WO2020181107A1 - Microrna mimics and uses thereof - Google Patents

Microrna mimics and uses thereof Download PDF

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Publication number
WO2020181107A1
WO2020181107A1 PCT/US2020/021223 US2020021223W WO2020181107A1 WO 2020181107 A1 WO2020181107 A1 WO 2020181107A1 US 2020021223 W US2020021223 W US 2020021223W WO 2020181107 A1 WO2020181107 A1 WO 2020181107A1
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sequence
strand
strand comprises
mir
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PCT/US2020/021223
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French (fr)
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Kurt Vagle
Christina Marie DALBY
Subhadeep ROY
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MiRagen Therapeutics, Inc.
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Publication of WO2020181107A1 publication Critical patent/WO2020181107A1/en

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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • MicroRNAs are naturally occurring, short RNA molecules that are part of a ribonucleoprotein complex known as RISC (RNA-induced silencing complex) and act to repress translation of specific mRNA molecules.
  • RISC RNA-induced silencing complex
  • levels of miRNAs have been demonstrated to be depressed below their normal levels in a variety of diseased states. Therefore a potential mode of therapy in these indications is the introduction of synthetic miRNAs to mimic the activity of natural miRNAs (miRNA mimics).
  • synthetic RNA molecules that are chemically identical to naturally occurring RNAs are not effective drugs due to their poor stability towards nucleases present in serum.
  • the present disclosure provides synthetic, modified miRNA mimics that increase miRNA activity.
  • the present disclosure provides synthetic, modified mimics of miR-29, miR-96, miR-182, and miR-183, as well as methods of making and uses thereof.
  • the modified miR-29 mimics of the disclosure are useful for reducing collagen deposition and for the treatment and prevention of associated conditions, such as fibrosis.
  • the modified miR-96, miR-182, and miR-183 mimics of the disclosure are useful for treating various sensory indications including ophthalmological indications, otic indications, anosmia and pain.
  • double stranded miR-29 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 1-16.
  • double stranded miR-29 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected SEQ ID Nos: 17-31.
  • double stranded miR-29 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; b) the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; c) the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; d) the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; e) the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; f) the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g) the second strand comprises the sequence of SEQ ID NO:
  • double stranded miR-29 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; b) the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; c) the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; d) the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; e) the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; f) the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g) the second strand comprises the sequence of SEQ ID NO:
  • double stranded miR-96 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence of SEQ ID NO: 32 or 35.
  • double stranded miR-96 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 38-41.
  • double stranded miR-96 mimics comprising a first strand a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; b) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39; c) the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 40; or d) the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 41.
  • double stranded miR-96 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; or b) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39.
  • double stranded miR-182 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 33 and 36.
  • double stranded miR-182 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 42 and 43.
  • double stranded miR-182 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42; or b) the second strand comprises the sequence of SEQ ID NO: 36, and the first strand comprises the sequence of SEQ ID NO: 43.
  • double stranded miR-182 mimics comprising a first strand and a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42.
  • double stranded miR-183 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 34 and 37.
  • double stranded miR-183 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID NOs: 44 and 45.
  • double stranded miR-183 mimics comprising a first strand a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44; or b) the second strand comprises the sequence of SEQ ID NO: 37, and the first strand comprises the sequence of SEQ ID NO: 45.
  • double stranded miR-183 mimics comprising a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO:
  • the first strand comprises the sequence of SEQ ID NO: 44.
  • compositions comprising one or more of the mimics as described in the above recited aspects, and present throughout the disclosure.
  • provided herein are methods of treating a fibrotic condition or various sensory indications including ophthalmological indications, otic indications, anosmia and pain, in a subject in need thereof, the method comprising administering to the subject one or more of the mimics or the pharmaceutical compositions thereof as described in the above recited aspects, and present throughout disclosure.
  • FIGS. 1A-1E show graphs showing the downregulation of miR-29b target mRNAs, Col3al (FIG. 1A); Colla2 (FIG. IB); Col4a5 (FIG. 1C); Collal (FIG. ID); and Fbnl (FIG. IE), upon active transfection of miR-29b mimics into IMR90 cells.
  • the sense and antisense strand of each of the transfected double stranded miRNAs are indicated on the graph.
  • ‘U’ and‘M’ refer to untreated and mock-transfected controls.
  • FIGS. 2A-2D show graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 2A); Col3al (FIG. 2B); Col4a5 (FIG. 2C); and Fbnl (FIG. 2D), upon active transfection of miR-29b mimics into IMR90 cells.
  • the sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph.
  • ‘U’ and‘M’ refer to untreated and mock-transfected controls.
  • FIGS. 3A-3E show graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 3A); Colla2 (FIG. 3B); Col3al (FIG. 3C); Col4a5 (FIG. 3D); and Fbnl (FIG. 3E), upon active transfection of miR-29b mimics into IMR90 cells.
  • the sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph.
  • ‘U’ and‘M’ refer to untreated and mock-transfected controls.
  • FIGS. 4A-4E shows graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 4A); Colla2 (FIG. 4B); Col3al (FIG. 4C); Col4a5 (FIG. 4D); and Fbnl (FIG. 4E), upon active transfection of miR-29b mimics into IMR90 cells.
  • the sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph.
  • ‘U’ and‘M’ refer to untreated and mock-transfected controls.
  • FIGS. 5A-5B shows graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 5A) and ACTA2 (FIG. 5B), upon passive delivery of miR-29b mimics into normal human lung fibroblasts (NHLF) cells.
  • the sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph.
  • FIGS. 6A-6B show representative images showing localization of miR-29b mimics to alkali burned corneas 6 hours after a single drop topical administration. The antisense and sense strands of the each of the miR-29b mimics are indicated on the image.
  • FIGS. 7A-7B show representative images showing the localization of the indicated fluorescently labelled miR-29b mimics delivered via intravitreal injection. The antisense and sense strands of the each of the miR-29b mimics are indicated on the image.
  • FIGS. 8A-8B show representative images showing the localization of the indicated fluorescently labeled miR-29b mimics delivered via subconjunctival injections. The antisense and sense strands of the each of the miR-29b mimics are indicated on the image.
  • FIGS. 9A-9B show graphs showing the downregulation of the indicated miR-96 target mRNAs at day 2 (FIG. 9A) and day 3 (FIG. 9B) following treatment of HeLa cells with miR-96 mimics.
  • the sense and antisense strands of each of the miR-96 mimics is indicated on the graph.
  • FIGS. 10A-10B show graphs showing the downregulation of the indicated miR-182 target mRNAs at day 2 (FIG. 10A) and day 3 (FIG. 10B) following treatment of HeLa cells with the indicated miR- 182 mimics.
  • the sense and antisense strands of each of the miR- 182 mimics is indicated on the graph.
  • FIGS. 1 lA-1 IB show graphs showing the downregulation of the indicated miR- 183 target mRNAs at day 2 (FIG. 11A) and day 3 (FIG. 1 IB) following treatment of HeFa cells with the indicated miR- 183 mimics.
  • the sense and antisense strands of each of the miR- 183 mimics is indicated on the graph.
  • miRNA microRNA
  • microRNA mimic may be used interchangeably with the terms“promiR,”“miR agonist,”“microRNA agonist,”“microRNA mimetic,”“miRNA mimic,” or“miR mimic;” the term“first strand” may be used interchangeably with the terms “antisense strand” or“guide strand”; the term“second strand” may be used interchangeably with the term“sense strand” or“passenger strand;” and the term“miR antagonist” may be used interchangeably with the terms“oligonucleotide inhibitor,”“antimiR”“antisense
  • oligonucleotide “miR antagomir” or“anti-microRNA oligonucleotide.”
  • a double stranded miR-29 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-29a, miR-29b, or miR-29c sequence, and the second strand comprises a sequence that is substantially complementary to the first strand. Exemplary mimics are either partially, heavily, or fully modified.
  • a double stranded miR-96 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-96 sequence, and the second strand comprises a sequence that is substantially complementary to the first strand. Exemplary mimics are either partially, heavily, or fully modified.
  • a double stranded miR-182 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-182 sequence, and the second strand comprises a sequence that is substantially complementary to the first strand.
  • Exemplary mimics are either partially, heavily, or fully modified.
  • a double stranded miR-183 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-183 sequence, and the second strand comprises a sequence that is substantially complementary to the first strand.
  • Exemplary mimics are either partially, heavily, or fully modified.
  • the nucleotides that form the first and the second strand of the double stranded microRNA mimics may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof.
  • the first strand and the second strand of the microRNA mimic comprise ribonucleotides and/or modified ribonucleotides.
  • modified nucleotide means a nucleotide where the nucleobase and/or the sugar moiety is modified relative to unmodified nucleotides.
  • a double stranded microRNA mimic of the disclosure comprises a first strand and a second strand, wherein the first strand comprises at least about 24 ribonucleotides and the second strand comprises at least about 22 ribonucleotides, wherein the second strand comprises a sequence that is substantially complementary to the first strand, and wherein the second strand comprises at least one stretch of three consecutive ribonucleotides comprising a 2’ fluoro-ribonucleotide.
  • a double stranded microRNA mimic of the disclosure comprises: (a) a first strand comprising about 21 nucleotides, and (b) a second strand comprising at least about 23 nucleotides, wherein the first strand comprises about 4 to about 9
  • the miRNA mimics of the disclosure have a first strand, whose sequence is identical to all or part of a mature miRNA sequence, and a second strand or a sense strand whose sequence is about 70% to about 100% complementary to the sequence of the first strand.
  • the first strand of the miRNA mimic is at least about 75, 80, 85, 90, 95, or 100% identical, including all integers there between, to the entire sequence of a mature, naturally occurring miRNA sequence.
  • the first strand is about or is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the sequence of a mature, naturally-occurring miRNA, such as the mouse, human, or rat miRNA sequence.
  • the first strand may comprise 20, 21, 22, or 23 nucleotide positions in common with a mature, naturally-occurring miRNA as compared by sequence alignment algorithms and methods well known in the art.
  • the sequence of the first strand is considered to be identical to the sequence of a mature miRNA even if the first strand includes a modified nucleotide instead of a naturally-occurring nucleotide.
  • the first strand of the mimic may comprise a modified cytidine nucleotide, such as 2’-fluoro-cytidine, at the corresponding position or if a mature, naturally -occurring miRNA sequence comprises a uridine nucleotide at a specific position, the miRNA region of the first strand of the mimic may comprise a modified uridine nucleotide, such as 2’-fluoro-uridine, 2’-O-methyl -uridine, 5-fluorouracil, or 4-thiouracil at the corresponding position.
  • the sequence of the first strand is considered to be identical to the mature, naturally-occurring miRNA sequence.
  • the first strand may include a modification of the 5’- terminal residue.
  • the first strand may have a 5’-terminal monophosphate.
  • the first strand does not contain a 5’-terminal monophosphate.
  • the second strand of the microRNA mimic is partially complementary to the sequence of the first strand.
  • the sequence of the second strand is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, inclusive of all values therebetween, complementary to the sequence of the first strand.
  • the second strand is substantially complementary to the sequence of the first strand.
  • the second strand is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, inclusive of all values therebetween, complementary to the sequence of the first strand.
  • the sequence of the second strand may be fully complementary to the first strand. In certain embodiments, about 19, 20, 21, 22, or 23 nucleotides of the complementary region of the second strand may be complementary to the first strand.
  • the second strand may comprise a modified cytidine nucleotide, such as 2’-0-methyl-cytidine, at the corresponding position.
  • the second strand comprises about 1, 2, 3, 4, 5, or 6 mismatches relative to the first strand. That is, up to 1, 2, 3, 4, 5, or 6 nucleotides between the first strand and the second strand may not be complementary.
  • the mismatches are not consecutive and are distributed throughout the second strand. In another embodiment, the mismatches are consecutive and may create a bulge.
  • the first and/or the second strand of the mimic may comprise an overhang on the 5’ or 3’ end of the strands.
  • the first strand comprises a 3’ overhang, i.e., a single-stranded region that extends beyond the duplex region, relative to the second strand.
  • the 3’ overhang of the first strand may range from about one nucleotide to about four nucleotides.
  • the 3’ overhang of the first strand may comprise 1 or 2 nucleotides.
  • the nucleotides comprising the 3’ overhang in the first strand are linked by phosphorothioate linkages.
  • the nucleotides comprising the 3’ overhang in the first strand may include ribonucleotides, deoxyribonucleotides, modified nucleotides, or combinations thereof.
