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

Abstract

The present invention provides a composition for treating diseases or symptoms related to the hair cycle (such as hair cycle-related diseases). [Solution] A composition for treating alopecia comprising a compound for miR-520d-5p polynucleotide upregulation, wherein the compound for miR-520d-5p polynucleotide upregulation is selected from the group consisting of vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacrine, bisoprolol, alexidine, bumetanide, and glycopyrrolate. The compound for miR-520d-5p polynucleotide upregulation can revert hair cycle-related genes to the wild type in dermal papilla cells, skin-derived precursor cells, bulge stem cells, or stem cells such as iPS cells that can differentiate into such cells.

Description

Technical Field

[0001] Reference to Related Applications This application claims priority to Singapore Patent Application No. 10202114489Q, filed on December 29, 2021, the content of which is incorporated herein by reference.

[0002] The present invention relates to miR-520d-5p polynucleotides and compounds for upregulating miR-520d-5p, particularly their novel uses. The present invention also relates to methods and compositions for treating diseases or conditions related to the hair cycle (e.g., alopecia).

Background Art

[0003] In WO2012 / 008302A, the inventor disclosed that by using miR-520d-5p, it is possible to induce somatic cells or malignant tumor cells into pluripotent stem cells (i.e., induction method or inducer), and it is also possible to treat malignant tumors (i.e., treatment method or therapeutic agent).

[0004] In WO2017 / 073689A, the inventor disclosed that by using miR-520d-5p, it is possible to improve DNA damage caused by ultraviolet rays (i.e., composition for improving DNA damage or composition for improving ultraviolet damage), and it is possible to suppress the decrease in collagen production due to ultraviolet rays (i.e., composition for promoting collagen production or composition for suppressing the decrease in collagen production).

[0005] In WO2018 / 203553A, the inventor disclosed that by using miR-520d-5p, it is possible to improve the quality of differentiated cells obtained from stem cells such as iPS cells (i.e., method for improving the quality of differentiated cells).

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

[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 dermal papillary cells (and those related to their proliferation or function), as well as signaling pathways. Furthermore, the following references describe genes expressed in bulge stem cells (and functioning as cellular markers for them): 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).

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

[0009] While various means (treatment methods, treatment compositions, etc.) have been proposed to treat diseases or symptoms related to the hair cycle (e.g., alopecia), there is still a need for novel methods to meet the demands of patients who do not achieve sufficient results with known means.

[0010] The object of this invention is to provide a novel means for treating diseases or symptoms related to the hair cycle (such as hair cycle-related diseases).

[0011] The inventors have discovered that by using miR-520d-5p, mutations in hair cycle-related genes (hair cycle-related genes) in hair cycle-related cells (hair cycle-related cells) can be restored to the wild type. The inventors have also discovered that several compounds have the effect of upregulating the expression of miR-520d-5p, and that mutations in hair cycle-related genes can also be restored to the wild type by using such miR-520d-5p upregulating compounds. Furthermore, it has been found that miR-520d-5p is presumed to suppress or regulate the expression of several hair cycle-related genes or other genes in hair cycle-related cells. Accordingly, the inventors have discovered that miR-520d-5p polynucleotide, or the polynucleotide encoding it, or compounds for upregulating miR-520d-5p can be used as activity enhancers for treating hair cycle-related diseases, etc., and have completed the present invention.

[0012] In other words, the present invention provides at least the following:

[0013] [Section 1] In one aspect, The following are provided: a miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation, which is used to revert mutations in intracellular hair cycle-related genes back to the wild type. Here, polynucleotides are, (a1) The base sequence of Sequence 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 Sequence ID No. 1. Includes. Sequence ID 1 (single-stranded miRNA sequence) 5'-CUACAAAGGGAAGCCCUUUC-3'

[0014] [Section 2] The cells are dermal papilla cells, skin-derived progenitor cells, or bulge stem cells, or stem cells capable of differentiating into such cells, as described in item 1, the miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation.

[0015] [Section 3] The hair cycle-related gene is at least one gene selected from the group consisting of the following 142 genes, the miR-520d-5p polynucleotide or the polynucleotide encoding it, or the miR-520d-5p upregulating compound described in item 1 or 2: 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] [Item 4] Used to revert the mutations of the hair cycle-related genes in cells to the wild type and suppress or regulate the expression of at least one gene selected from the group consisting of the following 18 genes, the miR-520d-5p polynucleotide according to any one of Items 1 to 3 or the polynucleotide encoding the same, or a compound for upregulating miR-520d-5p: AR, DIwO2, EDN3, FGF7 (KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTHLH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

[0017] [Item 5] The cell is a dermal papilla cell or a stem cell that can differentiate into such a cell, and The hair cycle-related gene is at least one gene selected from the group consisting of the following 26 genes, the miR-520d-5p polynucleotide or the polynucleotide encoding the same according to any one of Items 1 to 4, or the compound for upregulating miR-520d-5p: 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] [Item 6] A miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation, as described in any one of items 1 to 5, used to revert intracellular hair cycle-related gene mutations 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 aforementioned cells are bulge stem cells or stem cells capable of differentiating into such cells, and The hair cycle-related gene is at least one gene selected from the group consisting of the following 18 genes, and is a miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation, as described in any one of items 1 to 6: 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] A miR-520d-5p polynucleotide or a polynucleotide encoding it, or a miR-520d-5p upregulating compound, used to revert intracellular hair cycle-related gene mutations 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, as described in any one of items 1 to 7.

