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WO2018047148A1 - Composés pour inhibition du miarn - Google Patents

Composés pour inhibition du miarn Download PDF

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Publication number
WO2018047148A1
WO2018047148A1 PCT/IB2017/055499 IB2017055499W WO2018047148A1 WO 2018047148 A1 WO2018047148 A1 WO 2018047148A1 IB 2017055499 W IB2017055499 W IB 2017055499W WO 2018047148 A1 WO2018047148 A1 WO 2018047148A1
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mirna
sequence
mirna inhibitor
mir
inhibitor
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PCT/IB2017/055499
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English (en)
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Jeremy Baryza
Neil KUBICA
Steven Patrick MULLIN
Susanne SWALLEY
Jan Weiler
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Novartis Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--

Definitions

  • the invention relates generally to drug, bio-affecting and body treating compositions, generally to organic compounds, particularly to nucleic acid expression inhibitors, and more particularly to anti-miRNA oligonucleotides.
  • MicroRNAs are small, naturally-occurring, double-stranded RNA molecules that participate in gene regulation. They are non-coding, but target the mRNA of protein-coding genes for cleavage or repression of translation. miRNAs appear in many organisms, including plants, invertebrates such as nematodes, and vertebrates, from fish such as zebrafish to humans. They are a family of molecules, differing from each other in sequence. Many hundreds have been identified in humans, and there may be as many as 1000 or more. Up to 60% or more of human genes may be regulated, at least in part, by miRNAs. Friedman et al. 2009 Genome Res. 19: 92-105.
  • miRNAs are sequence-specific, though this specificity is imperfect.
  • the key recognition site is the seed region sequence (positions 2-8 of the antisense strand).
  • a single miRNA can regulate several genes with slightly different sequences.
  • a single gene can be regulated by several miRNAs.
  • miRNAs are known to be overexpressed in many cancers.
  • Several miRNAs are located at the sites of translocation breakpoints or deletions linked to human leukemias. Calin et al. 2004 Proc. Natl. Acad. Sci. USA 101 : 2999-3004. Altered miRNA expression has been reported in leukemia, lung cancer, and colon cancer. Calin et al. 2002 Proc. Natl. Acad. Sci. USA 99: 15524-9; Calin et al. 2004 Proc. Natl. Acad. Sci. USA 101 : 1 1755-60; and Michael et al. 2003 Mol. Cancer Res. 1 : 889-91 .
  • miRNA miR-21 blocks expression of critical apoptosis-related genes in human glioblastoma tissues; knockdown of this miRNA leads to increased apoptotic cell death. Chan et al. 2005 Cancer Res. 65: 6029-6033. Over-expression of miR- 21 , which is also frequently highly expressed in haematological malignancies, results in a pre-B malignant lymphoid-like phenotype; miR-21 inactivation leads to apoptosis and tumor regression. Medina et al. 2010 Nat. 467: 86-90.
  • the miRNA inhibitor comprises a polymer, e.g., a molecule comprising multiple mers (subunits), wherein a mer can be a nucleotide (nt), modified nucleotide or nucleotide analogue.
  • a polymer e.g., a molecule comprising multiple mers (subunits), wherein a mer can be a nucleotide (nt), modified nucleotide or nucleotide analogue.
  • the polymer is about 6 to about 21 mers long.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region (or a portion thereof) of a pri-miRNA hairpin or pre-miRNA hairpin, wherein (a) the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the first to fifth nt counting from the 5' end of the Dicer-excised region; or (b) the region of complementarity is at the 5' end of the miRNA inhibitor, and the mer at the 5' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • the invention provides an miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region (or a portion thereof) of a pri-miRNA hairpin or pre-miRNA hairpin, wherein the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • the miRNA inhibitor anneals to a pri-miRNA and/or pre-miRNA in a sequence-dependent manner and comprises a sequence which can straddle a Dicer or Drosha cleavage site and/or anneal to the Dicer-excised region of the pri-miRNA and/or pre- miRNA and reduce production of the miRNA from the pri-miRNA and/or pre-miRNA.
  • the entire miRNA inhibitor is complementary to the Dicer- excised region (or a portion thereof); thus, the miRNA inhibitor consists of a region of complementarity.
  • the region of complementarity has 1 to 3 mismatches to the Dicer-excised region or Drosha-excised region.