  • the 3’ overhang in the first strand comprises two ribonucleotides.
  • the 3’ overhang of the first strand comprises two uridine nucleotides linked through a phosphorothioate linkage.
  • the first strand may not contain an overhang.
  • the nucleotides in the second/sense strand of miR A mimics of the disclosure are linked by phosphodiester linkages and the nucleotides in the first/antisense strand are linked by phosphodiester linkages except for the last three nucleotides at the 3’ end which are linked to each other via phosphorothioate linkages.
  • miRNA mimics of the present disclosure comprise a plurality of modified nucleotides.
  • the first strand of the mimic comprises one or more 2’-fluoro nucleotides.
  • the first strand may not include any modified nucleotide.
  • the second strand comprises one or more 2’-O-methyl modified nucleotides.
  • the modified nucleotides that may be used in the microRNA mimics of the disclosure can include nucleotides with a base modification or substitution.
  • the natural or unmodified bases in RNA are the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U) (DNA has thymine (T)).
  • modified bases also referred to as heterocyclic base moieties
  • modified bases include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted a
  • the microRNA mimics can have nucleotides with modified sugar moieties.
  • modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2’, 3’ or 4’ positions and sugars having substituents in place of one or more hydrogen atoms of the sugar.
  • the sugar is modified by having a substituent group at the 2’ position.
  • the sugar is modified by having a substituent group at the 3’ position.
  • the sugar is modified by having a substituent group at the 4’ position.
  • a sugar may have a modification at more than one of those positions, or that an RNA molecule may have one or more nucleotides with a sugar modification at one position and also one or more nucleotides with a sugar modification at a different position.
  • Sugar modifications contemplated in the miRNA mimics include, but are not limited to, a substituent group selected from: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl.
  • a substituent group selected from: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkyn
  • miRNA mimics have a sugar substituent group selected from the following: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , Cl, Br, CN, OCN, CF 3 , OCF 3 , SOCH 3 , SO2CH 3 , ONO2, NO2, N 3 , NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, or similar substituents.
  • a sugar substituent group selected from the following: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , Cl, Br, CN, OCN, CF 3 , OCF 3 , SOCH 3 , SO2CH 3 , ONO2, NO2, N 3 ,
  • the modification includes 2’-methoxy ethoxy (2’-0-CH2CH20CH3, which is also known as 2’-0-(2-methoxyethyl) or 2’-MOE), that is, an alkoxyalkoxy group.
  • Another modification includes 2’-dimethylaminooxy ethoxy, that is, a 0(CH2)20N(CH3)2 group, also known as 2’-DMAOE and 2’-dimethylaminoethoxy ethoxy (also known in the art as 2 -0- dimethyl-amino-ethoxy-ethyl or 2’-DMAEOE), that is, 2’-0-CH2-0-CH2-N(CH 3 )2.
  • Sugar substituent groups on the 2’ position (2’-) may be in the arabino (up) position or ribo (down) position.
  • One 2’-arabino modification is 2’-F.
  • Other similar modifications may also be made at other positions on the sugar moiety, particularly the 3’ position of the sugar on the 3’ terminal nucleoside or in 2’-5’ linked oligonucleotides and the 5’ position of 5’ terminal nucleotide.
  • the sugar modification is a 2’-0-alkyl (e.g. 2’-0-methyl, 2’-0- methoxyethyl), 2’-halo (e.g., 2’-fluoro, 2’-chloro, 2’-bromo), and 4’ thio modifications.
  • the first strand of the miR-29a, miR-29b, or miR-29c mimic comprises one or more 2’ fluoro nucleotides.
  • the first strand of the mimics has no modified nucleotides.
  • the second strand of miR-29a, miR-29b, or miR-29c mimic comprises one or more 2’-0-methyl modified nucleotides.
  • the first and the second strand of microRNA mimics of the disclosure can also include backbone modifications, such as one or more phosphorothioate, phosphorodithioate, phosphotriester, boranophosphate, alkylphosphonates, phosphoramidates, phosphordiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, or
  • the first and/or the second strand of microRNA mimics of the disclosure can also include modifications on the sugar residue, such as ribose modification/ replacement.
  • the first and/or the second strand of the microRNA mimics of the disclosure may include one or more morpholino, peptide nucleic acids, serinol nucleic acids, locked nucleic acids (LNA), and unlocked nucleic acids.
  • the microRNA mimics are conjugated to an agent such as a steroid (cholesterol), a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand to facilitate targeting, delivery, and/or stability.
  • the agent is attached to the first strand or second strand of the microRNA mimic at its 3’ or 5’ end through a linker or a spacer group.
  • the agent is cholesterol, a cholesterol derivative, cholic acid or a cholic acid derivative.
  • miR-29, miR-96, miR-182, and miR-183 mimics according to the present disclosure comprise the exemplary first and second strands listed in Tables 1-4 below. Definitions of the modifications are presented in Table 5.
  • Table 1 Sense/Passenger/Second strands of exemplary miR-29 mimics, with exemplary duplex combinations
  • a double stranded miR-29 mimic of the disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 1-16.
  • a double stranded miR-29 mimic of the disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence selected SEQ ID NOs: 17-31.
  • a double stranded miR-29 mimic of the disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26;
  • the second strand comprises the sequence of SEQ ID NO: 2
  • the first strand comprises the sequence of SEQ ID NO: 17;
  • the second strand comprises the sequence of SEQ ID NO: 3
  • the first strand comprises the sequence of SEQ ID NO: 26;
  • the second strand comprises the sequence of SEQ ID NO: 4
  • the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19;
  • the second strand comprises the sequence of SEQ ID NO: 5
  • the first strand comprises the sequence of SEQ ID NO: 17;
  • the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27;
  • the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; h. the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29;
  • the second strand comprises the sequence of SEQ ID NO: 9
  • the first strand comprises the sequence of SEQ ID NO: 30;
  • the second strand comprises the sequence of SEQ ID NO: 10
  • the first strand comprises the sequence of SEQ ID NO: 31;
  • the second strand comprises the sequence of SEQ ID NO: 11
  • the first strand comprises the sequence of SEQ ID NO: 17;
  • the second strand comprises the sequence of SEQ ID NO: 12
  • the first strand comprises the sequence of SEQ ID NO: 17 or 21;
  • the second strand comprises the sequence of SEQ ID NO: 13
  • the first strand comprises the sequence of SEQ ID NO: 20.
  • a double stranded miR-29 mimic comprising a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26;
  • the second strand comprises the sequence of SEQ ID NO: 2
  • the first strand comprises the sequence of SEQ ID NO: 17;
  • the second strand comprises the sequence of SEQ ID NO: 3
  • the first strand comprises the sequence of SEQ ID NO: 26;
  • the second strand comprises the sequence of SEQ ID NO: 4
  • the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19;
  • the second strand comprises the sequence of SEQ ID NO: 5
  • the first strand comprises the sequence of SEQ ID NO: 17;
  • the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27;
  • the second strand comprises the sequence of SEQ ID NO: 7
  • the first strand comprises the sequence of SEQ ID NO: 28;
  • the second strand comprises the sequence of SEQ ID NO: 8
  • the first strand comprises the sequence of SEQ ID NO: 29
  • the second strand comprises the sequence of SEQ ID NO: 9
  • the first strand comprises the sequence of SEQ ID NO: 30;
  • the second strand comprises the sequence of SEQ ID NO: 10
  • the first strand comprises the sequence of SEQ ID NO: 31;
  • the second strand comprises the sequence of SEQ ID NO: 11
  • the first strand comprises the sequence of SEQ ID NO: 17;
  • the second strand comprises the sequence of SEQ ID NO: 12
  • the first strand comprises the sequence of SEQ ID NO: 17 or 21; or
  • the second strand comprises the sequence of SEQ ID NO: 13
  • the first strand comprises the sequence of SEQ ID NO: 20.
  • a double stranded miR-29 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; d. the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; e.
  • the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; f. the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; h. the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29; i. the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30; j.
  • the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31; k. the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; l. the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21; or m. the second strand comprises the sequence of SEQ ID NO: 13, and the first strand comprises the sequence of SEQ ID NO: 20.
  • a double stranded miR-29 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand
  • the second strand comprises the sequence of SEQ ID NO: 2
  • the first strand comprises the sequence of SEQ ID NO: 26; b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand
  • the second strand comprises the sequence of SEQ ID NO: 3
  • the first strand comprises the sequence of SEQ ID NO: 17; c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand
  • the second strand comprises the sequence of SEQ ID NO: 4, and the first strand
  • the second strand comprises the sequence of SEQ ID NO: 5
  • the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand
  • the second strand comprises the sequence of SEQ ID NO: 11, and the first strand
  • the second strand comprises the sequence of SEQ ID NO: 12, and the first strand
  • a double stranded miR-96 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence of SEQ ID NO: 32 or 35.
  • a double stranded miR-96 mimic of this disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence from SEQ ID NOs: 38-41.
  • a double stranded miR-96 mimic of this disclosure comprises a first strand a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; b. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39; c. the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 40; or d. the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 41.
  • a double stranded miR-96 mimic of this disclosure comprises a first strand a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; or b. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39.
  • a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 33 and 36.
  • a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID NOs: 42 and 43.
  • a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 33, and the first strand
  • the second strand comprises the sequence of SEQ ID NO: 36, and the first strand
  • a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42.
  • a double stranded miR-183 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 34 and 37.
  • a double stranded miR-183 mimic of this disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID NOs: 44 and 45.
  • a double stranded miR-183 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44; or b. the second strand comprises the sequence of SEQ ID NO: 37, and the first strand comprises the sequence of SEQ ID NO: 45.
  • a double stranded miR-183 mimic of this disclosure comprises a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44.
  • kits for treating a fibrotic condition, and/or various sensory indications including ophthalmological indications, otic indications, anosmia and pain comprising administering any one or more of the double stranded mimics provided herein in a subject in need thereof.
  • the term“subject” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees, cynomologous monkeys, and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rabbits, rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like).
  • the subject is a mammal.
  • the subject is a human.
  • the term“subject” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees, cynomologous monkeys, and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rabbits, rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like).
  • the subject is a mammal.
  • the subject is a human. i. Therapeutic Indications
  • the present disclosure provides methods of treating, ameliorating, or preventing one or more conditions in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein.
  • the present disclosure provides methods of treating, ameliorating, or preventing fibrotic conditions in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of a miR-29a, miR- 29b, and/or miR-29c mimics described herein.
  • Fibrotic conditions that may be treated using miR-29 mimics of the disclosure include, but are not limited to, tissue fibrosis such as pulmonary fibrosis, cardiac fibrosis, hepatic fibrosis, kidney fibrosis, diabetic fibrosis, skeletal muscle fibrosis, and ocular fibrosis; and dermal/cutaneous fibrosis such as keloids, cutaneous sclerosis, systemic sclerosis (scleroderma), hypertrophic scars, hand/joint/tendon fibrosis, and Peyronie’s disease.
  • the fibrotic condition treated using the miR-29 mimics of the disclosure is idiopathic pulmonary fibrosis.
  • miR-29 agonists in treating certain fibrotic conditions is described in U.S. Patent No. 8,440,636, which is hereby incorporated by reference herein.
  • the miR-29 mimics of the disclosure are also useful for regulating the expression of extracellular matrix genes in a cell and treating associated conditions, such as tissue fibrosis, dermal fibrosis, including the uses and conditions described in WO 2009/018493, which is hereby incorporated by reference in its entirety.
  • administration of miR-29 mimics of the present disclosure reduces the expression or activity one or more extracellular matrix genes in cells of the subject. In other embodiments, administration of miR-29 mimics of the present disclosure reduces the expression or activity one or more collagen synthesis genes in cells of the subject. In yet other embodiments, administration of miR-29 mimics up-regulates the expression or activity one or more genes involved in the skin development, epidermis development, ectoderm development and cellular homeostasis. Cells of the subject where the expression or activity of various genes is regulated by miR-29 mimics of the disclosure include fibroblasts and epidermal cells.
  • administration of miR-29 mimics down-regulates inflammatory responses associated with fibrosis.
  • administration of miR-29 mimics reduces the levels of pro-inflammatory cytokines such as IL-12, IL-4, GCSF, and TNF-a in fibrosis patients.
  • miR-29 mimics may also reduce infiltration of immune effector cells such as neutrophils, lymphocytes, monocytes, and macrophages in fibrotic tissues or organs.