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

[0022] [Section 10] The aforementioned miR-520d-5p polynucleotide, (b1) The base sequence of Sequence ID No. 2, or (b2) Polynucleotides having a base sequence in which 1 to 3 bases are deleted, substituted, inserted, or added in the base sequence of Sequence ID No. 2, (c1) The base sequence of Sequence ID No. 3, or (c2) A double-stranded polynucleotide composed of a polynucleotide having a base sequence in which 1 to 3 bases are deleted, substituted, inserted, or added in the base sequence of Sequence ID No. 3, or (d1) The base sequence of Sequence 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 Sequence ID No. 4. The miR-520d-5p polynucleotide described in item 1, or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation. Sequence ID 2 (guide strand sequence) 5'-UCUACAAAGGGAAGCCCUUUCUG-3' Sequence ID 3 (Passenger Chain Sequence) 5'-AAAGUGCUUCUCUUUGGUGGGU-3' Sequence ID 4 (Pre-miRNA sequence) 5'-UCUCAAGCUGUGAGUCUACAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCUCUUUGGUGGGUUACGGUUUGAGA-3'

[0023] [Section 11] The aforementioned miR-520d-5p upregulation compound is selected from the group consisting of the following 13 compounds, i.e., the miR-520d-5p polynucleotide or the polynucleotide encoding it, or the miR-520d-5p upregulation compound described in any one of items 1 to 10: Vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacrine, bisoprolol, candesartan, alexidine, bumetanide, and glycopyrrolate.

[0024] [Section 12] In another embodiment (1) A step of culturing cells using the miR-520d-5p polynucleotide or a polynucleotide encoding it as described in any one of items 1 to 11, or a compound for miR-520d-5p upregulation, and (2) A step to confirm whether the mutation in the hair cycle-related gene within the cell has reverted to the wild type. A method for producing cells, including the following, is provided.

[0025] [Section 13] In another embodiment (I) The miR-520d-5p polynucleotide or a polynucleotide encoding it as described in any one of items 1 to 11, or a compound for miR-520d-5p upregulation, (II) Cells produced by the method described in claim 12. A composition is provided, which includes, for use in treating hair cycle-related diseases or symptoms.

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

[0027] [Section 15] Each of the miR-520d-5p upregulating compounds is administered to the following maximum effective blood concentration, or applied topically to the following concentrations, in the composition according to item 13 or 14: Vancomycin: Administered at a dose of 30 μg / ml or less. Metoprolol: Administered, ≤ 41.8 ng / ml Benzethonium: For external use, 0.2% or less Chlorpropamide: Administered at a dose of 30 μg / ml or less. Betaxolol: Administered, 41.8 ng / ml or less Colistin: Administered at a dose of 4.4 μg / ml or less. Oxprenolol: Administered at a dose of 30.18 μg / ml or less. Ethacrine: Administered at a dose of 100 μg / ml or less. Bisoprolol: Administration, 100 ng / ml or less Candesartan: Administered, 75 ng / ml or less Alexidine: Bumetanide: Administered at a dose of 0.4 μg / ml or less. Glycopyrrolates: Administered at a concentration of 800 pg / ml or less.

[0028] Those skilled in the art should note that the categories of the above inventions or other inventions included herein can be changed to create different inventions. For example, “miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation, for use in returning mutations in hair cycle-related genes to the wild type within cells” may be changed to the invention of “a method for returning mutations in hair cycle-related genes within cells to the wild type using miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation.” Similarly, “miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation, or cells produced by the method of the present invention” may be changed to the invention of “a method for treating hair cycle-related diseases or symptoms using (e.g., administering) miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation, or cells produced by the method of the present invention.” A person skilled in the art can read the matters relating to the original invention described herein as matters relating to the modified invention. [Effects 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 created from somatic cells and mesenchymal stem cells derived from iPS cells are accompanied by many mutations and epigenetic modifications. By using miR-520d-5p, it is possible to revert hair cycle-related genes in these cells to the wild type and normalize the function of hair cycle-related cells. 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 with complementary base sequences, such as 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 miR-520d-5p upregulating compound according to the present invention, one of the above effects, preferably a synergistic effect of both, can be shown to have a favorable effect in treating hair cycle-related diseases (e.g., alopecia). [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows an overview of 178 hair cycle-related genes that are expressed during the growth, regression, resting, and shedding phases of the hair cycle. [Figure 2] Figure 2 shows an overview of the genes expressed in dermal papilla cells (DP cells) in Example 1. [Figure 3] Figure 3 shows an overview of the genes expressed in bulge stem cells in Example 2. [Figure 4] Figure 4 is a graph showing the expression levels induced by the miR-520d-5p upregulating compound in Example 2. [Modes for carrying out the invention]

[0031] In various embodiments, miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation (collectively referred to herein as "miR-520d-5p-related substances") are used to revert mutations in intracellular hair cycle-related genes to the wild type.

[0032] Using miR-520d-5p-related substances means increasing the expression level of miR-520d-5p in cells beyond the normal level (when miR-520d-5p-related substances are not used). Depending on the amount of miR-520d-5p-related substance introduced into the cells, the expression level of miR-520d-5p can be increased to a desired level. By using miR-520d-5p-related substances, 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, and 20 times (2000%). As mentioned above, the levels can be 30 times (3000%) or more, 40 times (4000%) or more, or 50 times (5000%) or more compared to normal levels.

[0033] In this invention, the term "hair cycle-related gene" refers to a gene that is expressed in cells related to the hair cycle (hair cycle-related cells) and is related to the hair cycle. The hair cycle includes the growth phase (a period in which hair matrix cells divide actively and hair grows rapidly, usually 2 to 6 years), the regression phase (a period in which the function of dermal papilla cells that support the division of hair matrix cells decreases, and dermal papilla cells separate from hair matrix cells, slowing down hair growth, usually 2 to 3 weeks), the resting phase (a period in which dermal papilla cells shrink and hair does not grow, usually 3 to 4 months), and the shedding phase (a period in which hair falls out, usually 2 to 5 months). Hair cycle-related genes are not particularly limited as long as the effect of returning the mutation to the wild type is achieved by miR-520d-5p related substances.

[0034] In various embodiments, 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 capable of differentiating 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 retain the potential to differentiate into other cell lineages important for promoting hair growth, such as blood cells, fibroblasts, adipocytes, and nerve cells. Hair cycle-related cells may consist of one or more types of cells.

