Use of MIRNA-485 inhibitors to induce hair growth

Abstract

The present disclosure includes the use of miRNA inhibitors to induce hair growth, increase hair density, increase hair follicle density, increase hair shaft thickness, increase hair length, prevent hair loss, reduce hair loss, or perform any combination of the above in a subject in need thereof. In some embodiments, the subject has one or more diseases selected from the group consisting of alopecia totalis, androgenetic alopecia, alopecia areata, alopecia universalis, regressive alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial alopecia, scarring alopecia, scalp hair loss, hair shaft abnormalities, infectious hair diseases, genetic diseases, and chemotherapy, hormonal imbalance, fungal infection, medication, chemical hair treatment, or age-related hair loss.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 130,121, filed December 23, 2020, which is incorporated by reference in its entirety herein.

[0002] References to sequence listings submitted electronically via EFS-WEB The entire electronically submitted sequence listing, submitted with this application in the form of an ASCII text file (filename: Sequence-Listing-4366-037PC02_SL_ST25, size: 76,935 bytes, created on: December 21, 2021), is hereby incorporated by reference.

[0003] The present disclosure provides for the use of miR-485 inhibitors (eg, polynucleotides encoding a nucleotide molecule comprising at least one miR-485 binding site) to induce hair growth. [Background technology]

[0004] In modern society, the number of people at risk of hair loss and alopecia is increasing due to the increasing elderly population and high stress. Hair loss and alopecia is medically harmless, but can cause a lot of emotional and psychosocial pressure for patients and their families. Causes of hair loss include part of the natural aging process, slow hair growth cycle, genetic predisposition, unwanted side effects of drugs and / or medical treatments, environmental factors such as allergies, infections, stress, lack of proper diet and sleep, and / or other systemic diseases.

[0005] It is estimated that 40% of men in the United States have some significant hair loss by age 35. This situation worsens as men get older. By age 60, it is estimated that 65% of adult men in the United States have significant hair loss. Women also experience hair loss. Unlike men, hair loss in women is usually accompanied by significant thinning of the hair across the scalp and hairline. The American Academy of Dermatology reports that 40% of women have significant hair loss by age 40. See, e.g., U.S. Patent No. 9,173,921. Therefore, new and more effective approaches to induce hair growth are highly desirable. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Pat. No. 9,173,921 Summary of the Invention [Means for solving the problem]

[0007] Provided herein is a method of inducing hair growth in a subject in need thereof, the method comprising administering to the subject a compound that inhibits miR-485 (a "miRNA inhibitor").

[0008] Also provided herein is a method of increasing hair density in a subject in need thereof, the method comprising administering to the subject a compound that inhibits miR-485 (an "miRNA inhibitor").

[0009] Also provided herein is a method of increasing hair follicle density in a subject in need thereof, the method comprising administering to the subject a compound that inhibits miR-485 (an "miRNA inhibitor").

[0010] Also provided herein is a method of increasing hair shaft thickness in a subject in need thereof, the method comprising administering to the subject a compound that inhibits miR-485 (an "miRNA inhibitor").

[0011] Also provided herein is a method of increasing hair length in a subject in need thereof, the method comprising administering to the subject a compound that inhibits miR-485 (an "miRNA inhibitor").

[0012] Also provided herein is a method for preventing hair loss in a subject at risk of hair loss, the method comprising administering to the subject a compound that inhibits miR-485 (a "miRNA inhibitor").

[0013] Also provided herein is a method of reducing hair loss in a subject in need thereof, the method comprising administering to the subject a compound that inhibits miR-485 (a "miRNA inhibitor").

[0014] In some embodiments, the subject has one or more disorders selected from the group consisting of alopecia totalis, androgenetic alopecia, alopecia areata, alopecia universalis, regressive alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial alopecia, scarring alopecia, scalp hair loss, hair shaft abnormalities, infectious hair disorders, genetic disorders, and hair loss due to chemotherapy, hormonal imbalance, fungal infection, medication, chemical hair treatment, or aging. In some embodiments, the subject is a human.

[0015] In some aspects, the miRNA inhibitors described herein induce autophagy and / or treat or prevent inflammation.

[0016] In some embodiments, the miRNA inhibitors described herein inhibit miR485-3p. In some embodiments, miR485-3p comprises 5'-GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1). In some embodiments, the miRNA inhibitor comprises a nucleotide sequence comprising 5'-UGUAUGA-3' (SEQ ID NO: 2), and the miRNA inhibitor is about 6 to about 30 nucleotides in length.

[0017] In some embodiments, the miRNA inhibitors described herein comprise at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 5' end of the nucleotide sequence. In some embodiments, the miRNA inhibitor comprises at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 3' end of the nucleotide sequence.

[0018] In some embodiments, the miRNA inhibitors described herein are selected from the group consisting of 5'-UGUAUGA-3' (SEQ ID NO:2), 5'-GUGUAUGA-3' (SEQ ID NO:3), 5'-CGUGUAUGA-3' (SEQ ID NO:4), 5'-CCGUGUAUGA-3' (SEQ ID NO:5), 5'-GCCGUGUAUGA-3' (SEQ ID NO:6), 5'-AGCCGUGUAUGA-3' (SEQ ID NO:7), 5'-GAGCCGUGUAUGA-3' (SEQ ID NO:8), 5'-AGAGCCGUGUAUGA -3' (SEQ ID NO: 9), 5'-GAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-GGAGAGCCGUGUAUGA-3' (SEQ ID NO: 11), 5'-AGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 12), 5'-GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 13), 5'-AGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 14), 5'-GAGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 15); 5'-UGUA UGAC-3' (SEQ ID NO: 16), 5'-GUGUAUGAC-3' (SEQ ID NO: 17), 5'-CGUGUAUGAC-3' (SEQ ID NO: 18), 5'-CCGUGUAUGAC-3' (SEQ ID NO: 19), 5'-GCCGUGUAUGAC-3' (SEQ ID NO: 20), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 21), 5'-GAGCCGUGUAUGAC-3' (SEQ ID NO: 22), 5'-AGAGCCGUGUAUGAC-3' (SEQ ID NO: 23), 5'-GAGAG CCGUGUAUGAC-3' (SEQ ID NO:24), 5'-GGAGAGCCGUGUAUGAC-3' (SEQ ID NO:25), 5'-AGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:26), 5'-GAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:27), 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:28), or 5'-GAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:29).

[0019] In some embodiments, the miRNA inhibitor is 5'-TGTATGA-3' (SEQ ID NO: 30), 5'-GTGTATGA-3' (SEQ ID NO: 51), 5'-CGTGTATGA-3' (SEQ ID NO: 52), 5'-CCGTGTATGA-3' (SEQ ID NO: 53), 5'-GCCGTGTATGA-3' (SEQ ID NO: 54), 5'-AGCCGTGTATGA-3' (SEQ ID NO: 55), 5'-GAGCCGTGTATGA-3' (SEQ ID NO: 35), 5'-AGAGCCGTGTATGA- 3' (SEQ ID NO:56), 5'-GAGAGCCGTGTATGA-3' (SEQ ID NO:57), 5'-GGAGAGCCGTGTATGA-3' (SEQ ID NO:58), 5'-AGGAGAGCCGTGTATGA-3' (SEQ ID NO:59), 5'-GAGGAGAGCCGTGTATGA-3' (SEQ ID NO:60), 5'-AGAGGAGAGCCGTGTATGA-3' (SEQ ID NO:61), 5'-GAGAGGAGAGCCGTGTATGA-3' (SEQ ID NO:62); 5'-TGTA TGAC-3' (SEQ ID NO: 63), 5'-GTGTATGAC-3' (SEQ ID NO: 64), 5'-CGTGTATGAC-3' (SEQ ID NO: 65), 5'-CCGTGTATGAC-3' (SEQ ID NO: 66), 5'-GCCGTGTATGAC-3' (SEQ ID NO: 67), 5'-AGCCGTGTATGAC-3' (SEQ ID NO: 68), 5'-GAGCCGTGTATGAC-3' (SEQ ID NO: 69), 5'-AGAGCCGTGTATGAC-3' (SEQ ID NO: 70), 5'-GAGAG CCGTGTATGAC-3' (SEQ ID NO: 71), 5'-GGAGAGCCGTGTATGAC-3' (SEQ ID NO: 72), 5'-AGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 73), 5'-GAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 74), 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 75), and 5'-GAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 76).

[0020] In some embodiments, the sequence of the miRNA inhibitor has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 77). In certain embodiments, the miRNA inhibitor has at least 90% similarity to 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 77). In some embodiments, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 77) with one substitution or two substitutions. In some embodiments, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 77). In some embodiments, the miRNA inhibitor described herein comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28).

[0021] In some embodiments, the miRNA inhibitors described herein comprise at least one modified nucleotide. In some embodiments, the at least one modified nucleotide is a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), an arabino nucleic acid (ABA), a bridged nucleic acid (BNA), and / or a peptide nucleic acid (PNA).

[0022] In some embodiments, the miRNA inhibitors described herein comprise a backbone modification. In certain embodiments, the backbone modification is a phosphorodiamidite morpholino oligomer (PMO) and / or a phosphorothioate (PS) modification.

[0023] In some embodiments, the miRNA inhibitors described herein are delivered in a delivery agent. In certain embodiments, the delivery agent is a micelle, an exosome, a lipid nanoparticle, an extracellular vesicle, or a synthetic vesicle.

[0024] In some embodiments, the miRNA inhibitor described herein is delivered by viral vector.In certain embodiments, the viral vector is AAV, adenovirus, retrovirus or lentivirus.In some embodiments, the viral vector is AAV with serotype of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or any combination thereof.

[0025] In some embodiments, the miRNA inhibitors described herein are delivered by a delivery agent. In certain embodiments, the delivery agent comprises a micelle, an exosome, a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, an extracellular vesicle, a synthetic vesicle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a conjugate, a viral vector, or a combination thereof.

[0026] In some embodiments, the delivery agent has the following formula: [WP]-L1-[CC]-L2-[AM] (Formula I) or [WP]-L1-[AM]-L2-[CC] (Formula II) (In the formula, WP is a water-soluble biopolymer moiety; CC is a positively charged carrier moiety, AM is the adjuvant moiety, L1 and L2 are independently an optional linker, Cationic carrier units form micelles when mixed with nucleic acids in an ionic ratio of about 1:1.

[0027] In some embodiments, the miRNA inhibitors described herein interact with the cationic carrier unit through ionic bonds. In some embodiments, the water-soluble polymer comprises poly(alkylene glycol), poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazoline ("POZ"), poly(N-acryloylmorpholine), or any combination thereof. In other embodiments, the water-soluble polymer comprises polyethylene glycol ("PEG"), polyglycerol, or poly(propylene glycol) ("PPG").

[0028] In some embodiments, the water soluble polymer has the following formula:

[0029] [ka] has.

[0030] In some embodiments, n is from 1 to 1000. In certain embodiments, n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141. In further embodiments, n is from about 80 to about 90, from about 90 to about 100, from about 100 to about 110, from about 110 to about 120, from about 120 to about 130, from about 140 to about 150, or from about 150 to about 160.

[0031] In some embodiments, the water-soluble polymer is linear, branched, or dendritic.

[0032] In some embodiments, the cationic carrier moiety comprises one or more basic amino acids. In certain embodiments, the cationic carrier moiety comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, At least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 basic amino acids. In certain embodiments, the cationic carrier moiety comprises from about 30 to about 50 basic amino acids.

[0033] In some embodiments, the basic amino acids comprise arginine, lysine, histidine, or any combination thereof, hi some embodiments, the cationic carrier moiety comprises about 40 lysine monomers.

[0034] In some embodiments, the adjuvant moiety can modulate the immune response, the inflammatory response, and / or the tissue microenvironment, hi certain embodiments, the adjuvant moiety comprises an imidazole derivative, an amino acid, a vitamin, or any combination thereof.

[0035] In some embodiments, the adjuvant moiety has the following formula:

[0036] [ka]

[0037] wherein each of G1 and G2 is H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring and n is 1-10.

[0038] In some embodiments, the adjuvant moiety comprises a nitroimidazole, hi certain embodiments, the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazole, azanidazole, benznidazole, or any combination thereof.

[0039] In some embodiments, the adjuvant moiety comprises an amino acid.

[0040] In some embodiments, the adjuvant moiety has the following formula:

[0041] [ka]

[0042] (Wherein, Ar is [ka] and

[0043] Each of Z1 and Z2 is H or OH.

[0044] In some embodiments, the adjuvant moiety comprises a vitamin, hi certain embodiments, the vitamin comprises a cyclic or heteroatom ring and a carboxyl or hydroxyl group.

[0045] In some embodiments, the vitamin has the following formula:

[0046] [ka]

[0047] wherein each of Y1 and Y2 is C, N, O, or S, and n is 1 or 2.

[0048] In some embodiments, the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H, and any combination thereof. For example, the vitamin can be vitamin B3.

[0049] In some embodiments, the adjuvant moiety comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 vitamin B3. In certain embodiments, the adjuvant moiety comprises about 10 vitamin B3.

[0050] In some embodiments, the delivery agent comprises a water soluble biopolymer portion having about 120 to about 130 PEG units, a cationic carrier portion comprising polylysine having about 30 to about 40 lysines, and an adjuvant portion having about 5 to about 10 vitamin B3.

[0051] In some embodiments, the delivery agent is conjugated with the miRNA inhibitor to form a micelle. For example, the conjugation can be covalent, non-covalent, or ionic.

[0052] In some embodiments, the cationic carrier units in the micelles and the miRNA inhibitor are mixed in solution such that the ionic ratio of the positive charges of the cationic carrier units to the negative charges of the miRNA inhibitor is about 1: 1. In some embodiments, the cationic carrier units can protect the miRNA inhibitor from enzymatic degradation.

[0053] In some embodiments, the miRNA inhibitors described herein are administered parenterally, intramuscularly, subcutaneously, by eye drop, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraventricularly, intraspinal, intraventricular, intrathecal, intracisternally, intracapsular, intratumoral, or any combination thereof.

[0054] In some embodiments, the miRNA inhibitors described herein are administered to the area of ​​the skin in need of promoting hair growth by painting, spraying, vaporizing, or injecting.

[0055] In some embodiments, the miRNA inhibitors described herein are administered locally to the area of ​​the skin where promotion of hair growth is required.

[0056] In some embodiments, the miRNA inhibitors described herein are formulated in a form selected from the group consisting of an ointment, a shampoo, a conditioner, a lotion, a tonic, a gel, and a mousse. In some embodiments, the administering step is performed by immersing or bathing the subject in the miRNA inhibitors described herein formulated in a form selected from the group consisting of an ointment, a shampoo, a conditioner, a lotion, a tonic, a gel, and a mousse. [Brief description of the drawings]

[0057] [Figure 1] 1 shows an exemplary structure of a carrier unit of the present disclosure. The example shown includes a cationic carrier moiety that can electrostatically interact with miRNA inhibitors (antimirs). In certain embodiments, a tissue-specific adjuvant moiety (AM) can be placed between a water-soluble biopolymer (WP) and a cationic carrier (CC). The CC and AM components are depicted in a linear arrangement for simplicity. However, the CC and AM can also be arranged in the form of a scaffold.

[0058] [Figure 2A]1 shows an evaluation of the hair growth effect of the miR-485 inhibitor (485ASO-001) on bald C57BL / 6J mice 5 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control). [Figure 2B] 1 shows an evaluation of the hair growth effect of the miR-485 inhibitor (485ASO-001) on bald C57BL / 6J mice 7 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control). [Figure 2C] 1 shows an evaluation of the hair growth effect of the miR-485 inhibitor (485ASO-001) on bald C57BL / 6J mice 9 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control). [Figure 2D] 1 shows an evaluation of the hair growth effect of the miR-485 inhibitor (485ASO-001) on bald C57BL / 6J mice 12 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control). [Figure 2E] 1 shows an evaluation of the hair growth effect of the miR-485 inhibitor (485ASO-001) on bald C57BL / 6J mice 14 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control). [Figure 2F]1 shows an evaluation of the hair growth effect of the miR-485 inhibitor (485ASO-001) on bald C57BL / 6J mice 16 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control).

[0059] [Figure 2G] Quantification of hair re-growth in depilated C57BL / 6J mice 5, 7, 9, 12, and 14 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of a miR-485 inhibitor (485ASO-001), or phosphate buffered saline (PBS) (negative control). The y-axis shows the hair re-growth quantification scale (0-6), with grade 0 = pink skin, no pigmentation, grade 1 = <30% skin area darkened but no visible hair, grade 2 = 30%-70% skin darkened and no visible hair, grade 3 = >70% skin area darkened or 30% visible hair, grade 4 = >70% skin area darkened and 30-70% visible hair, grade 5 = >70% skin area darkened and >70% visible hair, and grade 6 = >90% skin area darkened and >90% visible hair. Data are presented as mean (n=6) ± standard error of the mean (SEM). *p<0.05 and **p<0.01 for miR485-3p inhibitor (485ASO-001) treated group compared to PBS treated controls.

[0060] [Figure 3A] FIG. 1 shows the effect of the miR-485 inhibitor (485ASO-001) on hair shaft density in depilated ASO-001 / 6J mice 12 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate buffered saline (PBS) (negative control). [Figure 3B] FIG. 1 shows the effect of the miR-485 inhibitor (485ASO-001) on hair shaft density in depilated ASO-001 / 6J mice 16 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate buffered saline (PBS) (negative control).

[0061] [Figure 3C] Quantification of hair density in depilated C57BL / 6J mice 12 days (open bars) and 16 days (shaded bars) after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of the miR-485 inhibitor (485ASO-001), or phosphate-buffered saline (PBS) (negative control). The y-axis shows the assessment of hair density by analysis of images (magnification 200x, actual area = 3.6 mm2). Data are presented as mean (n = 6) ± standard error of the mean (SEM). *p<0.05 and **p<0.01 for 485ASO-001-treated groups compared to PBS-treated controls.

[0062] [Figure 4A] FIG. 1 shows the effect of the miRNA-485 inhibitor (485ASO-001) on hair length in depilated ASO-001 / 6J mice 16 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate buffered saline (PBS) (negative control). [Figure 4B] FIG. 1 shows the effect of the miRNA-485 inhibitor (485ASO-001) on hair length in depilated ASO-001 / 6J mice 21 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk), or phosphate buffered saline (PBS) (negative control).