  • the present disclosure provides methods of regulating an extracellular matrix gene in a cell comprising contacting the cell with a miR-29 mimic of the present disclosure.
  • the disclosure provides methods of regulating a collagen synthesis gene in a cell comprising contacting the cell with a miR-29 mimic of the present disclosure.
  • the miR-29 mimic reduces the expression or activity of the extracellular matrix gene or the collagen synthesis gene.
  • the present disclosure provides methods of treating, ameliorating, or preventing fibrotic conditions in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein.
  • Fibrotic conditions that may be treated using miR29 mimics of the disclosure include, but are not limited to, skeletal muscle fibrosis, diabetic fibrosis, pulmonary (lung) fibrosis, cardiac fibrosis, cutaneous fibrosis (skin, dermal), liver fibrosis, renal fibrosis, and ocular fibrosis.
  • pulmonary fibrosis includes or may be caused by idiopathic pulmonary fibrosis, scleroderma ILD, rheumatoid arthritis ILD, bronchiolitis obliterans syndrome, chronic obstructive pulmonary disease, bronchopulmonary dysplasia, or any combination thereof.
  • cutaneous fibrosis includes or may be caused by cutaneous sclerosis, systemic sclerosis (scleroderma), dystrophic epidermolysis bullosa, keloid scar, keloids, hypertrophic scar, hand/joint/tendon fibrosis, and Peyronie’s disease, or any combination thereof.
  • cardiac fibrosis includes or may be caused by myocardial infarction, congestive heart failure, myocardial fibrosis, or any combination thereof.
  • Liver fibrosis includes or may be caused by NASH, Cirrhosis, lViral (HBV/HCV), or any combination thereof.
  • renal fibrosis may include but is not limited to diabetic nephropathy, IgA nephropathy, lupus nephitis, Non-lupus chronic kidney disease, or any combination thereof.
  • ocular fibrosis includes or may be caused by fibrosis of the cornea, retina, trabecular meshwork and/or pterygium, Fuch’s endothelial comeal dystrophy, glaucoma/trabeculectomy bleb, age related macular degeneration, dieabetic retinopathy, or any combination thereof.
  • the present disclosure provides methods of treating, ameliorating, or preventing any one of the fibroses described herein, muscular dystrophy, Dupuytren’s contractures, tendinopathies, osteoarthritis, inflammatory bowel disease, or any combination thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein.
  • the present disclosure provides methods of treating, ameliorating, or preventing inflammation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein. In some embodiments, the present disclosure provides methods of treating, ameliorating, or preventing inflammatory bowel disease. ii. Regulation of Gene Expression
  • the miR29 mimics of the disclosure are also useful for regulating the expression of genes, e.g. extracellular matrix genes in a cell.
  • the present disclosure provides methods of regulating at least one gene in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the present disclosure provides methods of downregulating the expression of at least one gene associated with fibrosis in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the regulated gene is Collal, Colla2, Col3al, Eln, Tgfb2, Tgfb3, Mfap 2, Igfl, Ctgf, 1113, Ccr2, Itgal, Cdhl, Col4a5, Smad3, Itgb6, Wntl l, Mfap2, Sparc, Acta2, Plau, Ccr2, Col4al, Col2al, Plat, Col4a2, Egf, Eln, Col5a2, Ctgf, Thbs2, Ccl2, Plau, Fstll, Col5al, Fbnl, or any combination thereof.
  • the present disclosure provides methods of downregulating the expression of at least one gene associated with collagen synthesis in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the present disclosure provides methods of downregulating the expression of at least one growth factor gene in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the growth factor gene is TGF-b2, TGF-b3, EGF, IGF1, IGF2, IGFBP5, PDGFA, PDGFC, or any combination thereof.
  • the present disclosure provides methods of downregulating the expression of at least one collagen gene (regulating collagen transcription/translation) in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the collagen gene is COL1A1, 1A2, 2A1, 3A1, 4A1, 4A2, 4A5, 5A1, 5A2, 5A3, 6A4, 6A5, 6A6, 8A1, 8A2, 9A1, 11A1, 12A1, 14A1, 22A1, 28A1, or any combination thereof.
  • the present disclosure provides methods of downregulating the expression of at least one gene associated with post-translational modification and/or triple helix formation in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the gene associated with post-translational modification and triple helix formation is HSP47, P4HA2, P4HA3, PLOD2, or any combination thereof.
  • the present disclosure provides methods of downregulating the expression of at least one gene associated with N- and C-terminal cleavage and secretion in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the gene associated with N- and C-terminal cleavage and secretion is PCOLCE, PCOLCE2, or any combination thereof.
  • the present disclosure provides methods of downregulating the expression of at least one gene associated with fibril cross-linking in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • the gene associated with fibril cross-linking is LOX, LOXL2, or any combination thereof.
  • the present disclosure provides methods of downregulating mature collagen fibrils in a cell, comprising contacting the cell with one or more miR29 mimics disclosed herein.
  • administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression or activity one or more extracellular matrix genes in cells of the subject. In other embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression or activity one or more collagen synthesis genes in cells of the subject. In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression or activity of at least one gene associated with collagen synthesis in cells of the subject. In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces collagen transcription/translation in cells of the subject.
  • administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one growth factor gene in cells of the subject.
  • the growth factor gene is TGF-b2, TGF-b3, EGF, IGF1, IGF2, IGFBP5, PDGFA or PDGFC.
  • any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one collagen gene (e.g. collagen transcription/translation) in cells of the subject.
  • the collagen gene is COL1A1, 1A2, 2A1, 3A1, 4A1, 4A2, 4A5, 5A1, 5A2, 5A3, 6A4, 6A5, 6A6, 8A1, 8A2, 9A1,
  • administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one gene associated with post- translational modification and/or triple helix formation in cells of the subject.
  • the gene associated with post-translational modification and triple helix formation is HSP47, P4HA2, P4HA3, PLOD2, or any combination thereof.
  • administering any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one gene associated with N- and C- terminal cleavage and secretion in cells of the subject.
  • the gene associated with N- and C-terminal cleavage and secretion is PCOLCE, PCOLCE2, or any combination thereof.
  • administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one gene associated with fibril cross- linking in cells of the subject.
  • the gene associated with fibril cross- linking is LOX, LOXL2, or any combination thereof.
  • administering downregulates mature collagen fibrils in cells and/or genes associated with fibril cross-linking of the subject.
  • administration of miR29 mimics to a subject up-regulates the expression or activity one or more genes involved in the skin development, epidermis development, ectoderm development and cellular homeostasis.
  • Cells of the subject where the expression or activity of various genes is regulated by miR29 mimics of the disclosure include fibroblasts and epidermal cells.
  • administration of miR29 mimics down regulates inflammatory responses associated with fibrosis.
  • administration of miR29 mimics reduces the levels of pro-inflammatory cytokines such as IL-12, IL-4, GCSF, and TNF-a in fibrosis patients.
  • Administration of miR29 mimics may also reduce infiltration of immune effector cells such as neutrophils, lymphocytes, monocytes, and macrophages in fibrotic tissues or organs.
  • the present disclosure provides methods of regulating the expression of one or more extracellular matrix genes in a cell comprising contacting the cell with a miR29 mimic of the present disclosure. In certain embodiments, the present disclosure provides methods of regulating the expression of one or more extracellular matrix genes in a subject comprising administering to the subject a miR29 mimic of the present disclosure.
  • the extracellular matrix genes is elastin (ELN), fibrillin 1 (FBN1), collagen type I od (COL1A1), collagen type I a2 (COL1A2), collagen type III oil (COL3A1), collagen type IV a4 (COL4A4), collagen type V a3 (COL5A3), collagen type XI al (COL11A1), collagen type V al (COL5A1), or collagen type IV a5 (COL4A5).
  • the miR-96, miR-182, and miR-183 double stranded mimics of the disclosure are useful for treating, preventing or ameliorating various sensory indications including ophthalmological indications (e.g. retinitis pigmentosa), otic indications (e.g. hearing loss that it ototoxicity-induced; noise-induced; age related, genetic), anosmia and pain, such as retinal degeneration or retinitis pigmentosa comprising administering any of the double stranded miR- 96, miR-182, and/or miR-183 mimics, or pharmaceutical compositions thereof to a subject in need thereof.
  • ophthalmological indications e.g. retinitis pigmentosa
  • otic indications e.g. hearing loss that it ototoxicity-induced; noise-induced; age related, genetic
  • anosmia and pain such as retinal degeneration or retinitis pigmentosa
  • the invention also encompasses methods for improving or restoring visual acuity in a subject in need thereof comprising administering any of the double stranded miR-96, miR- 182, and/or miR-183 mimics or pharmaceutical compositions thereof to a subject in need thereof.
  • the present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of one or more microRNA mimics according to the disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least two microRNA mimics of the disclosure and a pharmaceutically acceptable carrier or excipient.
  • the disclosure also encompasses embodiments where additional therapeutic agents may be administered along with miRNA mimics.
  • the additional therapeutic agents may be administered concurrently but in separate formulations or sequentially. In other embodiments, additional therapeutic agents may be administered at different times prior to after administration of miRNA mimics.
  • the microRNA mimics of the disclosure, or the pharmaceutical compositions thereof may be administered via one or more of the following routes of administration: intravenous, intraocular, intravitreal intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, by implantation, by inhalation, intrathecal, intraventricular, via the ear, or intranasal.
  • kits comprising any one or more of the double stranded mimics described herein, or pharmaceutical compositions thereof.
  • compositions or kits described herein are provided.
  • Embodiment 1 A double stranded microRNA mimic, comprising a first strand and a second strand, wherein the first strand comprises at least about 24 ribonucleotides and the second strand comprises at least about 22 ribonucleotides, wherein the second strand comprises a sequence that is substantially complementary to the first strand, and wherein the second strand comprises at least one stretch of three consecutive ribonucleotides comprising a 2’ fluoro- ribonucleotide.
  • Embodiment 2 The double stranded microRNA mimic of embodiment 1, wherein 65% - 100% of the ribonucleotides of the first strand and/or at least about 65%-100% of the ribonucleotides of the second strand are modified ribonucleotides.
  • Embodiment 3 The double stranded microRNA mimic of embodiment 1 or 2, wherein all the ribonucleotides of the first strand and all the ribonucleotides of the second strand are modified ribonucleotides.
  • Embodiment 4 The double stranded microRNA mimic of any one of embodiments 1-3, wherein the first strand comprises a mature miR-96 sequence, a mature miR-182 sequence, or a mature miR-183 sequence.
  • Embodiment 5. The double stranded microRNA mimic of any one of embodiments 1-3, wherein the first strand comprises a mature miR-29a sequence, a mature miR-29b sequence, or a mature miR-29c sequence.
  • Embodiment 6 A double stranded microRNA mimic comprising: (a) a first strand comprising about 21 nucleotides, and (b) a second strand comprising at least about 23 nucleotides, wherein the first strand comprises about 4 to about 9 deoxyribonucleotides and about 16 to about 21 ribonucleotides, and wherein the second strand comprises about 6 to about 7 deoxyribonucleotides and about 16 to about 17 ribonucleotides.
  • Embodiment 7 The double stranded microRNA mimic of embodiment 6, wherein all the ribonucleotides of the first strand and all the ribonucleotides of the second strand are modified ribonucleotides.
  • Embodiment 8 The double stranded microRNA mimic of embodiments 6 and 7, wherein at least one deoxyribonucleotide of the first strand and/or at least one
  • deoxyribonucleotide of the second strand is a modified deoxyribonucleotide.
  • Embodiment 9 The double stranded microRNA mimic of any one of
  • the first strand comprises a mature miR-96 sequence, a mature miR- 182 sequence, or a mature miR-183 sequence.
  • Embodiment 10 The double stranded microRNA mimic of any one of embodiments 6-8, wherein the first strand comprises a mature miR-29a sequence, a mature miR-29b sequence, or a mature miR-29c sequence.
  • Embodiment 11 A double stranded miR-29 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 1-16.
  • Embodiment 12 A double stranded miR-29 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected SEQ ID Nos: 17-31.
  • Embodiment 13 A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; the second strand comprises the sequence of
  • Embodiment 14 A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; the second strand comprises the sequence of
  • Embodiment 15 A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; the second strand comprises the sequence of
  • Embodiment 16 A double stranded miR-96 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence of SEQ ID NO: 32 or 35.
  • Embodiment 17 A double stranded miR-96 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 38-41.
  • Embodiment 18 A double stranded miR-96 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39; the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 40; or the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 41.