[0035] The cells targeted for reverting mutations in hair cycle-related genes back to the wild type (hair cycle-related cells) may be those present in vivo or ex vivo (in vitro). For example, when performing the treatment method of the present invention (details described separately), the target cells are those present in vivo, while when performing the cell production method of the present invention (details described separately), the target cells are those present ex vivo.

[0036] In various embodiments, the hair cycle-related gene whose mutation is repaired is at least one gene selected from the group of 142 genes listed below: 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] As shown in Table 2 of Example 1 below, there are 178 types of genes related to the hair cycle. In other embodiments, at least one gene selected from the following 36 types of genes (genes with a gray background in Table 2), which include the above 178 types of genes but do not include the above 142 types of genes, may be targeted for mutation repair. ANGPT, APOE, ASCL4, BAX, CD200, CD34, CDH1, CDH3, CLCA2, CSTA, DIO2, DKK1, EDA2R, EDAR, ENG (CD105), FGF2, GLI1, GPRC5B, IFI™3, IL6, LAMA5 MKI67(Ki67) NANOGNB (HOXC14), NCOA4 PRSS53, PTGDS, PTHLH, RNF144A, SHISA2, SPECC2, SPP1, SPRY1, SRD5A1, STAR, STAT2 and WIF1.

[0038] In various embodiments, miR-520d-5p-related substances can repair mutations in hair cycle-related genes and repress or regulate the expression of genes (mRNA) that contain a nucleotide sequence with certain complementarity to miR-520d-5p, among genes expressed in cells, preferably hair cycle-related cells. The genes repressed or regulated by miR-520d-5p can be estimated using known miRNA target search databases or websites (e.g., miRBase, EumiR, miRanda, PicTar, TargetScanHuman 7.0). For example, genes with target scores of 50 or higher, 60 or higher, 70 or higher, 80 or higher, or 90 or higher may also be targeted. The genes whose expression is repressed or regulated may be hair cycle-related genes or other genes, for example, at least one gene selected from the group consisting of the following 18 genes: AR, DIO2, EDN3, FGF7(KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTHLH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

[0039] "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 miR-520d-5p related substance introduced into the cell, the expression level of the target gene can be suppressed to a desired degree. 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, "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 the cell, the expression level of the target gene can be adjusted to a desired extent. For example, it can be set to ±100%, ±50%, ±20%, ±10%, or ±5% or less compared to the expression level in normal cells. An embodiment in which miR-520d-5p (related substance) is applied to cells in which the target gene is overexpressing, thereby "suppressing" (reducing) gene expression and maintaining the intracellular expression level within a certain range, is equivalent to "suppressing or regulating" gene expression with 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 such 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 26 genes mentioned above, there are seven hair cycle-related genes (APOE, CLCA2, CSTA, DIO2, GPRC5B, RNF144A, WIF1) that are expressed in dermal papilla cells. In another embodiment, for example, when targeting cells cultured under different conditions than those disclosed herein and having different mutations, at least one of these seven genes may be targeted for mutation repair.

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

[0043] Furthermore, in the above embodiment where dermal papilla cells are the target cells, by using miR-520d-5p, it is possible to repair mutations in specific genes while simultaneously suppressing or regulating the expression of specific genes (mRNAs) among the genes expressed in dermal papilla cells. Examples of genes expressed in dermal papilla cells that may be subject to such 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 undergo both mutation repair and suppression or regulation of expression by miR-520d-5p. Among these, AR is an important hair cycle-related gene expressed in dermal papilla cells because it is overexpressed in male pattern baldness (AGA) and female pattern baldness (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 such 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 18 genes mentioned above, there are five other hair cycle-related genes (CDH3, EDAR, IFITM3, PTHLH, and WIF1) that are expressed in bulge stem cells. In another embodiment, for example, when targeting cells cultured under different conditions than those disclosed herein and having different mutations, at least one of these five genes may be targeted for mutation repair.

[0046] Furthermore, in the above embodiment using bulge stem cells as target cells, by using miR-520d-5p, it is possible to repair mutations in specific genes while simultaneously suppressing or regulating the expression of specific genes (mRNAs) among the genes expressed in dermal papilla cells. Examples of genes expressed in bulge stem cells that may be subject to such 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 may undergo both mutation repair and suppression or regulation of expression 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 such cells, and bulge stem cells or stem cells capable of differentiating into such 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 mutations in hair cycle-related genes to the wild type" means repairing the base sequence of a hair cycle-related gene that is different from the wild type (i.e., mutated) within a cell to be the same as the base sequence of the wild type. The number of mutated bases may be within a range determined by any lower and / or upper limit selected from the group consisting of 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 (the upper and / or lower limits themselves may or may not be included), for example, between 1 base and 100 bases. The bases to be converted may be all or some of the mutated bases. For example, the conversion rate (the ratio of the number of converted bases to the number of mutated bases) may be within a range determined by any lower 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 and lower limits themselves may or may not be included), for example, 1% to 100%. Such effects may be observed in a single cell (e.g., a hair cycle-related cell) or in a certain percentage (preferably 50% or more) of cells in a cell population containing a certain number of cells.

[0049] —miR-520d-5p polynucleotide / the polynucleotide that codes for it— In this specification, "miR-520d-5p polynucleotide" may refer to a polynucleotide having (a1) the base sequence of SEQ ID NO: 1, or (a2) a base sequence in which several bases are deleted, substituted, inserted, or added to the base sequence of SEQ ID NO: 1. In base sequence (a2), "several" is preferably 1 to 3, more preferably 1 to 2, and even more preferably 1.

[0050] Sequence ID 1 (single-stranded miRNA sequence) 5'-CUACAAAGGGAAGCCCUUUC-3'

[0051] In this invention, "a polynucleotide encoding the miR-520d-5p polynucleotide" refers to a polynucleotide that expresses a polynucleotide having the base sequence (a1) or (a2), that is, a polynucleotide capable of producing 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 be present transiently or persistently while replicating in the nucleus or cytoplasm, or may be permanently present by being incorporated into genomic DNA.