  • the miRNA inhibitor is at least about 6 mers long. In a ninth embodiment, the miRNA inhibitor is no more than about 21 mers long. In a tenth embodiment, the miRNA inhibitor is about 6 to about 21 . In an eleventh embodiment, the miRNA inhibitor is 6 to 21 mers long.
  • the miRNA inhibitor comprises a first strand (thus, the miRNA inhibitor is single-stranded). In a thirteenth embodiment, the miRNA inhibitor comprises a first and a second strand (thus, the miRNA inhibitor is double-stranded).
  • the miRNA inhibitor comprises the full sequence of, or at least about 15 contiguous positions of any miRNA inhibitor disclosed herein.
  • the sequence of the miRNA inhibitor is the sequence of any miRNA inhibitor disclosed herein, and the miRNA inhibitor is modified or unmodified, and comprises any combination of nucleotides, modified nucleotides and nucleotide analogues.
  • the invention provides a method of reducing the formation of a miRNA from a pri-miRNA and/or pre-miRNA, the method comprising the step of contacting the pri-miRNA and/or pre-miRNA with a miRNA inhibitor as described herein.
  • the invention provides a method of treating, preventing or ameliorating a disease or condition related to expression or over-expression a miRNA (e.g., a miRNA-related disease), wherein the method comprises the step of administering a therapeutically effective amount of a miRNA inhibitor of the invention.
  • the miRNA inhibitor is administered along with a pharmaceutically acceptable carrier.
  • the miRNA inhibitor inhibits miR-145, miR-208a, miR- 137 or miR-122.
  • the invention provides methods of using these miRNA inhibitors to inhibit the miRNA, and/or to treat, prevent or ameliorate a disease related to expression or over-expression of the miRNA.
  • the invention provides for the use of the miR-145 inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miR-145, including Marfan syndrome, Loeys-Dietz syndrome, Takayasu's arteritis, giant cell arteritis, liver fibrosis, kidney fibrosis, heart fibrosis, respiratory disease, pulmonary arterial hypertension (PAH), idiopathic pulmonary fibrosis (IPF), Asthma, Chronic Obstructive Pulmonary Disease
  • the invention provides for the use of the miR-208a inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miR-208a, including Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, heart disease, heart failure, acute coronary syndrome (ACS), hypertrophic heart dysfunction, acute myocardial infarction, obesity, fatty liver, metabolic syndrome, and Type II diabetes mellitus (T2DM).
  • NASH Non-alcoholic fatty liver disease
  • NASH Non-alcoholic steatohepatitis
  • cirrhosis cirrhosis
  • heart disease heart failure
  • ACS acute coronary syndrome
  • T2DM Type II diabetes mellitus
  • the invention provides for the use of the miRNA inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miRNA-137, or miRNA-137 variants with a single nucleotide polymorphism, including schizophrenia.
  • the invention provides for the use of the miRNA-122 inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miRNA-122, including viral infections, including HCV infection, and dysregulated homeostasis, including high cholesterol and fatty liver disease.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region (or a portion thereof) of a pri-miRNA hairpin or pre-miRNA hairpin, wherein (a) the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the first to fifth nt counting from the 5' end of the Dicer-excised region; or (b) the region of complementarity is at the 5' end of the miRNA inhibitor, and the mer at the 5' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region (or a portion thereof) of a pri-miRNA hairpin or pre-miRNA hairpin, wherein the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • the entire miRNA inhibitor is complementary to the Dicer-excised region (or a portion thereof); thus, the miRNA inhibitor consists of a region of complementarity.
  • the region of complementarity has 1 to 3 mismatches to the Dicer-excised region.
  • the miRNA inhibitor can anneal to a pri-miRNA and/or pre-miRNA in a sequence-dependent manner and comprises a sequence that can straddle a cleavage site and/or anneal to the Dicer-excised region of the pri-miRNA and/or pre-miRNA and reduce production of the miRNA from the pri-miRNA and/or pre-miRNA.
  • the miRNA inhibitors of the invention can mediate invasion of the stem loop of a miRNA, leading to repression of Dicer- or Drosha-mediated cleavage and thus decrease production of the mature miRNA.
  • the miRNA inhibitor inhibits miR-145, miR-208a, miR-137 or miR-122.
  • the invention provides methods of using these miRNA inhibitors to inhibit the miRNA, and/or to treat, prevent or ameliorate a disease related to expression or over-expression of the miRNA.
  • FIG. 1 shows an anti-miR oligonucleotide (AMO) walk.
  • AMO anti-miR oligonucleotide
  • FIG. 2 shows miR-145 stem loop hairpin binding and in vitro Dicer processing screens.