  • Embodiment 19 A double stranded miR-96 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; or the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39.
  • Embodiment 20 A double stranded miR-182 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 33 and 36.
  • Embodiment 21 A double stranded miR- 182 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 42 and 43.
  • Embodiment 22 A double stranded miR-182 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42; or the second strand comprises the sequence of SEQ ID NO: 36, and the first strand comprises the sequence of SEQ ID NO: 43.
  • Embodiment 23 A double stranded miR- 182 mimic comprising a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42.
  • Embodiment 24 A double stranded miR- 183 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 34 and 37.
  • Embodiment 25 A double stranded miR- 183 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 44 and 45.
  • Embodiment 26 A double stranded miR- 183 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44; or the second strand comprises the sequence of SEQ ID NO: 37, and the first strand comprises the sequence of SEQ ID NO: 45.
  • Embodiment 27 A double stranded miR- 183 mimic comprising a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44.
  • Embodiment 28 A pharmaceutical composition comprising any one or more of the double stranded microRNA mimics of embodiments 1-27.
  • Embodiment 29 Any one or more of the double stranded microRNA mimics of embodiments 1-27 or the pharmaceutical composition of embodiment 28, for use in the treatment of a fibrotic condition, or various sensory indications including ophthalmological indications, otic indications, anosmia and pain in a subject in need thereof.
  • Embodiment 30 A method of treating a fibrotic condition, or various sensory indications including ophthalmological indications, otic indications, anosmia and pain in a subject in need thereof, the method comprising administering to the subject any one or more of the mimics of embodiments 1-26, or the pharmaceutical composition of embodiment 27.
  • Example 1 In vitro studies of fully modified miR-29b mimics delivered by lipid transfection
  • miR-29b mimics were tested in IMR90 cell lines and were delivered via lipid transfection. IMR90 cells were plated in media containing serum at 8000 cells/well. 24 hours later, miR-29b were transfected in at dose levels of 50nM, 5nM, 0.5nM, and 0.05nM. After 72 hours of treatment with the miR-29b mimics, the cells were taken down and analyzed for levels of miR-29 target messenger RNAs by real-time PCR. Reduced mRNA levels of miR-29 target genes were observed for several of these miR-29b mimics demonstrating that these fully modified double stranded oligonucleotides were effective miR-29b mimics; see FIGS. 1, 2 and 3.
  • FIGS. 1 and 2 show the reduction in the mRNA levels of miR-29 target genes upon transfection of full modified miR-29b mimics, in which the sense/antisense strands are SEQ ID NO: 1/SEQ ID NO: 26, and SEQ ID NO: 2/SEQ ID NO: 17 (in FIG. 1); and SEQ ID NO: 3/SEQ ID NO: 26, and SEQ ID NO: 4/SEQ ID NO: 17 (in FIG. 2).
  • each of the sense strands SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 has a stretch of three consecutive 2’F modified nucleotides at different locations. See Table 1.
  • FIG. 3 shows the reduction in the mRNA levels of miR-29 target genes upon transfection of fully modified miR-29b mimics, in which the sense/antisense strands are either SEQ ID NO: 5/SEQ ID NO: 17, SEQ ID NO: 6/SEQ ID NO: 27, or SEQ ID NO: 7/SEQ ID NO: 28 ( see Table 1). Both strands are fully modified and each of the sense strands, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 has a stretch of three consecutive 2’F modified nucleotides at different locations.
  • FIGS. 3 and 4 further show the reduction in mRNA levels of miR-29 target genes upon transfection of miR-29b mimics having SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 as the sense strand, each of which comprises deoxyribomicleotide residues.
  • SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 are duplexed with SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively, each of which also comprises deoxyribomicleotide residues ( see Table 1).
  • FIG. 4 also shows the reduction in mRNA levels of miR-29 target genes upon transfection of miR-29b mimics having SEQ ID NO: 4 as the sense strand, and either SEQ ID NO: 18 or SEQ ID NO: 19 as the antisense strand.
  • SEQ ID NO: 18 and SEQ ID NO: 19 antisense strands comprise locked nucleic acid (LNA) residues.
  • results presented in FIGS. 1-4 show that all the miR-29b mimics tested are effective at repressing miR-29 target genes, Collal, Colla2, Col3al, Col4a5, and Fbnl.
  • the partially modified miR-29b mimic SEQ ID NO: 13/SEQ ID NO: 20
  • the partially modified miR-29b mimic localized primarily to the burned area 6 hours after administration of a single drop of the mimic.
  • the fully modified version (SEQ ID NO: 12/SEQ ID NO: 21) localized to other parts of the cornea, in addition to the area of the alkali bum.
  • IVT intravitreal injection
  • both the fully modified miR-29b mimic and the partially modified miR-29b mimic were detectable throughout the retina (FIG. 7). Oligonucleotide localization was seen throughout the plexiform layers of the retina and was most notable in the photoreceptor layer.
  • FIG. 9 shows the downregulation of target genes after day 2 (FIG. 9A) and day 3 (FIG. 9B) of treatment with the miR-96 mimics, in which the antisense/sense strands are either SEQ ID NO: 39/SEQ ID NO: 32; or SEQ ID NO: 38/SEQ ID NO: 32. Both strands of these miR-96 mimics are fully modified, and the sense strand, SEQ ID NO: 32, has a stretch of three consecutive 2’F nucleotides.
  • FIG. 9A shows the downregulation of target genes after day 2 (FIG. 9A) and day 3 (FIG. 9B) of treatment with the miR-96 mimics, in which the antisense/sense strands are either SEQ ID NO: 39/SEQ ID NO: 32; or SEQ ID NO: 38/SEQ ID NO: 32. Both strands of these miR-96 mimics are fully modified, and the sense strand, SEQ ID NO: 32, has a stretch of three consecutive 2’
  • FIG. 10 shows the downregulation of target genes after day 2 (FIG. 10A) and day 3 (FIG. 10B) of treatment with the miR-182 mimics, in which the antisense/sense strands are SEQ ID NO: 42/SEQ ID NO: 33; or SEQ ID NO: 43/SEQ ID NO: 36.
  • SEQ ID NO: 42 and SEQ ID NO: 33 are fully modified with the sense strand having a stretch of three consecutive 2’F nucleotides, whereas both SEQ ID NO: 43 and SEQ ID NO: 36 are partially modified strands.
  • FIG. 11 shows the downregulation of target genes after day 2 (FIG. 11A) and day 3 (FIG. 1 IB) of treatment with the miR-183 mimics, in which the antisense/sense strands are either SEQ ID NO: 44/SEQ ID NO: 34; or SEQ ID NO: 45/SEQ ID NO: 37.
  • Both SEQ ID NO: 44 and SEQ ID NO: 34 strands are fully modified with the sense strand having a stretch of three consecutive 2’F nucleotides, while both SEQ ID NO: 45 and SEQ ID NO: 37 are partially modified strands.

Abstract

Provided herein are synthetic, modified miRNA mimics that increase miRNA activity. In particular, the present disclosure provides synthetic, modified mimics of miR-29, miR-96, miR-182, and miR-183, as well as methods of making and uses thereof.

Description

MICRORNA MIMICS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/814,224, filed on March 5, 2019, the content of which is hereby incorporated by reference in its entirety for all purposes.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: MIRG_056_01WO_SeqList_ST25.txt, date recorded: March 5, 2020, file size 82.2 kilobytes).
BACKGROUND
[0003] MicroRNAs (miRNAs) are naturally occurring, short RNA molecules that are part of a ribonucleoprotein complex known as RISC (RNA-induced silencing complex) and act to repress translation of specific mRNA molecules. Levels of miRNAs have been demonstrated to be depressed below their normal levels in a variety of diseased states. Therefore a potential mode of therapy in these indications is the introduction of synthetic miRNAs to mimic the activity of natural miRNAs (miRNA mimics). However synthetic RNA molecules that are chemically identical to naturally occurring RNAs are not effective drugs due to their poor stability towards nucleases present in serum. Thus a variety of chemical modifications of the ribose sugar moiety and the phosphate linking group have been developed to confer nuclease stability on synthetic oligonucleotides. In the case of miRNAs, these chemical modifications need to be compatible with the various protein components that together produce an active RISC. More specifically the double stranded synthetic RNA molecule needs to be bound by the RISC followed by removal of the sense strand to produce an active RISC complex. Therefore, there remains a need for developing modified synthetic double-stranded RNA molecules that act as effective microRNA mimics, which can also allow removal of the sense strand. Provided herein are compositions and methods that address this need. SUMMARY
[0004] The present disclosure provides synthetic, modified miRNA mimics that increase miRNA activity. In particular, the present disclosure provides synthetic, modified mimics of miR-29, miR-96, miR-182, and miR-183, as well as methods of making and uses thereof. For example, the modified miR-29 mimics of the disclosure are useful for reducing collagen deposition and for the treatment and prevention of associated conditions, such as fibrosis. The modified miR-96, miR-182, and miR-183 mimics of the disclosure are useful for treating various sensory indications including ophthalmological indications, otic indications, anosmia and pain.
[0005] Accordingly in one aspect, provided herein are double stranded miR-29 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 1-16.
[0006] In another aspect, provided herein are double stranded miR-29 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected SEQ ID Nos: 17-31.
[0007] In another aspect, provided herein are double stranded miR-29 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; b) the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; c) the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; d) the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; e) the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; f) the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g) the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; h) the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29; i) the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30; j) the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31; k) the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; or 1) the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21.
[0008] In another aspect, provided herein are double stranded miR-29 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; b) the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; c) the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; d) the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; e) the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; f) the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g) the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; h) the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; or i) the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21.
[0009] In another aspect, provided herein are double stranded miR-96 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence of SEQ ID NO: 32 or 35.
[0010] In another aspect, provided herein are double stranded miR-96 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 38-41.
[0011] In another aspect, provided herein are double stranded miR-96 mimics comprising a first strand a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; b) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39; c) the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 40; or d) the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 41. [0012] In another aspect, provided herein are double stranded miR-96 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; or b) the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39.
[0013] In another aspect, provided herein are double stranded miR-182 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 33 and 36.
[0014] In another aspect, provided herein are double stranded miR-182 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 42 and 43.
[0015] In another aspect, provided herein are double stranded miR-182 mimics comprising a first strand and a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42; or b) the second strand comprises the sequence of SEQ ID NO: 36, and the first strand comprises the sequence of SEQ ID NO: 43.
[0016] In another aspect, provided herein are double stranded miR-182 mimics comprising a first strand and a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42.
[0017] In another aspect, provided herein are double stranded miR-183 mimics comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 34 and 37.
[0018] In another aspect, provided herein are double stranded miR-183 mimics comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID NOs: 44 and 45.
[0019] In another aspect, provided herein are double stranded miR-183 mimics comprising a first strand a second strand, wherein: a) the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44; or b) the second strand comprises the sequence of SEQ ID NO: 37, and the first strand comprises the sequence of SEQ ID NO: 45.
[0020] In another aspect, provided herein are double stranded miR-183 mimics comprising a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO:
34, and the first strand comprises the sequence of SEQ ID NO: 44.
[0021] In another aspect, provided herein are pharmaceutical compositions comprising one or more of the mimics as described in the above recited aspects, and present throughout the disclosure.
[0022] In another aspect, provided herein are methods of treating a fibrotic condition or various sensory indications including ophthalmological indications, otic indications, anosmia and pain, in a subject in need thereof, the method comprising administering to the subject one or more of the mimics or the pharmaceutical compositions thereof as described in the above recited aspects, and present throughout disclosure. In another aspect, provided herein are methods of making one or more of the mimics or the pharmaceutical compositions thereof, as described in the above recited aspects, and present throughout disclosure
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A-1E show graphs showing the downregulation of miR-29b target mRNAs, Col3al (FIG. 1A); Colla2 (FIG. IB); Col4a5 (FIG. 1C); Collal (FIG. ID); and Fbnl (FIG. IE), upon active transfection of miR-29b mimics into IMR90 cells. The sense and antisense strand of each of the transfected double stranded miRNAs are indicated on the graph. ‘U’ and‘M’ refer to untreated and mock-transfected controls.
[0024] FIGS. 2A-2D show graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 2A); Col3al (FIG. 2B); Col4a5 (FIG. 2C); and Fbnl (FIG. 2D), upon active transfection of miR-29b mimics into IMR90 cells. The sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph.‘U’ and‘M’ refer to untreated and mock-transfected controls.