[0063] [Figure 4C] Quantification of hair length in depilated C57BL / 6J mice 16 days (open bars) and 21 days (shaded bars) after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of the miRNA-485 inhibitor (485ASO-001), or phosphate-buffered saline (PBS) (negative control). The y-axis shows hair length in mm. Data are presented as mean (n=6) ± standard error of the mean (SEM). *p<0.05 and **p<0.01 for 485ASO-001-treated groups compared to PBS-treated controls.

[0064] [Figure 4D] 1 shows an evaluation of the hair regrowth effect of miRNA-485 inhibitor (485ASO-001) on C57BL / 6J mice 5 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control) by two intramuscular injections. [Figure 4E] 1 shows an evaluation of the hair regrowth effect of miRNA-485 inhibitor (485ASO-001) on C57BL / 6J mice 7 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control) by two intramuscular injections. [Figure 4F] 1 shows an evaluation of the hair regrowth effect of miRNA-485 inhibitor (485ASO-001) on C57BL / 6J mice 10 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control) by two intramuscular injections. [Figure 4G]1 shows an evaluation of the hair regrowth effect of miRNA-485 inhibitor (485ASO-001) on C57BL / 6J mice 12 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control) by two intramuscular injections. [Figure 4H] 1 shows an evaluation of the hair regrowth effect of miRNA-485 inhibitor (485ASO-001) on C57BL / 6J mice 14 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control) by two intramuscular injections. [Figure 4I] 1 shows an evaluation of the hair regrowth effect of miRNA-485 inhibitor (485ASO-001) on C57BL / 6J mice 16 days after administration of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control) by two intramuscular injections.

[0065] [Figure 4J]Quantification of hair re-growth in depilated C57BL / 6J mice 7, 10, 12, 14, and 16 days after two intramuscular injections of 0.1 mg / kg (mpk), 0.3 mg / kg (mpk), or 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001), or phosphate buffered saline (PBS) (negative control). The y-axis shows the hair regrowth quantification scale (0-6), with grade 0 = pink skin, no pigmentation, grade 1 = <30% skin area darkened but no visible hair, grade 2 = 30%-70% skin darkened and no visible hair, grade 3 = >70% skin area darkened or 30% visible hair, grade 4 = >70% skin area darkened and 30-70% visible hair, grade 5 = >70% skin area darkened and >70% visible hair, and grade 6 = >90% skin area darkened and >90% visible hair. Data are reported as mean ± SEM based on 5 mice per group.

[0066] [Diagram 5] FIG. 1 shows the effect of the miRNA-485 inhibitor (485ASO-001) on expression levels of 485-3p (FW7_mimic) 10 days (A) and 16 days (B) post-depilation (PD) following administration of two intramuscular injections of the miRNA-485 inhibitor (485ASO-001 or "ASO") at 0.3 mg / kg (mpk) (middle bar), or 0.6 mg / kg (mpk) (right bar), or phosphate-buffered saline (PBS) (negative control) (left bar).

[0067] [Figure 6A] 1 shows the results of H&E staining of mouse dorsal skin at hair follicle cycle 10 days post-depilation (pd). Longitudinal and transverse sections of mouse dorsal skin treated with phosphate buffered saline (PBS) (negative control) and positive control (minoxidil) after 10 days of treatment are shown. [Figure 6B]1 shows the results of H&E staining of mouse dorsal skin at hair follicle cycle 10 days after depilation (pd). Longitudinal and transverse sections of mouse dorsal skin treated with two intramuscular injections of miRNA-485 inhibitor (485ASO-001) at 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk) after 10 days of treatment.

[0068] [Figure 6C] FIG. 1 shows the effect of miRNA-485 inhibitor (485ASO-001) on dermal thickness (μm) in depilated C57BL / 6J mice on pd10. [Figure 6D] FIG. 1 shows the effect of miRNA-485 inhibitor (485ASO-001) on subcutaneous tissue thickness (μm) in depilated C57BL / 6J mice on pd10. [Figure 6E] FIG. 1 shows the effect of miRNA-485 inhibitor (485ASO-001) on hair follicle length (μm) in depilated C57BL / 6J mice on pd10. [Figure 6F] FIG. 1 shows the effect of miRNA-485 inhibitor (485ASO-001) on hair bulb diameter (μm) in depilated C57BL / 6J mice on pd10. [Figure 6G] FIG. 1 shows the effect of miRNA-485 inhibitor (485ASO-001) on hair follicle density in the dermis in depilated C57BL / 6J mice on day 10 pd. [Figure 6H] Figure 6 shows the effect of miRNA-485 inhibitor (485ASO-001) on hair follicle density in the subcutaneous tissue in depilated C57BL / 6J mice at pd 10. Different treatment groups are the same as those described in Figures 6A and 6B.

[0069] [Figure 7A] 1 shows the results of H&E staining of mouse dorsal skin on day 16 p.d. Longitudinal and transverse sections of mouse dorsal skin treated with phosphate buffered saline (PBS) (negative control) and positive control (minoxidil) after 16 days of treatment. [Figure 7B]1 shows the results of H&E staining of mouse dorsal skin at hair follicle cycle 16 days after depilation (pd). Longitudinal and transverse sections of mouse dorsal skin treated with two intramuscular injections of miRNA-485 inhibitor (485ASO-001) at 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk) after 16 days of treatment.

[0070] [Figure 7C] Effect of miRNA-485 inhibitor (485ASO-001) on subcutaneous tissue thickness (μm) in depilated C57BL / 6J mice on pd16 after two intramuscular injections of 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control) and positive control (minoxidil) on pd16. n = 3–10 mice at each time point. [Figure 7D] Effect of miRNA-485 inhibitor (485ASO-001) on dermal thickness (μm) in depilated C57BL / 6J mice on pd16 after two intramuscular injections of 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control) and positive control (minoxidil) on pd16. n = 3–10 mice at each time point. [Figure 7E] Effect of miRNA-485 inhibitor (485ASO-001) on hair follicle length (μm) in depilated C57BL / 6J mice on pd16 after two intramuscular injections of 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control) and positive control (minoxidil) on pd16. n = 3–10 mice at each time point. [Figure 7F]Effect of miRNA-485 inhibitor (485ASO-001) on hair bulb diameter (μm) in depilated C57BL / 6J mice on pd16 after two intramuscular injections of 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control) and positive control (minoxidil) on pd16. n = 3–10 mice at each time point. [Figure 7G] Effect of miRNA-485 inhibitor (485ASO-001) on hair follicle density within the dermis in depilated C57BL / 6J mice on pd16 after two intramuscular injections of 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control) and positive control (minoxidil) on pd16. n=3–10 mice for each time point. [Figure 7H] Effect of miRNA-485 inhibitor (485ASO-001) on hair follicle density in subcutaneous tissue in depilated C57BL / 6J mice on pd16 after two intramuscular injections of 0.3 mg / kg (mpk) and 0.6 mg / kg (mpk), or phosphate-buffered saline (PBS) (negative control) and positive control (minoxidil) on pd16. n=3–10 mice for each time point.

[0071] [Figure 8A] Western blotting analysis of CD36 and vascular endothelial growth factor (VEGF-A) expression is shown.

[0072] [Figure 8B]Quantification of CD36 protein expression on day 10 pd after treatment with 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control). Relative expression of each protein was normalized using β-actin. Values ​​are mean ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001. [Figure 8C] Quantification of VEGF-A protein expression at day 10 pd after treatment with 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control). Relative expression levels of each protein were normalized using β-actin. Values ​​are mean ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001.

[0073] [Figure 9A] Western blotting analysis of Wnt3a and β-catenin expression is shown.

[0074] [Figure 9B] Quantification of Wnt3a protein expression at day 10 pd after treatment with 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control). Relative expression levels of each protein were normalized using β-actin. Values ​​are mean ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001. [Figure 9C] Quantification of β-catenin protein expression at day 10 pd after treatment with 0.6 mg / kg (mpk) of miRNA-485 inhibitor (485ASO-001) or phosphate-buffered saline (PBS) (negative control). Relative expression levels of each protein were normalized using β-actin. Values ​​are means ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001.

[0075] [Figure 10]Immunofluorescence staining of CD36 is shown. CD36 enriched dermal sheath (DS) expression is observed in the perivascular regions of hair follicles in PBS-treated controls (A) and in hair follicles treated with miR-485 inhibitor (485ASO-001) (B). Scale bar = 50 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] The present disclosure relates to the use of a miRNA-485 inhibitor to induce hair growth, increase hair density, increase hair follicle density, increase hair shaft thickness, increase hair length, prevent hair loss, reduce hair loss, or any combination thereof in a subject in need thereof, comprising a nucleotide sequence encoding a nucleotide molecule comprising at least one miRNA-485 binding site, wherein the nucleotide molecule does not encode a protein.

[0077] Prior to describing the disclosure in more detail, it should be understood that the disclosure is not limited to specific compositions or process steps, as the specific compositions or process steps described may of course vary. As will be apparent to one skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has separate components and features that can be easily separated from or combined with the features of any of the other aspects without departing from the scope or spirit of the disclosure. Any method described can be carried out in the order of events described or in any other order that is logically possible.

[0078] The headings provided herein are not intended to be limitations of the various aspects of the disclosure, which aspects may be defined by reference to the entire specification. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only, and not for the purpose of limitation, since the scope of the disclosure is limited only by the appended claims.

[0079] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise stated herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0080] It should be noted that an entity designated with the term "a" or "an" refers to one or more of that entity, e.g., "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, "a" (or "an"), as well as "one or more" and "at least one" may be used interchangeably herein. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement shall serve as a predicate to the use of exclusive language such as "only", "only" and the like in connection with the recitation of claim elements, or to the use of limitation by negative.

[0081] Furthermore, "and / or" as used herein is to be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (only), B (only), and C (only).

[0082] Whenever an embodiment is described in this specification using the term "comprising," it should be understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0083] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary for many of the terms used in this disclosure.

[0084] Units, prefixes, and symbols are indicated in their International System of Units (SI) accepted format. Numeric ranges are intended to be inclusive of the numbers defining the range. When a range of numerical values ​​is described, it is to be understood that each intervening integer value between the stated upper and lower limits of the range, and each fraction thereof, is also specifically disclosed, along with each subrange between such values. Although the upper and lower limits of any range may be independently included or excluded from the range, each range in which either limit is included, neither limit is included, or both limits are included is also encompassed by the disclosure. Thus, ranges described herein are understood to be shorthand notations of all values ​​within the range, including the stated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0085] When a value is explicitly stated, it should be understood that values ​​that are approximately the same number or amount as the stated value are also included within the scope of the disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is included within the scope of the disclosure. Conversely, when different elements or groups of elements are disclosed separately, their combinations are also disclosed. When any element of a disclosure is disclosed as having multiple options, examples of the disclosure in which each option is excluded, alone or in any combination with other options, are also disclosed herein. In other words, multiple elements of a disclosure can have such an exclusion, and all combinations of elements with such exclusion are disclosed herein.

[0086] As used herein, the term "hair" is used to mean hair on the scalp, head, face and / or body, including, but not limited to, head hair, eyelashes, eyebrows, moustache, beard, ear hair, nose hair, chest hair, pubic hair and underarm hair.

[0087] As used herein, the term "hair growth" is used to mean the induction of early growth of new hairs, the prolongation of the active growth phase (anagen) of the hair cycle, the increase in the rate of hair growth, and / or the increase in hair shaft width, including, but not limited to, the induction of hair growth and making hair more visible.

[0088] As used herein, the terms "hair loss" and "thinning hair" refer to a decrease in normal hair density and / or shortening of the normal growth phase (anagen) of the hair cycle and / or a decrease in the width of the hair shaft and a decrease in the number of hairs, which may be caused by aging, genetic predisposition, and / or other causes, and may be present in men or women, young or old. The terms "hair loss", "alopecia", "baldness", and "pattern hair loss" are used interchangeably herein.

[0089] As used herein, the terms "inducing hair growth", "promoting hair growth", "stimulating hair growth", "promoting hair growth" or "increasing hair growth" are used to mean at least one of the following results: an increase in the number and / or length and / or thickness of hairs on at least a portion of the affected skin (or scalp) surface.

[0090] Nucleotides are represented by the commonly accepted single-letter symbols for nucleotides. Unless otherwise noted, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are represented herein by the commonly known single-letter symbols for nucleotides recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, "a" represents adenine, "c" represents cytosine, "g" represents guanine, "t" represents thymine, and "u" represents uracil.

[0091] Amino acid sequences are written from left to right in the amino to carboxyl terminus direction. Amino acids are represented herein by the commonly known three letter or one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0092] The term "about" is used herein to mean approximately, roughly, roughly, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above and below the stated value, for example, by a variance of 10 percent (higher or lower) above or below.

[0093] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, the AAV serotypes and phyla disclosed in Gao et al. (J. Virol. 78:6381 (2004)) and Morris et al. (Virol. 33:375 (2004)), and any other AAV now known or hereafter discovered (see, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV" includes derivatives of known AAVs. In some embodiments, "AAV" includes modified or artificial AAVs.

[0094] The terms "administration", "administering" and grammatical variations thereof refer to the introduction of a composition, such as a miRNA inhibitor of the present disclosure, into a subject by a pharma- ceutically acceptable route. The introduction of a composition, such as a micelle comprising a miRNA inhibitor of the present disclosure, into a subject is by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, or topical. Administration includes self-administration and administration by another. The appropriate route of administration allows the composition or agent to perform its intended function. For example, if the appropriate route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0095] As used herein, the term "approximately" as applied to one or more values ​​of interest refers to a value that is comparable to a specified reference value. In certain embodiments, the term "approximately" refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (more than or less than a number) in either direction of a specified reference value, unless otherwise specified or otherwise clear from the context (except when such number exceeds 100% of the possible values).

[0096] As used herein, the term "conserved" refers to nucleotides or amino acid residues of polynucleotide or polypeptide sequences, respectively, that are invariably found in the same position in two or more sequences that are being compared. Relatively conserved nucleotides or amino acids are those that are more conserved between related sequences than nucleotides or amino acids that occur in other parts of the sequences.

[0097] In some embodiments, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, regions, or features thereof.

[0098] As used herein, the term "derived from" refers to a component that is isolated or produced from a specific molecule or organism using a specific molecule or organism or information (e.g., amino acid or nucleic acid sequence). For example, a nucleic acid sequence derived from a second nucleic acid sequence may contain a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of a nucleotide or polypeptide, the derived species may be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. The mutagenesis used to derive a nucleotide or polypeptide may be intentionally directed or intentionally random, or a combination of each. The mutagenesis of a nucleotide or polypeptide to create a different nucleotide or polypeptide derived from the first one may be a random event (e.g., caused by the infidelity of polymerase), and the identification of the derived nucleotide or polypeptide may be made, for example, by a suitable screening method described herein.In some embodiments, a nucleotide or amino acid sequence derived from a second nucleotide or amino acid sequence has a similar sequence identity to the second nucleotide or amino acid sequence, respectively, of at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least %, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.

[0099] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA) is not normally translated into an amino acid but can be considered part of the coding region. However, all flanking sequences, e.g., promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are generally determined by a start codon at the 5'-terminus, which encodes the amino-terminus of the resulting polypeptide, and a translation stop codon at the 3'-terminus, which encodes the carboxy-terminus of the resulting polypeptide.

[0100] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each containing a nucleobase sequence) related to each other by Watson-Crick base pairing rules, or between an oligomer and a target gene. For example, the nucleobase sequence "TGA(5'→3')" is complementary to the nucleobase sequence "ACT(3'→5')". Complementarity may be "partial", in which fewer than all of the nucleobases of a given nucleobase sequence match another nucleobase sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleobase sequence and another nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Thus, in certain embodiments, the term "complementarity" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity or complementarity with a target nucleic acid sequence (e.g., a nucleic acid sequence of miR-485). Or, as an example, "perfect" or "complete" (100%) complementarity may exist between a given nucleobase sequence and another nucleobase sequence. In some embodiments, the degree of complementarity between nucleobase sequences significantly affects the efficiency and strength of hybridization between sequences.

[0101] The term "downstream" refers to a nucleotide sequence that is located 3' of a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to a sequence that follows the transcription start point. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0102] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound, such as the miRNA inhibitors of the disclosure.

[0103] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide. Expression includes, but is not limited to, transcription of a polynucleotide into a microRNA binding site, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), or any other RNA product. Expression includes, but is not limited to, transcription of a polynucleotide into a messenger RNA (mRNA), and translation of the mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be a nucleic acid, such as an RNA, produced by transcription of a gene. As used herein, a gene product can be a nucleic acid, an RNA or miRNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides having post-translational modifications, such as phosphorylation, methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0104] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules. In general, the term "homology" refers to the evolutionary relationship of two molecules. Thus, two homologous molecules have a common evolutionary ancestor. In the context of this disclosure, the term homology encompasses both identity and similarity.

[0105] In some embodiments, polymer molecules are considered to be "homologous" to one another if at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the monomers in the molecule are identical (exactly the same monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide sequences).

[0106] In the context of the present disclosure, substitutions (even when they are referred to as amino acid substitutions) are made at the nucleic acid level, i.e., the replacement of an amino acid residue with an alternative amino acid residue is made by replacing a codon encoding a first amino acid with a codon encoding a second amino acid.

[0107] As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules, e.g., between polynucleotide molecules. The term "identical" without any additional modifier, e.g., "polynucleotide A is identical to polynucleotide B," means that the polynucleotide sequences are 100% identical (100% sequence identity). For example, describing two sequences as "70% identical" is equivalent to describing them as having, e.g., "70% sequence identity."

[0108] The calculation of the percent identity (%) of two polypeptide or polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second polypeptide or polynucleotide sequences for optimal alignment, and non-identical sequences can be ignored for comparison). In certain embodiments, the length of the aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions, or bases in the case of polynucleotides, are then compared.

[0109] When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using a mathematical algorithm.

[0110] Suitable software programs that can be used to align different sequences (e.g., polynucleotide sequences) are available from a variety of sources. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST package of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm to perform a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, whereas BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite and available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0111] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, and the like.

[0112] Different regions in a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent sequence identity is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Note that length values ​​are always integers.