  • A In vitro binding of AMOs (analytes) was assessed by surface plasmon resonance assay (SPR) and plotted as association (report point) versus the mapped position (5' end of AMO) on the miR-145 stem loop hairpin (ligand).
  • B Gel-based in vitro Dicer enzymatic assay assessing processing of the pre-miR-145 hairpin (substrate) ⁇ preincubation with AMO inhibitors. Processing is reported as densitometry of the miR-145 duplex (product) relative to total intensity in the lane and normalized to product production in the absence of AMO inhibitors.
  • D, E Mapping of 05AF (2'MOE chemistry) (D) and 15MB (2'F/2'MOE mixmer chemistry) (E) as representative examples of the two major 'hot spots' for binding and biogenesis inhibition. Alignments are consistent with AMO nucleation in the single-stranded terminal loop from the 5' and 3' ends, respectively and disruption of the miR-145 hairpin structure at the distal Dicer cut site.
  • FIG. 3 shows confirmation of miR-145 stem loop hairpin invasion using biochemical and cell-based assays.
  • A Full kinetic analysis by SPR of 15MB and LXH874 (a peptide nucleic acid (PNA) targeting the same nucleotide sequence as 15MB) binding to miR-145 stem loop hairpin ligand.
  • B Gel-based in vitro Dicer processing assay highlighting binding in the absence of Dicer (Lane 1 vs. Lane2; gel shift) and processing inhibition in the presence of Dicer (Lane 3 vs. Lane 4; duplex production) for 15MB and the pre-miR-145 stem-loop hairpin substrate.
  • PNA peptide nucleic acid
  • C Comparison of 15MB and LXH874 with respect to binding (gel-shift) and processing inhibition (product production) in the presence of Dicer.
  • D Inhibition of TGFpi -dependent 'phenotypic switching' in primary human pulmonary arterial smooth muscle cells (PASMCs) using an a-smooth muscle actin immunofluorescence assay. 20HB and 15MB are highlighted as representative examples of inactive and active AMOs, respectively.
  • FIG. 4 shows miR-208a stem loop hairpin binding and in vitro Dicer processing screens.
  • A In vitro binding of AMOs (analytes) was assessed by SPR and plotted as association (report point) versus the mapped position (5' end of AMO) on the miR-208a stem loop hairpin (ligand).
  • B Gel-based in vitro Dicer enzymatic assay assessing processing of the pre-miR-208a hairpin (substrate) ⁇ pre-incubation with AMO inhibitors. Processing is reported as densitometry of the miR-208a duplex (product) relative to total intensity in the lane and normalized to product production in the absence of AMO inhibitors.
  • D, E Mapping of 57MF (2'MOE chemistry) (D) and 68ME (2'MOE chemistry) (E) as representative examples of the two major 'hot spots' for binding and biogenesis inhibition. Alignments are consistent with AMO nucleation in the single-stranded terminal loop from the 5' and 3' ends, respectively and disruption of the miR- 208a hairpin structure at the distal Dicer cut site.
  • FIG. 5 shows confirmation of miR-208a stem loop hairpin invasion using biochemical assays.
  • A Full kinetic analysis by SPR of 57MF and 68ME to miR-208a stem loop hairpin ligand.
  • B Comparison of 57MF and 68ME with respect to binding (gel-shift) and processing inhibition (product production) in the presence of Dicer.
  • FIG. 6 shows the AMO inhibition of pre-miR-137 processing by Dicer.
  • A Plot of the amount of remaining miR-137 hairpin after incubation with Dicer and AMO molecules. The amount of pre-miRNA hairpin remaining is compared to the amount of pre-miRNA incubated without Dicer enzyme. A representative gel showing the pre-miRNA protection in the presence of AMOs is beneath the plotted data.
  • B Structure of pri-miR-137 hairpin with mature miR-137 sequence in red, the loop sequence in blue, and the passenger strand in yellow.
  • C Mapping of AMOs to the pri-miR-137 hairpin to show binding regions.
  • FIG. 7 shows the AMO inhibition of pre-miR-122 processing by Dicer.
  • A Plot of the amount of mature miR-122 produced by Dicer in the presence of different AMOs. The production of mature miR-122 is compared to the processing in the absence of any inhibitor.
  • B Structure of pri-miR-122 hairpin with mature miR-122 sequence in red, the loop sequence in blue, and the passenger strand in yellow.
  • C Mapping of AMOs to the pri-miR- 122 hairpin to show binding regions.