[0025] FIGS. 3A-3E show graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 3A); Colla2 (FIG. 3B); Col3al (FIG. 3C); Col4a5 (FIG. 3D); and Fbnl (FIG. 3E), upon active transfection of miR-29b mimics into IMR90 cells. The sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph.‘U’ and‘M’ refer to untreated and mock-transfected controls.
[0026] FIGS. 4A-4E shows graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 4A); Colla2 (FIG. 4B); Col3al (FIG. 4C); Col4a5 (FIG. 4D); and Fbnl (FIG. 4E), upon active transfection of miR-29b mimics into IMR90 cells. The sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph. ‘U’ and‘M’ refer to untreated and mock-transfected controls.
[0027] FIGS. 5A-5B shows graphs showing the downregulation of miR-29b target mRNAs, Collal (FIG. 5A) and ACTA2 (FIG. 5B), upon passive delivery of miR-29b mimics into normal human lung fibroblasts (NHLF) cells. The sense and antisense strands of each of the transfected double stranded miRNAs are indicated on the graph.
[0028] FIGS. 6A-6B show representative images showing localization of miR-29b mimics to alkali burned corneas 6 hours after a single drop topical administration. The antisense and sense strands of the each of the miR-29b mimics are indicated on the image.
[0029] FIGS. 7A-7B show representative images showing the localization of the indicated fluorescently labelled miR-29b mimics delivered via intravitreal injection. The antisense and sense strands of the each of the miR-29b mimics are indicated on the image.
[0030] FIGS. 8A-8B show representative images showing the localization of the indicated fluorescently labeled miR-29b mimics delivered via subconjunctival injections. The antisense and sense strands of the each of the miR-29b mimics are indicated on the image.
[0031] FIGS. 9A-9B show graphs showing the downregulation of the indicated miR-96 target mRNAs at day 2 (FIG. 9A) and day 3 (FIG. 9B) following treatment of HeLa cells with miR-96 mimics. The sense and antisense strands of each of the miR-96 mimics is indicated on the graph.
[0032] FIGS. 10A-10B show graphs showing the downregulation of the indicated miR-182 target mRNAs at day 2 (FIG. 10A) and day 3 (FIG. 10B) following treatment of HeLa cells with the indicated miR- 182 mimics. The sense and antisense strands of each of the miR- 182 mimics is indicated on the graph.
[0033] FIGS. 1 lA-1 IB show graphs showing the downregulation of the indicated miR- 183 target mRNAs at day 2 (FIG. 11A) and day 3 (FIG. 1 IB) following treatment of HeFa cells with the indicated miR- 183 mimics. The sense and antisense strands of each of the miR- 183 mimics is indicated on the graph.
DETAILED DESCRIPTION
[0034] Over the last decade great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA by using synthetic miRNA mimics have been lagging behind.
[0035] Stability and increased efficacy are desired characteristics for improved miRNA mimics. Accordingly certain partially, heavily and fully modified miRNA mimics provided herein satisfy this need.
[0036] Throughout the disclosure, the term“microRNA mimic” may be used interchangeably with the terms“promiR,”“miR agonist,”“microRNA agonist,”“microRNA mimetic,”“miRNA mimic,” or“miR mimic;” the term“first strand” may be used interchangeably with the terms “antisense strand” or“guide strand”; the term“second strand” may be used interchangeably with the term“sense strand” or“passenger strand;” and the term“miR antagonist” may be used interchangeably with the terms“oligonucleotide inhibitor,”“antimiR”“antisense
oligonucleotide,”“miR antagomir” or“anti-microRNA oligonucleotide.”
I. Double Stranded MicroRNA Mimics
[0037] A double stranded miR-29 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-29a, miR-29b, or miR-29c sequence, and the second strand comprises a sequence that is substantially complementary to the first strand. Exemplary mimics are either partially, heavily, or fully modified. [0038] A double stranded miR-96 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-96 sequence, and the second strand comprises a sequence that is substantially complementary to the first strand. Exemplary mimics are either partially, heavily, or fully modified.
[0039] A double stranded miR-182 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-182 sequence, and the second strand comprises a sequence that is substantially complementary to the first strand. Exemplary mimics are either partially, heavily, or fully modified.
[0040] A double stranded miR-183 mimic according to the disclosure comprises a first strand and a second strand, wherein the first strand comprises a mature miR-183 sequence, and the second strand comprises a sequence that is substantially complementary to the first strand. Exemplary mimics are either partially, heavily, or fully modified.
[0041] The nucleotides that form the first and the second strand of the double stranded microRNA mimics may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. In certain embodiments, the first strand and the second strand of the microRNA mimic comprise ribonucleotides and/or modified ribonucleotides. The term “modified nucleotide” means a nucleotide where the nucleobase and/or the sugar moiety is modified relative to unmodified nucleotides.
[0042] In some embodiments, a double stranded microRNA mimic of the disclosure comprises a first strand and a second strand, wherein the first strand comprises at least about 24 ribonucleotides and the second strand comprises at least about 22 ribonucleotides, wherein the second strand comprises a sequence that is substantially complementary to the first strand, and wherein the second strand comprises at least one stretch of three consecutive ribonucleotides comprising a 2’ fluoro-ribonucleotide.
[0043] In some embodiments, a double stranded microRNA mimic of the disclosure comprises: (a) a first strand comprising about 21 nucleotides, and (b) a second strand comprising at least about 23 nucleotides, wherein the first strand comprises about 4 to about 9
deoxyribonucleotides and about 16 to about 21 ribonucleotides, and wherein the second strand comprises about 6 to about 7 deoxyribonucleotides and about 16 to about 17 ribonucleotides. [0044] In certain embodiments, the miRNA mimics of the disclosure have a first strand, whose sequence is identical to all or part of a mature miRNA sequence, and a second strand or a sense strand whose sequence is about 70% to about 100% complementary to the sequence of the first strand. In one embodiment, the first strand of the miRNA mimic is at least about 75, 80, 85, 90, 95, or 100% identical, including all integers there between, to the entire sequence of a mature, naturally occurring miRNA sequence. In certain embodiments, the first strand is about or is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the sequence of a mature, naturally-occurring miRNA, such as the mouse, human, or rat miRNA sequence.
Alternatively, the first strand may comprise 20, 21, 22, or 23 nucleotide positions in common with a mature, naturally-occurring miRNA as compared by sequence alignment algorithms and methods well known in the art.
[0045] It is understood that the sequence of the first strand is considered to be identical to the sequence of a mature miRNA even if the first strand includes a modified nucleotide instead of a naturally-occurring nucleotide. For example, if a mature, naturally-occurring miRNA sequence comprises a cytidine nucleotide at a specific position, the first strand of the mimic may comprise a modified cytidine nucleotide, such as 2’-fluoro-cytidine, at the corresponding position or if a mature, naturally -occurring miRNA sequence comprises a uridine nucleotide at a specific position, the miRNA region of the first strand of the mimic may comprise a modified uridine nucleotide, such as 2’-fluoro-uridine, 2’-O-methyl -uridine, 5-fluorouracil, or 4-thiouracil at the corresponding position. Thus, as long as the modified nucleotide has the same base-pairing capability as the nucleotide present in the mature, naturally-occurring miRNA sequence, the sequence of the first strand is considered to be identical to the mature, naturally-occurring miRNA sequence. In some embodiments, the first strand may include a modification of the 5’- terminal residue. For example, the first strand may have a 5’-terminal monophosphate. In some other embodiments, the first strand does not contain a 5’-terminal monophosphate.
[0046] In some embodiments, the second strand of the microRNA mimic is partially complementary to the sequence of the first strand. For example, the sequence of the second strand is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, inclusive of all values therebetween, complementary to the sequence of the first strand. In some other embodiments, the second strand is substantially complementary to the sequence of the first strand. For example, the second strand is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, inclusive of all values therebetween, complementary to the sequence of the first strand. In yet some other embodiments, the sequence of the second strand may be fully complementary to the first strand. In certain embodiments, about 19, 20, 21, 22, or 23 nucleotides of the complementary region of the second strand may be complementary to the first strand.
[0047] It is understood that the sequence of the second strand is considered to be
complementary to the first strand even if the second strand includes a modified nucleotide instead of a naturally-occurring nucleotide. For example, if the first strand sequence comprises a guanosine nucleotide at a specific position, the second strand may comprise a modified cytidine nucleotide, such as 2’-0-methyl-cytidine, at the corresponding position.
[0048] In some embodiments, the second strand comprises about 1, 2, 3, 4, 5, or 6 mismatches relative to the first strand. That is, up to 1, 2, 3, 4, 5, or 6 nucleotides between the first strand and the second strand may not be complementary. In one embodiment, the mismatches are not consecutive and are distributed throughout the second strand. In another embodiment, the mismatches are consecutive and may create a bulge.
[0049] In some embodiments, the first and/or the second strand of the mimic may comprise an overhang on the 5’ or 3’ end of the strands. In certain embodiments, the first strand comprises a 3’ overhang, i.e., a single-stranded region that extends beyond the duplex region, relative to the second strand. The 3’ overhang of the first strand may range from about one nucleotide to about four nucleotides. In certain embodiments, the 3’ overhang of the first strand may comprise 1 or 2 nucleotides. In some embodiments, the nucleotides comprising the 3’ overhang in the first strand are linked by phosphorothioate linkages. The nucleotides comprising the 3’ overhang in the first strand may include ribonucleotides, deoxyribonucleotides, modified nucleotides, or combinations thereof. In certain embodiments, the 3’ overhang in the first strand comprises two ribonucleotides. In one embodiment, the 3’ overhang of the first strand comprises two uridine nucleotides linked through a phosphorothioate linkage. In some embodiments, the first strand may not contain an overhang.
[0050] In some embodiments, the nucleotides in the second/sense strand of miR A mimics of the disclosure are linked by phosphodiester linkages and the nucleotides in the first/antisense strand are linked by phosphodiester linkages except for the last three nucleotides at the 3’ end which are linked to each other via phosphorothioate linkages.
[0051] In various embodiments, miRNA mimics of the present disclosure comprise a plurality of modified nucleotides. For instance, in one embodiment, the first strand of the mimic comprises one or more 2’-fluoro nucleotides. In another embodiment, the first strand may not include any modified nucleotide. In one embodiment, the second strand comprises one or more 2’-O-methyl modified nucleotides.
[0052] The modified nucleotides that may be used in the microRNA mimics of the disclosure can include nucleotides with a base modification or substitution. The natural or unmodified bases in RNA are the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U) (DNA has thymine (T)). In contrast, modified bases, also referred to as heterocyclic base moieties, include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines), 7-methylguanine and 7- methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
[0053] In some embodiments, the microRNA mimics can have nucleotides with modified sugar moieties. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2’, 3’ or 4’ positions and sugars having substituents in place of one or more hydrogen atoms of the sugar. In certain embodiments, the sugar is modified by having a substituent group at the 2’ position. In additional embodiments, the sugar is modified by having a substituent group at the 3’ position. In other embodiments, the sugar is modified by having a substituent group at the 4’ position. It is also contemplated that a sugar may have a modification at more than one of those positions, or that an RNA molecule may have one or more nucleotides with a sugar modification at one position and also one or more nucleotides with a sugar modification at a different position. [0054] Sugar modifications contemplated in the miRNA mimics include, but are not limited to, a substituent group selected from: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl.
[0055] In some embodiments, miRNA mimics have a sugar substituent group selected from the following: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, Cl, Br, CN, OCN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, or similar substituents. In one embodiment, the modification includes 2’-methoxy ethoxy (2’-0-CH2CH20CH3, which is also known as 2’-0-(2-methoxyethyl) or 2’-MOE), that is, an alkoxyalkoxy group. Another modification includes 2’-dimethylaminooxy ethoxy, that is, a 0(CH2)20N(CH3)2 group, also known as 2’-DMAOE and 2’-dimethylaminoethoxy ethoxy (also known in the art as 2 -0- dimethyl-amino-ethoxy-ethyl or 2’-DMAEOE), that is, 2’-0-CH2-0-CH2-N(CH3)2.
[0056] Sugar substituent groups on the 2’ position (2’-) may be in the arabino (up) position or ribo (down) position. One 2’-arabino modification is 2’-F. Other similar modifications may also be made at other positions on the sugar moiety, particularly the 3’ position of the sugar on the 3’ terminal nucleoside or in 2’-5’ linked oligonucleotides and the 5’ position of 5’ terminal nucleotide.