[0113] In certain embodiments, the percentage identity (%ID) or identity of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID=100×(Y / Z), where Y is the number of amino acid residues (or nucleic acid bases) that are evaluated as perfect matches in an alignment of the first and second sequences (aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is greater than the length of the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0114] Those skilled in the art will understand that the generation of sequence alignment for calculating the sequence identity percentage is not limited to binary sequence comparison performed only by primary sequence data. It will also be understood that sequence alignment can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., protein crystal structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is available at www.tcoffee.org, alternatively, for example, T-Coffee available from EBI. It will also be understood that the final alignment used to calculate the sequence identity percentage can be managed either automatically or manually.

[0115] As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably and refer to a preparation of a desired composition of the disclosure, e.g., a miRNA inhibitor of the disclosure, that has been subjected to one or more purification processes. In some embodiments, isolation or purification, as used herein, is a process of removing, partially removing (e.g., a fraction of), a composition of the disclosure, e.g., a miRNA inhibitor of the disclosure, from a sample containing contaminants.

[0116] In some embodiments, the isolated composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is below an acceptable level or amount. In other embodiments, the isolated composition has an amount and / or concentration of the desired composition of the present disclosure that is above an acceptable amount and / or concentration and / or activity. In other embodiments, the isolated composition is enriched compared to the starting material from which the composition is obtained. This enrichment can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or more than 99.9999% compared to the starting material.

[0117] In some embodiments, the isolated preparation is substantially free of residual biological products. In some embodiments, the isolated preparation is 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, or at least about 90% free of any contaminating biological material. The residual biological products may include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

[0118] As used herein, the term "linked" refers to a first amino acid sequence or polynucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence may be directly linked or juxtaposed to the second amino acid or polynucleotide sequence, or alternatively, an intervening sequence may be covalently linked from the first sequence to the second sequence. The term "linked" does not only mean the fusion of the first polynucleotide sequence to the second polynucleotide sequence at the 5' or 3' end, but also includes the insertion of the entire first polynucleotide sequence (or the second polynucleotide sequence, respectively) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence). The first polynucleotide sequence may be linked to the second polynucleotide sequence by a phosphodiester bond or a linker. The linker may be, for example, a polynucleotide.

[0119] As used herein, "miRNA inhibitor" refers to a compound that can reduce, change, and / or regulate the expression, function, and / or activity of miRNA. The miRNA inhibitor can be a polynucleotide sequence that is at least partially complementary to a target miRNA nucleic acid sequence, whereby the miRNA inhibitor hybridizes with the target miRNA sequence. For example, the miR-485 inhibitor of the present disclosure comprises a nucleotide sequence that encodes a nucleotide molecule that is at least partially complementary to a target miR-485 (e.g., miRNA-485-3p) nucleic acid sequence, whereby the miR-485 inhibitor hybridizes with the miR-485 sequence. In a further aspect, the hybridization of miR-485 to the miR-485 inhibitor sequence reduces, changes, and / or regulates the expression, function, and / or activity of miR-485 (e.g., hybridization results in increased expression of SIRT1 protein and / or SIRT1 gene).

[0120] The terms "miRNA", "miR" and "microRNA" are used interchangeably and refer to microRNA molecules found in eukaryotes that are involved in gene regulation by RNA. The term is used to refer to single-stranded RNA molecules processed from precursors. In some aspects, the term "antisense oligomer" can also be used to describe the microRNA molecules of the present disclosure. The names of miRNAs and their sequences relevant to the present disclosure are provided herein. MicroRNAs recognize and bind to target mRNAs through imperfect base pairing, resulting in destabilization or translational inhibition of the target mRNA, thereby downregulating target gene expression. Conversely, targeting of miRNAs with molecules that contain miRNA binding sites (generally molecules that contain sequences complementary to the seed region of the miRNA) can reduce or inhibit the translational inhibition induced by the miRNA, resulting in upregulation of the target gene.

[0121] The term "mismatch" or "multiple mismatches" refers to one or more nucleobases (whether contiguous or separated) in an oligomeric nucleobase sequence (e.g., miR-485 inhibitor) that do not match the target nucleic acid sequence (e.g., miR-485 inhibitor) according to base pairing rules. In many cases, perfect complementarity is desired, but in some embodiments, one or more (preferably 6, 5, 4, 3, 2, or 1) mismatches to the target nucleic acid sequence can occur. Variations at any position within the oligomer are included. In certain embodiments, the antisense oligomers (e.g., miR-485 inhibitors) of the present disclosure include nucleobase sequence variations near the ends, variations internally, and when present, are usually present within about 6, 5, 4, 3, 2, or 1 subunit of the 5' and / or 3' ends. In some embodiments, one, two, or three nucleobases may be removed and still provide on-target binding.

[0122] As used herein, the terms "modulate", "modify" and grammatical variations thereof, when applied to a particular concentration, level, expression, function, or behavior, generally refer to the ability to increase or decrease, e.g., directly or indirectly, promote / stimulate / upregulate or interfere with / inhibit / downregulate, a particular concentration, level, expression, function, or behavior. In some cases, a modulator may increase and / or decrease a particular concentration, level, activity, or function compared to a control, or compared to a generally expected average level of activity, or compared to a control level of activity. In some embodiments, the miRNA inhibitors disclosed herein, e.g., miR-485 inhibitors, can modulate (e.g., decrease, change, or eliminate) the expression, function, and / or activity of miR-485, thereby modulating the expression and / or activity of SIRT1 protein or gene.

[0123] "Nucleic acid", "nucleic acid molecule", "nucleotide sequence", "polynucleotide", and grammatical variations thereof, are used interchangeably and refer to ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule") in the phosphate polymeric form, either in single-stranded form or double-stranded, or any phosphoester analogs thereof, such as phosphorothioates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The terms nucleic acid molecule, and particularly DNA or RNA molecule, refer only to the primary and secondary structure of the molecule and are not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When describing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the normal convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). A "recombinant DNA molecule" is a DNA molecule that has been subjected to molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids as described herein.

[0124] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", and grammatical variations thereof, include any agent approved by a regulatory agency of the U.S. Federal government for use in animals, including humans, or listed in the U.S. Pharmacopeia, as well as any carrier or diluent that does not cause the production of undesirable physiological effects to an extent that would prohibit administration of the composition to a subject, and does not inhibit the biological activity and properties of the compound being administered. Included are excipients and carriers that are generally safe, non-toxic, and desirable, and that are useful in preparing pharmaceutical compositions.

[0125] As used herein, the term "pharmaceutical composition" refers to one or more of the compounds described herein, such as, for example, the miRNA inhibitors of the present disclosure, mixed or admixed with or suspended in one or more other chemical components, such as pharma- ceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration of a formulation containing the miRNA inhibitors of the present disclosure to a subject.

[0126] As used herein, the term "polynucleotide" refers to a polymer of any length of nucleotides that can include ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof.

[0127] In some embodiments, the term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). The term also includes modified polynucleotides, for example, by alkylation and / or capping, as well as unmodified forms of the polynucleotide.

[0128] In some embodiments, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), whether spliced ​​or unspliced, including tRNA, rRNA, shRNA, siRNA, miRNA, and mRNA, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers that contain non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other sequence-specific synthetic nucleic acid polymers, provided that they contain nucleobases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA.

[0129] In some embodiments of the present disclosure, polynucleotide can be an oligonucleotide, such as an antisense oligonucleotide. In some embodiments, oligonucleotide is RNA. In some embodiments, RNA is synthetic RNA. In some embodiments, synthetic RNA comprises at least one non-natural nucleobase. In some embodiments, all nucleobases of a certain type are replaced with non-natural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be replaced with non-natural nucleobases, such as 5-methoxyuridine).

[0130] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, for example, encoded by the SIRT1 gene. The polymers may contain modified amino acids. These terms also encompass amino acid polymers that are naturally modified or modified by intervention, such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), and other modifications known in the art, are also included within the definition. As used herein, the term "polypeptide" refers to proteins, polypeptides, and peptides of any size, structure, or function.

[0131] Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.

[0132] Polypeptides can be single polypeptides or multi-molecular complexes such as dimers, trimers, or tetramers. They can also include single-chain or multi-linked polypeptides. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be about 50 amino acids or less in length, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids in length.

[0133] As used herein, the terms "prevent", "preventing", and variations thereof, refer to partially or completely delaying the onset of a disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical signs of a particular disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or signs of a particular disease, disorder, and / or condition; partially or completely delaying the progression of a particular disease, disorder, and / or condition; and / or reducing the risk of developing a pathology associated with a disease, disorder, and / or condition. In some embodiments, prevention of an outcome is achieved by prophylactic treatment.

[0134] As used herein, the terms "preventing hair loss," "prevention of hair loss," "preventing hair thinning," and "prevention of hair thinning" refer to the effect of reducing any hair loss or thinning prior to this disclosure.

[0135] As used herein, the terms "reducing hair loss," "reducing hair loss," "reducing hair thinning," and "reducing hair thinning" refer to the effect of reducing any hair loss or thinning as described herein in a subject in need of reducing hair loss or thinning.

[0136] As used herein, the terms "promoter" and "promoter sequence" are interchangeable and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. A promoter may be derived in its entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or even may include synthetic DNA segments. Those skilled in the art will appreciate that different promoters can induce expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types most of the time is generally referred to as a "constitutive promoter." A promoter that causes a gene to be expressed in a particular cell type is generally referred to as a "cell-specific promoter" or "tissue-specific promoter." A promoter that causes a gene to be expressed at a particular stage of development or cell differentiation is generally referred to as a "development-specific promoter" or "cell differentiation-specific promoter." A promoter that is induced to express a gene following exposure or treatment of cells with a drug, biological molecule, chemical, ligand, light, etc. that induces the promoter is generally referred to as an "inducible promoter" or "regulatable promoter." It will be further recognized that since in many cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have the same promoter activity.

[0137] The boundaries of a promoter sequence are the transcription initiation site at its 3' end and extend upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. Within the promoter sequence, not only will a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1) be found, but also protein binding domains (consensus sequences) involved in the binding of RNA polymerase. In some embodiments, promoters that can be used with the present disclosure include tissue-specific promoters.

[0138] As used herein, "prophylaxis" refers to a therapeutic action or course of action used to prevent the onset of a disease or condition or to prevent or delay the symptoms associated with a disease or condition.

[0139] As used herein, "prophylaxis" refers to measures taken to maintain health, prevent or delay the onset of a disease or condition, or prevent or delay symptoms associated with a disease or condition.

[0140] As used herein, the term "gene regulatory region" or "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region that influences the transcription, RNA processing, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and / or a transcription termination sequence is usually located 3' to the coding sequence.

[0141] In some aspects, the miR-485 inhibitors disclosed herein (e.g., polynucleotides comprising RNA comprising one or more miR-485 binding sites) can include a promoter and / or other expression (e.g., transcription) control elements functionally associated with one or more coding regions. In a functional association, the coding region of a gene product is associated with one or more regulatory regions such that expression of the gene product is under the influence or control of the regulatory regions. For example, a coding region and a promoter are "functionally associated" when induction of promoter function results in transcription of the mRNA encoded by the coding region, and the nature of the association between the promoter and the coding region does not interfere with the ability of the promoter to induce expression of the gene product or the ability of the DNA template to be transcribed. Other expression control elements other than promoters, such as enhancers, operators, repressors, and transcription termination signals, can also be functionally associated with a coding region to induce expression of a gene product.

[0142] As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, for example between polynucleotide molecules (e.g., miRNA molecules).The calculation of the percentage similarity (%) between polymer molecules to each other can be performed in the same manner as the calculation of percentage identity (%), except that the calculation of percentage similarity (%) takes into account conservative substitutions as understood in the art.It is understood that percentage similarity (%) depends on the comparison scale used, i.e., whether nucleic acids are compared according to, for example, their evolutionary closeness, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or a combination thereof.

[0143] The terms "subject," "patient," "individual," and "host," and variations thereof, are used interchangeably herein and refer to any mammalian subject for which diagnosis, treatment, or therapy is desired, including, but not limited to, humans, domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), particularly humans. The methods described herein are applicable to both human therapy and veterinary applications.

[0144] As used herein, the terms "subject in need thereof" and "subject at risk of hair loss" include subjects, such as mammalian subjects, who will benefit from administration of a miRNA inhibitor (e.g., a miR-485 inhibitor) of the present disclosure, for example, to induce hair growth. In some embodiments, the subject has one or more disorders selected from the group consisting of alopecia totalis, androgenetic alopecia, alopecia areata, alopecia universalis, regressive alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial alopecia, scarring alopecia, scalp hair thinning, hair shaft abnormalities, infectious hair disorders, genetic disorders, and hair loss due to chemotherapy, hormonal imbalance, fungal infection, medication, chemical hair treatment, or aging. In some embodiments, the subject is a human.

[0145] As used herein, the term "therapeutically effective amount" refers to an amount of a reagent or pharmaceutical compound, including a miRNA inhibitor of the present disclosure, sufficient to produce a desired therapeutic, pharmacological, and / or physiological effect in a subject in need thereof. A therapeutically effective amount may be a "prophylactically effective amount" where prevention may be considered treatment.

[0146] As used herein, the terms "treat", "treatment", or "treating" refer to, for example, reducing the severity of a disease or condition, reducing the duration of a disease course, improving or eliminating one or more symptoms associated with a disease or condition (e.g., diabetes), providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition. The term also includes prophylaxis or prevention of a disease or condition or a symptom thereof.

[0147] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence.

[0148] A "vector" refers to any carrier for cloning and / or introducing a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached to effect replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo (i.e., capable of replicating under its own control). The term "vector" includes viral and non-viral carriers for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. Numerous vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector with complementary cohesive termini.

[0149] Vectors can be engineered to encode a selectable marker or reporter that allows for the selection or identification of cells that have taken up the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that have taken up and expressed other coding regions contained in the vector. Examples of selectable markers known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., as well as genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentenyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered as reporters.

[0150] II. How to use Provided herein is a method of inducing hair growth in a subject in need of induction of hair growth (e.g., a subject at risk of hair loss), comprising administering to the subject a compound that inhibits miR-485 ("miRNA inhibitor"). Also provided herein is a method of reducing hair loss in a subject in need of reducing hair loss (e.g., a subject at risk of hair loss), comprising administering to the subject a compound that inhibits miR-485. Also provided herein is a method of increasing hair density in a subject in need of increasing hair density (e.g., a subject at risk of hair loss), comprising administering to the subject a compound that inhibits miR-485. Also provided herein is a method of increasing hair length in a subject in need of increasing hair length (e.g., a subject at risk of hair loss), comprising administering to the subject a compound that inhibits miR-485. Also provided herein is a method of increasing hair follicle density in a subject in need of increasing hair follicle density (e.g., a subject at risk of hair loss), comprising administering to the subject a compound that inhibits miR-485. Also provided herein is a method of increasing hair shaft thickness in a subject in need thereof (e.g., a subject at risk of hair loss), comprising administering to the subject a compound that inhibits miR-485. Also provided herein is a method of preventing hair loss in a subject at risk of hair loss, comprising administering to the subject a compound that inhibits miR-485 (a miRNA inhibitor).

[0151] The underlying causes of hair loss are not always the same from one individual to another. Also, the process of hair growth involves several stages, and there are many factors that can alter the normal vigorous growth of hair. The hair growth cycle is divided into three stages: anagen, during which a very high level of cell proliferation occurs in the hair follicle and the hair actively grows; catagen, during which the proliferative activity of the hair follicle temporarily slows down and new hairs are formed; and telogen, during which the growth of the hair follicle simply stops and reverses until the hair falls out and a new anagen phase begins. Of course, it is entirely normal for the average person to lose a lot of hair every day, and therefore this cycle is usually repeated continuously throughout life to replace the hairs that have fallen out. However, this cycle slows down in all people with age, and the normal hair is gradually replaced by thinner hairs (vellus hairs), shortening the cycle. Early to mid-stage hair loss is almost always manifested as a thinning of the hair shafts of a large proportion of hair follicles and a slowing of hair shaft growth, with the remaining hairs being thinner, lighter and closer to the scalp, while the hair follicle density and hair shafts per follicle remain essentially unchanged. In people suffering from abnormal hair loss, the normal cyclic process is somehow disrupted, whether by an abnormal acceleration of the process or other changes. This ultimately leads to a more rapid transition to the telogen phase, gradually more vellus hairs growing, and finally baldness. It is only in the final stage of baldness that the hair follicles become irreversibly inactive (see, for example, International Publication WO2004 / 078117A2).

[0152] Hair thickness or density D can be quantified as the product of three factors: (1) hair follicle density F, measured in follicles per square centimeter, (2) the average number of hair shafts per follicle N, and (3) the average hair shaft thickness T. The product of these three quantities, which indicates hair density D, is given by: D=F*N*T

[0153] Another possible objective measure of thinning can be the percentage of hair width outside two standard deviations of a selected subset of hairs from the head.

[0154] Another alternative measure of hair density is the thickness (diameter) of a hair, expressed as the cross-sectional area of ​​the hair, A = πT2 / 4. An overall feeling or index of the "fullness" of a person's hair may include the length of the hair, L, giving a hair volume parameter, V=F*N*A*L. Since the feeling of fullness of hair can be increased by growing the remaining hair, V acts as a metric of appearance but does not represent thinning of hair. Thus, D=FNA is the objective measure used.

[0155] The typical hair follicle unit density is 60-120cm -2 N ranges from 1 to 2, with each follicle typically containing one or two hair shafts, but rarely more than two of different ages. Hair shaft thickness can be classified as thick, medium, or thin, with the average hair shaft thickness varying from about 40 microns in width for fine hair, to an average of about 90 microns in width for thick hair. N is generally a number between 1 and 2, more commonly 1 to 1.25. N can be omitted from density determination, but may need to be considered in certain rare cases.

[0156] The normalized hair density measure for each individual is the ratio of the density at the crown and / or front to the density at the left and / or right sides. The hair thinning ratio for an individual is therefore R = 100 × (D 頭頂部 ) / D 側頭部 , where D for each corresponding region 頭頂部 and D. 側頭部 = F*N*T. See, for example, U.S. Patent Application Publication No. 2009 / 0036800, which is incorporated herein by reference in its entirety.