  • a mature miRNA is formed from a pre-miRNA, which is in turn formed from a pri- miRNA.
  • This multi-step process requires cleavage by Drosha and Dicer, and involves various other biological components.
  • Cleavage by Drosha and Dicer excise RNA adjacent to, but not a part of, the mature miRNA.
  • the region excised by Dicer is termed herein the "Dicer-excised region” (and the like), and is a region that is not part of the mature miRNA.
  • the region excised by Drosha is termed herein the "Drosha-excised region” (and the like), and is also a region that is not part of the mature miRNA.
  • the miRNA inhibitors of the invention prevent or reduce formation of the miRNA inhibitor from the pri-miRNA and/or pre- miRNA.
  • the miRNA inhibitors of the invention can invade the stem loop hairpin structure of a pri-miRNA and/or pre-miRNA, binding to the loop or other part of the Dicer-excised region or Drosha-excised region and/or straddling and preventing or reducing cleavage at a Dicer cleavage site and/or a Drosha cleavage site.
  • the miRNA inhibitors of the invention bind to the Dicer-excised region of the pre-miRNA or pri-miRNA. This prevents cleavage and formation of the mature miRNA.
  • the miRNA inhibitor of the invention is a polymer, a molecule comprising multiple mers or subunits, wherein each mer is a nucleotide, modified nucleotide, or nucleotide analogue.
  • the miRNA inhibitor of the invention comprises a sequence of 15 contiguous positions of or comprises the sequence of any miRNA inhibitor disclosed herein. In a thirty-sixth embodiment, the sequence of the miRNA inhibitor of the invention has the sequence of any miRNA inhibitor disclosed herein.
  • the invention provides a method of reducing the formation of a miRNA from a pri-miRNA and/or pre-miRNA, the method comprising the step of contacting the pri-miRNA and/or pre-miRNA with a miRNA inhibitor of the invention as described herein.
  • the invention provides specific miRNA inhibitors that inhibit miR-145, miR-208a, miR-137 or miR-122.
  • the invention provides methods of using these miR-145, miR-208a, miR-137 or miR-122 miRNA inhibitors in treating or ameliorating diseases related to expression or over-expression of these miRNAs.
  • microRNA or “miRNA” or “miR” or the like mean small RNA molecules that naturally participate in gene regulation; these are naturally produced in the cells of many organisms, including plants, and invertebrates and vertebrates, including humans.
  • a miRNA comprises an antisense strand and (in some contexts) a sense strand.
  • a miRNA antisense strand is generally 18-25 nt long and binds to fully or partially complementary target mRNAs, thereby targeting them for degradation or translational inhibition.
  • the antisense strand can be fully or partially complementary to the sense strand. There may be as many as four mismatches or more between an antisense and a corresponding sense strand.
  • RNA refers to the combination of the antisense strand and the complementary sense strand. In other scientific references, the term “miRNA” refers only to the antisense strand. In still other references, the
  • miRNA / miRNA* is used to refer to the combination of a miRNA antisense and sense strand, wherein the term “miRNA” refers to the antisense strand and the term
  • RNA* refers to the sense strand. More recent research has shown that the sense strand is sometimes active (e.g., incorporated into the miRISC). Thus, the terms “5p” and “3p” are sometimes used to refer to strands that derive from the 5' side or the 3' side of the pre- miRNA hairpin, respectively.
  • the antisense strand is termed the "guide strand”
  • the sense strand is accordingly termed the "passenger strand”.
  • the term "miRNA” will usually indicate the double-stranded molecule, including both anti-sense and sense strand.
  • pri-miRNA means an intermediate in miRNA biogenesis. miRNA biogenesis is complex. The genes encoding miRNAs are much longer than the processed mature miRNA. Many miRNAs are known to reside in introns of their host genes and share their regulatory elements and primary transcript, and have a similar expression profile. Some miRNA genes are transcribed from their own promoters. A microRNA is transcribed by RNA polymerase II as a large RNA precursor that is called a "pri-miRNA" and comprises a 5' cap and a poly-A tail.
  • the pri-miRNA is processed in the nucleus by the microprocessor complex, comprising the RNase III enzyme Drosha, and the double-stranded-RNA-binding protein, Pasha/DGCR8.
  • the result is the "pre-miRNA", which is approximately 70 nucleotides in length and folded into an imperfect stem-loop structure.
  • the pre-miRNA is then exported into the cytoplasm by the karyopherin exportin 5 (exp5) and Ran-GTP complex.