[0052] In various embodiments, the “polynucleotide” may consist of nucleotides that make up natural RNA or DNA molecules, namely adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T), or it may consist of nucleotides and nucleotide analogs, nucleotide derivatives, and other nucleotide equivalents. For example, the polynucleotide may contain nucleotide analogs or nucleotide derivatives for purposes such as improving the nuclease resistance of the polynucleotide, stabilizing it, increasing its affinity with complementary nucleic acids, avoiding off-target effects, improving transport efficiency or cell permeability to target cells, or making it visible.

[0053] Nucleotide analogs can be either natural or unnatural molecules, and examples include nucleoside-modified nucleotide analogs, sugar-modified nucleotide analogs, and phosphate diester-modified nucleotide analogs.

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

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

[0056] Specific examples of phosphate diester bond-modified nucleotide analogs include nucleotide analogs in which the phosphate diester 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 base sequence (a1) or (a2), or it may be a polynucleotide containing nucleotides corresponding to base sequence (a1) or (a2) and nucleotides having a different base sequence. For example, if the miR-520d-5p polynucleotide takes the form of a mature miRNA or Pre-miRNA as described later, the miR-520d-5p may include a portion consisting of nucleotides corresponding to base sequence (a1) or (a2) and a portion consisting of nucleotides corresponding to a different base sequence contained in the mature miRNA or Pre-miRNA.

[0059] A polynucleotide consisting of nucleotides corresponding to base sequence (a1) or (a2) is incorporated into the RISC complex as a single-stranded miRNA and degrades mRNA with a base sequence 100% complementary to the single-stranded miRNA. Alternatively, the polynucleotide inhibits the translation of mRNA with a base sequence that is not completely 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 with sequence number 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 base sequence of SEQ ID NO: 2 (guide strand sequence), or (b2) a polynucleotide having a base sequence in which several bases are deleted, substituted, inserted, or added to the base sequence of SEQ ID NO: 2, and (c1) a polynucleotide having the base sequence of SEQ ID NO: 3 (passenger strand sequence), or (c2) a polynucleotide having a base sequence in which several bases are deleted, substituted, inserted, or added to the base sequence of SEQ ID NO: 3. In (b2) and (c2), "several" is preferably 1 to 3, more preferably 1 to 2, and even more preferably 1.

[0061] Sequence ID 2 (guide strand sequence) 5'-UCUACAAAGGGAAGCCCUUUCUG-3' Sequence ID 3 (Passenger Chain Sequence) 5'-AAAGUGCUUCUCUUUGGUGGGU-3'

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

[0063] Sequence ID 4 (Pre-miRNA sequence) 5'-UCUCAAGCUGUGAGUCUACAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCUCUUUGGUGGGUUACGGUUUGAGA-3'

[0064] The base length of the miR-520d-5p polynucleotide is not particularly limited and can be adjusted within a range that produces the desired effect. For example, if the miR-520d-5p polynucleotide consists of nucleotides corresponding to base sequences (a1), (b1), (c1), or (d1), the polynucleotide may have a base length of 20, 23, 22, or 87 as per the respective base sequences. If it consists of nucleotides corresponding to base sequences (a2), (b2), (c2), or (d2), the polynucleotide may have a base length derived from 20, 23, 22, or 87 with several deletions, substitutions, insertions, or additions.

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

[0066] Specific examples of compounds for miR-520d-5p upregulation include the following 13 compounds: vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacric 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 miR-520d-5p upregulation may form pharmaceutically acceptable salts. Pharmaceutically acceptable salts may be selected from, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with basic or acidic amino acids, halides, etc., taking into consideration the chemical structure of the miR-520d-5p upregulation compound. 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 chlorides, bromides, and iodides. A preferred salt of vancomycin is, for example, hydrochloride. A preferred salt of metoprolol is, for example, tartrate. A preferred salt of benzethonium is, for example, chloride. A preferred salt of betaxolol is, for example, hydrochloride. A preferred salt of colistin is, for example, sulfate. A preferred salt of oxprenolol is, for example, hydrochloride. Preferred bisoprolol salts include, for example, fumarate.

[0068] The compounds used for miR-520d-5p upregulation or their pharmaceutically acceptable salts may be solvates. Among the compounds used for miR-520d-5p upregulation or their pharmaceutically acceptable salts, the solvates may change depending on the solvent in which the compound is dissolved or the environment in which the compound is placed (for example, in air with a relatively constant humidity). Examples of such solvates include hydrates (monohydrate, dihydrate, trihydrate, etc.), ethanol solvates, acetic acid solvates, etc.

[0069] —Methods for cell production— In various embodiments, the method for producing cells includes (1) a step of culturing cells using the miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation (miR-520d-5p related substance) (hereinafter referred to as the "culturing step"), and (2) a step of confirming whether the mutations in the hair cycle-related genes within the cells have returned to the wild type (hereinafter referred to as the "confirmation step").

[0070] The culture process can be carried out by culturing target cells (hair cycle-related cells) in a culture medium supplemented with miR-520d-5p-related substances. The duration of the culture process can be adjusted within a range that achieves the effect of returning the cells to the wild type or partially returning them to the wild type, depending on the type of target cells, and is not particularly limited. The duration of the culture process 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 (the upper limit and / or lower limit itself may or may not be included), for example, 6 days or more (1 week) and 21 days (3 weeks) or less (22 days). The culture medium to which the miR-520d-5p-related substance is added may be used for the entire duration of the culture process, or the culture medium to which the miR-520d-5p-related substance is added may be used for a portion of the culture process, and the culture medium without the miR-520d-5p-related substance may be used for the remainder of the culture process.

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

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

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

[0074] The verification process involves determining the base sequence (target base sequence) of a hair cycle-related gene within the cell, comparing the target base sequence with the wild-type base sequence, and determining that the target base sequence has reverted to the wild type if the two base sequences match. The base sequence of the hair cycle-related gene can be determined using known methods. For the wild-type base sequence, a database can be referenced.

