to repair the mutated base to the wild type by treating cells with miR-520d-5p. However, for 6 of the 36 genes, the mutated base was not originally present in iPSCs (253G1) compared to the wild type.

[0109] Cells treated with pCDH / lenti / GFP, used as a control, did not induce any genomic transformation, and transfection with miR-520d-5p did not show any obvious adverse changes at the nucleotide level. The average transformation rate of 178 hair cycle-related genes was 54.4%.

[0110] [Table 2] JPEG2025501641000004.jpg252134JPEG2025501641000005.jpg250134JPEG2025501641000006.jpg245134

[0111] Next, the effects of converting the base sequences of 39 genes (see Non-Patent Document 1, Figure 2) associated with the growth or function of dermal papilla cells (DP cells) were analyzed in more detail using miR-520d-5p.

[0112] miR-520d-5p improved the sequence status of 35 of 39 genes in DP cells. Based on a target score of more than 50%, which corresponds to a 7-base match (complementary) with the mRNA sequence, miR-520d-5p is estimated to potentially target the mRNA of 15 genes, but miR-520d-5p improved the sequence of 13 of those 15 genes. Interestingly, although miR-520d-5p strongly targets the mRNA of the AR gene, AR and ARA55 (TGFB1-induced transcript 1) are overexpressed in the hair area or hair papilla of patients with androgenetic alopecia (AGA) and female pattern hair loss (FAGA), and it is thought that the function of AR due to its overexpression can be suppressed. At the same time, miR-520d-5p converts the mutations accumulated in the second exon of the AR gene to the wild type. In DR cells, the AR gene is an important factor related to (F)AGA and is regulated through various signaling pathways. The AR gene is promoted by EGF, TGFβ1, NCOA4 and RUNX1 signaling and inhibited by the Notch pathway (Non-Patent Documents 2 and 3), and it has been found that miR-520d-5p can improve the mutations of these genes.

[0113] The effect of converting the base sequences using miR-520d-5p was further analyzed in detail for 23 genes related to the growth or function of bulge stem cells (see FIG. 3). As a result, it was found that the sequence status of 21 of these genes was improved. Based on a target score of more than 50%, miR-520d-5p potentially targets the mRNA of three bulge stem cell-related genes, SOX9, THLH, and SMOC2, and suppresses or regulates the expression of these genes. SOX9, NFATC1, LHX2, and KRT15 are known as adult stem cell markers of bulge stem cells (see Non-Patent Documents 4-8), and it was found that miR-520d-5p can improve the mutations of these genes.

[0114] Based on the results of Example 1 above, it is supported that the action and effect of introducing the miR-520d-5p polynucleotide or a polynucleotide encoding it (or a compound for upregulating miR-520d-5p) into hair cycle-related cells such as hair papilla cells, skin-derived progenitor cells, and bulge stem cells can repair mutations in hair cycle-related genes or other genes expressed in these cells, and further suppress the expression of mRNA of some hair cycle-related genes.

[0115] [Example 2] We investigated whether the expression of miR-520d-5p could be upregulated by introducing miR-520d-5p upregulating compounds into normal human dermal fibroblasts (NHDFs) in vitro according to the following procedure.

[0116] Solutions were prepared using the test compounds and solvents shown in Table 3. The solutions were added to the medium so that the final concentrations in the medium were those shown in Table 3, and NHDF cells were cultured for 7 to 14 days. Adult NHDF cells were purchased from TAKARA BIO Inc. (Tokyo, Japan).

[0117] [Table 3]

[0118] The expression level of miR-520d-5p in the cultured NHDF was measured by adding a linker to the expressed miRNA (miR-520d-5p) and detecting it by RT-PCR using a specific primer set. The expression levels of miR-520d-5p of the test compounds 1 to 13 used were converted to relative values ​​when the expression level of miR-520d-5p of the control was set to 1.