[0157] In some embodiments, the subject has one or more disorders selected from the group consisting of alopecia totalis, androgenetic alopecia, alopecia areata, alopecia universalis, regressive alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial alopecia, scarring alopecia, scalp hair loss, hair shaft abnormalities, infectious hair disorders, genetic disorders, and hair loss due to chemotherapy, hormonal imbalance, fungal infection, medication, chemical hair treatment, or aging. In some embodiments, the subject is a human.

[0158] In some embodiments, the miR-485 inhibitor disclosed herein can be administered by any suitable route known in the art.In some embodiments, the miR-485 inhibitor is administered supplementally, intramuscularly, subcutaneously, by eye drop, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraventricularly, intrathecally, intracisternally, intracapsularly, intratumorally, locally, or any combination thereof.In certain embodiments, the miR-485 inhibitor is administered intramuscularly.

[0159] In some embodiments, the miRNA-485 inhibitor is administered by painting, spraying, vaporizing, or injecting onto the area of ​​the skin in which promotion of hair growth is required.

[0160] In some embodiments, the miRNA-485 inhibitor is administered locally to an area of ​​the skin where promotion of hair growth is required.

[0161] In some embodiments, the miRNA-485 inhibitor is formulated in a form selected from the group consisting of an ointment, shampoo, conditioner, lotion, tonic, gel, and mousse. In some embodiments, the administering step is performed by immersing or bathing the subject in the miRNA inhibitor formulated in a form selected from the group consisting of an ointment, shampoo, conditioner, lotion, tonic, gel, and mousse.

[0162] In some embodiments, the miR-485 inhibitor of the present disclosure can be used in combination with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent and the miR-485 inhibitor are administered simultaneously. In certain embodiments, the additional therapeutic agent and the miR-485 inhibitor are administered sequentially.

[0163] In some embodiments, the miR-485 inhibitor disclosed herein does not cause side effects.In certain embodiments, the miR-485 inhibitor disclosed herein does not adversely affect body weight when administered to a subject.In some embodiments, the miR-485 inhibitor disclosed herein does not cause increased mortality and does not cause pathological abnormalities when administered to a subject.

[0164] In some embodiments, the miR-485 inhibitors disclosed herein can exert a therapeutic effect (e.g., induce hair growth or prevent hair loss) by regulating the expression and / or activity of one or more genes. In some embodiments, the miRNA-485 inhibitors disclosed herein can regulate the expression and / or activity of a gene selected from CTBP1, TRIP6, SIRT1, CD36, PGC1-a, or a combination thereof.

[0165] Regulation of CTBP1 In some aspects, contacting a cell with a miRNA-485 inhibitor described herein can increase the expression of CTBP1 protein and / or the CTBP1 gene in the cell.

[0166] C-terminal binding protein 1 (CTBP1) is a protein that in humans is encoded by the CTBP1 gene. CTBP1 is a regulatory protein that binds to sequence-specific DNA binding proteins and helps turn off genes, for example, by recruiting histone-modifying enzymes that add repressive histone marks and remove activating marks. CTBP1 protein can also self-assemble, assembling gene regulatory complexes.

[0167] In humans, the CTBP1 gene is located on chromosome 4 (nucleotides 1,211,444 to 1,250,355 (negative orientation) of GenBank accession number NC_000004.12). Synonyms for the CTBP1 gene and its encoded protein are known and include "brefeldin A ribosylation substrate", "BARS", "HADDTS", "CtBP1", or "CTBP".

[0168] There are at least two known isoforms of the human CTBP1 protein that arise from alternative splicing. CTBP1 isoform 1 (UniProt Identification Number: Q13363-1, SEQ ID NO: 31) consists of 440 amino acids and has been selected as the canonical sequence. CTBP1 isoform 2 (UniProt Identification Number: Q13363-2, SEQ ID NO: 32) consists of 429 amino acids and differs from the canonical sequence in the following ways: 1-13: MGSSHLLNKGLPL→MS. The sequences of the two CTBP1 isoforms are shown in Table 1 below. [Table 1]

[0169] As used herein, the term "CTBP1" includes any variant or isoform of CTBP1 naturally expressed by a cell. Thus, in some embodiments, miR-485 inhibitors disclosed herein can increase the expression of CTBP1 isoform 1. In some embodiments, miR-485 inhibitors disclosed herein can increase the expression of CTBP1 isoform 2. In further embodiments, miR-485 inhibitors disclosed herein can increase the expression of CTBP1 isoform 1 and isoform 2. Unless otherwise specified, both isoform 1 and isoform 2 are collectively referred to herein as "CTBP1."

[0170] In some embodiments, contacting a cell with a miRNA-485 inhibitor increases the expression and / or activity of CTBP1 protein and / or CTBP1 gene in the cell by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold, compared to expression and / or activity in a reference cell (e.g., a corresponding cell that has not been contacted with a miRNA-485 inhibitor).

[0171] Without being bound to any one theory, in some embodiments, the miR-485 inhibitors disclosed herein increase the expression of CTBP1 protein and / or the CTBP1 gene by reducing the expression and / or activity of miR-485, e.g., miR-485-3p.

[0172] Regulation of TRIP6 In some aspects, contacting a cell with a miRNA-485 inhibitor described herein can increase the expression of TRIP6 protein and / or the TRIP6 gene in the cell.

[0173] Thyroid receptor-interacting protein 6 (TRIP6) is a protein that in humans is encoded by the TRIP6 gene. The TRIP6 protein is a member of the zyxin family and contains three LIM zinc-binding domains. TRIP6 protein has been shown to localize at focal adhesion sites and along actin stress fibers.

[0174] In humans, the TRIP6 gene is located on chromosome 7 (nucleotides 100,867,387 to 100,873,454 (positive strand orientation) of GenBank accession number NC_000007.14). Synonyms for the TRIP6 gene and its encoded protein include "thyroid hormone receptor interacting factor 6," "ZRP-1," "OIP1," "thyroid hormone receptor interacting protein 6," "OPA interacting protein 1," and "zyxin-related protein 1."

[0175] There are at least three known isoforms of the human TRIP6 protein that arise from alternative splicing: TRIP6 isoform 1 (UniProt Identification No. Q15654-1, SEQ ID NO: 78) consists of 4766 amino acids and has been selected as the canonical sequence; TRIP6 isoform 2 (UniProt Identification No. Q15654-2, SEQ ID NO: 79) consists of 106 amino acids and differs from the canonical sequence in the following ways: (i) 37 to 106: ALQPHPRVNF...IDLLSSTLAE → VLPGPRGTGG...CVTATRPTGI, (ii) 107 to 476: deletion. TRIP6 isoform 3 (UniProt identification number: Q15654-2, SEQ ID NO: 80) consists of 80 amino acids and differs from the canonical sequence in the following ways: (i) 37–80: ALQPHPRVNF...SHGVLQHTQG→GAPCRQGGPS...CVTATRPTGI; (ii) 81–476: deletion. Table 2 below shows the sequences of the different TRIP6 protein isoforms. [Table 2]

[0176] As used herein, the term "TRIP6" includes any variant or isoform of TRIP6 naturally expressed by a cell. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of TRIP6 isoform 1. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of TRIP6 isoform 2. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of TRIP6 isoform 3. In further embodiments, the miR-485 inhibitors disclosed herein can increase the expression of TRIP6 isoform 1, isoform 2, and isoform 3. Unless otherwise specified, isoform 1, isoform 2, and isoform 3 are collectively referred to herein as "TRIP6".

[0177] In some embodiments, contacting a cell with a miRNA-485 inhibitor increases the expression and / or activity of TRIP6 protein and / or TRIP6 gene in the cell by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold, compared to expression and / or activity in a reference cell (e.g., a corresponding cell that has not been contacted with a miRNA-485 inhibitor).

[0178] In some aspects, miR-485 inhibitors may also increase the expression and / or activity of other LIM domain-containing proteins.

[0179] Without being bound to any one theory, in some aspects, the miR-485 inhibitors disclosed herein increase the expression of TRIP6 protein and / or the TRIP6 gene by reducing the expression and / or activity of miR-485, e.g., miR-485-3p.

[0180] Regulation of SIRT1 In some aspects, contacting a cell (e.g., a neural stem cell) with a miRNA-485 inhibitor described herein can increase expression of SIRT1 protein and / or the SIRT1 gene in the cell.

[0181] Sirtuin 1 (SIRT1), also known as NAD-dependent deacetylase sirtuin 1, is a protein encoded by the SIRT1 gene in humans. The SIRT1 gene is located on human chromosome 10 (nucleotides 67,884,656 to 67,918,390 (positive strand orientation) of GenBank accession number NC_000010.11). Synonyms for the SIRT1 gene and its encoded protein include "regulatory protein SIR2 homolog 1," "silent mating-type information regulation 2 homolog 1," "SIR2," "SIR2-like protein 1," "SIR2L1," "SIR2α," "type 1 sirtuin," "hSIRT1," or "hSIR2."

[0182] There are at least two known isoforms of the human SIRT1 protein that arise from alternative splicing. SIRT1 isoform 1 (UniProt Identification Number: Q96EB6-1) consists of 747 amino acids and has been selected as the canonical sequence (SEQ ID NO: 31). SIRT1 isoform 2 (also known as "Δexon 8" (UniProt Identification Number: Q96EB6-2) consists of 561 amino acids and differs from the canonical sequence in the following ways: 454 to 639: deletion (SEQ ID NO: 32). The sequences of the two SIRT1 isoforms are shown in Table 3 below. [Table 3]

[0183] As used herein, the term "SIRT1" includes any variant or isoform of SIRT1 that is naturally expressed by a cell. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of SIRT1 isoform 1. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of SIRT1 isoform 2. In further embodiments, the miR-485 inhibitors disclosed herein can increase the expression of SIRT1 isoform 1 and isoform 2. Unless otherwise specified, both isoform 1 and isoform 2 are collectively referred to herein as "SIRT1."

[0184] In some embodiments, contacting a cell with a miRNA-485 inhibitor increases the expression and / or activity of SIRT1 protein and / or SIRT1 gene in the cell by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold, as compared to expression and / or activity in a reference cell (e.g., a corresponding cell that has not been contacted with a miRNA-485 inhibitor).

[0185] Without being bound to any one theory, in some embodiments, the miR-485 inhibitors disclosed herein increase the expression of SIRT1 protein and / or the SIRT1 gene by decreasing the expression and / or activity of miR-485, e.g., miR-485-3p.

[0186] Regulation of CD36 In some aspects, contacting a cell with an miRNA-485 inhibitor described herein can increase CD36 protein and / or CD36 gene expression in the cell.

[0187] Cluster determinant 36 (CD36), also known as platelet glycoprotein 4, is a protein that in humans is encoded by the CD36 gene. The CD36 gene is located on chromosome 7 (nucleotides 80,602,656 to 80,679,277 (positive strand orientation) of GenBank accession number NC_000007.14). Synonyms for the CD36 gene and its encoded protein are known and include "platelet glycoprotein IV", "fatty acid transferase", "scavenger receptor class B member 3", "glycoprotein 88", "glycoprotein IIIb", "glycoprotein IV", "thrombospondin receptor", "GPIIIB", "PAS IV", "GP3B", "GPIV", "FAT", "GP4", "BDPLT10", "SCARB3", "CHDS7", "PASIV", or "PAS-4".

[0188] There are at least four known isoforms of the human CD36 protein that arise from alternative splicing: CD36 isoform 1 (UniProt identification number: P16671-1) consists of 472 amino acids and has been selected as the canonical sequence (SEQ ID NO: 36). CD36 isoform 2 (also known as "ex8-del" (UniProt Identification Number: P16671-2) (SEQ ID NO: 37) consists of 288 amino acids and differs from the canonical sequence in the following ways: 274 to 288: SIYAVFESDVNLKGI → ETCVHFTSSFSVCKS, and 289 to 472: deletion. CD36 isoform 3 (also known as "ex6-7-del" (UniProt Identification Number: P16671-3) (SEQ ID NO: 38) consists of 433 amino acids and differs from the canonical sequence in the following ways: 234 to 272: deletion. CD36 isoform 4 (also known as "ex4-del" (UniProt Identification Number: P16671-4) (SEQ ID NO: 39) consists of 412 amino acids and differs from the canonical sequence in the following respects: 144-203: deletion. The sequences of the four CD36 isoforms are shown in Table 4 below. [Table 4]

[0189] As used herein, the term "CD36" includes any variant or isoform of CD36 naturally expressed by a cell. Thus, in some embodiments, miR-485 inhibitors disclosed herein can increase the expression of CD36 isoform 1. In some embodiments, miR-485 inhibitors disclosed herein can increase the expression of CD36 isoform 2. In some embodiments, miR-485 inhibitors disclosed herein can increase the expression of CD36 isoform 3. In some embodiments, miR-485 inhibitors disclosed herein can increase the expression of CD36 isoform 4. In further embodiments, miR-485 inhibitors disclosed herein can increase the expression of both isoform 1 and isoform 2, and / or isoform 3 and isoform 4, and / or isoform 1 and isoform 4, and / or isoform 2 and isoform 3 of CD36. In some embodiments, miR-485 inhibitors disclosed herein can increase the expression of all CD36 isoforms. Unless otherwise specified, isoform 1, isoform 2, isoform 3, and isoform 4 are collectively referred to herein as "CD36."

[0190] In some embodiments, contacting a cell with a miRNA-485 inhibitor increases the expression and / or activity of CD36 protein and / or CD36 gene in the cell by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold, compared to expression and / or activity in a reference cell (e.g., a corresponding cell that has not been contacted with a miRNA-485 inhibitor).

[0191] Without being bound to any one theory, in some aspects, the miR-485 inhibitors disclosed herein increase CD36 protein and / or CD36 gene expression by decreasing the expression and / or activity of miR-485, e.g., miR-485-3p.

[0192] Regulation of PGC1 In some aspects, contacting a cell with a miRNA-485 inhibitor described herein can increase expression of PGC-1α protein and / or the PGC-1α gene in the cell.

[0193] Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), also known as PPARG coactivator 1 alpha or ligand action modulator 6, is a protein encoded in humans by the PPARGC1A gene. The PGC-1α gene is located on human chromosome 4 (nucleotides 23,792,021 to 24,472,905 (positive strand orientation) of GenBank accession number NC_000004.12). Synonyms for the PGC-1α gene and its encoded protein are known and include "PPARGC1A", "LEM6", "PGC1", "PGC1A", "PGC-1v", "PPARGC1", "PGC1α", or "PGC-1(α)".

[0194] There are at least nine known isoforms of the human PGC1-α protein that arise from alternative splicing. PGC1-α isoform 1 (UniProt identification number: Q9UBK2-1) consists of 798 amino acids and has been selected as the canonical sequence (SEQ ID NO: 40). PGC1-α isoform 2 (also known as "isoform NT-7a" (UniProt Identification Number: Q9UBK2-2) (SEQ ID NO: 41) consists of 271 amino acids and differs from the canonical sequence in the following ways: 269 to 271: DPK → LFL; 272 to 798: deletion. PGC1-α isoform 3 (also known as "isoform B5" (UniProt Identification Number: Q9UBK2-3) (SEQ ID NO: 42) consists of 803 amino acids and differs from the canonical sequence in the following ways: 1 to 18: MAWDMCNQDSESVWSDIE → MDETSPRLEEDWKKVLQREAGWQ. PGC1-α isoform 4 (also known as "isoform B4" (UniProt Identification Number: Q9UBK2-4) (SEQ ID NO: 43) consists of 786 amino acids and differs from the canonical sequence in the following ways: 1 to 18: MAWDMCNQDSESVWSDIE→MDEGYF. PGC1-α isoform 5 (also known as "isoform B4-8a" (UniProt identification number: Q9UBK2-5) (SEQ ID NO: 44) consists of 289 amino acids and differs from the canonical sequence in the following ways: 1 to 18: MAWDMCNQDSESVWSDIE→MDEGYF, 294 to 301: LTPPTTPP→VKTNLISK, 302-798: deletion. PGC1-α isoform 6 (also known as "isoform B5-NT" (UniProt identification number: Q9UBK2-6) (SEQ ID NO: 45) consists of 276 amino acids and differs from the canonical sequence in the following ways: 1-18: MAWDMCNQDSESVWSDIE → MDETSPRLEEDWKKVLQREAGWQ, 269-271: DPK → LFL, 272-798: deletion.PGC1-α isoform 7 (also known as "B4-3ext" (UniProt Identification No. Q9UBK2-7) (SEQ ID NO: 46) consists of 138 amino acids and differs from the canonical sequence in the following ways: 1 to 18: MAWDMCNQDSESVWSDIE → MDEGYF, 144 to 150: LKKLLLA → VRTLPTV, 151 to 798: deletion. PGC1-α isoform 8 (also known as "isoform 8a" (UniProt Identification No. Q9UBK2-8) (SEQ ID NO: No. 47) consists of 301 amino acids and differs from the canonical sequence in the following ways: 294 to 301: LTPPTTPP → VKTNLISK, 302 to 798: deletion. PGC1-α isoform 9 (also known as "isoform 9" or "L-PGG-1α" (UniProt Identification No.: Q9UBK2-9) (SEQ ID NO: 48) consists of 671 amino acids and differs from the canonical sequence in the following ways: 1 to 127: deletion. The sequences of the nine PGC1-α isoforms are shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3]

[0195] As used herein, the term "PGC-1α" includes any variant or isoform of PGC-1α naturally expressed by a cell. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 1. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 2. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 1. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 2. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 3. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 4. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 5. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 6. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 7. In some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 8. Thus, in some embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 9. In further embodiments, the miR-485 inhibitors disclosed herein can increase the expression of PGC-1α isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, isoform 6, isoform 7, isoform 8, and isoform 9. Unless otherwise specified, both isoform 1 and isoform 2 are collectively referred to herein as "PGC-1α".

[0196] In some embodiments, contacting a cell with a miRNA-485 inhibitor increases the expression and / or activity of PGC-1α protein and / or PGC-1α gene in the cell by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold, compared to expression and / or activity in a reference cell (e.g., a corresponding cell that has not been contacted with a miRNA-485 inhibitor).

[0197] Without being bound to any one theory, in some aspects, the miR-485 inhibitors disclosed herein increase the expression of PGC-1α protein and / or the PGC-1α gene by reducing the expression and / or activity of miR-485, e.g., miR-485-3p.