[0057] In certain embodiments, the sugar modification is a 2’-0-alkyl (e.g. 2’-0-methyl, 2’-0- methoxyethyl), 2’-halo (e.g., 2’-fluoro, 2’-chloro, 2’-bromo), and 4’ thio modifications. For instance, in some embodiments, the first strand of the miR-29a, miR-29b, or miR-29c mimic comprises one or more 2’ fluoro nucleotides. In another embodiment, the first strand of the mimics has no modified nucleotides. In yet another embodiment, the second strand of miR-29a, miR-29b, or miR-29c mimic comprises one or more 2’-0-methyl modified nucleotides.
[0058] The first and the second strand of microRNA mimics of the disclosure can also include backbone modifications, such as one or more phosphorothioate, phosphorodithioate, phosphotriester, boranophosphate, alkylphosphonates, phosphoramidates, phosphordiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, or
phosphonocarboxylate linkages, where the linkage is the normal 3’-5’ linkage, 2’-5’ linked analog or inverted linkages such as 3’-3’, 5’-5’ and 2’-2\ The first and/or the second strand of microRNA mimics of the disclosure can also include modifications on the sugar residue, such as ribose modification/ replacement. For instance, the first and/or the second strand of the microRNA mimics of the disclosure may include one or more morpholino, peptide nucleic acids, serinol nucleic acids, locked nucleic acids (LNA), and unlocked nucleic acids.
[0059] In some embodiments, the microRNA mimics are conjugated to an agent such as a steroid (cholesterol), a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand to facilitate targeting, delivery, and/or stability. In some embodiments, the agent is attached to the first strand or second strand of the microRNA mimic at its 3’ or 5’ end through a linker or a spacer group. In some embodiments, the agent is cholesterol, a cholesterol derivative, cholic acid or a cholic acid derivative.
[0060] In various embodiments, miR-29, miR-96, miR-182, and miR-183 mimics according to the present disclosure comprise the exemplary first and second strands listed in Tables 1-4 below. Definitions of the modifications are presented in Table 5.
Table 1: Sense/Passenger/Second strands of exemplary miR-29 mimics, with exemplary duplex combinations
Figure imgf000014_0001
Figure imgf000015_0001
Table 2: Antisense/Guide/First strands of exemplary miR-29 mimics, with exemplary duplex combinations
Figure imgf000015_0002
Figure imgf000016_0001
Table 3: Sequences of sense/passenger/second strands of exemplary miR96/182/183 mimics, with exemplary duplex combinations
Figure imgf000016_0002
Figure imgf000017_0001
Table 4: Sequences of antisense/guide/first strands of exemplary miR96/182/183 mimics, with exemplary duplex combinations
Figure imgf000017_0002
Figure imgf000018_0001
Table 5: Definitions of Abbreviations
Figure imgf000018_0002
Figure imgf000019_0001
[0061] In some embodiments, a double stranded miR-29 mimic of the disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 1-16.
[0062] In some embodiments, a double stranded miR-29 mimic of the disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence selected SEQ ID NOs: 17-31.
[0063] In some embodiments, a double stranded miR-29 mimic of the disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26;
b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17;
c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26;
d. the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19;
e. the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17;
f. the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27;
g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; h. the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29;
i. the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30;
j . the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31;
k. the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17;
l. the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21;
m. the second strand comprises the sequence of SEQ ID NO: 13, and the first strand comprises the sequence of SEQ ID NO: 20.
[0064] A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26;
b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17;
c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26;
d. the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19;
e. the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17;
f. the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27;
g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28;
h. the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29; i. the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30;
j . the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31;
k. the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17;
l. the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21; or
m. the second strand comprises the sequence of SEQ ID NO: 13, and the first strand comprises the sequence of SEQ ID NO: 20.
[0065] In some embodiments, a double stranded miR-29 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; d. the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; e. the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; f. the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; h. the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29; i. the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30; j. the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31; k. the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; l. the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21; or m. the second strand comprises the sequence of SEQ ID NO: 13, and the first strand comprises the sequence of SEQ ID NO: 20.
[0066] In some embodiments, a double stranded miR-29 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand
comprises the sequence of SEQ ID NO: 26; b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand
comprises the sequence of SEQ ID NO: 17; c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand
comprises the sequence of SEQ ID NO: 26; d. the second strand comprises the sequence of SEQ ID NO: 4, and the first strand
comprises the sequence of SEQ ID NO: 17, 18 or 19; e. the second strand comprises the sequence of SEQ ID NO: 5, and the first strand
comprises the sequence of SEQ ID NO: 17; f. the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand
comprises the sequence of SEQ ID NO: 28; h. the second strand comprises the sequence of SEQ ID NO: 11, and the first strand
comprises the sequence of SEQ ID NO: 17; or i. the second strand comprises the sequence of SEQ ID NO: 12, and the first strand
comprises the sequence of SEQ ID NO: 17 or 21.
[0067] In some embodiments, a double stranded miR-96 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence of SEQ ID NO: 32 or 35.
[0068] In some embodiments, a double stranded miR-96 mimic of this disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence from SEQ ID NOs: 38-41.
[0069] In some embodiments, a double stranded miR-96 mimic of this disclosure comprises a first strand a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; b. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39; c. the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 40; or d. the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 41.
[0070] In some embodiments, a double stranded miR-96 mimic of this disclosure comprises a first strand a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; or b. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39.
[0071] In some embodiments, a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 33 and 36.
[0072] In some embodiments, a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID NOs: 42 and 43.
[0073] In some embodiments, a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 33, and the first strand
comprises the sequence of SEQ ID NO: 42; or b. the second strand comprises the sequence of SEQ ID NO: 36, and the first strand
comprises the sequence of SEQ ID NO: 43.
[0074] In some embodiments, a double stranded miR-182 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42.
[0075] In some embodiments, a double stranded miR-183 mimic of this disclosure comprises a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 34 and 37.
[0076] In some embodiments, a double stranded miR-183 mimic of this disclosure comprises a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID NOs: 44 and 45. [0077] In some embodiments, a double stranded miR-183 mimic of this disclosure comprises a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44; or b. the second strand comprises the sequence of SEQ ID NO: 37, and the first strand comprises the sequence of SEQ ID NO: 45.
[0078] In some embodiments, a double stranded miR-183 mimic of this disclosure comprises a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44.
II. Method of Use of Double Stranded microRNA Mimics
[0079] Provided herein are methods of use of the double stranded miRNA mimics provided herein.
[0080] For example, provided herein are methods of treating a fibrotic condition, and/or various sensory indications including ophthalmological indications, otic indications, anosmia and pain, comprising administering any one or more of the double stranded mimics provided herein in a subject in need thereof.
[0081] As used herein, the term“subject” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees, cynomologous monkeys, and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rabbits, rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal.
In exemplary embodiments, the subject is a human.
B. miR-29 Mimics
[0082] Provided herein are methods of use of the miR29 mimics provided herein. [0083] As used herein, the term“subject” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees, cynomologous monkeys, and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rabbits, rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal.
In exemplary embodiments, the subject is a human. i. Therapeutic Indications
[0084] The present disclosure provides methods of treating, ameliorating, or preventing one or more conditions in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein.
[0085] In various embodiments, the present disclosure provides methods of treating, ameliorating, or preventing fibrotic conditions in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of a miR-29a, miR- 29b, and/or miR-29c mimics described herein. Fibrotic conditions that may be treated using miR-29 mimics of the disclosure include, but are not limited to, tissue fibrosis such as pulmonary fibrosis, cardiac fibrosis, hepatic fibrosis, kidney fibrosis, diabetic fibrosis, skeletal muscle fibrosis, and ocular fibrosis; and dermal/cutaneous fibrosis such as keloids, cutaneous sclerosis, systemic sclerosis (scleroderma), hypertrophic scars, hand/joint/tendon fibrosis, and Peyronie’s disease. In one embodiment, the fibrotic condition treated using the miR-29 mimics of the disclosure is idiopathic pulmonary fibrosis. Use of miR-29 agonists in treating certain fibrotic conditions is described in U.S. Patent No. 8,440,636, which is hereby incorporated by reference herein.
[0086] The miR-29 mimics of the disclosure are also useful for regulating the expression of extracellular matrix genes in a cell and treating associated conditions, such as tissue fibrosis, dermal fibrosis, including the uses and conditions described in WO 2009/018493, which is hereby incorporated by reference in its entirety.
[0087] In some embodiments, administration of miR-29 mimics of the present disclosure reduces the expression or activity one or more extracellular matrix genes in cells of the subject. In other embodiments, administration of miR-29 mimics of the present disclosure reduces the expression or activity one or more collagen synthesis genes in cells of the subject. In yet other embodiments, administration of miR-29 mimics up-regulates the expression or activity one or more genes involved in the skin development, epidermis development, ectoderm development and cellular homeostasis. Cells of the subject where the expression or activity of various genes is regulated by miR-29 mimics of the disclosure include fibroblasts and epidermal cells. In some embodiments, administration of miR-29 mimics down-regulates inflammatory responses associated with fibrosis. For example, administration of miR-29 mimics reduces the levels of pro-inflammatory cytokines such as IL-12, IL-4, GCSF, and TNF-a in fibrosis patients.
Administration of miR-29 mimics may also reduce infiltration of immune effector cells such as neutrophils, lymphocytes, monocytes, and macrophages in fibrotic tissues or organs.
[0088] In certain embodiments, the present disclosure provides methods of regulating an extracellular matrix gene in a cell comprising contacting the cell with a miR-29 mimic of the present disclosure. In some embodiments, the disclosure provides methods of regulating a collagen synthesis gene in a cell comprising contacting the cell with a miR-29 mimic of the present disclosure. Upon treatment or contact, the miR-29 mimic reduces the expression or activity of the extracellular matrix gene or the collagen synthesis gene.
[0089] In some embodiments, the present disclosure provides methods of treating, ameliorating, or preventing fibrotic conditions in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein. Fibrotic conditions that may be treated using miR29 mimics of the disclosure include, but are not limited to, skeletal muscle fibrosis, diabetic fibrosis, pulmonary (lung) fibrosis, cardiac fibrosis, cutaneous fibrosis (skin, dermal), liver fibrosis, renal fibrosis, and ocular fibrosis.
[0090] In some embodiments, pulmonary fibrosis includes or may be caused by idiopathic pulmonary fibrosis, scleroderma ILD, rheumatoid arthritis ILD, bronchiolitis obliterans syndrome, chronic obstructive pulmonary disease, bronchopulmonary dysplasia, or any combination thereof.
[0091] In some embodiments, cutaneous fibrosis includes or may be caused by cutaneous sclerosis, systemic sclerosis (scleroderma), dystrophic epidermolysis bullosa, keloid scar, keloids, hypertrophic scar, hand/joint/tendon fibrosis, and Peyronie’s disease, or any combination thereof.
[0092] In some embodiments, cardiac fibrosis includes or may be caused by myocardial infarction, congestive heart failure, myocardial fibrosis, or any combination thereof.
[0093] In some embodiments, Liver fibrosis includes or may be caused by NASH, Cirrhosis, lViral (HBV/HCV), or any combination thereof.
[0094] In some embodiments, renal fibrosis may include but is not limited to diabetic nephropathy, IgA nephropathy, lupus nephitis, Non-lupus chronic kidney disease, or any combination thereof.
[0095] In some embodiments, ocular fibrosis includes or may be caused by fibrosis of the cornea, retina, trabecular meshwork and/or pterygium, Fuch’s endothelial comeal dystrophy, glaucoma/trabeculectomy bleb, age related macular degeneration, dieabetic retinopathy, or any combination thereof.
[0096] In some embodiments, the present disclosure provides methods of treating, ameliorating, or preventing any one of the fibroses described herein, muscular dystrophy, Dupuytren’s contractures, tendinopathies, osteoarthritis, inflammatory bowel disease, or any combination thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein.
[0097] In some embodiments, the present disclosure provides methods of treating, ameliorating, or preventing inflammation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one of the miR29 mimics described herein. In some embodiments, the present disclosure provides methods of treating, ameliorating, or preventing inflammatory bowel disease. ii. Regulation of Gene Expression
[0098] The miR29 mimics of the disclosure are also useful for regulating the expression of genes, e.g. extracellular matrix genes in a cell. In some embodiments, the present disclosure provides methods of regulating at least one gene in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein. [0099] In some embodiments, the present disclosure provides methods of downregulating the expression of at least one gene associated with fibrosis in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein. In particular embodiments, the regulated gene is Collal, Colla2, Col3al, Eln, Tgfb2, Tgfb3, Mfap 2, Igfl, Ctgf, 1113, Ccr2, Itgal, Cdhl, Col4a5, Smad3, Itgb6, Wntl l, Mfap2, Sparc, Acta2, Plau, Ccr2, Col4al, Col2al, Plat, Col4a2, Egf, Eln, Col5a2, Ctgf, Thbs2, Ccl2, Plau, Fstll, Col5al, Fbnl, or any combination thereof.