[0075] The hair cycle-related genes to be identified may be one type or two or more types, depending on the purpose of the identification step and / or the cell production method. For example, depending on the type of cell, in the specific embodiment described above, it may be all or part of the hair cycle-related genes. In various embodiments, the identification step involves determining the proportion of hair cycle-related genes that have reverted to the wild type from among the pre-set hair cycle-related genes in the target cells obtained in the culture step, and then confirming whether that 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 two or more. In various embodiments, the confirmation step uses a portion of the cell population obtained in the culture step as a sample, and determines the percentage of target cells contained therein in which the mutations in hair cycle-related genes have reverted to the wild type (or the percentage of cells in which the percentage of hair cycle-related genes that have reverted to the wild type among the pre-defined hair cycle-related genes has reached a certain level), and confirms whether that percentage has reached a certain level.

[0077] The cell production method may include steps other than the culture and harvesting steps, as needed. An example of such an optional step is the step of harvesting cells in which the target base sequence has been reverted to the wild-type base sequence (hereinafter referred to as the "harvesting step").

[0078] In the recovery process, cells whose 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 with mutations in intracellular hair cycle-related genes that have reverted to the wild type has reached a certain level (which can be determined by measuring using a sample) may be recovered.

[0079] In one embodiment, a method for repairing hair cycle-related genes is provided, which includes culturing cells using the miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for upregulating miR-520d-5p (miR-520d-5p-related substance), and restoring mutations in hair cycle-related genes to the wild type.

[0080] —Treatment Method / Composition— In various embodiments, a method for treating hair cycle-related diseases or symptoms includes administering (I) miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation (miR-520d-5p related substance), or (II) cells obtained by the present method.

[0081] In various embodiments, the composition is used to treat a hair cycle-related disease or condition (i.e., to carry out the treatment method described herein), and contains (I) a miR-520d-5p polynucleotide or a polynucleotide encoding it, or a compound for miR-520d-5p upregulation (miR-520d-5p related substance), or (II) cells obtained by the method described herein.

[0082] By administering the miR-520d-5p-related substance (I), the effect and efficacy of the miR-520d-5p-related substance in the body to revert to the wild type, or partially to the wild type, are exerted on hair cycle-related cells, thereby treating hair cycle-related diseases or symptoms. Furthermore, hair cycle-related diseases or symptoms can also be treated by administering cells (II) obtained by the method described above, i.e., cells that have already shown the effect of reverting mutations in cycle-related genes to the wild type. In the treatment method and composition of the present invention, the miR-520d-5p-related substance (I) may be one of the following: the miR-520d-5p polynucleotide or a polynucleotide encoding it, and a compound for miR-520d-5p upregulation, or both may be used (sequentially or simultaneously). Furthermore, either the miR-520d-5p-related substance (I) or cells (II) obtained by the method described above may be used, or both may be used (sequentially or simultaneously).

[0083] "Hair cycle-related diseases" refer to diseases associated with the growth phase, regression phase, resting phase, or shedding phase of the hair cycle, particularly diseases related to the expression (suppression or enhancement) of hair cycle-related genes in dermal papilla cells, skin-derived progenitor cells, bulge stem cells, or stem cells capable of differentiating into such cells. The hair cycle-related diseases that can be treated with this composition are not particularly limited, but alopecia areata are a typical example. Alopecia generally manifests as hair loss, vellus hair (where thick, long hair does not regenerate, and most hair becomes thin and short), and hair weakening due to a shortened growth phase (e.g., several months to one year) and a relatively longer regression and resting phase. Examples include male pattern baldness, female pattern baldness, alopecia areata, trichotillomania, telogen effluvium, endocrine abnormalities, nutritional deficiencies, skin infections, skin tumors, scarring alopecia, drugs / chemicals, and various hair deformities.

[0084] In this specification, the term "treatment" means treating a hair cycle-related disorder completely or partially therapeutically, curing (alleviating or improving) symptoms associated with a hair cycle-related disorder, slowing the progression of a hair cycle-related disorder or its symptoms, delaying its onset, preventing it, or reducing its risk, or a combination thereof.

[0085] The subjects of the treatment may be humans or non-human animals, and there are no particular limitations on sex or age. 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, cattle, monkeys, chimpanzees, pigs, horses, sheep, goats, dogs, cats, guinea pigs, and hamsters, preferably mammals that can serve as models for hair cycle-related diseases. The subjects of the treatment may be subjects of non-clinical or clinical trials.

[0086] The dosage of miR-520d-5p-related substances or cells obtained by the methods described herein can be adjusted within a range that produces a reversion or partial reversion effect to the wild type, and is not particularly limited.

[0087] In various embodiments, the miR-520d-5p upregulating compound can be administered in an amount that yields the maximum effective blood concentration shown in Table 1, or can be used as a topical preparation having the concentrations shown in Table 1. The maximum effective blood concentration of the miR-520d-5p upregulating compound upon administration or the concentration in the topical preparation can be the same as or less than the maximum effective blood concentration upon administration or the concentration in the topical preparation when the same compound is used as an active ingredient for other known indications (for example, it can be 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, or 0.01% or less).

[0088] [Table 1] JPEG2026053554000002.jpg9165

[0089] The amount (concentration) of the active ingredient contained in this composition, i.e., the miR-520d-5p related substance or the cells obtained by the method described herein, can be adjusted within a range that achieves the effect of returning to the wild type, or partially returning to the wild type, by considering, for example, the form of administration, the route of administration, the 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 herein may be determined by non-clinical studies (cell studies, animal studies) and clinical studies.

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

[0091] Examples of administration forms for topical skin preparations or topical mucosal preparations include liquids, sprays, ointments, creams, gels, and patches. 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, intra-arterial, intramuscular, intradermal, or subcutaneous. When administering cells obtained by the method described above, the composition may be administered as a cell preparation by infusion or injection.

[0093] Examples of oral drug administration forms include tablets, capsules, granules, powders, fine granules, syrups, pills, and liquids, and release may be controlled by methods such as sustained release.

