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WO2024228119A1 - Inhibiteurs de miarn-485 - Google Patents

Inhibiteurs de miarn-485 Download PDF

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Publication number
WO2024228119A1
WO2024228119A1 PCT/IB2024/054199 IB2024054199W WO2024228119A1 WO 2024228119 A1 WO2024228119 A1 WO 2024228119A1 IB 2024054199 W IB2024054199 W IB 2024054199W WO 2024228119 A1 WO2024228119 A1 WO 2024228119A1
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Prior art keywords
aso
aspects
mir
seq
vitamin
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Inventor
Jin-Hyeob RYU
Insun Kim
Yu Na Lim
Hyun Su Min
Young Jin Park
Hyo Jin Lee
Shahnaz BEGUM
Md Jamil HOSSAIN
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Biorchestra Co Ltd
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Biorchestra Co Ltd
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Publication of WO2024228119A1 publication Critical patent/WO2024228119A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • Oligonucleotides are nucleic acid polymers with the potential to treat or manage a wide range of diseases. Although the majority of oligonucleotide therapeutics have focused on gene silencing, other strategies are being pursued, including splice modulation and gene activation, expanding the range of possible targets beyond what is generally accessible to conventional pharmaceutical modalities. [0005] As of January 2020, ten oligonucleotide drugs have received regulatory approval from the FDA. However, a major obstacle preventing widespread usage of oligonucleotide therapeutics is the difficultly in achieving efficient delivery to target organs and tissues other than the liver.
  • oligonucleotide dugs BRIEF SUMMARY OF THE DISCLOSURE [0006]
  • ASO antisense oligonucleotide
  • a miR-485-3p ASO can have improved plasma stability and/or improved in vitro or in vivo cytotoxicity compared to a modified ASO.
  • the miR-485-3p ASO comprises at least two different sugar an unmodified nucleosides, e.g., LNA and 2’-O-methylethyl RNA monomer.
  • the miR-485-3p ASO has a phosphorothioate backbone structure. Accordingly, in some aspects, the miR-485 inhibitors provided herein are useful for treating various diseases or disorders. Additional aspects of the present disclosure are provided throughout the present application.
  • the disclosure is directed to an antisense oligonucleotide (ASO) comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that are complementary to a nucleic acid sequence within miRNA 485-3p (5'- GUCAUACACGGCUCUCCUCUCU -3' (SEQ ID NO: 1)), wherein the contiguous nucleotide sequence comprises at least two different nucleoside analogs.
  • the nucleoside analogs are sugar modified nucleosides.
  • the nucleoside analogs are for example two nucleoside analogs selected from Locked Nucleic Acid (LNA); 2'-O-alkyl-RNA; 2'-amino- DNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2'-O-methyl nucleic acid (2'- OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), and any combination thereof.
  • the nucoleoside analogs are bicyclic sugars.
  • the bicyclic sugars comprise cEt, 2',4'-constrained 2′-O-methoxyethyl (cMOE), LNA, ⁇ -L-LNA, ⁇ -D-LNA, 2'-O,4'-C-ethylene- bridged nucleic acids (ENA), amino-LNA, oxy-LNA, or thio-LNA.
  • the nucleoside analogs may be LNA and 2’-MOE.
  • any of the above ASOs comprise the contiguous nucleotide sequence of at least 5'-UGUAUGA-3'.
  • the above ASOs further 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' of the contiguous nucleotide sequence.
  • the ASOs further 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 3' of the contiguous nucleotide sequence.
  • the ASO has the sequence selected from the group consisting of: 5'-UGUAUGA-3', 5'-GUGUAUGA-3', 5'-CGUGUAUGA-3', 5'-CCGUGUAUGA-3' (SEQ ID NO: 2), 5'- GCCGUGUAUGA-3' (SEQ ID NO: 3), 5'-AGCCGUGUAUGA-3' (SEQ ID NO: 4), 5'- GAGCCGUGUAUGA-3' (SEQ ID NO: 5), 5'-AGAGCCGUGUAUGA-3' (SEQ ID NO: 6), 5'- GAGAGCCGUGUAUGA-3' (SEQ ID NO: 7), 5'-GGAGAGCCGUGUAUGA-3' (SEQ ID NO: 8), 5'- AGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 9), 5'-GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-AGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-AG
  • the ASO 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%, or at least about 95% sequence identity to 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49). In some aspects, the ASO disclosed herein has at least 90% sequence identity to 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'-AGAGAGGAGAGCCGTGTATGAC- 3' (SEQ ID NO: 49).
  • the ASO comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49) with one substitution or two substitutions. [0012] In some aspects, the ASO has the nucleotide sequence as set forth in 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49).
  • the ASO comprises the nucleotide sequence as set forth in 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), and wherein one or more of the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs (SEQ ID NO: 56).
  • the ASO comprises the nucleotide sequence as set forth in 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), and wherein one or more of the double underlined letters are 2’-MOEs (SEQ ID NO: 51). Any of the above ASOs can further comprise a backbone modification.
  • This backbone modification can comprise a phosphorodiamidate morpholino oligomer (PMO) and/or phosphorothioate (PS) modification.
  • PMO phosphorodiamidate morpholino oligomer
  • PS phosphorothioate
  • the nucleotides of the above ASOs can each be modified with PS modifications.
  • the ASO comprises the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs (SEQ ID NO: 52).
  • the ASO comprises the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’- MOEs (SEQ ID NO: 53). Any of the above ASOs may further comprise a backbone modification. [0014] In some aspects, the ASOs disclosed herein comprise one or more 5' methyl cytosine nucleobases.
  • the plasma stability of the ASO may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% recovery after one day, as measured by an HPLC.
  • the ASOs have plasma stability of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% recovery after about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, or about ten days, as measured by an HPLC.
  • the plasma stability of the ASO is at least about 90%, at least about 95%, or about 100% recovery after about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 days, as measured by an HPLC.
  • any of the ASO s above may be delivered in a delivery agent.
  • the delivery agent comprises for example 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 combinations thereof.
  • the delivery agent comprises a cationic carrier unit comprising: [WP]-L1-[CC]-L2-[AM] (formula I) or [WP]-L1-[AM]-L2-[CC] (formula II), wherein WP is a water-soluble biopolymer moiety, CC is a cationic carrier moiety, AM is an adjuvant moiety, and L1 and L2 are independently optional linkers.
  • the cationic carrier unit in the delivery agent and the isolated polynucleotide are capable of associating with each other to form a micelle when mixed together. The association may be via a covalent bond, or non-covalent bond.
  • the non-covalent bond may comprise an ionic bond.
  • the water-soluble biopolymer moiety comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ - hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines (“POZ”) poly(N-acryloylmorpholine), or any combinations thereof.
  • POZ polyoxazolines
  • the water-soluble biopolymer moiety comprises polyethylene glycol ("PEG”), polyglycerol, or poly(propylene glycol) (“PPG”). In some aspects, the water-soluble biopolymer moiety comprises: , (formula III), wherein n is 1-1000.
  • an ASO with a water-soluble biopolymer moiety of formula III wherein the 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.
  • the n in formula III may be 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.
  • the water-soluble biopolymer moiety is linear, branched, or dendritic.
  • the cationic carrier moiety comprises one or more basic amino acids.
  • the cationic carrier moiety comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at last about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50 basic amino acids.
  • the cationic carrier moiety comprises about 30 to about 50 basic amino acids.
  • the basic amino acid comprises arginine, lysine, histidine, or any combination thereof.
  • the cationic carrier moiety comprises about 40 lysine monomers.
  • the adjuvant moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue response.
  • the adjuvant moiety comprises an imidazole derivative, an amino acid, a vitamin, or any combination thereof.
  • the adjuvant moiety comprises: , (formula IV), wherein each of G 1 and G 2 is H, an aromatic ring, or 1-10 alkyl, or G 1 and G 2 together form an aromatic ring, and wherein n is 1-10.
  • the adjuvant moiety comprises nitroimidazole.
  • the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazol, azanidazole, benznidazole, or any combination thereof.
  • the adjuvant moiety comprises an amino acid.
  • the adjuvant moiety comprises , (formula V), wherein Ar is wherein each of Z1 and Z2 is H or OH.
  • the adjuvant moiety comprises a vitamin.
  • the vitamin comprises a cyclic ring or cyclic hetero atom ring and a carboxyl group or hydroxyl group. wherein the vitamin comprises: , (formula VI), wherein each of Y 1 and Y 2 is C, N, O, or S, and wherein n is 1 or 2.
  • 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.
  • the vitamin is vitamin B3.
  • the adjuvant moiety comprises at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, 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.
  • the adjuvant moiety comprises about 10 vitamin B3.
  • the ASO delivery agent comprises a water-soluble biopolymer moiety with about 120 to about 130 PEG units, a cationic carrier moiety comprising a poly-lysine with about 30 to about 40 lysines, and an adjuvant moiety with about 5 to about 10 vitamin B3.
  • the cationic carrier unit is capable of protecting the miR-485 inhibitor from enzymatic degradation.
  • the ASO can be administered intranasally, parenthetically, intramuscularly, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracerebroventricularly, intraspinally, intraventricular, intrathecally, intracistemally, intracapsularly, intratumorally, topically, or any combination thereof.
  • the delivery agent comprises a micelle.
  • the micelle comprises (i) about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines, each with an amine group, (iii) about 15 to about 20 lysines, each with a thiol group, and (iv) about 30 to about 40 lysines, each linked to vitamin B3.
  • the micelle comprises (i) about 120 to about 130 PEG units, (ii) about 32 lysines, each with an amine group, (iii) about 16 lysines, each with a thiol group, and (iv) about 32 lysines, each linked to vitamin B3.
  • a targeting moiety is further linked to the PEG units.
  • the targeting moiety is a LAT 1 targeting ligand. In some aspects, the targeting moiety comprises phenylalanine.
  • the present disclosure also provides a conjugate comprising the ASO disclosed herein, wherein the ASO is covalently attached to at least one non-nucleotide or non- polynucleotide moiety. In some aspects, the non-nucleotide or non-polynucleotide moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combinations thereof. [0025] In some aspects, the disclosure provides a pharmaceutical composition comprising the ASO disclosed herein or the conjugate disclosed herein and a pharmaceutically acceptable carrier.
  • the composition further comprises a therapeutic agent.
  • a kit comprising the ASO disclosed herein, the conjugate disclosed herein, or the composition disclosed herein, and instructions for use.
  • the disclosure is a diagnostic kit comprising the ASO disclosed herein, the conjugate disclosed herein, or the composition disclosed herein, and instructions for use.
  • a method of inhibiting or reducing miR-485-3p expression in a cell the method comprising administering the ASO disclosed herein, the conjugate disclosed herein, or the composition disclosed herein to the cell expressing miR-485-3p.
  • the ASO inhibits or reduces expression of miR-485-3p in the cell after the administration.
  • the expression of miR-485-3p is reduced by 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%, or about 100% after the administration compared to a cell not exposed to the ASO.
  • the ASO reduces expression of miR-485-3p in the cell after the administration by at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a cell not exposed to the ASO.
  • the cell is a neuron.
  • the disclosure is directed to a method for treating a disease or disorder in a subject in need thereof, comprising administering an effective amount of the ASO disclosed herein, the conjugate disclosed herein, or the composition disclosed herein to the subject.
  • the disease or disorder comprises a neurodegenerative disease.
  • the disease or condition comprises Alzheimer's disease.
  • the disease or condition comprises tauopathy, amyotrophic lateral sclerosis (ALS), Huntington’s disease, autism spectrum disorder, mental retardation, seizure, stroke, Parkinson's disease, spinal cord injury, or combinations thereof.
  • the subject is a human.
  • ASO antisense oligonucleotide
  • ASO an antisense oligonucleotide of 10 to 30 nucleotides in length comprising a contiguous nucleotide sequence that is complementary to a nucleic acid sequence within miRNA 485-3p (5'-GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1)), wherein the contiguous nucleotide sequence comprises at least two different nucleoside analogs.
  • Aspect 2 An antisense oligonucleotide of 10 to 30 nucleotides in length comprising a contiguous nucleotide sequence that is complementary to a nucleic acid sequence within miRNA 485-3p (5'-GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1)), wherein the contiguous nucleotide sequence comprises at least two different nucleoside analogs.
  • Aspect 3 The ASO of Aspect 1, wherein the nucleoside analogs are sugar modified nucleosides.
  • Aspect 3 The ASO of Aspect 1 or 2, wherein the nucleoside analogs are two nucleoside analogs selected from Locked Nucleic Acid (LNA); 2'-O-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2'-O-methyl nucleic acid (2'- OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), and any combination thereof.
  • LNA Locked Nucleic Acid
  • ANA hexitol nucleic acid
  • INA intercalating nucleic acid
  • INA intercalating nucleic acid
  • Aspect 4 The ASO of Aspect 1 or 2, wherein the nucoleoside analogs are bicyclic sugars.
  • Aspect 5. The ASO of Aspect 4, wherein the bicyclic sugars comprise cEt, 2',4'- constrained 2′-O-methoxyethyl (cMOE), LNA, ⁇ -L-LNA, ⁇ -D-LNA, 2'-O,4'-C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, or thio-LNA.
  • Aspect 6 The ASO of any one of Aspects 1 to 5, wherein the nucleoside analogs are LNA and 2’-MOE.
  • Aspect 8 The ASO of any one of Aspects 1 to 6, wherein the contiguous nucleotide sequence comprises 5'-UGUAUGA-3'.
  • Aspect 8 The ASO of any one of Aspects 1 to 7, wherein the ASO further 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 5' of the contiguous nu
  • Aspect 9 The ASO of any one of Aspects 1 to 8, wherein the ASO further 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' of the contiguous nucleotide sequence.
  • Aspect 10 The ASO of any one of Aspects 1 to 9, wherein the ASO has the sequence selected from the group consisting of: 5'-UGUAUGA-3', 5'-GUGUAUGA-3', 5'- CGUGUAUGA-3', 5'-CCGUGUAUGA-3' (SEQ ID NO: 2), 5'-GCCGUGUAUGA-3' (SEQ ID NO: 3), 5'- AGCCGUGUAUGA-3' (SEQ ID NO: 4), 5'-GAGCCGUGUAUGA-3' (SEQ ID NO: 5), 5'- AGAGCCGUGUAUGA-3' (SEQ ID NO: 6), 5'-GAGAGCCGUGUAUGA-3' (SEQ ID NO: 7), 5'- GGAGAGCCGUGUAUGA-3' (SEQ ID NO: 8), 5'-AGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 9), 5'- GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-AGAG
  • Aspect 11 The ASO of any one of Aspects 1 to 10, wherein the ASO has the sequence selected from the group consisting of: 5'-TGTATGA-3', 5'-GTGTATGA-3', 5'- CGTGTATGA-3', 5'-CCGTGTATGA-3' (SEQ ID NO: 26), 5'-GCCGTGTATGA-3' (SEQ ID NO: 27), 5'-AGCCGTGTATGA-3' (SEQ ID NO: 28), 5'-GAGCCGTGTATGA-3' (SEQ ID NO: 29), 5'- AGAGCCGTGTATGA-3' (SEQ ID NO: 30), 5'-GAGAGCCGTGTATGA-3' (SEQ ID NO: 31), 5'- GGAGAGCCGTGTATGA-3' (SEQ ID NO: 32), 5'-AGGAGAGCCGTGTATGA-3' (SEQ ID NO: 33), 5'- GAGGAGAGCCGTGTATGA-3' (SEQ ID NO: 34), 5'-AGAG
  • Aspect 12 The ASO of any one of Aspects 1 to 11, wherein the ASO 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'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49).