[0198] III. miRNA-485 Inhibitors Useful in the Disclosure The present disclosure discloses compounds that can inhibit miR-485 activity (miR-485 inhibitors).In some embodiments, the miR-485 inhibitors of the present disclosure comprise a nucleotide sequence that encodes or comprises a nucleotide molecule that comprises at least one miR-485 binding site, and the nucleotide molecule does not code for a protein.As described herein, in some embodiments, the miR-485 binding site is at least partially complementary to the target miRNA nucleic acid sequence (i.e., miR-485), so that the miR-485 inhibitor hybridizes with the nucleic acid sequence of miR-485.

[0199] In some embodiments, the miR-485 binding site of the miR inhibitor disclosed herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the nucleic acid sequence of miR-485, e.g., miRNA-485-3p. In certain embodiments, the miR-485 binding site is fully complementary to the nucleic acid sequence of miR-485, e.g., miRNA-485-3p.

[0200] The hairpin precursor of miR-485 can generate both miR-485-5p and miR-485-3p. In the context of this disclosure, "miR-485" encompasses both miR-485-5p and miR-485-3p, unless otherwise specified. Human mature miR-485-3p has the sequence 5'- GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1; miRBase Accession No. MIMAT0002176). The 5' end sequence of 5'-UCAUACA-3' (SEQ ID NO: 49) of miR-485-3p is the seed sequence. Human mature miR-485-5p has the sequence 5'-AGAGGCUGGCCGUGAUGAAUUC-3' (SEQ ID NO: 33; miRBase Accession No. MIMAT0002175). The 5'-end sequence of miR-485-5p, 5'-GAGGCUG-3' (SEQ ID NO: 50), is a seed sequence.

[0201] As will be apparent to those skilled in the art, human mature miR-485-3p has considerable sequence similarity with that of other species. For example, mouse mature miR-485-3p differs from human mature miR-485-3p by one amino acid at each of the 5' and 3' ends (i.e., an extra "A" at the 5' end and no "C" at the 3' end). Mouse mature miR-485-3p has the following sequence: [ka] (SEQ ID NO: 34; miRBase Accession No. MIMAT0003129; underlined portion corresponds to overlap with human mature miR-485-3p). The sequence of mouse mature miR-485-5p is identical to the human sequence: 5'-agaggcuggccgugaugaauuc-3' (SEQ ID NO: 33; miRBase Accession No. MIMAT0003128). In certain aspects, miR-485 inhibitors disclosed herein can bind to human and mouse miR-485-3p and / or miR-485-5p.

[0202] In some embodiments, the miR-485 binding site is a single-stranded polynucleotide sequence that is complementary (e.g., fully complementary) to the sequence of miR-485-3p (or a subsequence thereof). In some embodiments, the subsequence of miR-485-3p comprises a seed sequence. Thus, in certain embodiments, the miR-485 binding site has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity with the nucleic acid sequence set forth in SEQ ID NO:49. In certain embodiments, the miR-485 binding site is complementary to miR-485-3p except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In further embodiments, the miR-485 binding site is fully complementary to the nucleic acid sequence set forth in SEQ ID NO:1.

[0203] In some embodiments, the miR-485 binding site is a single-stranded polynucleotide sequence that is complementary (e.g., fully complementary) to the sequence of miR-485-5p (or a subsequence thereof). In some embodiments, the subsequence of miR-485-5p comprises a seed sequence. In certain embodiments, the miR-485 binding site has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity to the nucleic acid sequence set forth in SEQ ID NO:50. In certain embodiments, the miR-485 binding site is complementary to miR-485-5p except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

[0204] The seed region of a miRNA forms a tight duplex with the target mRNA. Many miRNAs base pair imperfectly with the 3' untranslated region (UTR) of the target mRNA, with the 5' proximal "seed" region of the miRNA providing most of the pairing specificity. Without being bound by any theory, it is believed that the first nine miRNA nucleotides (including the seed sequence) provide higher specificity, whereas the miRNA nucleotides 3' of this region provide lower sequence specificity, thereby allowing a higher degree of mismatched base pairing, with positions 2-7 being the most important. Thus, in certain embodiments of the present disclosure, the miR-485 binding site comprises a sequence that is fully complementary (i.e., 100% complementary) over the entire length of the seed sequence of miR-485.

[0205] miRNA sequences and miRNA binding sequences (inhibitors) that can be used in the context of the present disclosure include, but are not limited to, all or part of the sequences in the sequence listing set forth herein, as well as miRNA precursor sequences or the complements of one or more of these miRNAs, which sequences match at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about Also contemplated are any embodiment of the present disclosure that includes a particular miRNA or miRNA binding site by name to include an miRNA or its complementary sequence that is 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical.

[0206] In certain embodiments, the miRNA binding sequences of the present disclosure may include additional nucleotides at the 5' end, the 3' end, or both the 5' and 3' ends of the sequences shown in the sequence listing provided herein, so long as the modified sequence is still capable of specifically binding to miR-485. In some embodiments, the miRNA binding sequences of the present disclosure may differ in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides relative to the sequences shown in the sequence listing provided, so long as the modified sequence is still capable of specifically binding to miR-485.

[0207] It is also specifically contemplated that any methods and compositions described herein with respect to miRNA binding molecules or miRNAs can be practiced with respect to synthetic miRNA binding molecules. It will also be understood that disclosure relating to RNA sequences in the present disclosure is equally applicable to the corresponding DNA sequences.

[0208] In some embodiments, miRNA-485 inhibitors of the present disclosure comprise at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 5' end of the nucleotide sequence. In some embodiments, an miRNA-485 inhibitor comprises at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 3' end of the nucleotide sequence.

[0209] In some embodiments, the miR-485 inhibitors disclosed herein are about 6 to about 30 nucleotides in length. In certain embodiments, the miR-485 inhibitors disclosed herein are 7 nucleotides in length. In further embodiments, the miR-485 inhibitors disclosed herein are 8 nucleotides in length. In some embodiments, the miR-485 inhibitors disclosed herein are 9 nucleotides in length. In some embodiments, the miR-485 inhibitors of the present disclosure are 10 nucleotides in length. In certain embodiments, the miR-485 inhibitors are 11 nucleotides in length. In further embodiments, the miR-485 inhibitors are 12 nucleotides in length. In some embodiments, the miR-485 inhibitors disclosed herein are 13 nucleotides in length. In certain embodiments, the miR-485 inhibitors disclosed herein are 14 nucleotides in length. In some embodiments, the miR-485 inhibitors disclosed herein are 15 nucleotides in length. In further embodiments, the miR-485 inhibitors are 16 nucleotides in length. In certain embodiments, the miR-485 inhibitor of the present disclosure is 17 nucleotides in length. In some embodiments, the miR-485 inhibitor is 18 nucleotides in length. In some embodiments, the miR-485 inhibitor is 19 nucleotides in length. In certain embodiments, the miR-485 inhibitor is 20 nucleotides in length. In further embodiments, the miR-485 inhibitor of the present disclosure is 21 nucleotides in length. In some embodiments, the miR-485 inhibitor is 22 nucleotides in length.

[0210] In some embodiments, the miR-485 inhibitors disclosed herein comprise a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from SEQ ID NOs: 2-30. In certain embodiments, the miR-485 inhibitor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-30, where the nucleotide sequence may optionally comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

[0211] In some embodiments, the miRNA inhibitor is 5'-UGUAUGA-3' (SEQ ID NO:2), 5'-GUGUAUGA-3' (SEQ ID NO:3), 5'-CGUGUAUGA-3' (SEQ ID NO:4), 5'-CCGUGUAUGA-3' (SEQ ID NO:5), 5'-GCCGUGUAUGA-3' (SEQ ID NO:6), 5'-AGCCGUGUAUGA-3' (SEQ ID NO:7), 5'-GAGCCGUGUAUGA-3' (SEQ ID NO:8), 5'-AGAGCCGUGUAUGA-3' (SEQ ID NO:9), 5'-GCCGUGUAUGA-3' (SEQ ID NO:10), 5'-GCCGUGUAUGA-3' (SEQ ID NO:11), 5'-GCCGUGUAUGA-3' (SEQ ID NO:12), 5'-GCCGUGUAUGA-3' (SEQ ID NO:13), 5'-GCCGUGUAUGA-3' (SEQ ID NO:14), 5'-GCCGUGUAUGA-3' (SEQ ID NO:15), 5'-GCCGUGUAUGA-3' (SEQ ID NO:16), 5'-GCCGUGUAUGA-3' (SEQ ID NO:17), 5'-GCCGUGUAUGA-3' (SEQ ID NO:18), 5'-GCCGUGUAUGA-3' (SEQ ID NO:19), 5'-GCCGUGUAUGA-3' (SEQ ID NO:20), 5'-GCCGUGUAUGA-3' (SEQ ID NO:21), 5'-GCCGUGUAUGA-3' (SEQ ID NO:22), 5'-GCCGUGUAUGA-3' (SEQ ID NO:23), 5'-GCCGUGUAUGA-3' (SEQ ID NO:24), 5'-GCCGUGU (SEQ ID NO:9), 5'-GAGAGCCGUGUAUGA-3' (SEQ ID NO:10), 5'-GGAGAGCCGUGUAUGA-3' (SEQ ID NO:11), 5'-AGGAGAGCCGUGUAUGA-3' (SEQ ID NO:12), 5'-GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO:13), 5'-AGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO:14), or 5'-GAGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO:15).

[0212] In some embodiments, the miRNA inhibitor is 5'-UGUAUGAC-3' (SEQ ID NO: 16), 5'-GUGUAUGAC-3' (SEQ ID NO: 17), 5'-CGUGUAUGAC-3' (SEQ ID NO: 18), 5'-CCGUGUAUGAC-3' (SEQ ID NO: 19), 5'-GCCGUGUAUGAC-3' (SEQ ID NO: 20), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 21), 5'-GAGCCGUGUAUGAC-3' (SEQ ID NO: 22), 5'-AGAGCCGUGUAUGAC-3' (SEQ ID NO: 23), 5'-AGAGCCGUGUAUGAC-3' (SEQ ID NO: 24), 5'-AGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 26), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 27), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 28), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 29), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 30), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 31), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 32), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 33), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 34), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 35), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 36), C-3' (SEQ ID NO:23), 5'-GAGAGCCGUGUAUGAC-3' (SEQ ID NO:24), 5'-GGAGAGCCGUGUAUGAC-3' (SEQ ID NO:25), 5'-AGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:26), 5'-GAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:27), 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:28), or 5'-GAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:29).

[0213] In some embodiments, the miRNA inhibitor is 5'-TGTATGA-3' (SEQ ID NO: 30), 5'-GTGTATGA-3' (SEQ ID NO: 51), 5'-CGTGTATGA-3' (SEQ ID NO: 52), 5'-CCGTGTATGA-3' (SEQ ID NO: 53), 5'-GCCGTGTATGA-3' (SEQ ID NO: 54), 5'-AGCCGTGTATGA-3' (SEQ ID NO: 55), 5'-GAGCCGTGTATGA-3' (SEQ ID NO: 35), 5'-AGAGCCGTGTATGA- 3' (SEQ ID NO:56), 5'-GAGAGCCGTGTATGA-3' (SEQ ID NO:57), 5'-GGAGAGCCGTGTATGA-3' (SEQ ID NO:58), 5'-AGGAGAGCCGTGTATGA-3' (SEQ ID NO:59), 5'-GAGGAGAGCCGTGTATGA-3' (SEQ ID NO:60), 5'-AGAGGAGAGCCGTGTATGA-3' (SEQ ID NO:61), 5'-GAGAGGAGAGCCGTGTATGA-3' (SEQ ID NO:62); 5'-TGTA TGAC-3' (SEQ ID NO: 63), 5'-GTGTATGAC-3' (SEQ ID NO: 64), 5'-CGTGTATGAC-3' (SEQ ID NO: 65), 5'-CCGTGTATGAC-3' (SEQ ID NO: 66), 5'-GCCGTGTATGAC-3' (SEQ ID NO: 67), 5'-AGCCGTGTATGAC-3' (SEQ ID NO: 68), 5'-GAGCCGTGTATGAC-3' (SEQ ID NO: 69), 5'-AGAGCCGTGTATGAC-3' (SEQ ID NO: 70), 5'-GAGAG CCGTGTATGAC-3' (SEQ ID NO: 71), 5'-GGAGAGCCGTGTATGAC-3' (SEQ ID NO: 72), 5'-AGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 73), 5'-GAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 74), 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 75), and 5'-GAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 76).

[0214] In some embodiments, the miRNA inhibitors disclosed herein (i.e., miR-485 inhibitors) comprise a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:77). In some embodiments, the miRNA inhibitor comprises a nucleotide sequence that has at least 90% similarity to 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO:77). In some embodiments, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 77) with one substitution or two substitutions. In particular embodiments, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28) or 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 77). In particular embodiments, the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28).

[0215] In some embodiments, the miR-485 inhibitor of the present disclosure comprises a sequence disclosed herein, e.g., any one of SEQ ID NOs: 2-30, and at least one, at least two, at least three, at least four, or at least five additional nucleic acids at the N-terminus, at least one, at least two, at least three, at least four, or at least five additional nucleic acids at the C-terminus, or both. In some embodiments, the miR-485 inhibitor of the present disclosure comprises a sequence disclosed herein, e.g., any one of SEQ ID NOs: 2-30, and at least one additional nucleic acid at the N-terminus and / or at least one additional nucleic acid at the C-terminus. In some embodiments, the miR-485 inhibitor of the present disclosure comprises a sequence disclosed herein, e.g., any one of SEQ ID NOs: 2-30, and at least one or two additional nucleic acids at the N-terminus and / or at least one or two additional nucleic acids at the C-terminus. In some embodiments, the miR-485 inhibitor of the present disclosure comprises a sequence disclosed herein, e.g., any one of SEQ ID NOs: 2-30, and 1-3 additional nucleic acids at the N-terminus and / or 1-3 additional nucleic acids at the C-terminus. In some embodiments, the miR-485 inhibitor comprises 5'-GAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 29).

[0216] In some embodiments, the miR-485 inhibitor of the present disclosure comprises one miR-485 binding site. In further embodiments, the miR-485 inhibitor disclosed herein comprises at least two miR-485 binding sites. In certain embodiments, the miR-485 inhibitor comprises three miR-485 binding sites. In some embodiments, the miR-485 inhibitor comprises four miR-485 binding sites. In some embodiments, the miR-485 inhibitor comprises five miR-485 binding sites. In certain embodiments, the miR-485 inhibitor comprises six or more miR-485 binding sites. In some embodiments, all the miR-485 binding sites are the same. In some embodiments, all the miR-485 binding sites are different. In some embodiments, at least one miR-485 binding site is different. In some embodiments, all the miR-485 binding sites are miR-485-3p binding sites. In other embodiments, all of the miR-485 binding sites are miR-485-5p binding sites. In further embodiments, the miR-485 inhibitor comprises at least one miR-485-3p binding site and at least one miR-485-5p binding site.

[0217] III.a. Chemically Modified Polynucleotides In some embodiments, the miR-485 inhibitors disclosed herein include polynucleotides that include at least one chemically modified nucleoside and / or nucleotide. When a polynucleotide of the present disclosure is chemically modified, the polynucleotide can be referred to as a "modified polynucleotide."

[0218] "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or derivative thereof, along with an organic base (e.g., purine or pyrimidine) or derivative thereof (also referred to herein as "nucleobase"). "Nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method, for example, chemically, enzymatically, or recombinantly, to include one or more modified non-natural nucleosides.

[0219] A polynucleotide may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester bonds, in which case the polynucleotide comprises a region of nucleotides.

[0220] Modified polynucleotides disclosed herein may contain a variety of different modifications.In some embodiments, modified polynucleotides contain one, two or more (optionally different) nucleoside or nucleotide modifications.In some embodiments, modified polynucleotides may exhibit one or more desirable properties, such as improved thermal or chemical stability, reduced immunogenicity, reduced degradation, increased binding to target microRNA, reduced non-specific binding to other microRNA or other molecules, compared to unmodified polynucleotides.

[0221] In some embodiments, the polynucleotides (e.g., miR-485 inhibitors) of the present disclosure are chemically modified. As used herein, with respect to polynucleotides, the term "chemical modification" or, as appropriate, "chemically modified" refers to modifications to one or more of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides, including, but not limited to, their nucleobase, sugar, backbone, or any combination thereof, in their position, pattern, percentage, or population.

[0222] In some embodiments, the polynucleotides (e.g., miR-485 inhibitors) of the present disclosure may have uniform chemical modifications of all or any of the same nucleoside types, or groups of modifications generated by downward titration of the same starting modifications on all or any of the same nucleoside types, or a measured percentage of all or any of the same nucleoside types except with random incorporation. In further embodiments, the polynucleotides (e.g., miR-485 inhibitors) of the present disclosure may have uniform chemical modifications of two, three, or four of the same nucleoside types throughout the polynucleotide (e.g., all uridines and all cytosines are modified in the same way, etc.).

[0223] Modified nucleotide base pairing encompasses not only standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases, or between two complementary non-standard base structures. One example of such non-standard base pairing is base pairing between the modified nucleobase inosine and adenine, cytosine, or uracil. Any combination of bases / sugars or linkers can be incorporated into the polynucleotides of the present disclosure.

[0224] Those skilled in the art will understand that, unless otherwise noted, the polynucleotide sequences described in this application recite "T" in representative DNA sequences, but when the sequence represents RNA, "T" is replaced with "U." For example, the TDs of the present disclosure can be administered as RNA, as DNA, or as hybrid molecules containing both RNA and DNA units.

[0225] In some embodiments, a polynucleotide (e.g., a miR-485 inhibitor) comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20 or more) modified nucleobase combinations.

[0226] In some embodiments, the nucleobases, sugars, backbone linkages, or any combination thereof in a polynucleotide are at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% modified.

[0227] (i) Base modification In certain embodiments, chemical modifications are present in the nucleobases in the polynucleotides of the present disclosure (e.g., miR-485 inhibitors). In some embodiments, at least one chemically modified nucleoside is a modified uridine (e.g., pseudouridine (ψ), 2-thiouridine (s2U), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), or 5-methoxy-uridine (mo5U)), a modified cytosine (e.g., 5-methyl-cytidine (m5C)), a modified adenosine (e.g., 1-methyl-adenosine (m1A), N6-methyl-adenosine (m6A), or 2-methyl-adenine (m2A)), a modified guanosine (e.g., 7-methyl-guanosine (m7G) or 1-methylguanosine (m1G)), or a combination thereof.