[0119] The results are shown in FIG. 4. It can be seen that all of the 13 test compounds used in this example have the effect of upregulating miR-520d-5p in NDHF. Combining the results of Example 2 and Example 1, it can be estimated that even when a compound for upregulating miR-520d-5p is introduced into hair cycle-related cells, it will have the effect of returning the cells to the wild type or partially returning them to the wild type, that is, the expression of -520d-5p is enhanced, and as a result, the mutations of hair cycle-related genes and other genes expressed by hair cycle-related cells can be repaired. In addition, it can be estimated that when a compound for upregulating miR-520d-5p (the 13 test compounds and other compounds) is used to return a mutation of a hair cycle-related gene to the wild type or to treat a hair cycle-related disease or symptom, the effect of returning the cells to the wild type may be lower than when the compound is used as an active ingredient for other indications.

Claims

1. A miR-520d-5p polynucleotide or a polynucleotide encoding a miR-520d-5p polynucleotide, or a compound for upregulating a miR-520d-5p polynucleotide, for use in reverting a mutation in a hair cycle-related gene in a cell to a wild type; wherein the miR-520d-5p polynucleotide is (a1) the nucleotide sequence of SEQ ID NO: 1, or (a2) A nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 1 Includes.

2. The miR-520d-5p polynucleotide or a polynucleotide encoding the miR-520d-5p polynucleotide, or a compound for upregulating the miR-520d-5p polynucleotide according to claim 1, wherein the cell is a hair papilla cell, a skin-derived progenitor cell or a bulge stem cell, or a stem cell capable of differentiating into said cell.

3. The miR-520d-5p polynucleotide or the polynucleotide encoding the miR-520d-5p polynucleotide, or the compound for upregulating the miR-520d-5p polynucleotide according to claim 1, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 142 types of genes: ACT1, ALPL, ALX4, AQP3, AR, ASCL1, BCL2, BDNF, BMI1, BMP2, BMP4, BMP6, CCHCR1, CD271, CDKN1C, CDKN2A, Chr.20 1122 PAX1, CPE, CRHR2, CTGF, CTNNB1, CYP1B1, DDR2, DKK2, DKK4, DLL1, EDN3, EFNA, EGF, EGR1, EGR3, FBN2, FGF5, FGF7 (KGF), FGF18, FN1, FOXE1, FOXN1, FOXO3, FZD1, FZD4, FZD8, GADD45G, GATA3, GLI2, GLI3, GRID1, GUCY1A3, HES1, HES5, HEY1, HEY2, HEYL, HGF, HR (HAIRLESS), HSD3B1, IGF1, IRF4, ITGB1, JAG1, JAG2, KAT2A, KAT2B, KCNJ8 (KIR6), KDR, KRT5, KRT14, KRT15, ​​KRT19, LAMC3, LRIG1, LATS2, LEF1, LFNG, LGR5, LHX2, LMO1, LPL, LRP4, LRP5, MAPK1, NCAM1, NCOA3, NDP, NELL2, NFATC1, NFKB, NOG, NOS3, NOTCH2, NOTCH3, NOTCH4, NTRK, PDGFA, PTCH1, RBPJ, RGS2, RHOB, RUNX1, SDKN2A, SERPINE2, SHH, SIRT7, SLC25A37 (MSCP), SMAD4, SMAD6, SMO, SMOC2, SMTN, SOSTDC1, SOX2, SOX9, SPON1, SPRY2, SP5, SRD5A2, STAT3, STS, SUR2 (ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1 (TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

4. The miR-520d-5p polynucleotide or a polynucleotide encoding the miR-520d-5p polynucleotide, or a compound for upregulating the miR-520d-5p polynucleotide according to claim 1, which is used to revert a mutation of a hair cycle-related gene in a cell to a wild type and to suppress or regulate the expression of at least one gene selected from the group consisting of the following 18 types of genes: A.R., DIO2, EDN3, FGF7 (KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTH LH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

5. The cells are hair papilla cells or stem cells capable of differentiating into said cells, and The miR-520d-5p polynucleotide or a polynucleotide encoding the miR-520d-5p polynucleotide, or a compound for upregulating the miR-520d-5p polynucleotide according to claim 1, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 26 types of genes: ALX4, AR, BMP2, BMP4, CPE, EDN3, FGF7 (KGF), GUCY1A3, HEY1, HEY2, LAMC3, LEF1, LPL, LRP4, NDP, NOG, NTRK, SOSTDC1, SPON1, TFAP2A, TFAP2C, TRPS1, TWIST1, VAV3, VCAN and WNT5A.