  • Aspect 13 Aspect 13
  • Aspect 14 The ASO of any one of Aspects 1 to 11, wherein the ASO comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49) with one substitution or two substitutions. [0044] Aspect 15.
  • Aspect 16 The ASO of any one of Aspects 1 to 11, wherein the ASO has the nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49).
  • Aspect 16 The ASO of Aspect 15, wherein the ASO comprises the nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), and wherein one or more of the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs (SEQ ID NO: 56).
  • Aspect 15 or 16 wherein the nucleotide sequence comprises 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), and wherein one or more of the double underlined letters are 2’-MOEs (SEQ ID NO: 51).
  • Aspect 18 An ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs (SEQ ID NO: 52).
  • ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs (SEQ ID NO: 53).
  • Aspect 20 The ASO of any one of Aspects 1 to 17, wherein the ASO further comprises a backbone modification.
  • Aspect 21 The ASO of Aspect 20, wherein the backbone modification is a phosphorodiamidate morpholino oligomer (PMO) and/or phosphorothioate (PS) modification.
  • PMO phosphorodiamidate morpholino oligomer
  • PS phosphorothioate
  • Aspect 22 The ASO of Aspect 20 or 21, wherein each of the nucledotides are modified with PS modifications.
  • Aspect 23. The ASO of any one of Aspects 1 to 22, wherein the ASO comprises one or more 5' methyl cytosine nucleobases.
  • Aspect 24. The ASO of any one of Aspects 1 to 23, wherein the plasma stability of the ASO is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% recovery after one day, as measured by an HPLC.
  • Aspect 26 Aspect 26.
  • Aspect 28 The ASO of any one of Aspects 1 to 26, wherein the ASO is delivered in a delivery agent.
  • Aspect 28 The ASO of Aspect 27, wherein 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 combinations thereof.
  • Aspect 29 The ASO of Aspect 27, wherein 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
  • WP is a water-soluble biopolymer moiety
  • CC is a cationic carrier moiety
  • AM is an adjuvant moiety;
  • L1 and L2 are independently optional linkers.
  • Aspect 30 The ASO of Aspect 29, wherein the cationic carrier unit and the isolated polynucleotide are capable of associating with each other to form a micelle when mixed together.
  • Aspect 30 The ASO of Aspect 30, wherein the association is via a covalent bond.
  • Aspect 32 The ASO of Aspect 30, wherein the association is via a non-covalent bond.
  • Aspect 33 The ASO of Aspect 32, wherein the non-covalent bond comprises an ionic bond.
  • Aspect 34 The ASO of Aspect 34.
  • the ASO of any one of Aspects 29 to 33, wherein the water-soluble biopolymer moiety comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines (“POZ”) poly(N-acryloylmorpholine), or any combinations thereof.
  • POZ polyoxazolines
  • ASO of any one of Aspects 29 to 34, wherein the water-soluble polymer comprises polyethylene glycol ("PEG”), polyglycerol, or poly(propylene glycol) (“PPG”).
  • PEG polyethylene glycol
  • PPG poly(propylene glycol)
  • Aspect 36 The ASO of any one of Aspects 29 to 35, wherein the water-soluble biopolymer moiety comprises: , (formula III), wherein n is 1-1000.
  • Aspect 37 Aspect 37.
  • 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.
  • Aspect 38 The ASO of Aspect 36, wherein the 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.
  • Aspect 39 The ASO of any one of Aspects 29 to 38, wherein the water-soluble biopolymer moiety is linear, branched, or dendritic.
  • Aspect 40 The ASO of any one of Aspects 29 to 39, wherein the cationic carrier moiety comprises one or more basic amino acids.
  • Aspect 41 Aspect 41.
  • the ASO of Aspect 40 wherein the cationic carrier moiety comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at last about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50 basic amino acids.
  • Aspect 42 The ASO of Aspect 41, wherein the cationic carrier moiety comprises about 30 to about 50 basic amino acids.
  • Aspect 43 The ASO of any one of Aspects 40 to 42, wherein the basic amino acid comprises arginine, lysine, histidine, or any combination thereof.
  • Aspect 44 The ASO of any one of Aspects 29 to 43, wherein the cationic carrier moiety comprises about 40 lysine monomers.
  • Aspect 45 The ASO of any one of Aspects 29 to 44, wherein the adjuvant moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue response.
  • Aspect 46 The ASO of any one of Aspects 29 to 44, wherein the adjuvant moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue response.
  • Aspect 47 The ASO of Aspect 46, wherein the adjuvant moiety comprises: , (formula IV), wherein each of G 1 and G 2 is H, an aromatic ring, or 1-10 alkyl, or G 1 and G 2 together form an aromatic ring, and wherein n is 1-10.
  • Aspect 48 The ASO of Aspect 46, wherein the adjuvant moiety comprises nitroimidazole.
  • Aspect 49 The ASO of Aspect 46, wherein the adjuvant moiety comprises nitroimidazole.
  • Aspect 46 wherein the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazol, azanidazole, benznidazole, or any combination thereof.
  • Aspect 50 The ASO of any one of Aspects 29 to 49, wherein the adjuvant moiety comprises an amino acid.
  • Aspect 51 The ASO of Aspect 50, wherein the adjuvant moiety comprises (formula V), wherein Ar is wherein each of Z1 and Z2 is H or OH.
  • Aspect 52 The ASO of Aspect 50, wherein the adjuvant moiety comprises (formula V), wherein Ar is wherein each of Z1 and Z2 is H or OH.
  • Aspect 53 The ASO of Aspect 52, wherein the vitamin comprises a cyclic ring or cyclic hetero atom ring and a carboxyl group or hydroxyl group.
  • Aspect 54 The ASO of Aspect 52 or 53, wherein the vitamin comprises: (formula VI), wherein each of Y 1 and Y 2 is C, N, O, or S, and wherein n is 1 or 2.
  • Aspect 55 The ASO of Aspect 52 or 53, wherein the vitamin comprises: (formula VI), wherein each of Y 1 and Y 2 is C, N, O, or S, and wherein n is 1 or 2.
  • Aspect 56 The ASO of Aspect 55, wherein the vitamin is vitamin B3.
  • Aspect 57 The ASO of Aspect 57.
  • ASO of Aspect 55 or 56 wherein the adjuvant moiety comprises at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, 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.
  • Aspect 58 The ASO of any one of Aspects 55 to 57, wherein the adjuvant moiety comprises about 10 vitamin B3.
  • Aspect 59 The ASO of any one of Aspects 55 to 57, wherein the adjuvant moiety comprises about 10 vitamin B3.
  • Aspect 60 The ASO of any one of Aspects 29 to 59, wherein the cationic carrier unit is capable of protecting the miR-485 inhibitor from enzymatic degradation.
  • Aspect 61 Aspect 61.
  • Aspect 62 The ASO of any one of Aspects 28 to 61, wherein the delivery agent is a micelle.
  • the ASO of Aspect 62 wherein the micelle comprises (i) about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines, each with an amine group, (iii) about 15 to about 20 lysines, each with a thiol group, and (iv) about 30 to about 40 lysines, each linked to vitamin B3.
  • Aspect 64 The ASO of Aspect 63, wherein the micelle comprises (i) about 120 to about 130 PEG units, (ii) about 32 lysines, each with an amine group, (iii) about 16 lysines, each with a thiol group, and (iv) about 32 lysines, each linked to vitamin B3.
  • Aspect 65 The ASO of Aspect 63 or 64, wherein a targeting moiety is further linked to the PEG units.
  • Aspect 66 The ASO of Aspect 65, wherein the targeting moiety is a LAT 1 targeting ligand.
  • Aspect 67 The ASO of Aspect 66, wherein the targeting moiety is phenylalanine.
  • Aspect 68 A conjugate comprising the ASO of any one of Aspects 1 to 67, wherein the ASO is covalently attached to at least one non-nucleotide or non-polynucleotide moiety.
  • Aspect 69 Aspect 69.
  • Aspect 70 A pharmaceutical composition comprising the ASO of any one of Aspects 1 to 67 or the conjugate of Aspect 68 or 69 and a pharmaceutically acceptable carrier.
  • Aspect 71 The composition of Aspect 70, which further comprises a therapeutic agent.
  • Aspect 72 A kit comprising the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71, and instructions for use.
  • Aspect 73 A diagnostic kit comprising the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71, and instructions for use.
  • Aspect 74 A method of inhibiting or reducing miR-485-3p expression in a cell, the method comprising administering the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71 to the cell expressing miR-485-3p.
  • Aspect 75 The method of Aspect 74, wherein the ASO inhibits or reduces expression of miR-485-3p in the cell after the administration.
  • Aspect 76 Aspect 76.
  • Aspect 74 or 75 wherein the expression of miR-485-3p is reduced by 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%, or about 100% after the administration compared to a cell not exposed to the ASO.
  • Aspect 77 The method of any one of Aspects 74 to 76, wherein the ASO reduces expression of miR-485-3p in the cell after the administration by at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a cell not exposed to the ASO.
  • Aspect 78 The method of Aspect 74 or 75, wherein the expression of miR-485-3p is reduced by 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%, or about 100% after the administration compared to a cell not exposed to the ASO.
  • Aspect 79 A method of inducing hair growth in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • Aspect 80 A method of increasing the follicular density in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • Aspect 81 A method of increasing the follicular density in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • a method of increasing the hair shaft thickness in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • Aspect 82 A method of increasing the hair length in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • Aspect 83 A method of increasing the hair shaft thickness in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • a method for preventing hair loss in a subject at risk of hair loss comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • Aspect 84 A method for reducing hair loss in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71.
  • Aspect 85 Aspect 85.
  • a method of upregulating CD36 protein in a dermal sheath of hair follicle in a subject in need thereof comprising administering to the subject the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71. [0115] Aspect 86.
  • any one of claims 79 to 85 wherein the subject has one or more disorders comprising alopecia greata, androgenic alopecia, alopecia areata, alopecia universalis, involutional alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial, alopecia, scarring alopecia, scalp thinning, hair shaft abnormalities, infectious hair disorders, genetic disorders, and hair loss due to chemotherapy, hormonal imbalance, fungal infection, medication intake, chemical hair treatment, aging, or any combination thereof.
  • Aspect 87 Aspect 87.
  • a method for treating a disease or disorder in a subject in need thereof comprising administering an effective amount of the ASO of any one of Aspects 1 to 67, the conjugate of Aspect 68 or 69, or the composition of Aspect 70 or 71 to the subject. [0117] Aspect 88.
  • Aspect 87 wherein the disease or disorder comprises alopecia greata, androgenic alopecia, alopecia areata, alopecia universalis, involutional alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial, alopecia, scarring alopecia, scalp thinning, hair shaft abnormalities, infectious hair disorders, genetic disorders, and hair loss due to chemotherapy, hormonal imbalance, fungal infection, medication intake, chemical hair treatment, aging, or any combination thereof.
  • Aspect 89 The method of Aspect 87, wherein the disease or disorder comprises a neurodegenerative disease.
  • Aspect 90 The method of Aspect 87, wherein the disease or disorder comprises a neurodegenerative disease.
  • Aspect 87 wherein the disease or condition comprises Alzheimer's disease.
  • Aspect 91 The method of Aspect 87, wherein the disease or condition comprises tauopathy, amyotrophic lateral sclerosis (ALS), Huntington’s disease, autism spectrum disorder, mental retardation, seizure, stroke, Parkinson's disease, spinal cord injury, or combinations thereof.
  • Aspect 89 The method of any one of Aspects 79 to 88, wherein the subject is a human.
  • Aspect 90 The method of any one of Aspects 79 to 88, wherein the subject is a human.
  • FIG.1 shows a schematic diagram of modified ASOs.
  • FIG. 2 shows various nucleotide modifications, e.g., backbone modification, nucleobase modification, 2’ ribose substitution, 2’-ribose modification, and alternative chemistries.
  • FIGs. 3A-3F show various miR-485 inhibitors described herein.
  • FIG. 3A is an unmodified antisense oligonucleotide targeting miR485-3p (miR-485-3p ASO-A, ASO-A).
  • FIG. 3B is a modified antisense oligonucleotide targeting miR-485-3p.
  • the three nucleosides at the 5’ terminus and one nucleoside at the 3’ terminus are modified with LNA monomers.
  • the 12 th and 13 th nucleosides are 2’-O-methoxyethyl RNA monomers.
  • the backbone structure of the ASO is a phosphorothioate backbone.
  • FIG. 3C is a modified antisense oligonucleotide targeting miR- 485-3p.
  • the three nucleosides at the 5’ terminus and one nucleoside at the 3’ terminus are modified with LNA monomers.
  • the 12 th , 13 th , 15 th , 17 th , and 19 th nucleosides are modified with 2’-O- methoxyethyl RNA monomers.
  • the backbone structure of the ASO is a phosphorothioate backbone.
  • the unmodified nucleotides i.e., 4 th to 11 th , 14 th , 16 th , 18 th , 20 th , and 21 st are RNAs (miR-485-3p ASO-C, ASO-C).
  • FIG. 3D is a modified antisense oligonucleotide targeting miR- 485-3p.
  • the three nucleosides at the 5’ terminus, the one nucleoside at the 3’ terminus, and the 12 th , 13 th , 15 th , 17 th , and 19 th nucleosides are modified with 2’-O-methoxyethyl RNA monomers.
  • the backbone structure of the ASO is a phosphorothioate backbone.
  • the unmodified nucleotides, i.e., 4 th to 11 th , 14 th , 16 th , 18 th , 20 th , and 21 st are RNAs (miR-485-3p ASO-D, ASO-D).
  • FIG.3E is a modified antisense oligonucleotide targeting miR-485-3p.
  • the three nucleosides at the 5’ terminus and one nucleoside at the 3’ terminus are modified with LNA monomers.
  • the backbone structure of the ASO is a phosphorothioate backbone.
  • the unmodified nucleotides i.e., 4 th to 21 st , are DNAs (miR-485-3p ASO-E, ASO-E).
  • FIG. 3F is a modified antisense oligonucleotide targeting miR-485-3p.