[0228] In some aspects, the polynucleotides (e.g., miR-485 inhibitors) of the present disclosure are uniformly modified (e.g., completely modified, modified throughout the entire sequence) for a particular modification.For example, polynucleotides can be uniformly modified with the same type of base modification, such as 5-methyl-cytidine (m5C), which means that all cytosine residues in the polynucleotide sequence are replaced with 5-methyl-cytidine (m5C).Similarly, polynucleotides can be uniformly modified with any type of nucleoside residue present in the sequence by replacing it with a modified nucleoside, such as any of those described above.

[0229] In some embodiments, the polynucleotides (e.g., miR-485 inhibitors) of the present disclosure comprise a combination of at least two (e.g., two, three, four, or more) modified nucleobases. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of one type of nucleobase in the polynucleotides (e.g., miR-485 inhibitors) of the present disclosure are modified nucleobases.

[0230] (ii) Backbone Modification In some embodiments, the polynucleotides (i.e., miR-485 inhibitors) of the present disclosure can include any useful linkage between nucleosides. Such linkages, including backbone modifications, useful in the compositions of the present disclosure include, but are not limited to, 3'-alkylene phosphonates, 3'-amino phosphoramidates, alkene-containing backbones, aminoalkyl phosphoramidates, aminoalkyl phosphotriesters, boranophosphates, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene(methylimino), methyleneformacetate, and the like. thioformacetyl and thioformacetyl backbones, methyleneimino and methylenehydrazino backbones, morpholino linkages, -N(CH3)-CH2-CH2-, oligonucleosides with heteroatom internucleoside linkages, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioate, phosphotriesters, PNAs, siloxane backbones, sulfamate backbones, sulfide, sulfoxide, and sulfone backbones, sulfonate and sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates. [ka]

[0231] In some aspects, the presence of the backbone linkages disclosed above increases the stability and resistance to degradation of the polynucleotides (eg, miR-485 inhibitors) of the present disclosure.

[0232] In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the backbone linkages in a polynucleotide (e.g., a miR-485 inhibitor) of the disclosure are modified (e.g., all of them are phosphorothioate).

[0233] In some aspects, backbone modifications that can be included in polynucleotides (i.e., miR-485 inhibitors) of the disclosure include phosphorodiamidite morpholino oligomer (PMO) and / or phosphorothioate (PS) modifications.

[0234] (iii) Sugar modification Modified nucleosides and nucleotides that may be incorporated into the polynucleotides (e.g., miR-485 inhibitors) of the present disclosure may be modified on the sugar of the nucleic acid. In some embodiments, the sugar modification increases the affinity of the binding of the miR-485 inhibitor to the miR-485 nucleic acid sequence. The length and / or size of the miR-485 inhibitor may be reduced by incorporating affinity-improving nucleotide analogs, such as LNA or 2'-substituted sugars, into the miR-485 inhibitor.

[0235] In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the nucleotides in a polynucleotide of the disclosure (i.e., a miR-485 inhibitor) contain a sugar modification (e.g., LNA).

[0236] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotide units in a polynucleotide of the disclosure are sugar modified (e.g., LNA).

[0237] Generally, RNA comprises the sugar group ribose, which is a five-membered ring with oxygen. Non-limiting exemplary modified nucleotides include substitution of oxygen in ribose (e.g., with S, Se, or alkylene, e.g., methylene or ethylene); addition of a double bond (e.g., replacement of ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., formation of a four-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., formation of a six- or seven-membered ring with an additional carbon or heteroatom, e.g., anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and phospho. morpholino, which also has a roamidate backbone; polycyclic forms (e.g., tricyclo); and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where the ribose is replaced with a glycol unit linked to a phosphodiester bond), threose nucleic acid (TNA, where the ribose is replaced with α-L-threofuranosyl-(3'→2')), and peptide nucleic acid (PNA, where a 2-amino-ethyl-glycine bond replaces the ribose and phosphodiester backbone). The sugar group may also contain one or more carbons that have the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, a polynucleotide molecule may contain nucleotides that contain, for example, arabinose as the sugar.

[0238] The 2' hydroxyl group (OH) of ribose can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted C 1~6 Alkyl; optionally substituted C 1~6 Alkoxy; optionally substituted C 6~10 Aryloxy; optionally substituted C 3~8 Cycloalkyl; optionally substituted C 3~8 Cycloalkoxy; optionally substituted C 6~10 Aryloxy; optionally substituted C 6~10 Aryl-C 1~6 Alkoxy, optionally substituted C1~12 (heterocyclyl)oxy; sugar (e.g., ribose, pentose, or any described herein); polyethylene glycol (PEG), -O(CH2CH2O) n CH2CH2OR, where R is H or an optionally substituted alkyl and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20); "locked" nucleic acids (LNAs) (wherein the 2'-hydroxy is 1~6 Alkylene or C 1~6 They are linked to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge, where exemplary bridges include methylene, propylene, ether, amino bridge, aminoalkyl, aminoalkoxy, amino, and amino acid.

[0239] In some embodiments, the nucleotide analogs present in the polynucleotides (i.e., miR-485 inhibitors) of the present disclosure include, for example, 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-O-alkyl-SNA, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabinonucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units, 2'MOE units, or any combination thereof. In some embodiments, the LNA is, for example, oxyLNA (e.g., β-D-oxy-LNA or α-L-oxy-LNA), aminoLNA (e.g., β-D-amino-LNA or α-L-amino-LNA), thioLNA (e.g., β-D-thio-LNA or α-L-thio-LNA), ENA (e.g., β-D-ENA or α-L-ENA), or any combination thereof. In further aspects, nucleotide analogs that can be included in the polynucleotides (i.e., miR-485 inhibitors) of the present disclosure include locked nucleic acids (LNA), unlocked nucleic acids (UNA), arabino nucleic acids (ABA), bridged nucleic acids (BNA), and / or peptide nucleic acids (PNA).

[0240] In some embodiments, the polynucleotides (i.e., miR-485 inhibitors) of the present disclosure may include both modified RNA nucleotide analogs (e.g., LNA) and DNA units. In some embodiments, the miR-485 inhibitors are gapmers. See, e.g., U.S. Patent Nos. 8,404,649, 8,580,756, 8,163,708, and 9,034,837, all of which are incorporated herein by reference in their entirety. In some embodiments, the miR-485 inhibitors are micromers. See, U.S. Patent Publication No. US20180201928, which is incorporated herein by reference in its entirety.

[0241] In some embodiments, the polynucleotides (at least miR-485 inhibitors) of the present disclosure can include modifications to prevent rapid degradation by endonucleases and exonucleases. Modifications include, but are not limited to, (a) end modifications, such as 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkage, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkage, etc.), (b) base modifications, such as modified bases, stabilized bases, destabilized bases, or bases that base pair with a broader repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, and (d) internucleoside bond modifications, including phosphodiester bond modifications or replacements.

[0242] IV. Vectors and Delivery Systems In some embodiments, the miR-485 inhibitor of the present disclosure can be administered to, for example, a subject at risk of hair loss by using any relevant delivery system known in the art.In certain embodiments, the delivery system is a vector.Thus, in some embodiments, the present disclosure provides a vector that comprises the miR-485 inhibitor of the present disclosure.

[0243] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector or an adeno-associated viral vector. In some embodiments, the viral vector is an AAV with serotypes AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any combination thereof. In some embodiments, the adenoviral vector is a third generation adenoviral vector. ADEASY™ is by far the most common method for making adenoviral vector constructs. This system is composed of two types of plasmids: a shuttle (or transfer) vector and an adenoviral vector. The transgene of interest is cloned into the shuttle vector, verified, and linearized with the restriction enzyme PmeI. This construct is then transformed into ADEASIER-1 cells, which are BJ5183 E. coli cells containing PADEASY™. PADEASY™ is an approximately 33Kb adenoviral plasmid that contains the adenoviral genes necessary for virus production. The shuttle vector and adenoviral plasmid have matching left and right homology arms that facilitate homologous recombination of the transgene into the adenoviral plasmid. Standard BJ5183 can also be co-transformed with supercoiled PADEASY™ and shuttle vector, but this method results in a high background of non-recombinant adenoviral plasmid. The recombinant adenoviral plasmid is then verified for size and appropriate restriction digestion patterns to ensure that the transgene has been inserted into the adenoviral plasmid and no other patterns of recombination have occurred. Once verified, the recombinant plasmid is linearized with PacI to generate a linear dsDNA construct flanked by ITRs. 293 or 911 cells are transfected with the linearized construct and virus can be harvested after approximately 7-10 days. In addition to this method, methods for producing adenoviral vector constructs known in the art at the time this application was filed can be used to practice the methods disclosed herein.

[0244] In some embodiments, the viral vector is a retroviral vector, such as a lentiviral vector (e.g., a third or fourth generation lentiviral vector). Lentiviral vectors are typically generated in a transient transfection system in which one cell line is transfected with three separate plasmid expression systems. These include a transfer vector plasmid (part of the HIV provirus), a packaging plasmid or construct, and a plasmid containing the heterologous envelope gene (env) of a different virus. The three plasmid components of the vector are placed into a packaging cell, which then inserts into the HIV shell. The viral portion of the vector contains an insertion sequence, so the virus cannot replicate in the cell line. Current third generation lentiviral vectors only encode three of the nine HIV-1 proteins (Gag, Pol, Rev), which are expressed from separate plasmids to prevent the generation of replication-competent viruses by recombination. In fourth generation lentiviral vectors, the retroviral genome is further reduced (see, e.g., TAKARA® LENTI-X™ fourth generation packaging system).

[0245] Any AAV vector known in the art can be used in the methods disclosed herein. The AAV vector can include known vectors, and can include variants, fragments, or fusions thereof. In some embodiments, the AAV vector is selected from the group consisting of AAV type 1 (AAV1), AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AVV, primate AAV, non-primate AAV, bovine AAV, shrimp AVV, snake AVV, and any combination thereof.

[0246] In some aspects, the AAV vector is derived from an AAV vector selected from the group consisting of AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof.

[0247] In some aspects, the AAV vector is a chimeric vector derived from at least two AAV vectors selected from the group consisting of AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof.

[0248] In certain embodiments, the AAV vector comprises regions of at least two different AAV vectors known in the art.

[0249] In some embodiments, the AAV vector comprises a terminal end derived from a first AAV (e.g., AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, or any combination thereof). and a second terminal inverted sequence derived from a second AAV (e.g., AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, or any combination thereof).

[0250] In some embodiments, the AAV vector comprises a portion of an AAV vector selected from the group consisting of AAV1, AAV2, AAV3A, AVV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof. In some embodiments, the AAV vector comprises AAV2.

[0251] In some aspects, the AAV vector comprises a splice acceptor site. In some aspects, the AAV vector comprises a promoter. Any promoter known in the art can be used in the AAV vector of the present disclosure. In some embodiments, the promoter is an RNA Pol III promoter. In some aspects, the RNA Pol III promoter is selected from the group consisting of U6 promoter, H1 promoter, 7SK promoter, 5S promoter, adenovirus 2 (Ad2) VAI promoter, and any combination thereof. In some aspects, the promoter is a cytomegalovirus immediate early gene (CMV) promoter, EF1a promoter, Sv40 promoter, PGK1 promoter, Ubc promoter, human β-actin promoter, CAG promoter, TRE promoter, UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, GAL1 promoter, GAL10 promoter, TEF promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, or Ubi promoter. In certain aspects, the promoter comprises a U6 promoter.

[0252] In some embodiments, the AAV vector comprises a constitutively active promoter (constitutive promoter). In some embodiments, the constitutive promoter is selected from the group consisting of hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta actin promoter, cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, retroviral long terminal repeat (LTR), murine stem cell virus (MSCV), and herpes simplex virus thymidine kinase promoter.

[0253] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tissue-specific promoter. In certain embodiments, the tissue-specific promoter induces transcription of the coding region of the AAV vector in neuronal cells, glial cells, or both neuronal and glial cells.

[0254] In some embodiments, the AAV vector comprises one or more enhancers. In some embodiments, the one or more enhancers are present in the AAV alone or with the promoters disclosed herein. In some embodiments, the AAV vector comprises a poly(A) tail sequence in the 3'UTR. In some embodiments, the poly(A) tail sequence in the 3'UTR is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In some embodiments, the poly(A) tail sequence in the 3'UTR comprises bGH poly(A).

[0255] In some embodiments, the miR-485 inhibitor disclosed herein is administered with a delivery agent.Non-limiting examples of delivery agents that can be used include exosomes, lipidoids, liposomes, lipoplexes, lipid nanoparticles, extracellular vesicles, polymeric compounds, peptides, proteins, cells, nanoparticle mimics, nanotubes, micelle vectors, viral vectors, or conjugates.

[0256] Thus, in some embodiments, the present disclosure also provides a composition comprising a miRNA inhibitor (i.e., a miR-485 inhibitor) of the present disclosure and a delivery agent. In some embodiments, the delivery agent comprises, for example, a carrier unit that can self-assemble into a micelle or can be incorporated into a micelle. In some embodiments, the delivery agent has the following formula: [WP]-L1-[CC]-L2-[AM] (Formula I) or [WP]-L1-[AM]-L2-[CC] (Formula II) (In the formula, WP is a water-soluble biopolymer moiety; CC is a positively charged (i.e., cationic) carrier moiety; AM is the adjuvant moiety, L1 and L2 are independently an optional linker, The cationic carrier units form micelles when mixed with a nucleic acid at an ionic ratio of about 1: 1. Thus, in some embodiments, the miRNA inhibitors and the cationic carrier units can bind to each other (e.g., via covalent or non-covalent bonds) when mixed together.

[0257] In some embodiments, compositions comprising an miRNA inhibitor (ie, a miR-485 inhibitor) of the present disclosure interact with a cationic carrier unit via ionic bonds.

[0258] In some embodiments, the water soluble polymer comprises a poly(alkylene glycol), a poly(oxyethylated polyol), a poly(olefinic alcohol), a poly(vinylpyrrolidone), a poly(hydroxyalkyl methacrylamide), a poly(hydroxyalkyl methacrylate), a poly(saccharide), a poly(α-hydroxy acid), a poly(vinyl alcohol), a polyglycerol, a polyphosphazene, a polyoxazoline ("POZ"), a poly(N-acryloylmorpholine), or any combination thereof. In some embodiments, the water soluble polymer comprises a polyethylene glycol ("PEG"), a polyglycerol, or a poly(propylene glycol) ("PPG"). In some embodiments, the water soluble polymer comprises a poly(alkylene glycol) having the following formula: [ka] (wherein n is 1 to 1000).

[0259] In some embodiments, n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141. In some embodiments, n is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 140 to about 150, or about 150 to about 160.

[0260] In some embodiments, the water-soluble polymer is linear, branched, or dendritic. In some embodiments, the cationic carrier moiety comprises one or more basic amino acids. In some embodiments, the cationic carrier moiety comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 basic amino acids. In some embodiments, the cationic carrier moiety comprises about 30 to about 50 basic amino acids. In some embodiments, the basic amino acids comprise arginine, lysine, histidine, or any combination thereof. In some embodiments, the cationic carrier moiety comprises about 40 lysine monomers.

[0261] In some embodiments, the adjuvant moiety can modulate the immune response, the inflammatory response, and / or the tissue microenvironment. In some embodiments, the adjuvant moiety comprises an imidazole derivative, an amino acid, a vitamin, or any combination thereof. In some embodiments, the adjuvant moiety has the following formula: [ka] wherein each of G1 and G2 is H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring and n is 1-10.

[0262] In some embodiments, the adjuvant moiety comprises a nitroimidazole. In some embodiments, the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazole, azanidazole, benznidazole, or any combination thereof. In some embodiments, the adjuvant moiety comprises an amino acid.

[0263] In some embodiments, the adjuvant moiety has the following formula: [ka] (Wherein, Ar is [ka] and Each of Z1 and Z2 is H or OH.

[0264] In some embodiments, the adjuvant moiety comprises a vitamin. In some embodiments, the vitamin comprises a cyclic or heteroatom ring and a carboxyl or hydroxyl group. In some embodiments, the vitamin has the following formula: [ka] wherein each of Y1 and Y2 is C, N, O, or S, and n is 1 or 2.

[0265] In some embodiments, the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H, and any combination thereof. In some embodiments, the vitamin is vitamin B3.

[0266] In some embodiments, the adjuvant moiety comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 vitamin B3. In some embodiments, the adjuvant moiety comprises about 10 vitamin B3.

[0267] In some embodiments, the composition comprises a water soluble biopolymer portion having about 120 to about 130 PEG units, a cationic carrier portion comprising polylysine having about 30 to about 40 lysines, and an adjuvant portion having about 5 to about 10 vitamin B3.

[0268] In some embodiments, the composition comprises (i) a water-soluble biopolymer moiety having about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines having an amine group (e.g., about 32 lysines), (iii) about 15 to 20 lysines each having a thiol group (e.g., about 16 lysines each having a thiol group), and (iv) about 30 to 40 lysines fused to vitamin B3 (e.g., about 32 lysines each fused to vitamin B3). In some embodiments, the composition further comprises a solubility targeting moiety, such as a LAT1 targeting ligand, such as phenylalanine, attached to the water-soluble polymer. In some embodiments, the thiol groups in the composition form disulfide bonds.

[0269] In some embodiments, the composition comprises (1) a micelle comprising (i) about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines (e.g., about 32 lysines) having an amine group, (iii) about 15 to 20 lysines (e.g., about 16 lysines each having a thiol group), and (iv) about 30 to 40 lysines (e.g., about 32 lysines each fused to vitamin B3) fused to vitamin B3, and (2) a miR485 inhibitor (e.g., SEQ ID NO: 28), wherein the miR485 inhibitor is encapsulated within the micelle. In some embodiments, the composition further comprises a targeting moiety, e.g., a LAT1 targeting ligand, e.g., phenylalanine, attached to the PEG unit. In some embodiments, the thiol groups in the micelle form a disulfide bond.

[0270] The present disclosure also provides a micelle comprising an miRNA inhibitor of the present disclosure (ie, a miR-485 inhibitor), wherein the miRNA inhibitor and a delivery agent are linked to one another.