6. The miR-520d-5p polynucleotide or a polynucleotide encoding the miR-520d-5p polynucleotide, or a compound for upregulating the miR-520d-5p polynucleotide according to claim 1, which is used to revert a mutation in a hair cycle-related gene in a cell to the wild type and to suppress or regulate the expression of at least one gene selected from the group consisting of VAV3, NTRK (SLITRK2, 4, 6), HEY2, RORA, VCAN, HEY1, AR, TRPS1, TWIST1, LEF1, EDN3, SOSTDC1, FGF7 (KGF), DIO2 and RNF144A.

7. The cells are bulge stem cells or stem cells capable of differentiating into such cells; and The miR-520d-5p polynucleotide or the polynucleotide encoding the miR-520d-5p polynucleotide, or the compound for upregulating the miR-520d-5p polynucleotide according to claim 1, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 18 types of genes: ASCL1, BMP2, DKK4, GADD45G, KRT15, LHX2, LMO1, LRP5, NFATC1, SHH, SMOC2, SOSTDC1, SOX2, SOX9, SP5, SRD5A2, STAT3, STS, SUR2 (ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1 (TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

8. The miR-520d-5p polynucleotide or a polynucleotide encoding the miR-520d-5p polynucleotide, or a compound for upregulating the miR-520d-5p polynucleotide according to claim 7, which is used to revert a mutation in a hair cycle-related gene in a cell to the wild type and to suppress or regulate the expression of at least one gene selected from the group consisting of PTHLH, SOX9 and SMOC2.

9. The cells are hair papilla cells or stem cells capable of differentiating into said cells, and bulge stem cells or stem cells capable of differentiating into said cells, The miR-520d-5p polynucleotide or the polynucleotide encoding the miR-520d-5p polynucleotide, or the compound for upregulating the miR-520d-5p polynucleotide according to claim 1, wherein the hair cycle-related gene is selected from the group consisting of the following two types of genes: BMP2 and SOSTDC1.

10. The miR-520d-5p polynucleotide is (b1) the nucleotide sequence of SEQ ID NO: 2, or (b2) a polynucleotide having a base sequence in which 1 to 3 bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2; and (c1) the nucleotide sequence of SEQ ID NO: 3, or (c2) A polynucleotide having a base sequence in which 1 to 3 bases have been deleted, substituted, inserted or added in the base sequence of SEQ ID NO:

3. or a double-stranded polynucleotide consisting of wherein the polynucleotide encoding the miR-520d-5p polynucleotide is (d1) the nucleotide sequence of SEQ ID NO: 4, or (d2) A base sequence in which 1 to 6 bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO:

4.

2. The miR-520d-5p polynucleotide according to claim 1, or a polynucleotide encoding the miR-520d-5p polynucleotide, or a compound for upregulating the miR-520d-5p polynucleotide.

11. The miR-520d-5p polynucleotide or the polynucleotide encoding the miR-520d-5p polynucleotide, or the compound for upregulating miR-520d-5p polynucleotide according to claim 1, wherein the compound for upregulating miR-520d-5p is selected from the group consisting of the following 13 types of compounds: Vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacrynic acid, bisoprolol, candesartan, alexidine, bumetanide and glycopyrrolate.

12. 1) culturing cells using a miR-520d-5p polynucleotide or a polynucleotide encoding a miR-520d-5p polynucleotide according to any one of claims 1 to 11, or a compound for upregulating a miR-520d-5p polynucleotide; and 2) confirming whether the mutation in the hair cycle-related gene in the cell has reverted to the wild type; A method for producing a cell comprising the steps of:

13. (I) a polynucleotide encoding the miR-520d-5p polynucleotide or a compound for upregulating the miR-520d-5p polynucleotide according to any one of claims 1 to 11, or (II) Cells produced by the method according to claim 12.