  • the three nucleosides at the 5’ terminus and one nucleoside at the 3’ terminus are modified with 2’-O-methoxyethyl monomers.
  • the backbone structure of the ASO is a phosphorothioate backbone.
  • the unmodified nucleotides, i.e., 4 th to 21 st are DNAs (miR-485-3p ASO-F, ASO-F).
  • FIG.4 shows a work flow for antisense oligonucleotide synthesis.
  • FIGs.5A-5F show HPLC results of synthesized ASOs. The figures show the purity of miR-485-3p ASO-A, B, C, D, E and F by the HPLC spectrum, respectively. Each ASO was synthesized with a purity of over 99%.
  • FIG.6A shows photographs of female mice subcutaneously injected on day 0 and day 7 post depilation (0.6mpk) with either miR-485-3p ASO-A or miR-485-3p ASO-C encapsulated in a cationic micelle, compared with PBS-administered control female mice.
  • FIG. 6B shows quantitative analysis of hair growth scores after subcutaneous injection with miR-485-3p ASO-A and miR-485-3p ASO-C encapsulated in a cationic micelle, compared with PBS-administered control female mice. Data is based on 4 mice per group and reported as a mean ⁇ SEM. Scores are analyzed for days 10, 12, and 14 post depilation (PD). The left bar shows control, the middle bar shows ASO-A, and the right bar shows ASO-C. [0129] FIG.
  • FIG. 7A shows photographs of male mice intramuscularly injected on day 0 and day 7 post depilation (0.6mpk) with either miR-485-3p ASO-A or miR-485-3p ASO-C encapsulated in a cationic micelle, compared with PBS-administered control male mice. Photographs were taken on days 3, 7, 10, 12, and 14 post depilation (PD). The left column shows control, the middle column shows ASO-A, and the right column shows ASO-C.
  • FIG. 7B shows quantitative analysis of hair growth scores after intramuscular injection of miR-485-3p ASO-A and miR-485-3p ASO-C encapsulated in a cationic micelle, compared with PBS-administered control male mice.
  • FIG. 8A shows photographs of male mice subcutaneously injected on day 0 and day 7 post depilation (0.6mpk) with either miR-485-3p ASO-A or miR-485-3p ASO-C encapsulated in a cationic micelle, compared with PBS-administered control male mice. Photographs were taken on days 7, 10, 12, 14, and 17 post depilation (PD).
  • FIG. 8B shows quantitative analysis of hair growth scores after subcutaneous injection of miR-485-3p ASO-A and miR-485-3p ASO-C encapsulated in a cationic micelle, compared with PBS-administered control male mice. Data is based on 3-4 mice per group and reported as a mean ⁇ SEM. Scores are analyzed for days 10, 12, and 14 post depilation (PD). The left bar shows control, the middle bar shows ASO-A, and the right bar shows ASO-C. [0131] FIG. 9A shows the experimental schedule used to induce androgenetic alopecia (AGA) in male mice (male mice model).
  • AGA androgenetic alopecia
  • FIG.9B shows photographs of male mice subcutaneously injected with both DHT (1 mg/50 ⁇ l) every other day PD and with miR 485-3p ASO-C encapsulated in a cationic micelle (0.6 mpk) on day 0 and day 7 PD, compared with PBS- administered control male mice, and male mice injected with either DHT (1 mg/50 ⁇ l) every other day PD or miR-485-3p ASO-C encapsulated in a cationic micelle (0.6 mpk) on day 0 and day 7 PD. Photographs were taken on days 7, 10, 12, 14, 17, and 19 post depilation (PD).
  • PD post depilation
  • FIG.9C shows quantitative analysis of hair growth scores for male mice subcutaneously injected with both DHT (1 mg/50 ⁇ l) every other day PD and with miR 485-3p ASO-C encapsulated in a cationic micelle (0.6 mpk) on day 0 and day 7 PD, compared with PBS- administered control male mice, and male mice injected with either DHT (1 mg/50 ⁇ l) every other day PD or miR-485-3p ASO-C encapsulated in a cationic micelle (0.6 mpk) on day 0 and day 7 PD.
  • the present disclosure is generally directed to a miR-485 targeting antisense oligonucleotide (ASO) used to treat a disease or disorder.
  • ASO antisense oligonucleotide
  • a miR-485-3p ASO can have an improved plasma stability and/or improved in vitro or in vivo cytotoxicity compared to an unmodified ASO.
  • the miR-485-3p comprises at least two different sugar modified nucleosides, e.g., LNA and 2’-O-methylethyl RNA monomer.
  • the miR-485-3p ASO has a phosphorothioate backbone structure. Accordingly, in some aspects, the miR-485 inhibitors provided herein are useful for treating various diseases or disorders. Additional aspects of the present disclosure are provided throughout the present application. [0133] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, which can vary.
  • ranges recited are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints.
  • a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • nucleotide sequences are written left to right in 5' to 3' orientation.
  • Specific nucleotides (nucleotide residues) in a sequence may be referred to by their numeric position in the sequence with nucleotide 1 (nucleotide residue 1) being the first nucleotide on the 5’ end, nucleotide 2 (nucleotide residue 2) being the second nucleotide on the 5’ end, and so forth, until reaching the 3’ end.
  • Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • 'a' represents adenine
  • 'c' represents cytosine
  • 'g' represents guanine
  • 't' represents thymine
  • 'u' represents uracil.
  • Amino acid sequences are written left to right in amino to carboxy orientation. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the term "about” is used herein to mean approximately, roughly, around, or in the regions of. 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.
  • administering refers to introducing a composition, such as a miRNA inhibitor of the present disclosure, into a subject via a pharmaceutically acceptable route.
  • a composition such as a micelle comprising a miRNA inhibitor of the present disclosure
  • introduction of a composition is by any suitable route, including intratumorally, orally, pulmonarily, intranasally, parenterally (intravenously, intra- arterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly or topically.
  • Administration includes self-administration and the administration by another.
  • a suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.
  • abnormal level refers to a level (expression and/or activity) that differs (e.g., increased) from a reference subject who does not suffer from a disease or condition described herein).
  • an abnormal level e.g., inflammasomes
  • an abnormal level refers to a level that is increased by at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, at least about 0.4-fold, at least about 0.5-fold, at least about 0.6-fold, at least about 0.7-fold, at least about 0.8-fold, at least about 0.9-fold, 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 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 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about
  • the term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some aspects, the term “approximately” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
  • two or more sequences are said to be “completely conserved” or “identical” if they are 100% identical to one another.
  • 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 one another. In some aspects, 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 one another. In some aspects, 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 one another.
  • 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.
  • Conservation of sequence can apply to the entire length of a polynucleotide or polypeptide or can apply to a portion, region or feature thereof.
  • the term "derived from,” as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism.
  • a nucleic acid sequence that is derived from a second nucleic acid sequence can include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence.
  • the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis.
  • the mutagenesis used to derive nucleotides or polypeptides can be intentionally directed or intentionally random, or a mixture of each.
  • the mutagenesis of a nucleotide or polypeptide to create a different nucleotide or polypeptide derived from the first can be a random event (e.g., caused by polymerase infidelity) and the identification of the derived nucleotide or polypeptide can be made by appropriate screening methods, e.g., as discussed herein.
  • a nucleotide or amino acid sequence that is derived from a second nucleotide or amino acid sequence has a sequence identity 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 8
  • a "coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.
  • a coding region typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.
  • the terms "complementary” and “complementarity” refer to two or more oligomers (i.e., each comprising a nucleobase sequence), or an oligomer and a target gene, that are related with one another by Watson-Crick base-pairing rules.
  • nucleobase sequence "T- G-A (5' ⁇ 3'),” is complementary to the nucleobase sequence "A-C-T (3' ⁇ 5').”
  • Complementarity can be “partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules.
  • complementarity between a given nucleobase sequence and the other nucleobase sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • the term "complementary” 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% match or complementarity to a target nucleic acid sequence (e.g., miR-485 nucleic acid sequence). Or, there can be “complete” or “perfect” (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example. In some aspects, the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.
  • downstream refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence.
  • downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
  • excipient and “carrier” are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound, e.g., a miRNA inhibitor of the present disclosure.
  • RNA or a polypeptide refers to a process by which a polynucleotide produces a gene product, e.g., RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into a molecule comprising a micro RNA (miRNA) binding site, small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product. It includes, without limitation, transcription of the polynucleotide into messenger RNA (mRNA), and the translation of mRNA into a polypeptide.
  • miRNA micro RNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • a gene product can be, e.g., a nucleic acid, such as an RNA produced by transcription of a gene.
  • a gene product can be either a nucleic acid, RNA or miRNA produced by the transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., phosphorylation, methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • homology refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules. Generally, the term “homology” implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both to identity and similarity.
  • polymeric 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 monomer) or are similar (conservative substitutions).
  • the term "homologous” necessarily refers to a comparison between at least two sequences (e.g., polynucleotide sequences).
  • substitutions are conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.
  • identity refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules.
  • identity without any additional qualifiers, e.g., polynucleotide A is identical to polynucleotide B, implies the polynucleotide sequences are 100% identical (100% sequence identity).
  • the length of a sequence aligned 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. [0161] When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Suitable software programs that can be used to align different sequences are available from various sources.
  • One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov).
  • Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data.
  • a suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g., from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.
  • isolating or purifying as used herein is the process of removing, partially removing (e.g., a fraction) of a composition of the present disclosure, e.g., a miRNA inhibitor of the present disclosure from a sample containing contaminants.
  • an isolated composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount.
  • an isolated composition has an amount and/or concentration of desired composition of the present disclosure, at or above an acceptable amount and/or concentration and/or activity.
  • the isolated composition is enriched as compared to the starting material from which the composition is obtained.
  • This enrichment can be by 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 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material.
  • isolated preparations are substantially free of residual biological products.
  • the isolated preparations are 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological matter.
  • Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.
  • the term "linked" as used herein refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively.
  • the first amino acid or polynucleotide sequence can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence.
  • the term "linked" means not only a fusion of a first polynucleotide sequence to a second polynucleotide sequence at the 5'-end or the 3'-end, but also includes insertion of the whole first polynucleotide sequence (or the second polynucleotide sequence) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence, respectively).
  • the first polynucleotide sequence can be linked to a second polynucleotide sequence by a phosphodiester bond or a linker.
  • the linker can be, e.g., a polynucleotide.
  • a "miRNA inhibitor,” as used herein, refers to a compound that can decrease, alter, and/or modulate miRNA expression, function, and/or activity.
  • the miRNA inhibitor can be a polynucleotide sequence (e.g., antisense oligonucleotide, ASO) that is at least partially complementary to the target miRNA nucleic acid sequence, such that the miRNA inhibitor hybridizes to the target miRNA sequence.
  • ASO antisense oligonucleotide
  • a miR-485-3p inhibitor of the present disclosure comprises a nucleotide sequence encoding a nucleotide molecule that is at least partially complementary to the target miR-485-3p nucleic acid sequence, such that the miR- 485-3p inhibitor hybridizes to the miR-485-3p sequence.
  • the hybridization of the miR-485-3p to the miR-485-3p sequence decreases, alters, and/or modulates the expression, function, and/or activity of miR-485-3p (e.g., hybridization results in a decrease in the expression of one or more genes associated with inflammasomes).
  • miRNA inhibitor Unless indicated otherwise, the terms “miRNA inhibitor,” “miR-485 inhibitor,” and “miR-4853p inhibitor” can be used interchangeably.
  • miRNA miRNA
  • miR miR
  • microRNA a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. The term will be used to refer to the single-stranded RNA molecule processed from a precursor.
  • antisense oligomers can also be used to describe the microRNA molecules of the present disclosure. Names of miRNAs and their sequences related to the present disclosure are provided herein.
  • MicroRNAs recognize and bind to target mRNAs through imperfect base pairing leading to destabilization or translational inhibition of the target mRNA and thereby downregulate target gene expression. Conversely, targeting miRNAs via molecules comprising a miRNA binding site (generally a molecule comprising a sequence complementary to the seed region of the miRNA) can reduce or inhibit the miRNA-induced translational inhibition leading to an upregulation of the target gene.
  • a miRNA binding site generally a molecule comprising a sequence complementary to the seed region of the miRNA
  • mismatch refers to one or more nucleobases (whether contiguous or separate) in an oligomer nucleobase sequence (e.g., miR-485-3p inhibitor) that are not matched to a target nucleic acid sequence (e.g., miR-485-3p) according to base pairing rules. While perfect complementarity is often desired, in some aspects, one or more (e.g., 6, 5, 4, 3, 2, or 1 mismatches) can occur with respect to the target nucleic acid sequence. Variations at any location within the oligomer are included.
  • antisense oligomers of the disclosure include variations in nucleobase sequence near the termini, variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 subunit of the 5' and/or 3' terminus. In some aspects, one, two, or three nucleobases can be removed and still provide on- target binding.
  • the terms “modulate,” “modify,” and grammatical variants thereof, generally refer when applied to a specific concentration, level, expression, function or behavior, to the ability to alter, by increasing or decreasing, e.g., directly or indirectly promoting/stimulating/up-regulating or interfering with/inhibiting/down-regulating the specific concentration, level, expression, function or behavior, such as, e.g., to act as an antagonist or agonist.
  • a modulator can increase and/or decrease a certain concentration, level, activity or function relative to a control, or relative to the average level of activity that would generally be expected or relative to a control level of activity.
  • a miRNA inhibitor disclosed herein e.g., a miR-485-3p inhibitor
  • Nucleic acid refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • RNA molecules phosphate ester polymeric form of ribonucleosides
  • deoxyribonucleosides deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine
  • DNA molecules or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded
  • Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes.
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a 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 disclosure comprises one or more nucleic acids as described herein.
  • pharmaceutically acceptable carrier encompass any of the agents approved by a regulatory agency of the U.S. Federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause the production of undesirable physiological effects to a degree that prohibits administration of the composition to a subject and does not abrogate the biological activity and properties of the administered compound. Included are excipients and carriers that are useful in preparing a pharmaceutical composition and are generally safe, non-toxic, and desirable.
  • the term "pharmaceutical composition” refers to one or more of the compounds described herein, such as, e.g., a miRNA inhibitor of the present disclosure, mixed or intermingled with, or suspended in one or more other chemical components, such as pharmaceutically acceptable carriers and excipients.
  • a pharmaceutical composition is to facilitate administration of preparations comprising a miRNA inhibitor of the present disclosure to a subject.
  • polynucleotide refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof.
  • the term refers to the primary structure of the molecule.
  • the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, shRNA, siRNA, miRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids "PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • a polynucleotide can be, e.g., an oligonucleotide, such as an antisense oligonucleotide.
  • the oligonucleotide is an RNA.
  • the RNA is a synthetic RNA.
  • the synthetic RNA comprises at least one unnatural nucleobase.