[0271] In some embodiments, the bond is a covalent bond, a non-covalent bond, or an ionic bond. In some embodiments, the positive charge of the cationic carrier moiety of the cationic carrier unit is sufficient to form a micelle when mixed with the miR-485 inhibitor disclosed herein in solution, and the overall ionic ratio of the positive charge of the cationic carrier moiety of the cationic carrier unit to the negative charge of the miR-485 inhibitor (or vector containing the inhibitor) in solution is about 1:1.

[0272] In some embodiments, the cationic carrier unit can protect the miRNA inhibitors (i.e., miR-485 inhibitors) of the present disclosure from enzymatic degradation (see PCT Publication No. WO2020 / 261227, which is incorporated by reference in its entirety).

[0273] V. Pharmaceutical Compositions In some aspects, the present disclosure also provides pharmaceutical compositions comprising a miR-485 inhibitor (e.g., a polynucleotide or vector comprising a miR-485 inhibitor) disclosed herein suitable for administration to a subject. A pharmaceutical composition generally comprises a miR-485 inhibitor (e.g., a polynucleotide or vector) described herein and a pharma- ceutically acceptable excipient or carrier in a form suitable for administration to a subject. The pharma- ceutically acceptable excipient or carrier is determined in part by the particular composition to be administered and the particular method used to administer the composition.

[0274] Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising the miR-485 inhibitors of the present disclosure (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). Pharmaceutical compositions are generally formulated under sterile conditions and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0275] VI. Kit The present disclosure also provides a kit or product comprising the miRNA inhibitor of the present disclosure (e.g., a polynucleotide, vector, or pharmaceutical composition disclosed herein) and, optionally, instructions for use, e.g., instructions for use according to the methods disclosed herein. In some embodiments, the kit or product comprises a miR-485 inhibitor (e.g., a vector, e.g., an AAV vector, polynucleotide, or pharmaceutical composition disclosed herein) in one or more containers. In some embodiments, the kit or product comprises a miR-485 inhibitor (e.g., a vector, e.g., an AAV vector, polynucleotide, or pharmaceutical composition disclosed herein) and a pamphlet. Those skilled in the art will readily recognize that the miR-485 inhibitor disclosed herein (e.g., a vector, polynucleotide, and pharmaceutical composition disclosed herein, or a combination thereof) can be easily incorporated into one of the established kit formats well known in the art. EXAMPLES

[0276] The following examples are offered by way of illustration and not by way of limitation. Working Example Example 1: Preparation of miR-485 inhibitors

[0277] (a) Synthesis of alkyne-modified tyrosine: Alkyne-modified tyrosine was prepared as an intermediate for the synthesis of the tissue-specific targeting portion (TM, see FIG. 1 ) of the cationic carrier unit to target the micelles of the present disclosure to the LAT1 transporter at the BBB.

[0278] A mixture of N-(tert-butoxycarbonyl)-L-tyrosine methyl ester (Boc-Tyr-OMe) (0.5 g, 1.69 mmol) and K2CO3 (1.5 eq, 2.54 mmol) in acetonitrile (4.0 ml) was added dropwise to propargyl bromide (1.2 eq, 2.03 mmol). The reaction mixture was heated at 60° C. overnight. After the reaction, the reaction mixture was extracted using water:ethyl acetate (EA). The organic layer was then washed using a brine solution. The crude was purified by flash column (10% EA in hexane). The obtained product was then dissolved in 1,4-dioxane (1.0 ml) and 6.0 M HCl (1.0 ml). The reaction mixture was heated at 100° C. overnight. Then, dioxane was removed and extracted with EA. An aqueous NaOH (0.5 M) solution was added to the mixture until the pH value was 7. The reaction mixture was concentrated in an evaporator and centrifuged at 12000 rpm at 0° C. The precipitate was washed with deionized water and freeze-dried.

[0279] (b) Synthesis of poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL): This synthesis step prepared the water-soluble biopolymer (WP) and cationic carrier (CC) of the cationic carrier unit of the present disclosure (see FIG. 1).

[0280] Poly(ethylene glycol)-b-poly(L-lysine) was synthesized by ring-opening polymerization of Lys(TFA)-NCA using monomethoxy PEG (MeO-PEG) as a macroinitiator. In brief, MeO-PEG (600 mg, 0.12 mmol) and Lys(TFA)-NCA (2574 mg, 9.6 mmol) were dissolved in DMF containing 1 M thiourea and DMF (or NMP) separately. The Lys(TFA)-NCA solution was added dropwise to the MeO-PEG solution by a microsyringe, and the reaction mixture was stirred at 37 °C for 4 days. The reaction bottle was purged with argon and vacuum. All reactions were carried out under an argon atmosphere. After the reaction, the mixture was precipitated in excess diethyl ether. The precipitate was redissolved in methanol and precipitated again in cold diethyl ether. The precipitate was then filtered and a white powder was obtained after drying under reduced pressure. To deprotect the TFA group of PEG-PLL(TFA), the following steps were carried out.

[0281] MeO-PEG-PLL(TFA) (500 mg) was dissolved in methanol (60 mL) and 1N NaOH (6 mL) was added dropwise to the polymer solution under stirring. The mixture was kept at 37° C. for 1 day with stirring. The reaction mixture was dialyzed against 10 mM HEPES 4 times and against distilled water. A white powder of PEG-PLL was obtained after lyophilization.

[0282] (b) Synthesis of azido-poly(ethylene glycol)-b-poly(L-lysine) (N3-PEG-PLL): This synthetic step prepared the water-soluble biopolymer (WP) and cationic carrier (CC) of the cationic carrier unit of the present disclosure (see FIG. 1).

[0283] Azide-poly(ethylene glycol)-b-poly(L-lysine) was synthesized by ring-opening polymerization of Lys(TFA)-NCA with azide-PEG (N3-PEG). Briefly, N3-PEG (300 mg, 0.06 mmol) and Lys(TFA)-NCA (1287 mg, 4.8 mmol) were dissolved in DMF and DMF (or NMP) containing 1 M thiourea, respectively. The Lys(TFA)-NCA solution was added dropwise to the N3-PEG solution by a microsyringe, and the reaction mixture was stirred at 37°C for 4 days. The reaction bottle was purged with argon and vacuum. All reactions were carried out under an argon atmosphere. After the reaction, the mixture was precipitated in excess diethyl ether. The precipitate was redissolved in methanol and precipitated again in cold diethyl ether. The precipitate was then filtered, and a white powder was obtained after drying under reduced pressure. To deprotect the TFA group of PEG-PLL(TFA), the following steps were carried out.

[0284] N3-PEG-PLL (500 mg) was dissolved in methanol (60 mL) and 1N NaOH (6 mL) was added dropwise to the polymer solution with stirring. The mixture was kept at 37° C. for 1 day with stirring. The reaction mixture was dialyzed against 10 mM HEPES four times and against distilled water. A white powder of N3-PEG-PLL was obtained after lyophilization.

[0285] (c) Synthesis of (methoxy or) azido-poly(ethylene glycol)-b-poly(L-lysine / nicotinamide / mercaptopropanamide) (N3-PEG-PLL(Nic / SH)): In this step, a tissue-specific adjuvant moiety (AM, see FIG. 1) was attached to the WP-CC component of the cationic carrier unit of the present disclosure. The tissue-specific adjuvant moiety (AM) used in the cationic carrier unit was nicotinamide (vitamin B3). This step results in the WP-CC-AM component of the cationic carrier unit shown in FIG. 1.

[0286] Azide-poly(ethylene glycol)-b-poly(L-lysine / nicotinamide / mercaptopropanamide) (N3-PEG-PLL(Nic / SH)) was synthesized by chemical modification of N3-PEG-PLL and nicotinic acid in the presence of EDC / NHS. N3-PEG-PLL (372 mg, 25.8 μmol) and nicotinic acid (556.7 mg, 1.02 equivalents relative to the NH2 of PEG-PLL) were dissolved separately in a mixture of deionized water and methanol (1:1). EDC·HCl (556.7 mg, 1.5 equivalents relative to the NH2 of N3-PEG-PLL) was added to the nicotinic acid solution, and NHS (334.2 mg, 1.5 equivalents relative to the NH2 of PEG-PLL) was added stepwise to the mixture.

[0287] The reaction mixture was added to the N3-PEG-PLL solution. The reaction mixture was kept at 37 °C for 16 h with stirring. After 16 h, 3,3'-dithiodipropionic acid (36.8 mg, 0.1 equiv.) was dissolved in methanol, EDC·HCl (40.3 mg, 0.15 equiv.), and NHS (24.2 mg, 0.15 equiv.) were dissolved in deionized water, respectively. NHS and EDC·HCl were then added sequentially to the 3,3'-dithiodipropionic acid solution. After the crude N3-PEG-PLL(Nic) solution was added, the mixture was stirred at 37 °C for 4 h.

[0288] For purification, the mixture was dialyzed against methanol for 2 h and activated for 30 min after addition of DL-dithiothreitol (DTT, 40.6 mg, 0.15 eq.).

[0289] To remove DTT, the mixture was dialyzed sequentially against methanol, 50% methanol in deionized water, and deionized water.

[0290] (d) Synthesis of phenylalanine-poly(ethylene glycol)-b-poly(L-lysine / nicotinamide / mercaptopropanamide) (Phe-PEG-PLL(Nic / SH)): In this step, a tissue-specific targeting moiety (TM) was attached to the WP-CC-AM component synthesized in the above step. The TM component (phenylalanine) was prepared by reaction of the intermediate prepared in step (a) with the product of step (c).

[0291] To target brain endothelial tissues in blood vessels, phenylalanine as LAT1-targeting amino acid was introduced by click reaction between N3-PEG-PLL(Nic / SH) and alkyne-modified tyrosine in the presence of copper catalyst. Briefly, N3-PEG-PLL(Nic / SH) (130 mg, 6.5 μmol) and alkyne-modified phenylalanine (5.7 mg, 4.0 equiv.) were dissolved in deionized water (or 50 mM sodium phosphate buffer). Then, CuSO4·H2O (0.4 mg, 25 mol%) and Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, 3.4 mg, 1.2 equiv.) were dissolved in deionized water and the N3-PEG-PLL(Nic / SH) solution was added. Sodium ascorbate (3.2 mg, 2.5 equiv.) was then added to the mixture. The reaction mixture was kept under stirring at room temperature for 16 h. After reaction, the mixture was transferred into a dialysis membrane (MWCO=7,000) and dialyzed against deionized water for 1 day. The final product was obtained after lyophilization.

[0292] (E) Polyion Complex (PIC) Micelle Formulations - Following preparation of the cationic carrier units of the present disclosure as described above, micelles were fabricated. The micelles described in this example consist of a cationic carrier unit combined with an antisense oligonucleotide payload.

[0293] Nano-sized PIC micelles were prepared by mixing MeO-PEG-PLL (Nic) or Phe-PEG-PLL (Nic) with miRNA. PEG-PLL (Nic) was dissolved in HEPES buffer (10 mM) at a concentration of 0.5 mg / mL. Then, miRNA solution (22.5 μM) in RNase-free water was mixed with the polymer solution at a miRNA inhibitor (SEQ ID NO: 28) (485ASO-001) to polymer ratio of 2:1 (v / v).

[0294] The mixing ratio of polymer and anti-miRNA was determined by optimizing the micelle formation conditions, i.e., the ratio of amine in the polymer (the carrier of the present disclosure) to phosphate in the anti-miRNA (payload). The mixture of polymer (carrier) and anti-miRNA (payload) was vigorously mixed at 3000 rpm by multi-vortex for 90 seconds and kept at room temperature for 30 minutes to stabilize the micelles.

[0295] Prior to use, micelles (10 μM anti-miRNA concentration) were stored at 4 °C. MeO-micelles or Phe-micelles were prepared using the same method, and micelles containing different amounts of Phe (25%-75%) were also prepared by mixing both polymers during micelle preparation.

[0296] Example 2: Materials and Methods Unless otherwise stated, the examples described below use one or more of the following materials and methods. In vivo hair growth assay in C57BL / 6J mice

[0297] Six-week-old C57BL / 6J mice were obtained from DBL Co.Ltd. Laboratory Animal Facility and were allowed to adapt to the new environment for 1 week. To observe synchronized telogen, the hair was removed from each mouse with clippers, and the back was depilated with a depilatory cream during telogen. Unless otherwise stated, mice received intramuscular (IM) injections of vehicle control or intramuscular injections of miR-485 inhibitor 485ASO-001 twice (0.3mpk and 0.6mpk) at (PD PD0 and PD7). The appearance of skin pigmentation and hair growth were observed and recorded by digital photomicrography with the experimenter(s) blinded to the treatment condition. Progression status was assigned values ​​from 0 to 6 based on pigmentation level and hair shaft density, with 0 being no hair growth (and no pigmentation) and increasing numbers indicating darker skin and denser hair growth area. Scoring was performed blinded. Images representing the different scores were shown as a hair re-growth quantification scale (0-6), with grade 0 = pink skin, no pigmentation, grade 1 = <30% skin area darkened but no visible hair, grade 2 = 30%-70% skin area darkened and no visible hair, grade 3 = >70% skin area darkened or 30% visible hair, grade 4 = >70% skin area darkened and 30-70% visible hair, grade 5 = >70% skin area darkened and >70% visible hair, and grade 6 = >90% skin area darkened and >90% visible hair.

[0298] Histological staining, measurement of the histological structure of skin and hair follicles Dorsal skin from all groups was fixed in 4% PFA on PD10 and PD16 and evaluated by hematoxylin and eosin (H&E) staining. The number of HFs in the dermis and subcutaneous tissue was counted (cross section). The thickness of the dermis and subcutaneous tissue was measured in three visible microscopic fields, with at least seven measurements. Hair follicle length and hair bulb diameter were measured in longitudinal and transverse sections of the dorsal skin by analyzing images with Motic images plus 2.0ML.

[0299] Real-time PCR Total RNA was isolated using the Isolation of small and large RNA kit (Macherey Nagel, Dren). The concentration of the collected RNA was measured with a nucleic acid quantification system (Nanodrop. Thermo Scientific Inc.). cDNA was synthesized with the miScript II RT Kit (Qiagen, Hilden, Germany). For the analysis, the expression of miR-485-3p was performed by TaqMan miRNA analysis using TOPREAL™ qPCR 2X PreMIX (Enzynomics, Korea) on a CFX Connect system (Bio-Rad). Real-time PCR measurements of individual cDNAs were performed using TaqMan probes to measure DNA formation with a Bio-Rad real-time PCR system. The following primers were used:

[0300] Probe: FAM-CGAGGTCGACTTCCTAGA-NFQ. miR-485 3p forward: 5'CATACACGGCTCTCCTCTCTAAA-3'. Relative gene expression was calculated using 2 -ΔΔC T The analysis was carried out by:

[0301] Western blot Skin samples were collected 10 and 16 days after depilation. Mouse skin tissue lysates were prepared by homogenization in ice-cold RIPA buffer (iNtRON Biotechnology) containing a protease / phosphatase inhibitor cocktail (Cell Signaling Technology, Cat#5872). Tissue debris was removed by centrifugation at 13000 rpm for 15 min at 4°C, and the supernatant was collected. Lysates were boiled for 5 min in 1x SDS (Fisher Scientific) loading buffer containing 5% β-mercaptoethanol (Fisher Scientific). Samples were then subjected to SDS-PAGE in MOPS SDS Running Buffer (Invitrogen) on PAGE 8%, 12% Bis-Tris gels (Invitrogen), transferred to PVDF membranes, and incubated with the following primary antibodies: rabbit anti-CD36 (Abcam, Cat. No. ab80080, 1:1000), rabbit anti-VEGF-A (Abcam, Cat. No. ab51745, 1:1000), rabbit anti-Wnt3a (IBL, Cat. No. 11088, 1:1000), rabbit anti-β-actin (Abcam, Cat. No. ab1997, 1:100), anti-actin (Santa Cruz, Cat. No. sc-47778). Results were visualized using an enhanced chemiluminescence system and quantified by densitometric analysis (Image J software, NIH). All experiments were performed at least three times independently.

[0302] Immunofluorescence staining C57BL / 6J mouse skin tissue was embedded in a frozen tissue embedding medium (OTC Compound, Sakura Finetek Japan Co., Ltd.), and frozen section slides were prepared using a frozen section preparation system (Cryostat, Leica Camera AG). After fixation with 4% PFA for 15 minutes, the tissue was washed with PBS and reacted for 1 hour with a blocking solution obtained by adding 5% skim milk, 1% donkey serum, and 0.1% Triton (registered trademark) X-100 to PBS. The tissue was then reacted for 1 hour at room temperature or overnight at 4°C with a primary antibody solution obtained by diluting a CD36 antibody solution (Invitrogen Cat. No. PA1-16813) 50-fold with the blocking solution. After washing three times with PBS, the tissue was reacted for 1 hour at room temperature with a secondary antibody solution obtained by diluting an FITC-labeled anti-mouse IgG antibody (Invitrogen Corp.) 200-fold with the blocking solution. After reacting with DAPI solution, the tissues were washed three times with PBS and mounted with antifade reagent (Prolong Gold Antifade Reagent). The tissues were observed using a fluorescence microscope (Olympus Corp.).

[0303] Example 3. Analysis of the dose-dependent hair growth promoting effect of miRNA-485 inhibitors To begin evaluating the hair regrowth effect of miRNA-485 inhibitor (485 ASO-001) (shown in Example 1) on hair-depilated mice, 6-week-old C57BL / 6J mice were purchased and adapted to a new environment for one week. To observe synchronized telogen phase, hair was removed from each mouse by depilatory cream. Then, the animals were divided into 5 groups (n=6) to evaluate the hair regrowth effect of miRNA-485 inhibitor (485ASO-001). Negative control (PBS) and 485 ASO-001 (0.1mpk, 0.3mpk, and 0.6mpk) were administered by one intramuscular injection. Digital photographs of the mice were taken post-depilation (pd) and on days 5 (Figure 2A), 7 (Figure 2B), 9 (Figure 2C), 12 (Figure 2D), 14 (Figure 2E), and 16 (Figure 2F) after treatment, and the hair growth area was analyzed by morphological observation with a score of 0 to 6 points (Figure 2G).

[0304] As shown in Figure 2G, mice treated with 485ASO-001 (0.3mpk and 0.6mpk) showed a significant increase in hair growth score compared to the PBS-treated control group on days 7 and 12. Furthermore, mice treated with 485ASO-001 (0.6mpk) showed more hair re-growth compared to mice treated with 485ASO-001 (0.1mpk and 0.3mpk) (Figure 2G).