[0100] In some embodiments, the present disclosure provides methods of downregulating the expression of at least one gene associated with collagen synthesis in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein.
[0101] In some embodiments, the present disclosure provides methods of downregulating the expression of at least one growth factor gene in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein. In particular embodiments, the growth factor gene is TGF-b2, TGF-b3, EGF, IGF1, IGF2, IGFBP5, PDGFA, PDGFC, or any combination thereof.
[0102] In some embodiments, the present disclosure provides methods of downregulating the expression of at least one collagen gene (regulating collagen transcription/translation) in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein. In particular embodiments, the collagen gene is COL1A1, 1A2, 2A1, 3A1, 4A1, 4A2, 4A5, 5A1, 5A2, 5A3, 6A4, 6A5, 6A6, 8A1, 8A2, 9A1, 11A1, 12A1, 14A1, 22A1, 28A1, or any combination thereof.
[0103] In some embodiments, the present disclosure provides methods of downregulating the expression of at least one gene associated with post-translational modification and/or triple helix formation in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein. In particular embodiments, the gene associated with post-translational modification and triple helix formation is HSP47, P4HA2, P4HA3, PLOD2, or any combination thereof.
[0104] In some embodiments, the present disclosure provides methods of downregulating the expression of at least one gene associated with N- and C-terminal cleavage and secretion in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein. In particular embodiments, the gene associated with N- and C-terminal cleavage and secretion is PCOLCE, PCOLCE2, or any combination thereof. [0105] In some embodiments, the present disclosure provides methods of downregulating the expression of at least one gene associated with fibril cross-linking in a cell comprising contacting the cell with one or more miR29 mimics disclosed herein. In particular embodiments, the gene associated with fibril cross-linking is LOX, LOXL2, or any combination thereof.
[0106] In some embodiments, the present disclosure provides methods of downregulating mature collagen fibrils in a cell, comprising contacting the cell with one or more miR29 mimics disclosed herein.
[0107] In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression or activity one or more extracellular matrix genes in cells of the subject. In other embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression or activity one or more collagen synthesis genes in cells of the subject. In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression or activity of at least one gene associated with collagen synthesis in cells of the subject. In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces collagen transcription/translation in cells of the subject.
[0108] In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one growth factor gene in cells of the subject. In particular embodiments, the growth factor gene is TGF-b2, TGF-b3, EGF, IGF1, IGF2, IGFBP5, PDGFA or PDGFC.
[0109] In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one collagen gene (e.g. collagen transcription/translation) in cells of the subject. In particular embodiments, the collagen gene is COL1A1, 1A2, 2A1, 3A1, 4A1, 4A2, 4A5, 5A1, 5A2, 5A3, 6A4, 6A5, 6A6, 8A1, 8A2, 9A1,
11A1, 12A1, 14A1, 22A1, 28A1, or any combination thereof.
[0110] In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one gene associated with post- translational modification and/or triple helix formation in cells of the subject. In particular embodiments, the gene associated with post-translational modification and triple helix formation is HSP47, P4HA2, P4HA3, PLOD2, or any combination thereof.
[0111] In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one gene associated with N- and C- terminal cleavage and secretion in cells of the subject. In particular embodiments, the gene associated with N- and C-terminal cleavage and secretion is PCOLCE, PCOLCE2, or any combination thereof.
[0112] In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject reduces the expression of at least one gene associated with fibril cross- linking in cells of the subject. In particular embodiments, the gene associated with fibril cross- linking is LOX, LOXL2, or any combination thereof.
[0113] In some embodiments, administration of any one of the miR29 mimics of the present disclosure to a subject downregulates mature collagen fibrils in cells and/or genes associated with fibril cross-linking of the subject.
[0114] In yet other embodiments, administration of miR29 mimics to a subject up-regulates the expression or activity one or more genes involved in the skin development, epidermis development, ectoderm development and cellular homeostasis. Cells of the subject where the expression or activity of various genes is regulated by miR29 mimics of the disclosure include fibroblasts and epidermal cells. In some embodiments, administration of miR29 mimics down regulates inflammatory responses associated with fibrosis. For example, administration of miR29 mimics reduces the levels of pro-inflammatory cytokines such as IL-12, IL-4, GCSF, and TNF-a in fibrosis patients. Administration of miR29 mimics may also reduce infiltration of immune effector cells such as neutrophils, lymphocytes, monocytes, and macrophages in fibrotic tissues or organs.
[0115] In certain embodiments, the present disclosure provides methods of regulating the expression of one or more extracellular matrix genes in a cell comprising contacting the cell with a miR29 mimic of the present disclosure. In certain embodiments, the present disclosure provides methods of regulating the expression of one or more extracellular matrix genes in a subject comprising administering to the subject a miR29 mimic of the present disclosure. In some embodiments, the extracellular matrix genes is elastin (ELN), fibrillin 1 (FBN1), collagen type I od (COL1A1), collagen type I a2 (COL1A2), collagen type III oil (COL3A1), collagen type IV a4 (COL4A4), collagen type V a3 (COL5A3), collagen type XI al (COL11A1), collagen type V al (COL5A1), or collagen type IV a5 (COL4A5).
B. miR-96, miR-182, miR-183 Mimics
[0116] The miR-96, miR-182, and miR-183 double stranded mimics of the disclosure are useful for treating, preventing or ameliorating various sensory indications including ophthalmological indications (e.g. retinitis pigmentosa), otic indications (e.g. hearing loss that it ototoxicity-induced; noise-induced; age related, genetic), anosmia and pain, such as retinal degeneration or retinitis pigmentosa comprising administering any of the double stranded miR- 96, miR-182, and/or miR-183 mimics, or pharmaceutical compositions thereof to a subject in need thereof. The invention also encompasses methods for improving or restoring visual acuity in a subject in need thereof comprising administering any of the double stranded miR-96, miR- 182, and/or miR-183 mimics or pharmaceutical compositions thereof to a subject in need thereof.
III. Pharmaceutical Compositions
[0117] The present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of one or more microRNA mimics according to the disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least two microRNA mimics of the disclosure and a pharmaceutically acceptable carrier or excipient.
IV. Routes of Administration
[0118] The disclosure also encompasses embodiments where additional therapeutic agents may be administered along with miRNA mimics. The additional therapeutic agents may be administered concurrently but in separate formulations or sequentially. In other embodiments, additional therapeutic agents may be administered at different times prior to after administration of miRNA mimics. [0119] The microRNA mimics of the disclosure, or the pharmaceutical compositions thereof may be administered via one or more of the following routes of administration: intravenous, intraocular, intravitreal intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, by implantation, by inhalation, intrathecal, intraventricular, via the ear, or intranasal.
V. Kits and Articles of Manufacture
[0120] The present disclosure provides kits comprising any one or more of the double stranded mimics described herein, or pharmaceutical compositions thereof.
[0121] The present disclosure also provides articles of manufacture comprising any one of the compositions or kits described herein.
ILLUSTRATIVE EMBODIMENTS
[0122] The disclosure may be further defined by reference to the following illustrative embodiments.
[0123] Embodiment 1. A double stranded microRNA mimic, comprising a first strand and a second strand, wherein the first strand comprises at least about 24 ribonucleotides and the second strand comprises at least about 22 ribonucleotides, wherein the second strand comprises a sequence that is substantially complementary to the first strand, and wherein the second strand comprises at least one stretch of three consecutive ribonucleotides comprising a 2’ fluoro- ribonucleotide.
[0124] Embodiment 2. The double stranded microRNA mimic of embodiment 1, wherein 65% - 100% of the ribonucleotides of the first strand and/or at least about 65%-100% of the ribonucleotides of the second strand are modified ribonucleotides.
[0125] Embodiment 3. The double stranded microRNA mimic of embodiment 1 or 2, wherein all the ribonucleotides of the first strand and all the ribonucleotides of the second strand are modified ribonucleotides.
[0126] Embodiment 4. The double stranded microRNA mimic of any one of embodiments 1-3, wherein the first strand comprises a mature miR-96 sequence, a mature miR-182 sequence, or a mature miR-183 sequence. [0127] Embodiment 5. The double stranded microRNA mimic of any one of embodiments 1-3, wherein the first strand comprises a mature miR-29a sequence, a mature miR-29b sequence, or a mature miR-29c sequence.
[0128] Embodiment 6. A double stranded microRNA mimic comprising: (a) a first strand comprising about 21 nucleotides, and (b) a second strand comprising at least about 23 nucleotides, wherein the first strand comprises about 4 to about 9 deoxyribonucleotides and about 16 to about 21 ribonucleotides, and wherein the second strand comprises about 6 to about 7 deoxyribonucleotides and about 16 to about 17 ribonucleotides.
[0129] Embodiment 7. The double stranded microRNA mimic of embodiment 6, wherein all the ribonucleotides of the first strand and all the ribonucleotides of the second strand are modified ribonucleotides.
[0130] Embodiment 8. The double stranded microRNA mimic of embodiments 6 and 7, wherein at least one deoxyribonucleotide of the first strand and/or at least one
deoxyribonucleotide of the second strand is a modified deoxyribonucleotide.
[0131] Embodiment 9. The double stranded microRNA mimic of any one of
embodiments 6-8, wherein the first strand comprises a mature miR-96 sequence, a mature miR- 182 sequence, or a mature miR-183 sequence.
[0132] Embodiment 10. The double stranded microRNA mimic of any one of embodiments 6-8, wherein the first strand comprises a mature miR-29a sequence, a mature miR-29b sequence, or a mature miR-29c sequence.
[0133] Embodiment 11. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 1-16.
[0134] Embodiment 12. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected SEQ ID Nos: 17-31.
[0135] Embodiment 13. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29; the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30; the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31; the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21; the second strand comprises the sequence of SEQ ID NO: 13, and the first strand comprises the sequence of SEQ ID NO: 20.
[0136] Embodiment 14. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29; the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30; the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31; the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21; or the second strand comprises the sequence of SEQ ID NO: 13, and the first strand comprises the sequence of SEQ ID NO: 20.
[0137] Embodiment 15. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26; the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19; the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17; the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27; the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28; the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; or the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21.
[0138] Embodiment 16. A double stranded miR-96 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence of SEQ ID NO: 32 or 35.
[0139] Embodiment 17. A double stranded miR-96 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 38-41.
[0140] Embodiment 18. A double stranded miR-96 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39; the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 40; or the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 41.
[0141] Embodiment 19. A double stranded miR-96 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; or the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39.
[0142] Embodiment 20. A double stranded miR-182 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 33 and 36.
[0143] Embodiment 21. A double stranded miR- 182 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 42 and 43.
[0144] Embodiment 22. A double stranded miR-182 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42; or the second strand comprises the sequence of SEQ ID NO: 36, and the first strand comprises the sequence of SEQ ID NO: 43.
[0145] Embodiment 23. A double stranded miR- 182 mimic comprising a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42.
[0146] Embodiment 24. A double stranded miR- 183 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 34 and 37.
[0147] Embodiment 25. A double stranded miR- 183 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 44 and 45.
[0148] Embodiment 26. A double stranded miR- 183 mimic comprising a first strand a second strand, wherein: the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44; or the second strand comprises the sequence of SEQ ID NO: 37, and the first strand comprises the sequence of SEQ ID NO: 45.
[0149] Embodiment 27. A double stranded miR- 183 mimic comprising a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44. [0150] Embodiment 28. A pharmaceutical composition comprising any one or more of the double stranded microRNA mimics of embodiments 1-27.
[0151] Embodiment 29. Any one or more of the double stranded microRNA mimics of embodiments 1-27 or the pharmaceutical composition of embodiment 28, for use in the treatment of a fibrotic condition, or various sensory indications including ophthalmological indications, otic indications, anosmia and pain in a subject in need thereof.
[0152] Embodiment 30. A method of treating a fibrotic condition, or various sensory indications including ophthalmological indications, otic indications, anosmia and pain in a subject in need thereof, the method comprising administering to the subject any one or more of the mimics of embodiments 1-26, or the pharmaceutical composition of embodiment 27.