  • all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine).
  • polypeptide polypeptide
  • peptide protein
  • protein polymers of amino acids of any length.
  • the polymer can comprise modified amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine
  • polypeptide refers to proteins, polypeptides, and peptides of any size, structure, or function.
  • Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides.
  • the term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • a "peptide" can be less than or equal to about 50 amino acids long, 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 long.
  • prevent refers partially or completely delaying onset of an disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some aspects, preventing an outcome is achieved through prophylactic treatment.
  • promoter and “promoter sequence” are interchangeable and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the 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.
  • Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue- specific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
  • the promoter sequence is typically bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a promoter that can be used with the present disclosure includes a tissue specific promoter.
  • prolactic refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.
  • a “prophylaxis” refers to a measure taken to maintain health and prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.
  • the term “gene regulatory region” or “regulatory region” refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non- coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, 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.
  • a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • a miR-485-3p inhibitor disclosed herein e.g., a polynucleotide encoding a RNA comprising one or more miR-485-3p binding site
  • a coding region for a gene product is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s).
  • a coding region and a promoter are "operably associated” if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • Other expression control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.
  • similarity refers to the overall relatedness between polymeric molecules, e.g.
  • polynucleotide molecules e.g. miRNA molecules
  • Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art. It is understood that percentage of similarity is contingent on the comparison scale used, i.e., whether the nucleic acids are compared, e.g., according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or combinations thereof.
  • subject refers to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • domestic animals e.g., dogs, cats and the like
  • farm animals e.g., cows, sheep, pigs, horses and the like
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like
  • the phrase "subject in need thereof” includes subjects, such as mammalian subjects, that would benefit from administration of a miRNA inhibitor of the disclosure (e.g., miR-485 inhibitor), e.g., to decrease abnormal inflammasome activity.
  • a miRNA inhibitor of the disclosure e.g., miR-485 inhibitor
  • the term "therapeutically effective amount” is the amount of reagent or pharmaceutical compound comprising a miRNA inhibitor of the present disclosure that is sufficient to a produce a desired therapeutic effect, pharmacologic and/or physiologic effect on a subject in need thereof.
  • a therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy.
  • treat refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
  • the term also includes prophylaxis or prevention of a disease or condition or its symptoms thereof.
  • upstream refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence.
  • a "vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell.
  • a vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the 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 replication under its own control.
  • the term “vector” includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector.
  • selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.
  • reporter known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase (LacZ), ⁇ -glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters. II.
  • miRNA-485 Inhibitors Useful for the Present Disclosure [0200] Disclosed herein is an improved antisense oligonucleotide that can inhibit miR-485 activity (miR-485 inhibitor).
  • a miR-485 inhibitor of the present disclosure comprises a nucleotide sequence encoding a nucleotide molecule that comprises at least one miR- 485 binding site, wherein the nucleotide molecule does not encode a protein and wherein the miR- 485 inhibitor comprises at least two different nucleoside analogs.
  • the miR-485 binding site is at least partially complementary to the target miRNA nucleic acid sequence (i.e., miR-485), such that the miR-485 inhibitor hybridizes to the miR-485 nucleic acid sequence.
  • the present disclosure comprises an antisense oligonucleotide (ASO) of 10 to 30 nucleotides in length comprising a contiguous nucleotide sequence that is complementary to a nucleic acid sequence within miRNA 485-3p (5'- GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1)), wherein the contiguous nucleotide sequence comprises at least two different nucleoside analogs.
  • ASO antisense oligonucleotide
  • the at least two different nucleoside analogs are sugar modified nucleosides.
  • the present disclosure comprises an antisense oligonucleotide (ASO) of 10 to 30 nucleotides in length comprising a contiguous nucleotide sequence that is complementary to a nucleic acid sequence within miRNA 485-3p (5'- GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1)), wherein the contiguous nucleotide sequence comprises at least two different nucleoside analogs selected from Locked Nucleic Acid (LNA); 2'- O-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2'-O-methyl nucleic acid (2'--
  • ASO antisense
  • the nucoleoside analogs are bicyclic sugars.
  • the bicyclic sugars in the miR-485-3p targeting ASO comprise cEt, 2',4'-constrained 2'-O- methoxyethyl (cMOE), LNA, ⁇ -L-LNA, ⁇ -D-LNA, 2'-O,4'-C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, or thio-LNA.
  • the present disclosure is directed to an antisense oligonucleotide (ASO) of 10 to 30 nucleotides in length comprising a contiguous nucleotide sequence that is complementary to a nucleic acid sequence within miRNA 485-3p (5'- GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1)), wherein the contiguous nucleotide sequence comprises at least two different nucleoside analogs that comprise LNA and 2’-MOE.
  • ASO antisense oligonucleotide
  • the miR-485 binding site of a 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 complementarity to the nucleic acid sequence of a miR-485.
  • the miR- 485 binding site is fully complementary to the nucleic acid sequence of a miR-485.
  • the human mature miR-485-3p has the sequence 5'- GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1; miRBase Acc. No. MIMAT0002176). A 5' terminal subsequence of miR-485-3p 5'-UCAUACA-3' is the seed sequence.
  • the human mature miR-485-3p has significant sequence similarity to that of other species. For instance, the mouse mature miR-485-3p differs from the human mature miR-485-3p by a single amino acid at each of the 5'- and 3'- ends (i.e., has an extra "A" at the 5'-end and missing "C" at the 3'-end).
  • the mouse mature miR-485-3p has the following sequence: 5'-AGUCAUACACGGCUCUCCUCUC-3' (SEQ ID NO: 50; miRBase Acc. No. MIMAT0003129; underlined portion corresponds to overlap to human mature miR-485-3p).
  • the miR-485 binding site is a single-stranded polynucleotide sequence that is complementary (e.g., fully complementary) to a sequence of a miR-485-3p (or a subsequence thereof).
  • the miR-485-3p subsequence comprises the seed sequence.
  • 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 5'-UCAUACA-3'.
  • 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.
  • the miR-485 binding site is fully complementary to the nucleic acid sequence set forth in SEQ ID NO: 1.
  • the seed region of a miRNA forms a tight duplex with the target mRNA.
  • Most miRNAs imperfectly base-pair with the 3' untranslated region (UTR) of target mRNAs, and the 5' proximal "seed" region of miRNAs provides most of the pairing specificity.
  • UTR 3' untranslated region
  • the 5' proximal "seed" region of miRNAs provides most of the pairing specificity.
  • the first nine miRNA nucleotides (encompassing the seed sequence) provide greater specificity whereas the miRNA ribonucleotides 3' of this region allow for lower sequence specificity and thus tolerate a higher degree of mismatched base pairing, with positions 2-7 being the most important.
  • the miR-485 binding site comprises a subsequence that is fully complementary (i.e., 100% complementary) over the entire length of the seed sequence of miR-485.
  • miRNA sequences and miRNA binding sequences that can be used in the context of the disclosure include, but are not limited to, all or a portion of those sequences in the sequence listing provided herein, as well as the miRNA precursor sequence, or complement of one or more of these miRNAs.
  • miRNAs or complementary sequences thereof whose sequences are 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 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 to the mature sequence of the specified miRNA sequence or complementary sequence
  • miRNA binding sequences of the present disclosure can include additional nucleotides at the 5′, 3′, or both 5′ and 3′ ends of the sequences in the sequence listing provided herein, as long as the modified sequence is still capable of specifically binding to miR- 485.
  • miRNA binding sequences of the present disclosure can differ in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides with respect to the sequences in the sequence listing provided, as long as the modified sequence is still capable of specifically binding to miR-485.
  • any methods and compositions discussed herein with respect to miRNA binding molecules or miRNA can be implemented with respect to synthetic miRNA binding molecules.
  • a miRNA-485 inhibitor of the present disclosure 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 5' of the nucleotide sequence.
  • a miRNA-485 inhibitor of the present disclosure 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' of the nucleotide sequence.
  • a miR-485 inhibitor disclosed herein is about 6 to about 30 nucleotides in length. In some aspects, a miR-485 inhibitor disclosed herein is 7 nucleotides in length. In further aspects, a miR-485 inhibitor disclosed herein is 8 nucleotides in length. In some aspects, a miR-485 inhibitor is 9 nucleotides in length. In some aspects, a miR-485 inhibitor of the present disclosure is 10 nucleotides in length. In some aspects, a miR-485 inhibitor is 11 nucleotides in length. In further aspects, a miR-485 inhibitor is 12 nucleotides in length. In some aspects, a miR-485 inhibitor disclosed herein is 13 nucleotides in length.
  • a miR- 485 inhibitor disclosed herein is 14 nucleotides in length. In some aspects, a miR-485 inhibitor disclosed herein is 15 nucleotides in length. In further aspects, a miR-485 inhibitor is 16 nucleotides in length. In some aspects, a miR-485 inhibitor of the present disclosure is 17 nucleotides in length. In some aspects, a miR-485 inhibitor is 18 nucleotides in length. In some aspects, a miR-485 inhibitor is 19 nucleotides in length. In some aspects, a miR-485 inhibitor is 20 nucleotides in length. In further aspects, a miR-485 inhibitor of the present disclosure is 21 nucleotides in length.
  • a miR-485 inhibitor is 22 nucleotides in length.
  • a miR-485 inhibitor disclosed herein comprises 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 the group consisting of 5'-UGUAUGA-3', 5'- GUGUAUGA-3', 5'-CGUGUAUGA-3', 5'-UGUAUGAC-3', 5'-GUGUAUGAC-3', and SEQ ID NOs: 2 to 25.
  • a miR-485 inhibitor comprises a nucleotide sequence selected from the group consisting of 5'-UGUAUGA-3', 5'-GUGUAUGA-3', 5'-CGUGUAUGA-3', 5'-UGUAUGAC-3', 5'- GUGUAUGAC-3', and SEQ ID NOs: 2 to 25, wherein the nucleotide sequence can optionally comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.
  • the miRNA inhibitor of the present disclosure has a plasma stability that exhibits at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% recovery after one day, as measured by an HPLC.
  • the miRNA inhibitor of the present disclosure has a plasma stability that exhibits at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% recovery after about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, or about ten days, as measured by an HPLC.
  • the miRNA inhibitor of the present disclosure has a plasma stability that exhibits at least about 90%, at least about 95%, or about 100% recovery after about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 days, as measured by an HPLC.
  • a miRNA inhibitor comprises 5'-UGUAUGA-3', 5'-GUGUAUGA-3', 5'-CGUGUAUGA-3', 5'-CCGUGUAUGA-3' (SEQ ID NO: 2), 5'-GCCGUGUAUGA-3' (SEQ ID NO: 3), 5'-AGCCGUGUAUGA-3' (SEQ ID NO: 4), 5'-GAGCCGUGUAUGA-3' (SEQ ID NO: 5), 5'- AGAGCCGUGUAUGA-3' (SEQ ID NO: 6), 5'-GAGAGCCGUGUAUGA-3' (SEQ ID NO: 7), 5'- GGAGAGCCGUGUAUGA-3' (SEQ ID NO: 8), 5'-AGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 9), 5'- GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-AGAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 11), or 5'-
  • the miRNA inhibitor has 5'-UGUAUGAC-3', 5'-GUGUAUGAC-3', 5'- CGUGUAUGAC-3' (SEQ ID NO: 13), 5'-CCGUGUAUGAC-3' (SEQ ID NO: 14), 5'- GCCGUGUAUGAC-3' (SEQ ID NO: 15), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 16), 5'- GAGCCGUGUAUGAC-3' (SEQ ID NO: 17), 5'-AGAGCCGUGUAUGAC-3' (SEQ ID NO: 18), 5'- GAGAGCCGUGUAUGAC-3' (SEQ ID NO: 19), 5'-GGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 20), 5'- AGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 21), 5'-GAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 22), 5'-AGAGGAGAGCCGUGUAUGAC-3
  • the miRNA inhibitor has a sequence selected from the group consisting of: 5'-TGTATGA-3', 5'-GTGTATGA-3', 5'-CGTGTATGA-3', 5'-CCGTGTATGA-3' (SEQ ID NO: 26), 5'-GCCGTGTATGA-3' (SEQ ID NO: 27), 5'-AGCCGTGTATGA-3' (SEQ ID NO: 28), 5'- GAGCCGTGTATGA-3' (SEQ ID NO: 29), 5'-AGAGCCGTGTATGA-3' (SEQ ID NO: 30), 5'- GAGAGCCGTGTATGA-3' (SEQ ID NO: 31), 5'-GGAGAGCCGTGTATGA-3' (SEQ ID NO: 32), 5'- AGGAGAGCCGTGTATGA-3' (SEQ ID NO: 33), 5'-GAGGAGAGCCGTGTATGA-3' (SEQ ID NO: 34), 5'-AGAGGAGAGCCGTGTATGA-3' (SEQ ID NO:
  • a miRNA inhibitor disclosed herein comprises 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'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49), wherein the miRNA inhibitor comprises at least two different nucleoside analogs, e.g., LNA and 2’ MOE.
  • the miRNA inhibitor comprises a nucleotide sequence that has at least 90% similarity to 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'-AGAGAGGAGAGCCGTGTATGAC- 3' (SEQ ID NO: 49), wherein the miRNA inhibitor comprises at least two different nucleoside analogs, e.g., LNA and 2’ MOE.
  • the miRNA inhibitor comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49) with one substitution or two substitutions, wherein the miRNA inhibitor comprises at least two different nucleoside analogs, e.g., LNA and 2’ MOE.
  • the miRNA inhibitor comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 49), wherein the miRNA inhibitor comprises at least two different nucleoside analogs, e.g., LNA and 2’ MOE.
  • the miRNA inhibitor comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), wherein the miRNA inhibitor comprises at least two different nucleoside analogs, e.g., LNA and 2’ MOE.
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), and wherein one or more of the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs (SEQ ID NO: 56).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), and wherein one or more of the double underlined letters are 2’-MOEs (SEQ ID NO: 51).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs (SEQ ID NO: 52).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), wherein one or more of the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs, and wherein one or more of the double underlined letters are 2’-MOEs (SEQ ID NO: 57).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs (SEQ ID NO: 53).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs and wherein the backbone structure is fully or partially phosphorothioate modified.
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs and wherein the backbone structure is fully phosphorothioate modified (SEQ ID NO: 54) (miR-485-3p ASO-C, ASO-C).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs and wherein the backbone structure is fully or partially phosphorothioate modified.
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs and the double underlined letters are 2’-MOEs and wherein the backbone structure is fully phosphorothioate modified (SEQ ID NO: 55) (miR-485-3p ASO-B, ASO-B).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGUGUAUGAC- 3' (SEQ ID NO: 25), and wherein the bolded letters (AGA at the 5’ terminus (residue nos.1, 2, and 3), residue nos. 12, 13, 15, 17, and 19, and C at the 3’ terminus (residue no.