  • Ran ras-related nuclear protein
  • Ran is a small GTP binding protein that belongs to the RAS superfamily and is essential for the translocation of RNA and proteins through the nuclear pore complex.
  • the Ran GTPase binds Exp5 and forms a nuclear heterotrimer with pre-miRNAs. Once in the cytoplasm, the pre-miRNA undergoes an additional processing step by the RNase III enzyme Dicer, generating the miRNA.
  • “pri-miRNA” or “pri-miRNA hairpin” is a naturally occurring RNA structure that is processed by Drosha to yield a pre-miRNA hairpin.
  • pre-miRNA or "pre-miRNA hairpin” mean a naturally occurring RNA hairpin that is processed by Dicer to yield the mature miRNA.
  • the "loop” (or “loop sequence”) is the RNA nucleotide sequence that is between the Dicer cleavage positions in a pre- miRNA hairpin, e.g., the sequence extending from the 5'-cleavage Dicer cleavage site to the 3'-Dicer cleavage site of a pre-miRNA. Additional information about miRNAs and their biogenesis is available in the scientific literature, for example: Lee et al. 2004 EMBO J. 23: 4051 -4060; Han et al. 2004 Genes Dev.
  • miRNA inhibitor means any composition or method capable of reducing the level, activity, stability and/or production of a miRNA.
  • miRNA inhibitors include, inter alia, anti-miRNA oligonucleotides (AMOs) and other types of inhibitors.
  • AMOs anti-miRNA oligonucleotides
  • Several authors have described methods of inhibiting miRNAs. General inhibition of miRNA production can be mediated by factors inhibiting components which produce miRNAs, such as factors which generally inhibit Dicer, where the inhibitor effect is global, rather than specific to a particular miRNA. Specific inhibitors targeting individual miRNAs have been described, including AMOs.
  • anti-miRNA oligonucleotide means a single or double-stranded molecule comprising nucleotides, modified nucleotides and/or nucleotide analogs, which recognizes (anneals to) a miRNA (or pre-miRNA or pri-miRNA) in a sequence-specific manner, and reduces the level, activity, stability and/or production of a miRNA.
  • This sequence specificity can be imperfect, as one or more mismatches may exist between the miRNA and the AMO.
  • An AMO can comprise an antisense strand, or an antisense strand and a sense strand. Many AMOs are reviewed in, for example: Lennox et al. 201 1 Gene Ther. 18: 1 1 1 1 1 - 1 120.
  • polynucleotide means a polymer of nucleotides.
  • nucleotide or is meant a molecule consisting of a phosphate, a sugar and a base.
  • a nucleotide can form a subunit of a polynucleotide such as a strand of RNA or DNA, or a subunit of a polymer or other molecule comprising one or more nucleotides and one or more of another type of molecule such as a modified nucleotide or nucleotide analogue.
  • a ribonucleotide In the case of a ribonucleotide, the sugar is a ribose; a ribonucleotide is a subunit of RNA. In the case of a deoxyribonucleotide, the sugar is deoxyribose; a deoxyribonucleotide is a subunit of DNA.
  • a polynucleotide can comprise RNA, DNA, or both; one or more nucleotide can be modified or unmodified.
  • nucleotide encompass unmodified or modified variants of these molecules (e.g., a modified nucleotide or modified polynucleotide); a polynucleotide can comprise any number of one or more nucleotide, modified nucleotide and/or nucleotide analogues.
  • modified nucleotide or “modified polynucleotide” and the like, as used herein, mean any nucleotide or polynucleotide with one or more modifications or substitutions of the phosphate, base, or sugar.
  • nucleotide analogue or “nucleotide analog” or “nt analog” or “nucleotide substitute” or the like, as used herein, mean a polymer, or individual component ("mer") of such a polymer, which is not a nucleotide, but which is capable of mediating sequence- specific annealing to a nucleotide.
  • minor groove binders mean any molecule which is capable of binding non-covalently to the minor groove of a nucleic acid double helix in either a sequence-specific or sequence-independent manner.
  • the outer surface of a double-helical nucleic acid such as DNA or RNA has two channels, namely the major and minor grooves. Both of these grooves contain chemical information by way of arrangements of hydrogen- bond donors and acceptors, electrostatic charges, dipoles, hydrophobic regions and so on.
  • the major groove contains approximately twice the information content of the minor groove in terms of the number of potential hydrogen-bonding contacts.