1. A composition for use in the treatment of a hair cycle-related disease or condition, comprising:

14. 14. The composition according to claim 13, which is used for the therapeutic treatment of alopecia.

15. 14. The composition of claim 13, wherein the compound for upregulating miR-520d-5p is administered to achieve the following maximum effective blood concentrations or applied topically at the following concentrations: Vancomycin: administered, 30 μg / ml or less Metoprolol: administered, 41.8 ng / ml or less Benzethonium: For external use, 0.2% or less Chlorpropamide: Administration, 30 μg / ml or less Betaxolol: administered, 41.8 ng / ml or less Colistin: Administration, 4.4 μg / ml or less Oxprenolol: Administration, 30.18 μg / ml or less Ethacrynic acid: Administration, 100 μg / ml or less Bisoprolol fumarate: administered, 100 ng / ml or less Candesartan: administered, 75 ng / ml or less Alexidine: Administration, 1 mM or less Bumetanide: Administration, 0.4 μg / ml or less Glycopyrrolate: Administer, below 800 pg / ml.

16. A method of reverting a mutation in a hair cycle-related gene to wild type in a cell of a patient in need thereof, comprising administering to the patient in need thereof: a) a miR-520d-5p polynucleotide; b) a polynucleotide encoding a miR-520d-5p polynucleotide; or c) Compounds for upregulating miR-520d-5p administering wherein the miR-520d-5p polynucleotide is: (a1) the nucleotide sequence of SEQ ID NO: 1; or (a2) A nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 1 A method comprising:

17. 17. The method of claim 16, wherein the cells of a patient in need thereof are dermal papilla cells, skin-derived progenitor cells, or bulge stem cells, or stem cells capable of differentiating into said cells.

18. The method according to claim 16, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 142 genes: ACT1, ALPL, ALX4, AQP3, AR, ASCL1, BCL2, BDNF, BMI1, BMP2, BMP4, BMP6, CCHCR1, CD271, CDKN1C, CDKN2A, Chr.20 1122 PAX1, CPE, CRHR2, CTGF, CTNNB1, CYP1B1, DDR2, DKK2, DKK4, DLL1, EDN3, EFNA, EGF, EGR1, EGR3, FBN2, FGF5, FGF7 (KGF), FGF18, FN1, FOXE1, FOXN1, FOXO3, FZD1, FZD4, FZD8, GADD45G, GATA3, GLI2, GLI3, GRID1, GUCY1A3, HES1, HES5, HEY1, HEY2, HEYL, HGF, HR (HAIRLESS), HSD3B1, IGF1, IRF4, ITGB1, JAG1, JAG2, KAT2A, KAT2B, KCNJ8 (KIR6), KDR, KRT5, KRT14, KRT15, ​​KRT19, LAMC3, LRIG1, LATS2, LEF1, LFNG, LGR5, LHX2, LMO1, LPL, LRP4, LRP5, MAPK1, NCAM1, NCOA3, NDP, NELL2, NFATC1, NFKB, NOG, NOS3, NOTCH2, NOTCH3, NOTCH4, NTRK, PDGFA, PTCH1, RBPJ, RGS2, RHOB, RUNX1, SDKN2A, SERPINE2, SHH, SIRT7, SLC25A37 (MSCP), SMAD4, SMAD6, SMO, SMOC2, SMTN, SOSTDC1, SOX2, SOX9, SPON1, SPRY2, SP5, SRD5A2, STAT3, STS, SUR2 (ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1 (TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

19. The method according to claim 16, wherein the administration of a miR-520d-5p polynucleotide or a polynucleotide encoding a miR-520d-5p polynucleotide or a compound for upregulating a miR-520d-5p polynucleotide suppresses or regulates the expression of at least one gene selected from the group consisting of the following 18 genes: A.R., DIO2, EDN3, FGF7 (KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTH LH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

20. The cells of the patient in need thereof are hair papilla cells or stem cells capable of differentiating into said cells; and The method according to claim 16, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 26 types of genes: ALX4, AR, BMP2, BMP4, CPE, EDN3, FGF7 (KGF), GUCY1A3, HEY1, HEY2, LAMC3, LEF1, LPL, LRP4, NDP, NOG, NTRK, SOSTDC1, SPON1, TFAP2A, TFAP2C, TRPS1, TWIST1, VAV3, VCAN and WNT5A.