[0305] In addition, C57BL / 6J mice were divided into 5 groups (n=5) to evaluate the hair regrowth effect of miRNA-485 inhibitor (485ASO-001). Negative control (PBS), positive control (2% minoxidil), and 485ASO-001 (0.3mpk, and 0.6mpk) were administered by intramuscular injection twice (PD0 and PD7). Digital photographs of the mice were taken at post-depilation (pd) and on the 5th (Figure 4D), 7th (Figure 4E), 10th (Figure 4F), 12th (Figure 4G), 14th (Figure 4H), and 16th (Figure 4I) days after treatment, and the hair regrowth area was analyzed by morphological observation with a score of 0 to 6 points (Figure 4J). Mice treated with two repeated intramuscular injections of 485ASO-001 (0.3 mpk and 0.6 mpk) showed a significant increase in hair regrowth scores compared to the PBS-treated control and the positive control minoxidil-treated groups on days 7, 10, 12, 14, and 16 (Figure 4J). Furthermore, mice treated with 485ASO-001 (2x at 0.6 mpk) showed more hair regrowth compared to mice treated with 485ASO-001 (2x at 0.3 mpk) (Figure 4J).

[0306] The above results demonstrate the dose-dependent efficacy of miRNA-485 inhibitor (485ASO-001) in inducing hair growth. For example, these results show that miRNA-485 inhibitor (485ASO-001) promoted early signs of anagen hair growth in telogen mice (see, e.g., Figures 4D-4J). In particular, mice treated with miRNA-485 inhibitor (485ASO-001) (2x at 0.6 mpk) were shown to strongly activate and maintain anagen hair growth compared to PBS-treated control and minoxidil-treated groups (Figures 4D-4J). These results suggest that miRNA-485 inhibitor (485ASO-001) activates the telogen-to-anagen transition, thereby promoting hair growth.

[0307] Example 4: Analysis of the dose-dependent effects of miRNA-485 inhibitors on hair density To further evaluate the hair re-growth effect of the miRNA-485 inhibitor (485ASO-001) prepared as shown in Example 1 on hair loss mice treated with PBS and 485ASO-001 as shown in Example 1, the hair density after treatment with 485ASO-001 (0.1 mpk, 0.3 mpk, 0.6 mpk) was compared with that of the PBS-treated control group. Dermoscopic images of each mouse were taken using Kong, Bomtech Electronics Co., Ltd., Seoul, Korea, at the same area of ​​interscapular skin (3.6 mm ) on day 12 ( FIG. 3A ) and day 16 ( FIG. 3B ). 2 The hair density was assessed by analyzing the images (magnification 200x, actual area = 3.6 mm). 2 ).

[0308] As shown in Figure 3C, mice treated with 485ASO-001 (0.6 mpk) showed a significant increase in hair density compared to the PBS-treated control group on days 12 and 16. Mice treated with 485ASO-001 (0.6 mpk) showed increased hair density compared to mice treated with 485ASO-001 (0.1 mpk) and 485ASO-001 (0.3 mpk).

[0309] The above results demonstrate the dose-dependent efficacy of miRNA-485 inhibitor (485ASO-001) in increasing hair density.

[0310] Example 5: Analysis of the dose-dependent effects of miRNA-485 inhibitors on hair length To further evaluate the hair regrowth effect of the miRNA-485 inhibitor (485ASO-001) prepared as shown in Example 1 on bald mice treated with PBS and 485ASO-001 as shown in Example 1, the hair length after treatment with 485ASO-001 (0.1 mpk, 0.3 mpk, 0.6 mpk) was compared with the PBS-treated control group. After treatment, hair was plucked from representative areas of the bald dorsal interscapular region on the back on days 16 (Figure 4A) and 21 (Figure 4B).

[0311] As shown in FIG. 4C, mice treated with 485ASO-001 (0.3 mpk and 0.6 mpk) showed a significant increase in hair length compared to the PBS-treated control group on day 16.

[0312] The above results demonstrate the dose-dependent efficacy of miRNA-485 inhibitor (485ASO-001) in increasing hair length.

[0313] Example 6: Expression of miR-485-3p in skin development and hair follicle cycling To test the effect of miRNA-485 inhibitor (485ASO-001) on the expression level of miRNA-485-3p (FW7_mimic), tissue samples from early to late anagen phase of the mouse hair growth cycle were obtained on pd10 (Figure 5A) and pd16 (Figure 5B). The expression of miRNA was decreased in late anagen phase compared to early anagen phase of the adult hair cycle. There was a significant decrease in the expression of miRNA on pd10 (p=0.01) and pd16 (p=0.03) in the mice treated with miRNA-485 inhibitor (485ASO-001) (2 times at 0.3 mpk) compared to the control group treated with (485ASO-001) (2 times at 0.3 mpk) and PBS. The above results show a dose-dependent downregulation of the expression of miRNA-485-3p by miRNA-485 inhibitor (485ASO-001).

[0314] Example 7: Analysis of dose-dependent effects of miRNA-485 inhibitors on skin and hair follicle density The effect of miRNA-485 (485ASO-001) on hair growth was further evaluated by H&E staining (Fig. 6A, 6B, 7A, 7B). On pd10, significant increases were observed in the dermis (p=0.03) (Fig. 6C), subcutaneous tissue (p=0.0001) (Fig. 6D), hair follicle diameter (p=0.001) (Fig. 6E), hair bulb diameter (p=0.01) (Fig. 6F), hair follicle density in the dermis (p<0.0009) (Fig. 6G), and hair follicle density in the subcutaneous tissue (p<0.0009) (Fig. 6H). Mice treated with 485ASO-001 (2 x 0.6 mpk) showed a significant increase in the number of hair follicles in the subcutaneous tissue (p=0.0003) (Figure 7C), hair follicle diameter (p=0.008) (Figure 7F), hair follicle density in the dermis (p<0.0001) (Figure 7G), and hair follicle density in the subcutaneous tissue (p<0.0001) (Figure 7H) compared to the PBS-treated control group. Mice treated with 485ASO-001 (0.6 mpk) showed an increase in the number of hair follicles in the subcutaneous tissue on pd10 (p<0.001) (Figure 6H) and in the dermis on pd16 (p=0.004) (Figure 7G) compared to the minoxidil-treated group. Treatment with the miRNA-485 inhibitor (485ASO-001) (0.3mpk and 0.6mpk) (p<0.001) increased hair follicle number in a dose-dependent manner compared to the control (Figures 6G, 6H, 7G, 7H). Minoxidil treatment also significantly (p<0.01) increased hair follicle number compared to the negative control, but this increase was lower than that with the miRNA-485 inhibitor (485ASO-001) (0.6mpk) treatment (Figures 6G, 6H). Treatment with the miRNA-485 inhibitor (485ASO-001) (0.6mpk) and minoxidil significantly increased dermal thickness compared to the control group (p<0.03 and p<0.04), but the increase in dermal and subcutaneous tissue thickness with minoxidil treatment was lower than that with the miRNA-485 inhibitor (485ASO-001) (0.6mpk) (p<0.001) treatment (Figure 6C, Figure 6D). Treatment with the miRNA-485 inhibitor (485ASO-001) (0.6mpk) significantly increased hair bulb diameter on pd10 compared to the control and minoxidil treatment groups (p<0.01).On pd16, the miRNA-485 inhibitor (485ASO-001) (0.3mpk and 0.6mpk) significantly increased subcutaneous tissue thickness compared to controls (p<0.001 and 0.0003).

[0315] Example 8: miRNA-485 inhibitors increased the expression of Wnt and β-catenin proteins The effect of miRNA-485 inhibitor (485ASO-001) on Wnt3a and β-catenin protein expression was evaluated by Western blotting. Specifically, Wnt / β-catenin signaling is involved in hair follicle morphogenesis, regeneration, and growth. Wnt3a induces hair growth by activating the Wnt / β-catenin signaling pathway in dermal papilla (DP) cells. β-catenin is expressed in the dermal papilla and promotes the induction and duration of anagen as well as the regulation and differentiation of keratinocytes. miRNA-485 inhibitor (485 ASO-001) (0.6 mpk) increased the expression of Wnt3a (Figure 9B) and β-catenin (Figure 9C). β-catenin has been shown to be involved in skin and hair follicle development and is an essential molecule in the Wnt signaling pathway. The above results indicate that the miRNA-485 inhibitor (485ASO-001) (0.6 mpk) significantly promotes hair shaft elongation and differentiation.

[0316] Example 9: miRNA-485 inhibitors increased CD36 and VEGF-A protein expression The effect of miRNA-485 inhibitor (485ASO-001) on CD36 and vascular endothelial growth factor A (VEGF-A) protein expression was evaluated by Western blotting. Treatment with miRNA-485 inhibitor (485ASO-001) (0.6mpk) increased the protein expression levels of CD36 (Figure 8B) and VEGF-A (Figure 8C) compared with the PBS-treated group. VEGF-A is the most potent and specific vascular growth factor and a key regulator in physiological and pathological angiogenesis (capillary formation). VEGF-A levels are regulated by transcriptional regulation and mRNA stability.

[0317] The mesenchyme of mouse hair follicles is composed of hair follicle-lining smooth muscle known as the dermal sheath (DS). The DS regulates the capillaries within the hair follicle along with hair cycling. Immunofluorescence experiments were performed to evaluate the effect of miRNA-485 inhibitor (485ASO-001) (0.6mpk) on the expression of CD36 in angiogenesis influenced by DS. The immunofluorescence results (Figure 10B) showed that CD36-expressing cells were increased in the DS of hair follicles treated with miRNA-485 inhibitor (485ASO-001). This can be associated with angiogenesis, especially in hair follicles in the anagen phase. ***

[0318] It should be appreciated that the "Detailed Description of the Invention" section is intended to be used in interpreting the claims, rather than the "Summary of the Invention" and "Abstract" sections. The Summary of the Invention and Abstract sections may set forth one or more, but not all, exemplary aspects of the disclosure contemplated by the inventor(s) and are therefore not intended to limit the scope of the disclosure and the appended claims in any way.

[0319] The present disclosure has been described above using functional building blocks that indicate the implementation of specific functions and relationships thereof. The boundaries of these functional elements have been arbitrarily defined herein for the convenience of description. Alternative boundaries may be defined as long as the specific functions and relationships thereof are appropriately implemented.

[0320] The above specific description of specific embodiments fully illustrates the general nature of the present disclosure, so that others can easily modify and / or adapt such specific embodiments to various applications without departing from the general concept of the present disclosure by applying knowledge within the skill of the art and without undue experimentation. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teachings and advice presented herein. The words or terms used herein are for the purpose of description and not for the purpose of limitation, and the words or terms used herein should be understood by those skilled in the art in light of the teachings and advice presented herein.

[0321] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0322] The entire contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated herein by reference for all purposes, and the references cited therein are similarly incorporated by reference.

Claims

1. 1. A pharmaceutical composition for use in inducing hair growth, increasing hair density, increasing hair follicle density, increasing hair shaft thickness, increasing hair length, increasing expression of CD36 protein in the dermal root sheath of hair follicles, preventing hair loss, reducing hair loss, or any combination thereof, in a subject in need thereof, said pharmaceutical composition comprising a compound that inhibits miR-485 (a "miRNA inhibitor").

2. 2. The pharmaceutical composition of claim 1, wherein the subject has one or more diseases including alopecia totalis, androgenetic alopecia, alopecia areata, alopecia universalis, regressive alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial alopecia, scarring alopecia, scalp hair thinning, hair shaft abnormalities, infectious hair diseases, genetic diseases, and hair loss due to chemotherapy, hormonal imbalance, fungal infection, medication, chemical hair treatment, or aging.

3. The pharmaceutical composition of claim 1, wherein the miRNA inhibitor inhibits miR485-3p, and the miR485-3p comprises 5'-GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1).

4. The pharmaceutical composition of claim 1, wherein the miRNA inhibitor comprises a nucleotide sequence comprising 5'-UGUAUGA-3' (SEQ ID NO:2), and the miRNA inhibitor is about 6 to about 30 nucleotides in length.

5. The miRNA inhibitor is 5'-UGUAUGA-3' (SEQ ID NO: 2), 5'-GUGUAUGA-3' (SEQ ID NO: 3), 5'-CGUGUAUGA-3' (SEQ ID NO: 4), 5'-CCGUGUAUGA-3' (SEQ ID NO: 5), 5'-GCCGUGUAUGA-3' (SEQ ID NO: 6), 5'-AGCCGUGUAUGA-3' (SEQ ID NO: 7), 5'-GAGCCGUGUAUGA-3' (SEQ ID NO: 8), 5'-AGAGCCGUGUAUGA-3' (SEQ ID NO: 9), 5'-GAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-GGAGAG 5'-CCGUGUAUGA-3' (SEQ ID NO:11), 5'-AGGAGAGCCGUGUAUGA-3' (SEQ ID NO:12), 5'-GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO:13), 5'-AGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO:14), 5'-GAGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO:15), 5'-UGUAUGAC-3' (SEQ ID NO:16), 5'-GUGUAUGAC-3' (SEQ ID NO:17), 5'-CGUGUAUGAC-3' (SEQ ID NO:18), 5'-CCGUGUAUGAC-3' (SEQ ID NO:19), No. 19), 5'-GCCGUGUAUGAC-3' (SEQ ID NO: 20), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 21), 5'-GAGCCGUGUAUGAC-3' (SEQ ID NO: 22), 5'-AGAGCCGUGUAUGAC-3' (SEQ ID NO: 23), 5'-GAGAGCCGUGUAUGAC-3' (SEQ ID NO: 24), 5'-GGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), 5'-AGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 26), 5'-GAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 27), ), 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 28), 5'-GAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 29), 5'-TGTATGA-3' (SEQ ID NO: 30), 5'-GTGTATGA-3' (SEQ ID NO: 51), 5'-CGTGTATGA-3' (SEQ ID NO: 52), 5'-CCGTGTATGA-3' (SEQ ID NO: 53), 5'-GCCGTGTATGA-3' (SEQ ID NO: 54), 5'-AGCCGTGTATGA-3' (SEQ ID NO: 55), 5'-GAGCCGTGTATGA-3' (SEQ ID NO: 35),5'-AGAGCCGTGTATGA-3' (SEQ ID NO: 56), 5'-GAGAGCCGTGTATGA-3' (SEQ ID NO: 57), 5'-GGAGAGCCGTGTATGA-3' (SEQ ID NO: 58), 5'-AGGAGAGCCGTGTATGA-3' (SEQ ID NO: 59), 5'-GAGGAGAGCCGTGTATGA-3' (SEQ ID NO: 60), 5'-AGAGGAGA GCCGTGTATGA-3' (SEQ ID NO: 61), 5'-GAGAGGAGAGCCGTGTATGA-3' (SEQ ID NO: 62); 5'-TGTATGAC-3' (SEQ ID NO: 63), 5'-GTGTATGAC-3' (SEQ ID NO: 64), 5'-CGTGTATGAC-3' (SEQ ID NO: 65), 5'-CCGTGTATGAC-3' (SEQ ID NO: 66), 5'-GCCGTGT ATGAC-3' (SEQ ID NO: 67), 5'-AGCCGTGTATGAC-3' (SEQ ID NO: 68), 5'-GAGCCGTGTATGAC-3' (SEQ ID NO: 69), 5'-AGAGCCGTGTATGAC-3' (SEQ ID NO: 70), 5'-GAGAGCCGTGTATGAC-3' (SEQ ID NO: 71), 5'-GGAGAGCCGTGTATGAC-3' (SEQ ID NO: 72) 5'-AGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 73), 5'-GAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 74), 5'-AGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 75), or 5'-GAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 76).

6. The pharmaceutical composition of any one of claims 1 to 3, wherein the miRNA inhibitor comprises the sequence set forth in 5'-AGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO:28).

7. 2. The pharmaceutical composition of claim 1, wherein the miRNA inhibitor comprises at least one modified nucleotide, including locked nucleic acid (LNA), unlocked nucleic acid (UNA), arabino nucleic acid (ABA), bridged nucleic acid (BNA), peptide nucleic acid (PNA), or any combination thereof.

8. 2. The pharmaceutical composition of claim 1, wherein the miRNA inhibitor comprises a backbone modification comprising a phosphorodiamidate morpholino oligomer (PMO), a phosphorothioate (PS) modification, or any combination thereof.

9. 2. The pharmaceutical composition of claim 1, wherein the miRNA inhibitor is delivered in a delivery agent comprising a micelle, an exosome, a viral vector, a lipid nanoparticle, an extracellular vesicle, or a synthetic vesicle.

10. The delivery agent is [WP]-L1-[CC]-L2-[AM] (Formula I) or [WP]-L1-[AM]-L2-[CC] (Formula II) (In the formula, WP is a water soluble biopolymer moiety; CC is a cationic carrier moiety; AM is the adjuvant moiety, L1 and L2 are independently an optional linker; The pharmaceutical composition of claim 9 , wherein the cationic carrier unit, when mixed with the miRNA inhibitor, forms a micelle with the miRNA inhibitor.

11. 11. The pharmaceutical composition of claim 10, wherein the water soluble polymer comprises poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazoline ("POZ"), poly(N-acryloylmorpholine), polyethylene glycol ("PEG"), or poly(propylene glycol) ("PPG"), or any combination thereof.

12. The pharmaceutical composition of claim 10 , wherein the cationic carrier moiety comprises one or more basic amino acids.

13. The pharmaceutical composition of claim 10, wherein the adjuvant moiety comprises an imidazole derivative, an amino acid, a vitamin, or any combination thereof.

14. The pharmaceutical composition of claim 10, wherein the delivery agent comprises a water-soluble biopolymer portion having about 120 to about 130 PEG units, a cationic carrier portion comprising polylysine having about 30 to about 40 lysines, and an adjuvant portion having about 5 to about 10 vitamin B3.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the miRNA inhibitor is formulated for parenteral administration, intramuscular administration, subcutaneous administration, ophthalmic administration, intravenous administration, intraperitoneal administration, intradermal administration, intraorbital administration, intracerebral administration, intracranial administration, intraventricular administration, intraspinal administration, intraventricular administration, intrathecal administration, intracisternal administration, intracapsular administration, intratumoral administration, topical administration, or any combination thereof.

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