  • the major groove is therefore the preferred recognition site for cellular proteins such as control proteins, promoters and repressors.
  • the minor groove is normally (with a few exceptions) relatively unoccupied. The vulnerability of the minor groove makes it a particularly useful target for compounds that bind to nucleic acids.
  • composition comprises a pharmaceutically effective amount of one or more miRNA inhibitor, a pharmaceutically acceptable carrier, and, optionally, an additional disease treatment which works synergistically with the miRNA inhibitor.
  • pharmacologically effective amount means the amount of a miRNA inhibitor effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective where there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • Such carriers include, but are not limited to, lipid nanoparticles, saline, buffered saline, dextrose, water, glycerol, ethanol, or any other carrier known in the art, and combinations thereof. Any appropriate pharmaceutical carrier known in the art can be used in conjunction with the miRNA inhibitors disclosed herein.
  • terminal loop means the single-stranded portion of the stem loop hairpin structure of a pri-miRNA and/or pre-miRNA, wherein the sequence immediately 5' to the terminal loop and the sequence immediately 3' to the terminal loop are complementary to each other and form a double-stranded region; the terminal loop is not the stem.
  • stem means the substantially double-stranded portion of the stem loop hairpin structure of a pri-miRNA and/or pre-miRNA, which double-stranded portion is not the terminal loop.
  • substantially double-stranded means that the stem is largely or mostly double-stranded, though it may comprise single-stranded regions.
  • the "Dicer-excised region (or portion or segment)" of a pre-miRNA or pri-miRNA is the region, portion, segment, etc., deleted after cleavage by Dicer. This region, portion, segment, etc., is not part of the mature miRNA, but is adjacent to it.
  • anneals means that a position (mer) of the miRNA inhibitor hydrogen bonds (e.g., base pairs) with the complementary base at a corresponding position of the target pre-miRNA or pri-miRNA.
  • G bonds to C, A to U.
  • position 37 of the example pre-miRNA is U
  • the mer of a miRNA inhibitor which anneals to position 37 would be A [or a modified or substitute base which hydrogen bonds to U, e.g., hypoxanthine (I)].
  • G can hydrogen bond with (anneal to) U, or I to A, forming a wobble base pair.
  • SNALP refers to a stable nucleic acid-lipid particle.
  • a SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid.
  • SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817.
  • ddles is meant that the sequence (or its complement comprises at least one nucleotide on either side of a cleavage site.
  • delay of progression means administration of the combination to patients being in a pre-stage or in an early phase, of the first manifestation or a relapse of the disease to be treated, in which patients, e.g., a pre-form of the
  • corresponding disease is diagnosed or which patients are in a condition, e.g., during a medical treatment or a condition resulting from an accident, under which a corresponding disease will likely develop.
  • “Jointly therapeutically active” or “joint therapeutic effect” means that the compounds may be given separately (in a chronically staggered manner, especially a sequence-specific manner) in such time intervals that they preferably, in the warm-blooded animal, especially human, to be treated, still show a (preferably synergistic) interaction (joint therapeutic effect). Whether this is the case, can inter alia be determined by following the blood levels, showing that both compounds are present in the blood of the human to be treated at least during certain time intervals. miRNA in human diseases or conditions
  • miRNAs are a family of molecules differing from each other in sequence. Many hundreds of them have been identified in humans, and there may be as many as 1000 or more. Up to 60% or more of human genes may be regulated, at least in part, by miRNAs. Friedman et al. 2009 Genome Res. 19: 92-105.
  • miRNAs have many important or essential functions in various biochemical processes. These include differentiation, apoptosis proliferation, stress response, and cell fate determination. Hornstein et al. 2010 Cell Cycle 9: 4041 -42. In Caenorhabditis elegans, miRNAs regulate temporal transitions between developmental stages. Feinbaum et al. 1992 Dev. Biol. 210: 87-95; Olsen et al. 1999 Dev. Biol. 216: 671 -80. In Drosophila, the miRNA bantam both prevents apoptosis and stimulates cell proliferation by suppressing the proapoptotic gene hid. Bennecke et al. 2003 Cell 1 13: 25-36.
  • miRNAs are also involved in insulin secretion. Poy et al. 2004 Nature 432: 226-30. miRNAs are precisely regulated and characteristic patterns of miRNA expression appear during brain development and neuronal differentiation. Krichevsky et al. 2003 RNA 9: 1274- 81 ; Miska et al. 2004 Genome Biol. 5: R68; and Sempere et al. 2004 Genome Biol. 5: R13.