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGTGTATGAC- 3' (SEQ ID NO: 49), and wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are LNAs, wherein the non-bolded letters are DNAs, and wherein the backbone structure in the ASO is fully phosphorothioate modified (SEQ ID NO: 59) (miR-485-3p ASO-E, ASO-E).
  • the miRNA inhibitor of the present disclosure is an ASO comprising the contiguous nucleotide sequence as set forth in 5'-AGAGAGGAGAGCCGTGTATGAC- 3' (SEQ ID NO: 49), and wherein the bolded letters (AGA at the 5’ terminus and C at the 3’ terminus) are 2’-MOEs, wherein the non-bolded letters are DNAs, and wherein the backbone structure in the ASO is fully phosphorothioate modified (SEQ ID NO: 60) (miR-485-3p ASO-F, ASO-F).
  • the miRNA inhibitor of the present disclosure comprises one or more 5' methyl cytosine nucleobases.
  • a miR-485 inhibitor of the present disclosure comprises one miR- 485 binding site.
  • a miR-485 inhibitor disclosed herein comprises at least two miR-485 binding sites.
  • a miR-485 inhibitor comprises three miR-485 binding sites.
  • a miR-485 inhibitor comprises four miR-485 binding sites.
  • a miR-485 inhibitor comprises five miR-485 binding sites.
  • a miR-485 inhibitor comprises six or more miR-485 binding sites.
  • all the miR-485 binding sites are identical.
  • all the miR-485 binding sites are different.
  • at least one of the miR-485 binding sites is different.
  • a miR-485 inhibitor comprises at least one miR-485-3p binding site and at least one miR-485-5p binding site.
  • II.a. Chemically Modified Polynucleotides [0233]
  • a miR-485 inhibitor disclosed herein comprises a polynucleotide which includes at least two different chemically modified nucleoside and/or nucleotide, e.g., LNA, 2’ MOE, and/ or phosphorothioate modifications.
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • a “nucleotide” refers to a nucleoside including a phosphate group. Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • the modified polynucleotides disclosed herein can comprise various distinct modifications. In some aspects, the modified polynucleotides contain two or more (optionally different) nucleoside or nucleotide modifications.
  • a modified polynucleotide can exhibit one or more desirable properties, e.g., improved thermal or chemical stability, reduced immunogenicity, reduced degradation, increased binding to the target microRNA, reduced non-specific binding to other microRNA or other molecules, as compared to an unmodified polynucleotide.
  • a polynucleotide of the present disclosure e.g., a miR-485 inhibitor
  • the terms "chemical modification” or, as appropriate, “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in one or more of their position, pattern, percent or population, including, but not limited to, its nucleobase, sugar, backbone, or any combination thereof.
  • a polynucleotide of the present disclosure can have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation
  • the polynucleotide of the present disclosure e.g., a miR-485 inhibitor
  • Modified nucleotide base pairing encompasses not only the standard adenine- thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the modified nucleobase inosine and adenine, cytosine or uracil.
  • any combination of base/sugar or linker can be incorporated into polynucleotides of the present disclosure.
  • polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA, the "T”s would be substituted for "U”s.
  • polynucleotides of the present disclosure can be administered as RNAs, as DNAs, or as hybrid molecules comprising both RNA and DNA units.
  • the polynucleotide (e.g., a miR-485 inhibitor) includes a combination of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20 or more) modified nucleobases.
  • the nucleobases, sugar, backbone linkages, or any combination thereof in a polynucleotide are modified by 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%.
  • the chemical modification is at nucleobases in a polynucleotide of the present disclosure (e.g., a miR-485 inhibitor).
  • the at least one chemically modified nucleoside is a modified uridine (e.g., pseudouridine ( ⁇ ), 2-thiouridine (s2U), 1-methyl- pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), 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-methyl-gua
  • a modified uridine e.g., pseudo
  • the polynucleotide of the present disclosure is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with the same type of base modification, e.g., 5-methyl-cytidine (m5C), meaning that all cytosine residues in the polynucleotide sequence are replaced with 5-methyl-cytidine (m5C).
  • m5C 5-methyl-cytidine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified nucleoside such as any of those set forth above.
  • the polynucleotide of the present disclosure includes a combination of at least two (e.g., 2, 3, 4 or more) modified nucleobases.
  • the polynucleotide of the present disclosure can include any useful linkage between the nucleosides.
  • linkages, including backbone modifications, that are useful in the composition of the present disclosure include, but are not limited to the following: 3'-alkylene phosphonates, 3'-amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, -CH 2 -O-N(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 -, -CH 2 -NH-CH 2 -, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene (methylimino), methylene formacetyl and thioformace
  • the presence of a backbone linkage disclosed above increase the stability and resistance to degradation of a polynucleotide of the present disclosure (i.e., miR-485 inhibitor).
  • 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 of the present disclosure (i.e., miR-485 inhibitor) are modified (e.g., all of them are phosphorothioate).
  • a backbone modification that can be included in a polynucleotide of the present disclosure comprises phosphorodiamidate morpholino oligomer (PMO) and/or phosphorothioate (PS) modification.
  • PMO phosphorodiamidate morpholino oligomer
  • PS phosphorothioate
  • sugar modifications [0250] The modified nucleosides and nucleotides which can be incorporated into a polynucleotide of the present disclosure (i.e., miR-485 inhibitor) can be modified on the sugar of the nucleic acid. In some aspects, the sugar modification increases the affinity of the binding of a miR-485 inhibitor to miR-485 nucleic acid sequence.
  • Incorporating affinity-enhancing nucleotide analogues in the miR-485 inhibitor, such as LNA or 2'-substituted sugars, can allow the length and/or the size of the miR-485 inhibitor to be reduced.
  • RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
  • modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.
  • GAA glyco
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.
  • the 2′ hydroxyl group (OH) of ribose can be modified or replaced with a number of different substituents.
  • substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 6-10 aryl-C 1-6 alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20,
  • nucleotide analogues present in a polynucleotide of the present disclosure comprise, e.g., 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-O- alkyl-SNA, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabino nucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units, 2'MOE units, or any combination thereof.
  • ANA arabino nucleic acid
  • INA intercalating nucleic acid
  • the LNA is, e.g., oxy-LNA (such as beta-D-oxy-LNA, or alpha-L-oxy-LNA), amino-LNA (such as beta-D-amino-LNA or alpha-L-amino-LNA), thio-LNA (such as beta-D-thio0-LNA or alpha-L-thio-LNA), ENA (such a beta-D-ENA or alpha-L-ENA), or any combination thereof.
  • oxy-LNA such as beta-D-oxy-LNA, or alpha-L-oxy-LNA
  • amino-LNA such as beta-D-amino-LNA or alpha-L-amino-LNA
  • thio-LNA such as beta-D-thio0-LNA or alpha-L-thio-LNA
  • ENA such a beta-D-ENA or alpha-L-ENA
  • nucleotide analogues that can be included in a polynucleotide of the present disclosure comprises 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).
  • LNA locked nucleic acid
  • UNA unlocked nucleic acid
  • ABA arabino nucleic acid
  • BNA bridged nucleic acid
  • PNA peptide nucleic acid
  • a polynucleotide of the present disclosure i.e., miR-485 inhibitor
  • a miR-485 inhibitor is a gapmer.
  • a miR-485 inhibitor is a micromir. See U.S. Pat. Appl. Publ. No. US20180201928, which is herein incorporated by reference in its entirety.
  • a polynucleotide of the present disclosure i.e., miR-485 inhibitor
  • Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.)
  • base modifications e.g., replacement with modified bases, stabilizing bases, de
  • the miR-485 inhibitors of the present disclosure can be administered, e.g., to a subject suffering from a disease or condition associated with abnormal (e.g., increased) inflammasome activity, using any relevant delivery system known in the art.
  • the delivery system is a vector.
  • the present disclosure provides a vector comprising a miR-485 inhibitor of the present disclosure.
  • the vector is viral vector.
  • the viral vector is an adenoviral vector or an adenoassociated viral vector.
  • the viral vector is an AAV that has a serotype of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any combination thereof.
  • the adenoviral vector is a third generation adenoviral vector.
  • ADEASYTM is by far the most popular method for creating adenoviral vector constructs. The system consists of two types of plasmids: shuttle (or transfer) vectors and adenoviral vectors. 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 PADEASYTM.
  • PADEASYTM is a ⁇ 33Kb adenoviral plasmid containing the adenoviral genes necessary for virus production.
  • the shuttle vector and the adenoviral plasmid have matching left and right homology arms which facilitate homologous recombination of the transgene into the adenoviral plasmid.
  • Recombinant adenoviral plasmids are then verified for size and proper restriction digest patterns to determine that the transgene has been inserted into the adenoviral plasmid, and that other patterns of recombination have not occurred. Once verified, the recombinant plasmid is linearized with PacI to create a linear dsDNA construct flanked by ITRs. 293 or 911 cells are transfected with the linearized construct, and virus can be harvested about 7-10 days later.
  • PacI PacI
  • 293 or 911 cells are transfected with the linearized construct, and virus can be harvested about 7-10 days later.
  • other methods for creating adenoviral vector constructs known in the art at the time the present application was filed can be used to practice the methods disclosed herein.
  • the viral vector is a retroviral vector, e.g., a lentiviral vector (e.g., a third or fourth generation lentiviral vector).
  • Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems. These include the transfer vector plasmid (portions of the HIV provirus), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (env) of a different virus.
  • the three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell.
  • the virus portions of the vector contain insert sequences so that the virus cannot replicate inside the cell system.
  • AAV vector Any AAV vector known in the art can be used in the methods disclosed herein.
  • the AAV vector can comprise a known vector or can comprise a variant, fragment, or fusion thereof.
  • 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, goat AVV, primate AAV, non-primate AAV, bovine AAV, shrimp AVV, snake AVV, and any combination thereof.
  • AAV1 AAV type 1
  • AAV2 AAV2
  • AAV3A AAV3A
  • AVV3B AAV4
  • AAV5 AAV6, AAV7, AAV8, AVV9, AVV10, AVV11, AVV12, AVV13, AAVrh.74
  • avian AAV bovine AAV
  • canine AAV equine AAV, goat AVV
  • 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, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof.
  • 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, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof.
  • the AAV vector comprises regions of at least two different AAV vectors known in the art.
  • the AAV vector comprises an inverted terminal repeat 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, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, or any derivative thereof) and a second inverted terminal repeat 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 A
  • the AVV 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, goat AVV, primate AAV, non-primate AAV, ovine AAV, shrimp AVV, snake AVV, and any combination thereof.
  • the AAV vector comprises AAV2.
  • the AVV vector comprises a splice acceptor site.
  • the AVV vector comprises a promoter. Any promoter known in the art can be used in the AAV vector of the present disclosure.
  • the promoter is an RNA Pol III promoter.
  • the RNA Pol III promoter is selected from the group consisting of the U6 promoter, the H1 promoter, the 7SK promoter, the 5S promoter, the adenovirus 2 (Ad2) VAI promoter, and any combination thereof.
  • the promoter is a cytomegalovirus immediate-early gene (CMV) promoter, an EF1a promoter, an SV40 promoter, a PGK1 promoter, a Ubc promoter, a human beta actin promoter, a CAG promoter, a TRE promoter, a UAS promoter, a Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a GAL1 promoter, a GAL10 promoter, a TEF promoter, a GDS promoter, a ADH1 promoter, a CaMV35S promoter, or a Ubi promoter.
  • the promoter comprises the U6 promoter.
  • the AAV vector comprises a constitutively active promoter (constitutive promoter).
  • the constitutive promoter is selected from the group consisting of hypoxanthine phosphoribosyl transferase (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, a retrovirus long terminal repeat (LTR), Murine stem cell virus (MSCV) and the thymidine kinase promoter of herpes simplex virus.
  • HPRT hypoxanthine phosphoribosyl transferase
  • CMV cytomegalovirus
  • simian virus e.g., SV40
  • papilloma virus adenovirus
  • the promoter is an inducible promoter.
  • the inducible promoter is a tissue specific promoter.
  • the tissue specific promoter drives transcription of the coding region of the AVV vector in a neuron, a glial cell, or in both a neuron and a glial cell.
  • the AVV vector comprises one or more enhancers.
  • the one or more enhancer are present in the AAV alone or together with a promoter disclosed herein.
  • the AAV vector comprises a 3'UTR poly(A) tail sequence.
  • the 3'UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In some aspects, the 3'UTR poly(A) tail sequence comprises bGH poly(A). [0271] In some aspects, a miR-485 inhibitor disclosed herein is administered with a delivery agent.
  • Non-limiting examples of delivery agents that can be used include 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 micelle, a viral vector, or a conjugate.
  • the present disclosure also provides a composition comprising a miRNA inhibitor of the present disclosure (i.e., miR-485 inhibitor) and a delivery agent.
  • the delivery agent comprises a carrier unit, e.g., that can self-assemble into micelles or be incorporated into micelles.
  • the delivery agent comprises a cationic carrier unit comprising [WP]-L1-[CC]-L2-[AM] (formula I) or [WP]-L1-[AM]-L2-[CC] (formula II) wherein WP is a water-soluble biopolymer moiety; CC is a positively charged (i.e., cationic) carrier moiety; AM is an adjuvant moiety; and, L1 and L2 are independently optional linkers, and wherein when mixed with a nucleic acid at an ionic ratio of about 1:1, the cationic carrier unit forms a micelle.
  • the miRNA inhibitor and the cationic carrier unit are capable of associating with each other (e.g., via a covalent bond or a non-valent bond) to form a micelle when mixed together.
  • a composition comprising a miRNA inhibitor of the present disclosure i.e., miR-485 inhibitor
  • the water-soluble biopolymer moiety comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ - hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines (“POZ”) poly(N-acryloylmorpholine), or any combinations thereof.
  • the water-soluble polymer comprises polyethylene glycol (“PEG”), polyglycerol, or poly(propylene glycol) (“PPG").
  • the water-soluble biopolymer moiety comprises: , (formula III), wherein n is 1-1000.
  • the 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.
  • the 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.
  • the water-soluble biopolymer moiety is linear, branched, or dendritic.
  • the cationic carrier moiety comprises one or more basic amino acids.
  • the cationic carrier moiety comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at last 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.
  • the cationic carrier moiety comprises about 30 to about 50 basic amino acids.
  • the basic amino acid comprises arginine, lysine, histidine, or any combination thereof.
  • the cationic carrier moiety comprises about 40 lysine monomers.
  • the adjuvant moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue microenvironment.
  • the adjuvant moiety comprises an imidazole derivative, an amino acid, a vitamin, or any combination thereof.
  • the adjuvant moiety comprises: , (formula IV), wherein each of G 1 and G 2 is H, an aromatic ring, or 1-10 alkyl, or G 1 and G 2 together form an aromatic ring, and wherein n is 1-10.