[0094] In addition to the active ingredient, the composition may also contain additives commonly used in the manufacture of pharmaceuticals, quasi-drugs, cosmetics, etc., preferably additives used in the manufacture of topical preparations, injections, oral preparations, etc., such as stabilizers, preservatives, buffers, pH adjusters, excipients, bases, etc. The amount of such additives can be appropriately adjusted by those skilled in the art.

[0095] Furthermore, in addition to the treatment methods and compositions described herein, 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 this composition, it is preferable that the miR-520d-5p related substance is subject to technology related to drug delivery systems (DDS) used in injectable preparations, topical preparations, etc. One specific example of a DDS is nanoparticle formation, i.e., encapsulating the miR-520d-5p related substance in nano-sized particles made of a specific compound to improve the delivery efficiency to target cells. The nanoparticles may contain only the miR-520d-5p polynucleotide or a polynucleotide encoding it, or only a compound for miR-520d-5p upregulation (e.g., benzethonium), or they may contain both the polynucleotide encoding the miR-520d-5p polynucleotide or a polynucleotide encoding it and a compound for miR-520d-5p upregulation. Alternatively, the nanoparticle formulation may contain multiple types of nanoparticles, for example, both nanoparticles containing miR-520d-5p polynucleotide or a polynucleotide encoding it, and nanoparticles containing a compound for miR-520d-5p upregulation. Various compounds constituting nanoparticles are known and are not particularly limited, and examples include biocompatible lipids (e.g., triglycerides, diglycerides, monoglycerides, fatty acids, steroids), surfactants (e.g., phospholipids), and polymers (e.g., polylactic acid: PLA, polylactic acid glycolic acid copolymer: PLGA, polyethylene glycol: PEG, etc.). By considering the type of miR-520d-5p related substance, its solubility (water-soluble / lipid-soluble), and other properties, and using appropriate raw materials and manufacturing methods, nanoparticles with an appropriate encapsulation rate and appropriate size (particle size distribution) can be produced.

[0097] In this composition, the miR-520d-5p related substance may be formulated in collagen, that is, it may be contained in a collagen-containing base (filler). In other words, this composition may take the form of a collagen preparation containing a base (filler) containing the miR-520d-5p related substance and collagen. Since the collagen preparation is gradually absorbed into the body over a certain period of time after being injected intradermally or subcutaneously, the miR-520d-5p related substance contained in the collagen can be gradually released. The collagen preparation may contain, as the miR-520d-5p related substance (I), only the miR-520d-5p polynucleotide or the polynucleotide encoding it, only the miR-520d-5p upregulation compound, or both the polynucleotide encoding the miR-520d-5p polynucleotide or the polynucleotide encoding it and the miR-520d-5p upregulation compound. 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 derived from humans when administered to humans), and optionally contains other components such as hyaluronic acid and gelatin (modified collagen).

[0098] This composition can be manufactured in accordance with the laws, regulations, and guidelines of each country, and can be classified as a pharmaceutical product, a cosmetic product, or a quasi-drug (medicinal cosmetic product) that falls somewhere between the two, and can claim various effects and efficacy.

[0099] In this specification, singular terms may be replaced with plural terms, and vice versa, unless otherwise stated or obvious to those skilled in the art, such replacements are not possible.

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

[0101] [Example 1] We investigated whether mutations in the genes of mesenchymal stem cells (MSCs) differentiated from iPS cells (iPSCs) could be converted to the wild type, i.e., whether the genomic sequence could be improved, by introducing miR-520d-5p into iPSCs in vitro, following the procedure below. This targeted 178 hair cycle-related genes involved in the hair cycle, including the growth, regression, resting, and shedding phases. The 178 hair cycle-related genes were identified 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 Research Institute, 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). Furthermore, 520d-5p-expressing lentiviral particles for transfecting hMSCs were generated using the human mesangium cell line 293FT (Invitrogen Japan KK, Tokyo, Japan). 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. Additionally, hMSCs were induced 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 construct 293FT cells (5 x 10 6The effects of miR-520d-5p overexpression were investigated by transfecting cells (10 cm culture dish) with 20 μg of pMIRNA1-miR-520d-5p / GFP (System Biosciences, Mountain View, CA, USA) or the mock vector pCDH. hMSCs expressing miR-520d-5p were investigated by centrifugation of cells at 170,000 × g at 4°C for 120 minutes and collection of virus particles produced in 293FT cell culture medium. Virus pellets were collected and processed using Lenti-X. TM The number of viral copies was measured using a qRT-PCR titration kit (Clontech, Mountain View, CA, USA). For infection of 293FT cells, 1 million lentiviral copies were used per 10 cm culture dish. 293FT cells were transfected with 50 nM synthetic oligonucleotides using FuGENE HD transfection reagent (Roche Diagnostics, Basel, Switzerland). Cells treated with pCDH / lenti / GFP were used as controls. Finally, differentiation from iPS cells to hMSCs was induced using a hiPSC-derived MSC differentiation system and reagents (Veritas Corporation, Tokyo, Japan). These hMSCs were then transfected with 520d-5p lentivirus to generate 520d-5p transfected hMSCs.

[0104] Next-generation sequencing (NGS) analysis iPSCs (253G1), 520d transfected 253G1-derived progenitor cells (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 for each group (n=3). Genomic DNA was processed using SureSelectXT Human All Exon v5 + UTR (Agilent Technologies, Inc.) and sequenced on the 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. The mapping files in SAM (Sequence Alignment / Map) format were converted to BAM (binary version of SAM) format and sorted using SAMtools-1.2. Local readjustment around known indels was performed on the sorted BAM files using GATK-Lite-2.3.0. PCR duplicates were removed using 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 at: http: / / sourceforge.net / projects / picard / files / picard-tools / ). Finally, the base quality score was recalibrated 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 equipped with 101 bp paired-end reads. Finally, paired-end reads in FastQ format were mapped to the reference genome HG19 using TopHat.