[0153] The following examples are included for illustrative purposes and are not intend to limit the scope of the disclosure.
[0154] All patent and non-patent documents referenced throughout this disclosure are incorporated by reference herein in their entirety for all purposes.
EXAMPLES
[0155] Sequences of the sense (passenger) and the corresponding antisense (guide) strands of the miR-29b mimics and miR- 183/182/96 mimics used in the Examples are listed in Tables 1-4.
Example 1 - In vitro studies of fully modified miR-29b mimics delivered by lipid transfection
[0156] miR-29b mimics were tested in IMR90 cell lines and were delivered via lipid transfection. IMR90 cells were plated in media containing serum at 8000 cells/well. 24 hours later, miR-29b were transfected in at dose levels of 50nM, 5nM, 0.5nM, and 0.05nM. After 72 hours of treatment with the miR-29b mimics, the cells were taken down and analyzed for levels of miR-29 target messenger RNAs by real-time PCR. Reduced mRNA levels of miR-29 target genes were observed for several of these miR-29b mimics demonstrating that these fully modified double stranded oligonucleotides were effective miR-29b mimics; see FIGS. 1, 2 and 3. The double stranded miR-29b mimics tested in this experiment are indicated in FIGS. 1, 2 and 3; and the sequences thereof are listed in Tables 1 and 2. [0157] FIGS. 1 and 2 show the reduction in the mRNA levels of miR-29 target genes upon transfection of full modified miR-29b mimics, in which the sense/antisense strands are SEQ ID NO: 1/SEQ ID NO: 26, and SEQ ID NO: 2/SEQ ID NO: 17 (in FIG. 1); and SEQ ID NO: 3/SEQ ID NO: 26, and SEQ ID NO: 4/SEQ ID NO: 17 (in FIG. 2). Both strands are fully modified, and each of the sense strands, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 has a stretch of three consecutive 2’F modified nucleotides at different locations. See Table 1.
[0158] FIG. 3 shows the reduction in the mRNA levels of miR-29 target genes upon transfection of fully modified miR-29b mimics, in which the sense/antisense strands are either SEQ ID NO: 5/SEQ ID NO: 17, SEQ ID NO: 6/SEQ ID NO: 27, or SEQ ID NO: 7/SEQ ID NO: 28 ( see Table 1). Both strands are fully modified and each of the sense strands, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 has a stretch of three consecutive 2’F modified nucleotides at different locations.
[0159] FIGS. 3 and 4 further show the reduction in mRNA levels of miR-29 target genes upon transfection of miR-29b mimics having SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 as the sense strand, each of which comprises deoxyribomicleotide residues. Also, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 are duplexed with SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively, each of which also comprises deoxyribomicleotide residues ( see Table 1).
[0160] FIG. 4 also shows the reduction in mRNA levels of miR-29 target genes upon transfection of miR-29b mimics having SEQ ID NO: 4 as the sense strand, and either SEQ ID NO: 18 or SEQ ID NO: 19 as the antisense strand. Both SEQ ID NO: 18 and SEQ ID NO: 19 antisense strands comprise locked nucleic acid (LNA) residues.
[0161] Thus, the results presented in FIGS. 1-4 show that all the miR-29b mimics tested are effective at repressing miR-29 target genes, Collal, Colla2, Col3al, Col4a5, and Fbnl.
Example 2 - In vitro studies of fully modified miR-29b mimics delivered by passive transfection
[0162] The activity of fully modified miR-29b mimics with and without cholesterol conjugates was studied in normal human lung fibroblasts (NHLF) upon passive delivery. As shown in FIGs. 5 A and 5B, the unconjugated fully modified miR-29b mimic (sense/ antisense = SEQ ID NO: 4/SEQ ID NO: 17) did not cause downregulation of the target genes studied. However, downregulation of the target genes was observed when the cholesterol conjugated fully modified miR-29b mimic (sense/ antisense = SEQ ID NO: 11/SEQ ID NO: 17 and SEQ ID NO: 12/SEQ ID NO: 17) were passively transfected. The sense strand in each of the miR-29b mimics used in this experiment is fully modified and has a stretch of three consecutive 2’F modified nucleotides at different locations.
Example 3 - In vivo studies with fully modified miR-29b mimics
[0163] Fluorescein conjugated miR-29b mimics that were fully modified (sense/ antisense = SEQ ID NO: 12/SEQ ID NO: 21) or partially modified (sense/ antisense = SEQ ID NO: 13/SEQ ID NO: 20) were delivered to the eye via multiple routes of administration including topical eye drops, subconjunctival injection and intravitreal injection. When administered topically to alkali burned eyes (FIG. 6), the partially modified miR-29b mimic (SEQ ID NO: 13/SEQ ID NO: 20) localized primarily to the burned area 6 hours after administration of a single drop of the mimic. In contrast, the fully modified version (SEQ ID NO: 12/SEQ ID NO: 21) localized to other parts of the cornea, in addition to the area of the alkali bum. In the case of administration via intravitreal injection (IVT) both the fully modified miR-29b mimic and the partially modified miR-29b mimic were detectable throughout the retina (FIG. 7). Oligonucleotide localization was seen throughout the plexiform layers of the retina and was most notable in the photoreceptor layer.
[0164] Pronounced differences between the mimics with different chemistries were observed with subconjunctival injection (FIG. 8). While the lightly modified miR-29b mimic (SEQ ID NO: 13/SEQ ID NO: 20) could not be detected in the conjunctiva, trabecular meshwork or cornea of the treated eye, the fully modified version (SEQ ID NO: 12/SEQ ID NO: 21) showed strong signals at the injection site as well as the trabecular meshwork and cornea adjacent to the injection site. Both compounds did however show uptake into the retina.
Example 4 - In vitro evaluation of fully-modified miR-183/182/96 mimics
[0165] Hela cells were incubated with fully modified and partially modified miR-183, 182 and 96 mimics (FIGS. 9-11) at 0.5, 1, 3 and 5 mM for 48 h. Cells were subsequently harvested to assess target engagement. Target genes previously tested in a rat retinal cell system as well as new targets identified by literature review were tested to see whether they were suitable to assess miRNA mimic activity. Eventually, 4 indirect target genes (SOCS3, GABARAP, B2M and Serping) of the cluster were chosen for subsequent studies. Those genes were expected to be down-regulated with miRNA mimic treatment.
[0166] FIG. 9 shows the downregulation of target genes after day 2 (FIG. 9A) and day 3 (FIG. 9B) of treatment with the miR-96 mimics, in which the antisense/sense strands are either SEQ ID NO: 39/SEQ ID NO: 32; or SEQ ID NO: 38/SEQ ID NO: 32. Both strands of these miR-96 mimics are fully modified, and the sense strand, SEQ ID NO: 32, has a stretch of three consecutive 2’F nucleotides. FIG. 9 also shows the downregulation of target genes after day 2 and day 3 of treatment with partially modified miR-96 mimics, in which the antisense/sense strands are either SEQ ID NO: 40/SEQ ID NO: 35; or SEQ ID NO: 41/SEQ ID NO: 35, respectively.
[0167] FIG. 10 shows the downregulation of target genes after day 2 (FIG. 10A) and day 3 (FIG. 10B) of treatment with the miR-182 mimics, in which the antisense/sense strands are SEQ ID NO: 42/SEQ ID NO: 33; or SEQ ID NO: 43/SEQ ID NO: 36. SEQ ID NO: 42 and SEQ ID NO: 33 are fully modified with the sense strand having a stretch of three consecutive 2’F nucleotides, whereas both SEQ ID NO: 43 and SEQ ID NO: 36 are partially modified strands.
[0168] FIG. 11 shows the downregulation of target genes after day 2 (FIG. 11A) and day 3 (FIG. 1 IB) of treatment with the miR-183 mimics, in which the antisense/sense strands are either SEQ ID NO: 44/SEQ ID NO: 34; or SEQ ID NO: 45/SEQ ID NO: 37. Both SEQ ID NO: 44 and SEQ ID NO: 34 strands are fully modified with the sense strand having a stretch of three consecutive 2’F nucleotides, while both SEQ ID NO: 45 and SEQ ID NO: 37 are partially modified strands.
[0169] Together, the results presented in FIGS. 9-11 show that fully modified miR-96, 182 and 183 mimics repress target genes to a greater degree when compared to the corresponding partially modified versions of these mimics.

Claims

1. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 1-16.
2. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected SEQ ID Nos: 17-31.
3. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26;
b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17;
c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26;
d. the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19;
e. the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17;
f. the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27;
g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28;
h. the second strand comprises the sequence of SEQ ID NO: 8, and the first strand comprises the sequence of SEQ ID NO: 29;
i. the second strand comprises the sequence of SEQ ID NO: 9, and the first strand comprises the sequence of SEQ ID NO: 30;
j . the second strand comprises the sequence of SEQ ID NO: 10, and the first strand comprises the sequence of SEQ ID NO: 31;
k. the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; or
l. the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21.
4. A double stranded miR-29 mimic comprising a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 1, and the first strand comprises the sequence of SEQ ID NO: 26;
b. the second strand comprises the sequence of SEQ ID NO: 2, and the first strand comprises the sequence of SEQ ID NO: 17;
c. the second strand comprises the sequence of SEQ ID NO: 3, and the first strand comprises the sequence of SEQ ID NO: 26;
d. the second strand comprises the sequence of SEQ ID NO: 4, and the first strand comprises the sequence of SEQ ID NO: 17, 18 or 19;
e. the second strand comprises the sequence of SEQ ID NO: 5, and the first strand comprises the sequence of SEQ ID NO: 17;
f. the second strand comprises the sequence of SEQ ID NO: 6, and the first strand comprises the sequence of SEQ ID NO: 27;
g. the second strand comprises the sequence of SEQ ID NO: 7, and the first strand comprises the sequence of SEQ ID NO: 28;
h. the second strand comprises the sequence of SEQ ID NO: 11, and the first strand comprises the sequence of SEQ ID NO: 17; or
i. the second strand comprises the sequence of SEQ ID NO: 12, and the first strand comprises the sequence of SEQ ID NO: 17 or 21.
5. A double stranded miR-96 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence of SEQ ID NO: 32 or 35.
6. A double stranded miR-96 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 38-41.
7. A double stranded miR-96 mimic comprising a first strand a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38;
b. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39; c. the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 40; or
d. the second strand comprises the sequence of SEQ ID NO: 35, and the first strand comprises the sequence of SEQ ID NO: 41.
8. A double stranded miR-96 mimic comprising a first strand and a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 38; or
b. the second strand comprises the sequence of SEQ ID NO: 32, and the first strand comprises the sequence of SEQ ID NO: 39.
9. A double stranded miR- 182 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID Nos: 33 and 36.
10. A double stranded miR- 182 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID Nos: 42 and 43.
11. A double stranded miR- 182 mimic comprising a first strand and a second strand,
wherein:
a. the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42; or
b. the second strand comprises the sequence of SEQ ID NO: 36, and the first strand comprises the sequence of SEQ ID NO: 43.
12. A double stranded miR- 182 mimic comprising a first strand and a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 33, and the first strand comprises the sequence of SEQ ID NO: 42.
13. A double stranded miR- 183 mimic comprising a first strand and a second strand, wherein the second strand comprises a sequence selected from SEQ ID NOs: 34 and 37.
14. A double stranded miR- 183 mimic comprising a first strand and a second strand, wherein the first strand comprises a sequence selected from SEQ ID NOs: 44 and 45.
15. A double stranded miR-183 mimic comprising a first strand a second strand, wherein: a. the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44; or
b. the second strand comprises the sequence of SEQ ID NO: 37, and the first strand comprises the sequence of SEQ ID NO: 45.
16. A double stranded miR-183 mimic comprising a first strand a second strand, wherein the second strand comprises the sequence of SEQ ID NO: 34, and the first strand comprises the sequence of SEQ ID NO: 44.
17. A pharmaceutical composition comprising any one or more of the mimics of claims 1-16.
18. A method of treating a fibrotic condition or various sensory indications including
ophthalmological indications, otic indications, anosmia and pain, in a subject in need thereof, the method comprising administering to the subject any one or more of the mimics of claims 1-16, or the pharmaceutical composition of claim 17.
19. The use of any one or more of the mimics of claims 1-16, or the pharmaceutical
composition of claim 17 for the treatment of a fibrotic condition or various sensory indications including ophthalmological indications, otic indications, anosmia and pain in a subject in need thereof.
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