21. The method of claim 16, wherein administration of a miR-520d-5p polynucleotide or a polynucleotide encoding a miR-520d-5p polynucleotide or a compound for upregulating a miR-520d-5p polynucleotide suppresses or regulates expression of at least one gene selected from the group consisting of VAV3, NTRK (SLITRK2, 4, 6), HEY2, RORA, VCAN, HEY1, AR, TRPS1, TWIST1, LEF1, EDN3, SOSTDC1, FGF7 (KGF), DIO2 and RNF144A.

22. the cells of a patient in need thereof are basal stem cells or stem cells capable of differentiating into said cells; and The method according to claim 16, wherein the hair cycle-related gene is at least one gene selected from the group consisting of the following 18 types of genes: ASCL1, BMP2, DKK4, GADD45G, KRT15, LHX2, LMO1, LRP5, NFATC1, SHH, SMOC2, SOSTDC1, SOX2, SOX9, SP5, SRD5A2, STAT3, STS, SUR2 (ABCC9), TCF7, TFAP2A, TFAP2C, TGFB1, TGFB2, TGFB1l1, THBS1 (TSP1), TIMP2, TRPS1, TWIST1, TWIST2, TYRO3, VAV3, VCAN, VDR, VEGFA, VIM, VSIG8, WNT4, WNT5A, WNT7B, WNT9B and WNT10B.

23. The method of claim 22, wherein administration of a miR-520d-5p polynucleotide or a polynucleotide encoding a miR-520d-5p polynucleotide or a compound for upregulating a miR-520d-5p polynucleotide suppresses or regulates expression of at least one gene selected from the group consisting of PTHLH, SOX9 and SMOC2.

24. The cells of a patient in need thereof include hair papilla cells or stem cells capable of differentiating into said cells, and bulge stem cells or stem cells capable of differentiating into said cells, and The method according to claim 16, wherein the hair cycle-related gene is selected from the group consisting of the following two types of genes: BMP2 and SOSTDC1.

25. The miR-520d-5p polynucleotide is (b1) the nucleotide sequence of SEQ ID NO: 2, or (b2) a polynucleotide having a base sequence in which 1 to 3 bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO: 2; and (c1) the nucleotide sequence of SEQ ID NO: 3, or (c2) A polynucleotide having a base sequence in which 1 to 3 bases have been deleted, substituted, inserted or added in the base sequence of SEQ ID NO:

3. or (d1) the nucleotide sequence of SEQ ID NO: 4, or (d2) A nucleotide sequence in which 1 to 6 nucleotides are deleted, substituted, inserted or added in the nucleotide sequence of SEQ ID NO: 4 is a polynucleotide having the formula: The method of claim 16.

26. The method of claim 1, wherein the compound for upregulating miR-520d-5p is selected from the group consisting of the following 13 compounds: Vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacrynic acid, bisoprolol, candesartan, alexidine, bumetanide and glycopyrrolate.

27. In the manufacture of a medicine for treating a hair cycle-related disease or condition a) a miR-520d-5p polynucleotide; b) a polynucleotide encoding a miR-520d-5p polynucleotide; or c) Compounds for upregulating miR-520d-5p Use of wherein the miR-520d-5p polynucleotide is (a1) the nucleotide sequence of SEQ ID NO: 1, or (a2) Includes a base sequence in which 1 to 3 bases are deleted, substituted, inserted or added in the base sequence of SEQ ID NO:

1.

28. Use of a miR-520d-5p polynucleotide or a polynucleotide encoding a miR-520d-5p polynucleotide or a compound for upregulating a miR-520d-5p polynucleotide according to any one of claims 1 to 11 in the manufacture of a medicament for treating a hair cycle-related disease or condition.

29. 29. The use according to claim 27 or 28, wherein the hair cycle related disorder comprises alopecia.

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