[0105] Mutation analysis The Samtools mpileup [options: -d 10000 -L 10000 -B -t DP, DV, SP, DP4, DPR] was piped with the bcftools call [options: -A -v -m -f GQ] to create a Variant Calling Format (VCF) file. The VCF file was further filtered to select variants with at least two supporting reads, a minimum allele frequency of 0.8, and a maximum read depth of 35 at the called site. For high-throughput studies, deep sequencing of selected cDNAs was followed by Sanger sequencing to screen for non-synonymous 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 Table 2 shows the results regarding the effect of sequence conversion using miR-520d-5p on hair cycle-related genes.

[0107] Of the 178 hair cycle-related genes, genomic conversion was observed in 142 genes by treating cells with miR-520d-5p, repairing mutated bases (or at least some of them) to the wild type. In 26 genes, all mutated bases were repaired to the wild type, resulting in a 100% conversion rate. For example, in the AR gene, 71.4% of the mutated bases (5 out of 7 bases) in iPSCs were restored to the wild type in 520d / hMSCs. However, the repeat sequences in the AR gene (22 CAG repeats and 17 GGC repeats) were identical in 520d / hMSCs to those in iPSCs. Furthermore, conversions of consecutive 2-base, 3-base, or 4-base sequences were 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 (indicated by the gray background in Table 2), no genomic transformation was observed in which mutated bases were repaired to the wild type by treating cells with miR-520d-5p. However, for 6 of these 36 genes, mutated bases relative to the wild type were not originally present in the iPSC(253G1).

[0109] Cells treated with pCDH / lenti / GFP, used as a control, did not induce genomic conversion. Furthermore, transfection with miR-520d-5p did not show any clearly degrading changes at the nucleotide level. The mean conversion rate for 178 hair cycle-related genes was 54.4%.

[0110] [Table 2] JPEG2026053554000004.jpg252134JPEG2026053554000005.jpg250134JPEG2026053554000006.jpg245134

[0111] Next, the effects of sequence conversion using miR-520d-5p were analyzed in more detail for 39 genes (see Non-Patent Literature 1, Figure 2) related to the growth or function of dermal papilla cells (DP cells).

[0112] miR-520d-5p improved the sequence status of 35 out of 39 genes in DP cells. Furthermore, based on a target score exceeding 50%, corresponding to a 7-base match (complementarity) with the mRNA sequence, miR-520d-5p is estimated to potentially target the mRNA of 15 genes, and miR-520d-5p improved the sequence of 13 of those 15 genes. Interestingly, miR-520d-5p strongly targets the mRNA of the AR gene, and since AR and ARA55 (TGFB1-induced transcript 1) are overexpressed in the hair follicles or dermal papillae of patients with male pattern baldness (AGA) and female pattern baldness (FAGA), it is thought that miR-520d-5p can suppress the function of AR caused by this overexpression. In addition, miR-520d-5p converts the mutations accumulated in the second exon of the AR gene back to the wild type. In DR cells, the AR gene is a crucial component 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 mutations in these genes.

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

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

[0115] [Example 2] We investigated whether miR-520d-5p expression is upregulated by introducing a compound for miR-520d-5p upregulation into normal human dermal fibroblasts (NHDFs) in vitro, following the procedure below.

[0116] Solutions were prepared using the test compounds and solvents shown in Table 3. The solutions were added to the culture medium to the final concentrations 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 cultured NHDF was measured by adding a linker to the expressed miRNA (miR-520d-5p) and detecting it using RT-PCR with a specific primer set. The expression levels of miR-520d-5p for test compounds 1 to 13 were converted to relative values ​​with the expression level of the control miR-520d-5p set to 1.

[0119] The results are shown in Figure 4. It can be seen that all 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 miR-520d-5p upregulating compounds are introduced into hair cycle-related cells, they will have the effect of returning to the wild type, or partially returning to the wild type. That is, the expression of -520d-5p will be enhanced, and as a result, mutations in hair cycle-related genes and other genes expressed by hair cycle-related cells can be repaired. In addition, it can be estimated that when miR-520d-5p upregulating compounds (13 test compounds and other compounds) are used to return mutations in hair cycle-related genes to the wild type, or to treat hair cycle-related diseases or symptoms, the effect may be lower than when used as an active ingredient for other indications.

Claims

1. A polynucleotide encoding miR-520d-5p polynucleotide, or a compound for upregulating miR-520d-5p polynucleotide, used to revert mutations in intracellular hair cycle-related genes back to the wild type. Here, the miR-520d-5p polynucleotide is (a1) The base sequence of Sequence 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 Sequence ID No.

1. Includes.

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

3. The hair cycle-related gene is at least one gene selected from the group consisting of the following 142 genes, according to claim 1, a polynucleotide encoding miR-520d-5p polynucleotide, or a compound for miR-520d-5p polynucleotide upregulating: 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. A polynucleotide encoding miR-520d-5p polynucleotide or a compound for upregulating miR-520d-5p polynucleotide according to claim 1, used to revert mutations in intracellular hair cycle-related genes to the wild type and to suppress or regulate the expression of at least one gene selected from the group consisting of the following 18 genes: AR, DIO2, EDN3, FGF7 (KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTHLH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

5. The cells are dermal papilla cells or stem cells capable of differentiating into such cells, and The hair cycle-related gene is at least one gene selected from the group consisting of the following 26 genes, according to claim 1, a polynucleotide encoding miR-520d-5p polynucleotide, or a compound for upregulating miR-520d-5p polynucleotide: 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. A polynucleotide encoding miR-520d-5p polynucleotide or a compound for upregulating miR-520d-5p polynucleotide according to claim 1, used to revert intracellular hair cycle-related gene mutations 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 aforementioned cells are bulge stem cells or stem cells capable of differentiating into such cells, and The hair cycle-related gene is at least one gene selected from the group consisting of the following 18 genes, according to claim 1, a polynucleotide encoding miR-520d-5p polynucleotide, or a compound for upregulating miR-520d-5p polynucleotide: 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. A polynucleotide encoding miR-520d-5p polynucleotide or a compound for upregulating miR-520d-5p polynucleotide according to claim 7, used to revert mutations in intracellular hair cycle-related genes 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 aforementioned cells are dermal papilla cells or stem cells capable of differentiating into such cells, and bulge stem cells or stem cells capable of differentiating into such cells. The hair cycle-related gene is selected from the group consisting of the following two types of genes, according to claim 1, the miR-520d-5p polynucleotide or the polynucleotide encoding the miR-520d-5p polynucleotide, or the compound for upregulating the miR-520d-5p polynucleotide: BMP2 and SOSTDC1.