  • miRNAs are also involved in disease states. Dysregulation of miRNAs have been implicated in inflammatory and autoimmune disease, neurological disorders, myocardial disease and several types of cancer. See, Haramati et al. 2010 Proc. Natl. Acad. Sci. USA 107: 131 1 1 -131 16; Thum et al. 2008 Nature 456: 980-984; and Garzon et al. 2010 Nat. Rev. Drug Disc. 9: 775-789. miRNAs are known to be overexpressed in many cancers. Some are important factors in the development or maintenance of the neoplastic state.
  • miRNA miR-21 is elevated in human glioblastoma tumor tissues, early- passage glioblastoma cultures and in six established glioblastoma cell lines. This miR contributes to the malignant phenotype by blocking expression of critical apoptosis-related genes. Knockdown of miR-21 triggers activation of caspases and leads to increased apoptotic cell death. Chan et al. 2005 Cancer Res. 65: 6029-6033. Additional information about miRNAs can be found in the literature, e.g., Bartel. 2004 Cell 1 16: 281 -297; Yi et al. 2003 Genes Dev. 17: 301 1 -3016; Lee et al.
  • miRNAs A variety of miRNAs are known. These include several which are described in more detail below, and to which AMOs are shown herein. These include: miR-145, miR-208a, miR-137 and miR-122. miR-145
  • miR-145 (also known as miRNA145 and MIRN145) is a miRNA involved in several diseases, including respiratory diseases, including pulmonary arterial hypertension (PAH). 2009 Nature 46, 705-710. miR-145 plasma expression is increased in PAH patients compared to matched controls. Inhibition of miR-145 will block pathophysiological vascular remodeling (SMA+ cell hyperplasia) in PAH. miR-145 is also involved in idiopathic pulmonary fibrosis (IPF). miR-145 expression increases in TGFpl -treated lung fibroblasts and IPF patient lung samples compared to normal lung.
  • PHA pulmonary arterial hypertension
  • miR-145 over-expression in lung fibroblasts represses KLF4 and increases SMA expression; miR-145 inhibition reduces TGF i -induced SMA expression.
  • miR-145 -/- mice are protected from bleomycin-induced pulmonary fibrosis. See, for example, 2013 FASEB J. 27: 2382-92.
  • miR-145 is also involved in Asthma/Chronic Obstructive Pulmonary Disease (COPD).
  • COPD Asthma/Chronic Obstructive Pulmonary Disease
  • TGF , IFN- ⁇ and IFN- ⁇ increase miR-145/SMA expression in human airway SMCs and/or fibroblasts.
  • miR-145 increases in house dust mite (HDM) acute asthma mouse model.
  • HDM house dust mite
  • miR-145 is also believed to be involved in chronic/scarless wound healing and/or liver fibrosis and primary open angle glaucoma (POAG). miR-145 is also associated with Marfan syndrome, Loeys-Dietz syndrome, Takayasu's arteritis, giant cell arteritis, liver fibrosis, kidney fibrosis, and heart fibrosis.
  • miR-145 is thus associated with various diseases, including Marfan syndrome, Loeys-Dietz syndrome, Takayasu's arteritis, giant cell arteritis, liver fibrosis, kidney fibrosis, heart fibrosis, respiratory disease, pulmonary arterial hypertension (PAH), idiopathic pulmonary fibrosis (IPF), Asthma, Chronic Obstructive Pulmonary Disease (COPD), chronic/scarless wound healing, and primary open angle glaucoma (POAG).
  • a miR-145 inhibitor can thus be used to treat any of these diseases, or any other disease known in the art related to expression or over-expression of miR-145.
  • Anti-miR-145 oligos can be designed and tested for the ability to decrease production of a mature miR-145 from the corresponding pre-miR-145.
  • the putative anti-miR-145 oligos are designed from simple walking through the pre-miR-145 sequence.
  • the efficacy of putative AMOs to miR-145 can be tested by various methods known in the art.
  • the efficacy of putative AMOs to miR-145 can be tested, for example, in a SPR screen (see Example 1 , for example).
  • anti-miR-145 oligos can be tested using 1 ° human pulmonary arterial smooth muscle cells (PASMCs) (See, for example, the
  • Example 1 The efficacy of putative AMOs to miR-145 can also be tested in vitro using a Dicer processing assay (see also, Example 1).