  • the adjuvant moiety comprises nitroimidazole.
  • the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazol, azanidazole, benznidazole, or any combination thereof.
  • the adjuvant moiety comprises an amino acid.
  • the adjuvant moiety comprises (formula V), wherein Ar is wherein each of Z1 and Z2 is H or OH.
  • the adjuvant moiety comprises a vitamin.
  • the vitamin comprises a cyclic ring or cyclic hetero atom ring and a carboxyl group or hydroxyl group.
  • the vitamin comprises: (formula VI), wherein each of Y 1 and Y 2 is C, N, O, or S, and wherein n is 1 or 2.
  • 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.
  • the vitamin is vitamin B3.
  • the adjuvant moiety comprises at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, 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.
  • the adjuvant moiety comprises about 10 vitamin B3.
  • the composition comprises a water-soluble biopolymer moiety with about 120 to about 130 PEG units, a cationic carrier moiety comprising a poly-lysine with about 30 to about 40 lysines, and an adjuvant moiety with about 5 to about 10 vitamin B3.
  • the composition comprises (i) a water-soluble biopolymer moiety with about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines with 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 with a thiol group), and (iv) about 30 to 40 lysines fused to vitamin B3 (e.g., about 32 lysines, each fused to vitamin B3).
  • an amine group e.g., about 32 lysines
  • a thiol group e.g., about 16 lysines, each with a thiol group
  • vitamin B3 e.g., about 32 lysines, each fused to vitamin B3
  • the composition further comprises a targeting moiety, e.g., a LAT1 targeting ligand, e.g., phenyl alanine, linked to the water soluble polymer.
  • a targeting moiety e.g., a LAT1 targeting ligand, e.g., phenyl alanine
  • the thiol groups in the composition form disulfide bonds.
  • the composition comprises (1) a micelle comprising (i) about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines with 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 with a thiol group), and (iv) about 30 to 40 lysines fused to vitamin B3 (e.g., about 32 lysines, each fused to vitamin B3), and (2) a miR485 inhibitor (e.g., SEQ ID NO: 25), wherein the miR485 inhibitor is encapsulated within the micelle.
  • a micelle comprising (i) about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines with an amine group (e.g., about 32 lysines), (iii) about 15 to 20 lysines, each having a thiol group (
  • the composition further comprises a targeting moiety, e.g., a LAT1 targeting ligand, e.g., phenyl alanine, linked to the PEG units.
  • a targeting moiety e.g., a LAT1 targeting ligand, e.g., phenyl alanine
  • the thiol groups in the micelle form disulfide bonds.
  • the present disclosure also provides a micelle comprising a miRNA inhibitor of the present disclosure (i.e., miR-485 inhibitor, e.g., SEQ ID NO: 25) wherein the miRNA inhibitor and the delivery agent are associated with each other.
  • the association is a covalent bond, a non-covalent bond, or an ionic bond.
  • 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 a solution, wherein the overall ionic ratio of the positive charges of the cationic carrier moiety of the cationic carrier unit and the negative charges of the miR-485 inhibitor (or vector comprising the inhibitor) in the solution is about 1: 1.
  • the cationic carrier unit is capable of protecting the miRNA inhibitor of the present disclosure (i.e., miR-485 inhibitor) from enzymatic degradation. See PCT Publication No. WO2020/261227, which is herein incorporated by reference in its entirety. IV.
  • compositions comprising a miR-485 inhibitor disclosed herein (e.g., a polynucleotide or a vector comprising the miR-485 inhibitor) that are suitable for administration to a subject.
  • the pharmaceutical compositions generally comprise a miR-485 inhibitor described herein (e.g., a polynucleotide or a vector) and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject.
  • Pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
  • compositions comprising a miR-485 inhibitor of the present disclosure.
  • the pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • Methods of Treatment Provided herein are the methods of inducing hair growth 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 ("miRNA inhibitor”) disclosed herein.
  • the process by which hair grows encompasses several phases and there are many contributory factors that can alter the normal vigorous growth of hair.
  • the hair growth cycle is divided into three phases: an anagen phase, in which the hair is growing actively, with a very substantial level of cell proliferation occurring in the hair follicle; a catagen phase, when the follicle slows down its proliferative activity temporarily to permit hair development; and a telogen phase, in which the follicle simply stops growing and regresses, until the hair is shed, and a new anagen phase begins. It is of course completely normal for the average person to lose many hairs on a daily basis, and therefore, this cycle is normally repeated continually throughout life, to replenish the hair that is lost.
  • the cycle does slow down with age in all individuals however, with the normal hairs gradually being replaced by progressively finer hair (vellus hair), and the cycles becoming shorter.
  • Early-to-mid stage hair loss is almost entirely manifested as thinning of the hair shafts of a large percentage of follicles and retarded shaft growth such that the hairs that are present are thin, light, and close to the scalp, however follicle density and shafts per follicle are basically unchanged.
  • follicle density and shafts per follicle are basically unchanged.
  • the normal cyclical process becomes disrupted in some fashion, whether it be through an abnormal acceleration or some other alteration of the process.
  • Hair thickness or density (D) can be quantified as the product of three factors: (1) follicular density (F) measured in follicles per square centimeter, (2) average number of hair shafts per follicle (N,) and (3) average thickness (T) of the hair shafts. The product of these three quantities indicates hair density (D) as measured by the equation below.
  • Typical follicular unit densities are in the range of 60-120 cm ⁇ 2 and each follicle generally contains one or two shafts, but rarely, more than two hair shafts of varying ages.
  • the hair shaft thickness can be classified as coarse, medium, or fine, and the mean value of the shaft thickness will vary from about 40 microns in width for fine hair, while coarse hair might average 90 microns in width.
  • N will generally be a number between 1 and 2, and more commonly between 1 and 1.25. When N is close to a value of 1 it can be eliminated from the density determination but may need to be considered in some rare cases.
  • a normalized hair density measurement for every individual is the ratio of top and/or front hair density to left and/or right side hair density.
  • the subject has one or more disorders selected from the group consisting of alopecia greata, androgenic alopecia, alopecia areata, alopecia universalis, involutional alopecia, trichotillomania, telogen effluvium, anagen effluvium, cicatricial, alopecia, scarring alopecia, scalp thinning, hair shaft abnormalities, infectious hair disorders, genetic disorders, and hair loss due to chemotherapy, hormonal imbalance, fungal infection, medication intake, chemical hair treatment, or aging.
  • the subject is a human.
  • a miR-485 inhibitor disclosed herein can be administered by any suitable route known in the art.
  • a miR-485 inhibitor is administered parenthetically, intramuscularly, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracerebroventricularly, intraspinally, intraventricular, intrathecally, intracistemally, intracapsularly, intratumorally, topically, or any combination thereof.
  • a miR-485 inhibitor is administered intramuscularly. [0300]
  • a miR-485 inhibitor is administered to a skin area where promoting hair growth is needed by spread, spray, steam, or injection.
  • a miR-485 inhibitor is administered topically to a skin area where promoting hair growth is needed.
  • a miR-485 inhibitor is 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.
  • the administering step is performed by soaking or bathing the subject in the miRNA inhibitor 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.
  • a miR-485 inhibitor of th present disclosure can be used in combination with one or more additional therapeutic agents.
  • the additional therapeutic agent and the miR-485 inhibitor are administered concurrently.
  • the additional therapeutic agent and the miR-485 inhibitor are administered sequentially.
  • the administration of a miR-485 inhibitor disclosed herein does not result in any adverse effects.
  • the miR-485 inhibitors of the present disclosure do not adversely affect body weight when administered to a subject.
  • the miR-485 inhibitors disclosed herein do not result in increased mortality or cause pathological abnormalities when administered to a subject.
  • the miR-485 inhibitors of the present disclosure can exert therapeutic effects (e.g., inducing hair growth or preventing hair loss) by regulating the expression and/or activity of one or more genes.
  • miR-485 inhibitors disclosed herein are capable of regulating the expression and/or activity of a gene selected from CTBP1, TRIP6, SIRT1, CD36, PGC1-a, or combinations thereof.
  • CTBP1 Regulation [0306] In some aspects, contacting a miR-485 inhibitor described herein with a cell can increase the expression of a CTBP1 protein and/or a CTBP1 gene in the cell.
  • C-terminal-binding protein 1 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 help turn genes off, e.g., by recruiting histone modifying enzymes that add repressive histone marks and remove activating marks.
  • CTBP1 protein can also self-associate and bring together gene regulatory complexes.
  • the CTBP1 gene is located on chromosome 4 (nucleotides 1,211,444 to 1,250,355 of GenBank Accession Number NC_000004.12, minus strand orientation).
  • CTBP1 Synonyms of the CTBP1 gene, and the encoded protein thereof, are known and include "Brefeldin A- Ribosylated Substrate,” “BARS,” “HADDTS,” CtBP1,” or “CTBP.” [0309] There are at least two known isoforms of human CTBP1 protein, resulting from alternative splicing.
  • CTBP1 isoform 1 (UniProt identifier: Q13363-1; SEQ ID NO: 61) consists of 440 amino acids and has been chosen as the canonical sequence.
  • CTBP1 isoform 2 (UniProt identifier: Q13363-2; SEQ ID NO: 62) consists of 429 amino acids and differs from the canonical sequence as follows: 1-13: MGSSHLLNKGLPL (SEQ ID NO: 63) ⁇ MS. Table 1 below provides the sequences for the two CTBP1 isoforms. Table 1.
  • CTBP1 Protein Isoforms Isoform 1 (UniProt: MGSSHLLNKGLPLGVRPPIMNGPLHPRPLVALLDGRDCTVEMPILKDVATVAFCDAQSTQ EIHEKVLNEAVGALMYHTITLTREDLEKFKALRIIVRIGSGFDNIDIKSAGDLGIAVCNV Q13363-1) (SEQ ID PAASVEETADSTLCHILNLYRRATWLHQALREGTRVQSVEQIREVASGAARIRGETLGII NO: 61) GLGRVGQAVALRAKAFGFNVLFYDPYLSDGVERALGLQRVSTLQDLLFHSDCVTLHCGLN EHNHHLINDFTVKQMRQGAFLVNTARGGLVDEKALAQALKEGRIRGAALDVHESEPFSFS QGPLKDAPNLICTPHAAWYSEQASIEMREEAAREIRRAITGRIPDSLKNCVNKDHLTAAT HWASMDPAVVHPELNGAAYRYPPGVVGVAPTGIPAAVEGI
  • a miR-485 inhibitor disclosed herein can increase the expression of CTBP1 isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of CTBP1 isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of both CTBP1 isoform 1 and isoform 2.
  • CTBP1 isoform 1 and isoform 2 are collectively referred to herein as "CTBP1.”
  • contacting a cell with a miR-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 or more, compared to the expression and/or activity in a reference cell (e.g., corresponding cell that has not been contacted with the miR-485 inhibitor).
  • a reference cell e.g., corresponding cell that has not been contacted with the miR
  • a miR-485 inhibitor disclosed herein increases the expression of CTBP1 protein and/or CTBP1 gene by reducing the expression and/or activity of miR-485, e.g., miR-485-3p.
  • TRIP6 Regulation [0313] In some aspects, contacting a miR-485 inhibitor described herein with a cell can increase the expression of a TRIP6 protein and/or a TRIP6 gene in the cell.
  • Thyroid receptor-interacting protein 6 (TRIP6) is a protein that in humans is encoded by the TRIP6 gene. TRIP6 protein is a member of the zyxin family and comprises three LIM zinc-binding domains.
  • TRIP6 protein has been shown to localize to focal adhesion sites and along actin stress fibers. [0315] In humans, the TRIP6 gene is located on chromosome 7 (nucleotides 100,867,387 to 100,873,454 of GenBank Accession Number NC_000007.14, plus strand orientation).
  • TRIP6 Synonyms of the TRIP6 gene, and the encoded protein thereof, are known and include: “Thyroid Hormone Receptor Interactor 6,” “ZRP-1,” “OIP1,” “Thyroid Hormone Receptor Interacting Protein 6,” “OPA-Interacting Protein 1,” and “Zyxin Related Protein 1.”
  • TRIP6 isoform 1 (UniProt identifier: Q15654-1; SEQ ID NO: 64) consists of 476 amino acids and has been chosen as the canonical sequence.
  • TRIP6 isoform 2 (UniProt identifier: Q15654-2; SEQ ID NO: 65) consists of 106 amino acids and differs from the canonical sequence as follows: (i) 37-106: ALQPHPRVNFCPLPSEQCYQAPGGPEDRGPAWVGSHGVLQHTQGLPADRGGLRPGSLDAEIDLLSSTLAE (SEQ ID NO: 67) ⁇ VLPGPRGTGGSGAGVGGVPWSTPAHAGAPCRQGGPSPWKPGRRDRLAEQHAGRAEWGSGSCVTATRPTGI (SEQ ID NO: 68); (ii) 107-476: Missing.
  • TRIP6 isoform 3 (UniProt identifier: Q15654-3; SEQ ID NO: 66) consists of 80 amino acids and differs from the canonical sequence as follows: (i) 37-80: ALQPHPRVNFCPLPSEQCYQAPGGPEDRGPAWVGSHGVLQHTQG (SEQ ID NO: 69) ⁇ GAPCRQGGPSPWKPGRRDRLAEQHAGRAEWGSGSCVTATRPTGI (SEQ ID NO: 70); (ii) 81-476: Missing. Table 2 below provides the sequences for the different TRIP6 protein isoforms. Table 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of TRIP6 isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of TRIP6 isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of TRIP6 isoform 3.
  • a miR-485 inhibitor disclosed herein can increase the expression of both TRIP6 isoform 1, isoform 2, and isoform 3.
  • contacting a cell with a miR-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 or more, compared to the expression and/or activity in a reference cell (e.g., corresponding cell that has not been contacted with the miR-485 inhibitor).
  • a reference cell e.g., corresponding cell that has not been contacted with the miR-485 inhibitor.
  • a miR-485 inhibitor can also increase the expression and/or activity of other LIM-domain containing proteins.
  • a miR-485 inhibitor disclosed herein increases the expression of TRIP6 protein and/or TRIP6 gene by reducing the expression and/or activity of miR-485, e.g., miR-485-3p.
  • SIRT1 Regulation [0321] In some aspects, contacting a miR-485 inhibitor described herein with a cell (e.g., neural stem cells) can increase the expression of a SIRT1 protein and/or a SIRT1 gene in the cell.
  • SIRT1 also known as NAD-dependent deacetylase sirtuin-1
  • SIRT1 is a protein that in humans is encoded by the SIRT1 gene.
  • the SIRT1 gene is located on chromosome 10 in humans (nucleotides 67,884,656 to 67,918,390 of GenBank Accession Number NC_000010.11, plus strand orientation).