10. The aforementioned miR-520d-5p polynucleotide, (b1) The base sequence of Sequence ID No. 2, or (b2) Polynucleotides having a base sequence in which 1 to 3 bases are deleted, substituted, inserted, or added in the base sequence of Sequence ID No. 2, (c1) The base sequence of Sequence ID No. 3, or (c2) Polynucleotides having a base sequence in which 1 to 3 bases are deleted, substituted, inserted, or added in the base sequence of Sequence ID No. 3 It is a double-stranded polynucleotide composed of, Here, the polynucleotide encoding the miR-520d-5p polynucleotide is (d1) The base sequence of Sequence 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 Sequence ID No.

4. 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 upregulation compound is selected from the group consisting of the following 13 compounds, as described in claim 1, for the miR-520d-5p polynucleotide or the polynucleotide encoding the miR-520d-5p polynucleotide, or the miR-520d-5p polynucleotide upregulation compound: Vancomycin, metoprolol, benzethonium, chlorpropamide, betaxolol, colistin, oxprenolol, ethacrine, bisoprolol, candesartan, alexidine, bumetanide, and glycopyrrolate.

12. 1) A step of culturing cells using the miR-520d-5p polynucleotide or a polynucleotide encoding miR-520d-5p polynucleotide described in any one of claims 1 to 11, or a compound for upregulating miR-520d-5p polynucleotide, and 2) A step to confirm whether the mutations in the hair cycle-related genes within the cell have reverted to the wild type. A method for producing cells, including the production of cells.

13. (I) The miR-520d-5p polynucleotide described in any one of claims 1 to 11, or a polynucleotide encoding the miR-520d-5p polynucleotide, or a compound for miR-520d-5p polynucleotide upregulation, (II) Cells produced by the method described in claim 12 A composition used for the treatment of hair cycle-related diseases or symptoms, including the following.

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

15. The composition according to claim 13, wherein the miR-520d-5p upregulating compound is administered to the following maximum effective blood concentrations, or applied topically at the following concentrations: Vancomycin: Administered at a dose of 30 μg / ml or less. Metoprolol: Administered, ≤ 41.8 ng / ml Benzethonium: For external use, 0.2% or less Chlorpropamide: Administered at a dose of 30 μg / ml or less. Betaxolol: Administered, ≤ 41.8 ng / ml Colistin: Administered at a dose of 4.4 μg / ml or less. Oxprenolol: Administered at a dose of 30.18 μg / ml or less. Ethacrine: Administered at a dose of 100 μg / ml or less. Bisoprolol fumarate: Administration, ≤ 100 ng / ml Candesartan: Administered, dose 75 ng / ml or less. Alexidine: Administered, less than 1 mM Bumetanide: Administered at a dose of 0.4 μg / ml or less. Glycopyrrolates: Administered at a concentration of 800 pg / ml or less.

16. A method for restoring mutations in hair cycle-related genes to the wild type in the cells of patients who require it, a) miR-520d-5p polynucleotide; b) A polynucleotide encoding the miR-520d-5p polynucleotide; or c) Compound for miR-520d-5p upregulation This includes administering, Here, the miR-520d-5p polynucleotide is: (a1) The nucleotide sequence of Sequence 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 Sequence ID No.

1. Methods that include...

17. The method according to claim 16, wherein the patient's cells required are dermal papillary cells, skin-derived progenitor cells, or bulge stem cells, or stem cells capable of differentiating into the aforementioned 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 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 modulates the expression of at least one gene selected from the group consisting of the following 18 genes: AR, DIO2, EDN3, FGF7 (KGF), HEY1, HEY2, LEF1, NTRK (SLITRK2, 4, 6), PTHLH, RNF144A, RORA, SMOC2, SOSTDC1, SOX9, TRPS1, TWIST1, VAV3 and VCAN.

20. The patients who require this treatment have cells that are dermal papilla cells or stem cells capable of differentiating into such cells, 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 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 according to 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 modulates 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.

22. The patients who require this treatment have cells that are basil stem cells or stem cells capable of differentiating into such 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 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 according to 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 modulates the expression of at least one gene selected from the group consisting of PTHLH, SOX9, and SMOC2.

24. Patients who require it need cells that differentiate into dermal papilla cells or stem cells capable of differentiating into such cells, and bulge stem cells or stem cells capable of differentiating into such cells, 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 aforementioned miR-520d-5p polynucleotide, (b1) The base sequence of Sequence ID No. 2, or (b2) Polynucleotides having a base sequence in which 1 to 3 bases are deleted, substituted, inserted, or added in the base sequence of Sequence ID No. 2, (c1) The base sequence of Sequence ID No. 3, or (c2) Polynucleotides having a base sequence in which 1 to 3 bases are deleted, substituted, inserted, or added in the base sequence of Sequence ID No. 3 It is a double-stranded polynucleotide composed of, or (d1) The base sequence of Sequence 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 Sequence ID No.

4. It is a polynucleotide having, The method according to claim 16.

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

27. In the manufacture of pharmaceuticals for treating hair cycle-related diseases or symptoms a) miR-520d-5p polynucleotide; b) A polynucleotide encoding the miR-520d-5p polynucleotide; or c) Compound for miR-520d-5p upregulation Use, Here, the miR-520d-5p polynucleotide is (a1) The base sequence of Sequence ID No. 1, or (a2) Includes a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted, or added in the nucleotide sequence of Sequence ID No.

1.

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

29. The use according to claim 27 or 28, wherein the hair cycle-related disease includes alopecia.

Images