  • SIRT1 Synonyms of the SIRT1 gene, and the encoded protein thereof, are known and include "regulatory protein SIR2 homolog 1,” “silent mating-type information regulation 2 homolog 1,” “SIR2,” “SIR2-Like Protein 1,” “SIR2L1,” “SIR2alpha,” “Sirtuin Type 1,” “hSIRT1,” or “hSIR2.”
  • SIRT1 isoform 1 (UniProt identifier: Q96EB6-1) consists of 747 amino acids and has been chosen as the canonical sequence (SEQ ID NO: 71).
  • SIRT1 isoform 2 (also known as "delta-exon8) (UniProt identifier: Q96EB6-2) consists of 561 amino acids and differs from the canonical sequence as follows: 454-639: missing (SEQ ID NO: 72). Table 3 below provides the sequences for the two SIRT1 isoforms. Table 3.
  • a miR-485 inhibitor disclosed herein can increase the expression of SIRT1 isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of SIRT1 isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of both SIRT1 isoform 1 and isoform 2.
  • SIRT1 isoform 1 and isoform 2 are collectively referred to herein as "SIRT1.”
  • contacting a cell with a miR-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 or more, compared to the expression and/or activity in a reference cell (e.g., corresponding cell that has not been contacted with the miR-485 inhibitor).
  • a reference cell e.g., corresponding cell that has not been contacted with the mi
  • a miR-485 inhibitor disclosed herein increases the expression of SIRT1 protein and/or SIRT1 gene by reducing the expression and/or activity of miR-485, e.g., miR-485-3p.
  • CD36 Regulation [0327] In some aspects, contacting a miR-485 inhibitor described herein with a cell can increase the expression of a CD36 protein and/or a CD36 gene in the cell.
  • Cluster determinant 36 (CD36) is 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 of GenBank Accession Number NC_000007.14, plus strand orientation).
  • Synonyms of the CD36 gene, and the encoded protein thereof, are known and include "platelet glycoprotein IV,” “fatty acid translocase,” “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.” [0329] There are at least four known isoform of human CD36 protein, resulting from alternative splicing.
  • CD36 isoform 1 (UniProt identifier: P16671-1) consists of 472 amino acids and has been chosen as the canonical sequence (SEQ ID NO: 73).
  • CD36 isoform 2 (also known as "ex8-del") (UniProt identifier: P16671-2) (SEQ ID NO: 74) consists of 288 amino acids and differs from the canonical sequence as follows: 274-288: SIYAVFESDVNLKGI (SEQ ID NO: 77) ⁇ ETCVHFTSSFSVCKS (SEQ ID NO: 78); and 289-472: missing.
  • CD36 Isoform 3 (also known as "ex6-7-del") (UniProt identifier: P16671-3) (SEQ ID NO: 75) consists of 433 amino acids and differs from the canonical sequence as follows: 234-272: missing.
  • CD36 isoform 4 (also known as "ex4-del” (UniProt identifier: P16671-4) (SEQ ID NO: 76) consists of 412 amino acids and differs from the canonical sequence as follows: 144-203: missing. Table 4 below provides the sequences for the four CD36 isoforms. Table 4.
  • CD36 Protein Isoforms Isoform 1 MGCDRNCGLIAGAVIGAVLAVFGGILMPVGDLLIQKTIKKQVVLEEGTIAFKNWVKTGTE VYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGA (UniProt: P16671- IFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLW 1) (SEQ ID NO: GYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKVAIIDTYKGKRNLSYWE SHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAF 73) ASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGL NPNEEEHRTYLDI
  • a miR-485 inhibitor disclosed herein can increase the expression of CD36 isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of CD36 isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of CD36 isoform 3.
  • a miR- 485 inhibitor disclosed herein can increase the expression of CD36 isoform 4.
  • a miR-485 inhibitor disclosed herein can increase the expression of both CD36 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.
  • a miR-485 inhibitor disclosed herein can increase the expression of all CD36 isoforms. Unless indicated otherwise, isoform 1, isoform 2, isoform 3, and isoform 4 are collectively referred to herein as "CD36.” [0331] In some aspects, contacting a cell with a miR-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 or more, compared to the expression and/or activity in a reference cell (e.g., corresponding cell
  • a miR-485 inhibitor disclosed herein increases the expression of CD36 protein and/or CD36 gene by reducing the expression and/or activity of miR-485, e.g., miR-485-3p.
  • PGC1 Regulation [0333] In some aspects, contacting a miR-485 inhibitor described herein with a cell can increase the expression of a PGC-1 ⁇ protein and/or a PGC-1 ⁇ gene in the cell.
  • PPC1- ⁇ Peroxisome proliferator-activated receptor gamma coactivator 1-alpha
  • PPARG Coactivator 1 Alpha or Ligand Effect Modulator-6 is a protein that in humans is encoded by the PPARGC1A gene.
  • the PGC1- ⁇ gene is located on chromosome 4 in humans (nucleotides 23,792,021 to 24,472,905 of GenBank Accession Number NC_000004.12, plus strand orientation).
  • PGC1- ⁇ gene Synonyms of the PGC1- ⁇ gene, and the encoded protein thereof, are known and include “PPARGC1A,” “LEM6,” “PGC1,” “PGC1A,” “PGC-1v,” “PPARGC1, “PGC1alpha,” or “PGC-1(alpha).”
  • PGC1- ⁇ isoform 1 (UniProt identifier: Q9UBK2-1) consists of 798 amino acids and has been chosen as the canonical sequence (SEQ ID NO: 79).
  • PGC1- ⁇ isoform 2 (also known as "Isoform NT-7a") (UniProt identifier: Q9UBK2-2) (SEQ ID NO: 80) consists of 271 amino acids and differs from the canonical sequence as follows: 269-271: DPK ⁇ LFL; 272-798: Missing.
  • PGC1- ⁇ isoform 3 (also known as "Isoform B5") (UniProt identifier: Q9UBK2-3) (SEQ ID NO: 81) consists of 803 amino acids and differs from the canonical sequence as follows: 1-18: MAWDMCNQDSESVWSDIE (SEQ ID NO: 88) ⁇ MDETSPRLEEDWKKVLQREAGWQ (SEQ ID NO: 89).
  • PGC1- ⁇ isoform 4 (also known as "Isoform B4") (UniProt identifier: Q9UBK2-4) (SEQ ID NO: 82) consists of 786 amino acids and differs from the canonical sequence as follows: 1-18: MAWDMCNQDSESVWSDIE (SEQ ID NO: 88) ⁇ MDEGYF (SEQ ID NO: 90).
  • PGC1- ⁇ isoform 5 (also known as "Isoform B4-8a") (UniProt identifier: Q9UBK2-5) (SEQ ID NO: 83) consists of 289 amino acids and differs from the canonical sequence as follows: 1-18: MAWDMCNQDSESVWSDIE (SEQ ID NO: 88) ⁇ MDEGYF (SEQ ID NO: 90); 294-301: LTPPTTPP (SEQ ID NO: 91) ⁇ VKTNLISK (SEQ ID NO: 92); 302-798: Missing.
  • PGC1- ⁇ isoform 6 (also known as "Isoform B5-NT") (UniProt identifier: Q9UBK2-6) (SEQ ID NO: 84) consists of 276 amino acids and differs from the canonical sequence as follows: 1-18: MAWDMCNQDSESVWSDIE (SEQ ID NO: 88) ⁇ MDETSPRLEEDWKKVLQREAGWQ (SEQ ID NO: 89); 269-271: DPK ⁇ LFL; 272-798: Missing.
  • PGC1- ⁇ isoform 7 (also known as "B4-3ext") (UniProt identifier: Q9UBK2-7) (SEQ ID NO: 85) consists of 138 amino acids and differs from the canonical sequence as follows: 1-18:MAWDMCNQDSESVWSDIE (SEQ ID NO: 88) ⁇ MDEGYF (SEQ ID NO: 90); 144-150: LKKLLLA (SEQ ID NO: 93) ⁇ VRTLPTV (SEQ ID NO: 94); 151- 798: Missing.
  • PGC1- ⁇ isoform 8 (also known as "Isoform 8a") (UniProt identifier: Q9UBK2-8) (SEQ ID NO: 86) consists of 301 amino acids and differs from the canonical sequence as follows: 294-301: LTPPTTPP (SEQ ID NO: 91) ⁇ VKTNLISK (SEQ ID NO: 92); 302-798: Missing.
  • PGC1- ⁇ isoform 9 also known as "Isoform 9" or "L-PGG-1alpha" (UniProt identifier: Q9UBK2- 9) (SEQ ID NO: 87) consists of 671 amino acids and differs from the canonical sequence as follows: 1-127: Missing.
  • PGC1- ⁇ Protein Isoforms Isoform 1 (UniProt: MAWDMCNQDSESVWSDIECAALVGEDQPLCPDLPELDLSELDVNDLDTDSFLGGLKWCS DQSEIISNQYNNEPSNIFEKIDEENEANLLAVLTETLDSLPVDEDGLPSFDALTDGDVT Q9UBK2-1) (SEQ ID TDNEASPSSMPDGTPPPQEAEEPSLLKKLLLAPANTQLSYNECSGLSTQNHANHNHRIR NO: 79) TNPAIVKTENSWSNKAKSICQQQKPQRRPCSELLKYLTTNDDPPHTKPTENRNSSRDKC TSKKKSHTQSQSQHLQAKPTTLSLPLTPESPNDPKGSPFENKTIERTLSVELSGTAGLT PPTTPPHKANQDNPFRASPKLKSSCKTVVPPPSKKPRYSESSGTQGNNSTKKGPEQ
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 3.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 4.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 5.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 6.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 7.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 8.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 9.
  • a miR-485 inhibitor disclosed herein can increase the expression of PGC1- ⁇ isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, isoform 6, isoform 7, isoform 8, and isoform 9. Unless indicated otherwise, both isoform 1 and isoform 2 are collectively referred to herein as "PGC1- ⁇ .”
  • contacting a cell with a miR-485 inhibitor increases the expression and/or activity of PGC1- ⁇ protein and/or PGC1- ⁇ 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
  • a miR-485 inhibitor disclosed herein increases the expression of PGC1- ⁇ protein and/or PGC1- ⁇ gene by reducing the expression and/or activity of miR-485, e.g., miR-485-3p.
  • the present disclosure also provides a method of treating a disease or condition that is associated with abnormal (e.g., reduced or increased) level of proteins and/or genes related to a neurodegenerative disease or disorder.
  • the term "neurodegenerative disease or disorder” refers to a disease or disorder caused by the progressive pathologic changes within the nervous system, particularly within the neurons of the brain.
  • Such progressive destruction of the nervous system can result in physical (e.g., ataxias) and/or mental (e.g., dementia) impairments.
  • Non-limiting examples of neurodegenerative diseases or disorders that can be treated with the present disclosure include Alzheimer's disease, Parkinson's disease, or any combination thereof.
  • Other diseases or conditions that can be treated with the present disclosure include, but are not limited to, autism spectrum disorder, mental retardation, seizure, stroke, spinal cord injury, or any combination thereof.
  • a disease or disorder that can be treated with the present disclosure comprises Alzheimer's disease.
  • Alzheimer's disease comprises pre-dementia Alzheimer's disease, early Alzheimer's disease, moderate Alzheimer's disease, advanced Alzheimer's disease, early onset familial Alzheimer's disease, inflammatory Alzheimer's disease, non-inflammatory Alzheimer's disease, cortical Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, or any combination thereof.
  • a disease or disorder that can be treated comprises a parkinsonism.
  • the term "parkinsonism" refers to a group of neurological disorders that causes a combination of the movement abnormalities seen in Parkinson's disease.
  • Non-limiting examples of such movement abnormalities include tremor, slow movement (bradykinesia), postural instability, loss of postural reflexes, flexed posture, freezing phenomenon (when the feet are transiently "glued” to the ground), impaired speech, muscle stiffness (rigidity), or combinations thereof.
  • parkinsonism comprises a Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticalbasal degeneration (CBD), normal pressure hydrocephalus (NSA), vascular parkinsonism (also known as cerebrovascular disease), diffuse Lewy body disease, Parkinson-dementia, X-linked dystonia-parkinsonism, secondary Parkinsonism (resulting from environmental etiology, e.g., toxins, drugs, post encephalitic, brain tumors, head trauma, normal pressure hydrocephalus), or combinations thereof.
  • a parkinsonism that can be treated with the present disclosure is a Parkinson's disease.
  • PD Parkinson's disease
  • PD refers to neurodegenerative disorder leading to motor and non-motor manifestations (i.e., symptoms) and characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system.
  • Non- limiting examples of motor and non-motor manifestations of PD are provided elsewhere in the present disclosure.
  • Proteinopathy ⁇ -synuclein abnormal aggregation
  • Other exemplary features of PD include dopaminergic neuron damage, mitochondrial dysfunction, neuroinflammation, protein homeostasis (e.g., autophagic clearance of damaged proteins and organelles glial cell dysfunction), and combinations thereof.
  • miR-485 inhibitors of the present disclosure can treat PD by improving one or more of these features of PD.
  • administering a miR-485 inhibitor of the present disclosure reduces the occurrence or risk of occurrence of one or more symptoms of cognitive impairments in a subject (e.g., suffering from a neurodegenerative disease) by 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%, or about 100% compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • administering a miR-485 inhibitor of the present disclosure reduces memory loss in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., memory loss in the subject prior to the administering).
  • administering a miR-485 inhibitor of the present disclosure reduces memory loss or the risk of occurrence of memory loss in a subject (e.g., suffering from a neurodegenerative disease) by 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%, or about 100% compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor.
  • administering a miR-485 inhibitor of the present disclosure improves memory retention in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., memory retention in the subject prior to the administering).
  • administering a miR-485 inhibitor of the present disclosure improves and/or increases memory retention in a subject (e.g., suffering from a neurodegenerative disease) by 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor
  • administering a miR-485 inhibitor of the present disclosure improves spatial working memory in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., spatial working memory in the subject prior to the administering).
  • a reference e.g., spatial working memory in the subject prior to the administering.
  • spatial working memory refers to the ability to keep spatial information activity in working memory over a short period of time.
  • spatial working memory is improved and/or increased by at least about 5%, 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor.
  • increasing and/or inducing neurogenesis is associated with increased proliferation, differentiation, migration, and/or survival of neural stem cells and/or progenitor cells.
  • administering a miR-485 inhibitor of the present disclosure can increase the proliferation of neural stem cells and/or progenitor cells in the subject.
  • the proliferation of neural stem cells and/or progenitor cells is increased by at least about 5%, 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • the survival of neural stem cells and/or progenitor cells is increased by at least about 5%, 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor.
  • increasing and/or inducing neurogenesis is associated with an increased number of neural stem cells and/or progenitor cells.
  • the number of neural stem cells and/or progenitor cells is increased by at least about 5%, 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor.
  • increasing and/or inducing neurogenesis is associated with increased axon, dendrite, and/or synapse development.
  • axon, dendrite, and/or synapse development is increased by at least about 5%, 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • administering a miR-485 inhibitor disclosed herein prevents and/or inhibits the development of an amyloid beta plaque load in a subject (e.g., suffering from a neurodegenerative disease).
  • administering a miR-485 inhibitor disclosed herein delays the onset of the development of an amyloid beta plaque load in a subject (e.g., suffering from a neurodegenerative disease). In some aspects, administering a miR-485 inhibitor of the present disclosure lowers the risk of development an amyloid beta plaque load in a subject (e.g., suffering from a neurodegenerative disease). [0351] In some aspects, administering a miR-485 inhibitor of the present disclosure increases dendritic spine density of a neuron in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., dendritic spine density of a neuron in the subject prior to the administering).
  • administering a miR-485 inhibitor of the present disclosure increases dendritic spine density of a neuron in a subject (e.g., suffering from a neurodegenerative disease) by 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR- 485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR- 485 inhibitor
  • administering a miR-485 inhibitor disclosed herein decreases the loss of dendritic spines of a neuron in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., loss of dendritic spines of a neuron in the subject prior to the administering).
  • administering a miR-485 inhibitor decreases the loss of dendritic spines of a neuron in a subject (e.g., suffering from a neurodegenerative disease) by 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%, or about 100% compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor.
  • Non-limiting examples of motor symptoms associated with Parkinson's disease include resting tremor, reduction of spontaneous movement (bradykinesia), rigidity, postural instability, freezing of gait, impaired handwriting (micrographia), decreased facial expression, and uncontrolled rapid movements.
  • Non-limiting examples of non-motor symptoms associated with Parkinson's disease include autonomic dysfunction, neuropsychiatric problems (mood, cognition, behavior, or thought alterations), sensory alterations (especially altered sense of smell), and sleep difficulties.
  • administering a miR-485 inhibitor of the present disclosure improves one or more motor symptoms in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., corresponding motor symptoms in the subject prior to the administering).
  • administering a miR-485 inhibitor of the present disclosure improves one or more motor symptoms in a subject (e.g., suffering from a neurodegenerative disease) by 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR- 485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR- 485 inhibitor
  • administering a miR-485 inhibitor of the present disclosure improves one or more non-motor symptoms in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., corresponding non-motor symptom in the subject prior to the administering).
  • administering a miR-485 inhibitor disclosed herein improves one or more non-motor symptoms in a subject (e.g., suffering from a neurodegenerative disease) by 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor
  • administering a miR-485 inhibitor disclosed herein improves synaptic function in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., synaptic function in the subject prior to the administering).
  • a reference e.g., synaptic function in the subject prior to the administering.
  • synaptic function refers to the ability of the synapse of a cell (e.g., a neuron) to pass an electrical or chemical signal to another cell (e.g., a neuron).
  • administering a miR-485 inhibitor of the present disclosure improves synaptic function in a subject (e.g., suffering from a neurodegenerative disease) by 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 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor
  • administering a miR-485 inhibitor of the present disclosure can prevent, delay, and/or ameliorate the loss of synaptic function in a subject (e.g., suffering from a neurodegenerative disease) compared to a reference (e.g., loss of synaptic function in the subject prior to the administering).
  • administering a miR-485 inhibitor prevents, delays, and/or ameliorates the loss of synaptic function in a subject (e.g., suffering from a neurodegenerative disease) by 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%, or about 100% compared to a reference (e.g., subjects that did not receive an administration of the miR-485 inhibitor).
  • a reference e.g., subjects that did not receive an administration of the miR-485 inhibitor.
  • a miR-485 inhibitor disclosed herein can be administered by any suitable route known in the art.
  • a miR-485 inhibitor is administered parenthetically, intramuscularly, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracerebroventricularly, intraspinally, intraventricular, intrathecally, intracistemally, intracapsularly, intratumorally, or any combination thereof.
  • a miR-485 inhibitor is administered intracerebroventricularly (ICV).
  • a miR-485 inhibitor is administered intravenously.
  • a miR-485 inhibitor of the present disclosure can be used in combination with one or more additional therapeutic agents.
  • the additional therapeutic agent and the miR-485 inhibitor are administered concurrently.
  • the additional therapeutic agent and the miR-485 inhibitor are administered sequentially.
  • the administration of a miR-485 inhibitor disclosed herein does not result in any adverse effects.
  • miR-485 inhibitors of the present disclosure do not adversely affect body weight when administered to a subject.
  • miR-485 inhibitors disclosed herein do not result in increased mortality or cause pathological abnormalities when administered to a subject.
  • kits or products of manufacture comprising a 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.
  • the kit or product of manufacture comprises a miR-485 inhibitor (e.g., vector, e.g., an AAV vector, a polynucleotide, or a pharmaceutical composition of the present disclosure) in one or more containers.
  • the kit or product of manufacture comprises miR-485 inhibitor (e.g., a vector, e.g., an AAV vector, a polynucleotide, or a pharmaceutical composition of the present disclosure) and a brochure.
  • miR-485 inhibitors disclosed herein e.g., vectors, polynucleotides, and pharmaceutical compositions of the present disclosure, or combinations thereof
  • the following examples are offered by way of illustration and not by way of limitation.
  • amidites and acetonitrile were dehydrated via incubation in a molecular sieve for 1 day (Thermo-Fisher).
  • Methylamine, ethanol and 1-methyl- 2-pyrrolidone (NMP) were purchased from Daejung Chemical and NMP was distillated prior to use.
  • Triethylamine and triethylamine tri-hydro fluoride were purchased from Sigma-Aldrich.
  • All oligonucleotides were automatically synthesized by oligo synthesizer (AKTA oligopilot10, GE Healthcare).
  • ribo C-300 133.78 mg, 40 ⁇ mol
  • the water contents, UV, and pressure were monitored until a 22-mer oligomer was synthesized.
  • the column was separated from the oligo synthesizer and washed with acetonitrile.
  • the CpG was dried in a centrifuge evaporator (EYELA) and separated from the oligonucleotide by the following step: Dried CpG was mixed with 4 mL of methylamine (40%) and ethanol mixture (3:1, v/v) solution and stirred for 2 hours at 25°C. The mixture was filtered using a syringe filter (0.45 ⁇ m) and dried in a centrifuge evaporator. The dried power was dissolved into NMP 7680ul.
  • Triethylamine 3840ul and triethylamine tri-hydro fluoride 5120ul were sequentially added into the solution.
  • the mixture solution was maintained with stirring for 2 hrs at 65°C.30mL of isopropyl alcohol and 3 mL of 3M sodium acetate were sequentially added into the solution.
  • the solution was stored at -20°C and allowed to precipitate for 12 hours.
  • the precipitate was separated by centrifuge (13,000 rpm, 30 min) and dried in a centrifuge evaporator.
  • Example 2 Preparation of Modified Oligonucleotides
  • modified oligonucleotide i.e., miR-485-3p ASO-B, miR-485- 3p ASO-C, miR-485-3p ASO-D, miR-485-3p ASO-E, and miR-485-3p ASO-F
  • each sequence (A, U, G, C, and T) of RNA and DNA amidite, Locked Nucleic Acid (LNA) amidite A, G, T, and 5- Me-2’-C
  • methoxyethyl(MOE) 5-Me-C-2’-MOE, G-2’-MOE, A-2’-MOE, and 5-Me-U-2’- MOE) were purchased from Glen research.
  • CpG As a Unylinker Support CpG was purchased from GE healthcare for oligonucleotide synthesis. Acetonitrile (synthesis quality, water content ⁇ 25 ppm) and Toluene was supplied from Acros. All other reagents for RNA synthesis such as Detritylation, Activator, Capping A, Capping B1, Capping B2 were purchased from Sigma-Aldrich. The 3-(N,N- dimethylaminomethylidene)amino-3H-1,2,4,-dithiazole-5-thion for sulfurizing was purchased from Glen research. The amidites and acetonitrile were dehydrated via incubation in a molecular sieve for 1 day (Thermo-Fisher).
  • Ammonium hydroxide for ASO deprotection was purchased from Duksan. 1-methyl-2-pyrrolidone (NMP) was purchased from Daejung Chemical and NMP was distillated prior to use. Triethylamine and triethylamine tri-hydro fluoride were purchased from Sigma-Aldrich. [0367] All oligonucleotides were automatically synthesized by an oligo synthesizer (AKTA oligopilot10, GE Healthcare). In brief, Primer Support 5G Unylinker 350 (58.14 mg, 20 ⁇ mol) was filled into the column and the sequence information was entered into the AKTA software.
  • the water contents, UV, and pressure were monitored until a 22-mer oligomer was synthesized.
  • the column was separated from the oligo synthesizer and washed with acetonitrile.
  • the CpG was dried in a centrifuge evaporator (EYELA) and separated from the oligonucleotides by the following steps: Dried CpG was mixed with 4 mL of ammonium hydroxide (25 ⁇ 28 %) solution and stirred for 2 hours at 65°C. The mixture was filtered using a syringe filter (0.45 ⁇ m) and dried in a centrifuge evaporator. The dried power was dissolved into NMP 5400ul.
  • Triethylamine 2700ul and triethylamine tri-hydro fluoride 3600ul were sequentially added into the solution.
  • the mixture solution was maintained with stirring for 2 hrs at 65°C.25mL of isopropyl alcohol and 1.5 mL of 3M sodium acetate were sequentially added into the solution.
  • the solution was stored at -20°C and allowed to precipitate for 12 hours.
  • the precipitate was separated by centrifuge (13,000 rpm, 30 min) and dried in a centrifuge evaporator.
  • Synthesized oligonucleotides (miR-485-3p ASO-B, miR-485-3p ASO-C, miR-485- 3p ASO-D, miR-485-3p ASO-E, and miR-485-3p ASO-F shown in FIGs. 3B-3F respectively) were purified using Prap Liquid Chromatography (Prap LCm Ultimate 3000, Thermo scientific) attached with a PLRP-S column (Agilent).
  • TEAA triethylamine acetate
  • ACN acetonitrile
  • Example 3 In vivo Efficacy Assay [0369] In vivo hair growth is described in FIGs.6A, 7A, and 8A.
  • FIG.6A shows that female mice subcutaneously injected with miR-485-3p ASO-C exhibit accelerated Telogen-to-Anagen transition and more rapid hair growth compared to PBS- injected control female mice. Speedy and synchronized anagen entry hair coat coverage was observed on the whole dorsal telogen skin of female mice treated with two subcutaneous injections of miR-485-3p ASO-C encapsulated in a cationic micelle. miR-485-3p ASO-C was observed to accelerate hair regrowth at a rate greater than that of miR-485-3p ASO-A.
  • FIG.7A shows that male mice intramuscularly injected with miR-485-3p ASO-C exhibit accelerated Telogen-to-Anagen transition and more rapid hair growth compared to PBS- injected control male mice. Speedy and synchronized anagen entry hair coat coverage was observed on the whole dorsal telogen skin of male mice treated with two intramuscular injections of miR-485-3p ASO-C encapsulated in a cationic micelle. miR-485-3p ASO-C was observed to accelerate hair regrowth at a rate greater than that of miR-485-3p ASO-A. [0372] FIG.
  • FIG.6B shows that female mice subcutaneously injected with miR-485-3p ASO-C had significantly greater hair coat coverage on the whole dorsal telogen skin (measured by hair growth scores) compared to female control mice at days 10, 12, and 14.
  • FIG. 7B shows that male mice intramuscularly injected with miR-485-3p ASO-C had significantly greater hair coat coverage on the whole dorsal telogen skin (measured by hair growth scores) compared to male control mice at days 10, 12, and 14.
  • FIG. 7B shows that male mice intramuscularly injected with miR-485-3p ASO-C had significantly greater hair coat coverage on the whole dorsal telogen skin (measured by hair growth scores) compared to male control mice at days 10, 12, and 14.
  • FIGs.9A-9C In vivo hair regrowth in an AGA (Androgenetic alopecia) mouse model is described in FIGs.9A-9C. To test hair regrowth efficacy in an AGA mouse model, mouse hair was shaved using electric clippers. Mice were subsequently treated with hair removal cream for one day before miR-485-3p-ASO injection.
  • DHT was subcutaneously injected one day before depletion and there after every other day (1mg/50 ⁇ l) during the experiment to mimic androgenetic hair loss condition.
  • miR-485-3p ASO-C was injected on days 0 and 7 by subcutaneous injection. The dorsal back of the mice was imaged three times per week to assess hair regrowth efficacy in the experimental group.
  • FIG. 9B shows that male mice subcutaneously injected with miR-485-3p ASO-C exhibit accelerated Telogen-to-Anagen transition and more rapid hair growth compared to PBS- injected control male mice.
  • FIG.9B also shows that DHT-induced AGA male mice subcutaneously injected with miR-485-3p ASO-C exhibit accelerated Telogen- to-Anagen transition and more rapid hair growth compared to DHT-induced AGA male mice not administered with miR-485-3p ASO-C.
  • FIG. 9C shows that male mice subcutaneously injected with miR-485-3p ASO-C had significantly greater hair coat coverage on the whole dorsal telogen skin (measured by hair growth scores) compared to male control mice at days 10, 12, and 14.
  • FIG. 9C shows that male mice subcutaneously injected with miR-485-3p ASO-C had significantly greater hair coat coverage on the whole dorsal telogen skin (measured by hair growth scores) compared to male control mice at days 10, 12, and 14.

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Abstract

La présente invention comprend un inhibiteur de miR-485 pour traiter une maladie ou un trouble associé au niveau accru de miR-485-3p. Selon certains aspects, l'inhibiteur de miR-485 peut réduire l'expression d'un gène et/ou d'une protéine associée à miR-485-3p.
PCT/IB2024/054199 2023-05-01 2024-04-30 Inhibiteurs de miarn-485 Pending WO2024228119A1 (fr)

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WO2021156831A1 (fr) * 2020-02-07 2021-08-12 Biorchestra Co., Ltd. Inhibiteur de miarn-485 pour régulation à la hausse de gènes
US20220105123A1 (en) * 2019-06-26 2022-04-07 Biorchestra Co., Ltd. Micellar nanoparticles and uses thereof
WO2022137188A1 (fr) * 2020-12-23 2022-06-30 Biorchestra Co., Ltd. Utilisation d'inhibiteurs d'arnmi-485 pour induire la croissance capillaire

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US20220105123A1 (en) * 2019-06-26 2022-04-07 Biorchestra Co., Ltd. Micellar nanoparticles and uses thereof
WO2021156831A1 (fr) * 2020-02-07 2021-08-12 Biorchestra Co., Ltd. Inhibiteur de miarn-485 pour régulation à la hausse de gènes
WO2022137188A1 (fr) * 2020-12-23 2022-06-30 Biorchestra Co., Ltd. Utilisation d'inhibiteurs d'arnmi-485 pour induire la croissance capillaire

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