  • the pre-miR-145 e.g., the substrate
  • Dicer processing assay see also, Example 1
  • the pre-miR-145 e.g., the substrate
  • Dicer Dicer processing assay
  • the various products are examined electrophoretically, and the presence is determined of: the complex of the pre-miR-145 with the putative AMO, the pre-miR-145 substrate; the mature miR-145; the AMO; and other products.
  • a decrease in the production of the mature miR from the pre-miR indicates that the AMO is effective.
  • Similar assays can be used to test the activity of other putative AMOs on their corresponding pre-miR.
  • AMOs to miR-145 can thus be used in methods of treating any of the indications related to miR-145 described herein.
  • miR-208a miR-208a
  • miR-208a (also known as miR208a, MIRNA208A, MIRN208a, and the like) is a miRNA specifically expressed in the heart and other organs, and is involved in various diseases. miR-208a can regulate the expression of myosin heavy chain (MHC) gene, is closed related to the differentiation of cardiac embryonic stem cells and involved in the myocardial fibrosis and hypertrophy via regulating subunits alpha and beta of MHC.
  • MHC myosin heavy chain
  • miR-208a is also implicated in obesity, and administration of Nebivolol (Neb), a ⁇ 1 adrenergic receptor blocker, suppressed miR-208a and is thought to underlie Neb-induced body weight loss in leptin-resistant rats. Mahmood et al. 2015 FASEB J. 29 (supp. 1 ): 716.15. Inhibiting miR-208a can be useful in de-bulking fatty liver. miR-208a- related indications also include metabolic syndrome and Type II diabetes mellitus (T2DM).
  • T2DM Type II diabetes mellitus
  • miR-208a is also associated with Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. miR-208a expression or over-expression is thus associated with several diseases, including Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, heart disease, heart failure, acute coronary syndrome (ACS), hypertrophic heart dysfunction, acute myocardial infarction, obesity, fatty liver, metabolic syndrome, and Type II diabetes mellitus (T2DM).
  • a miR-208a inhibitor can thus be used to treat any of these diseases or any other disease known in the art related to expression or over-expression of miR-208a.
  • miR-137 (also known as miR137 and MIRN137) is highly expressed in the brain, and is involved in various diseases, including schizophrenia. Non-coding variants of miR-137 increase schizophrenia risk with genome-wide significance. A study of induced human neurons harboring the minor alleles of four disease-associated single nucleotide
  • SNPs polymorphisms in miR-137 showed increased miR-137 levels compared to those in major allele-carrying cells.
  • miR-137 gain-of-function caused downregulation of the presynaptic target genes complexin-1 (Cplxl), Nsf and synaptotagmin-1 (Syt1).
  • Cplxl complexin-1
  • Nsf neurosepinephrine
  • SNPs in mRNAs can either create or destroy miRNA target sites. Any of these scenarios can be a source of increased disease risk.
  • miRNA-137 Abnormal expression of miR-137 affects vesicle release at presynaptic terminals and in turn alters hippocampal functioning. Han et al. 2015 Nature Neurosci. 18: 931 -933. See also, Yates et al. 2015 Nature Rev. 16, 373. miRNA-137, and miRNA-137 variants with a single nucleotide polymorphism, are thus associated with schizophrenia.
  • a miR-137 inhibitor can be used to treat this disease or any other disease known in the art related to expression or over-expression of miR-137.
  • miR-137 has been observed in various cancers such as colorectal cancer, gastric cancer, oral cancer, squamous cell carcinoma of the head and neck and breast cancer, as reviewed in Lee et al. Oncotarget, May 20 2015 and references cited therein. miR-122
  • miR-122 (also known as miR122 and MIRN122) is largely expressed in the liver, and is associated with various diseases, including various liver diseases. miR-122 is a completely conserved liver-specific miRNA in vertebrates, and is essential for the maintenance of liver homeostasis. This 22-nt miRNA regulates diverse functions, such as cholesterol, glucose and iron homeostasis, lipid metabolism and infection of hepatitis C virus (HCV) and the parasitic protozoa, Leishmania donovani.
  • HCV hepatitis C virus
  • miR-122 expression is reduced in non-alcoholic steatohepatitis (NASH) patients, and in a subset of hepatocellular carcinoma (HCC) patients including Hepatitis B virus (HB) positive patients with highly invasive and metastatic cancer.
  • NASH non-alcoholic steatohepatitis
  • HCC hepatocellular carcinoma
  • HB Hepatitis B virus
  • anti-miR-122 molecules may be useful in treating high cholesterol, fatty liver disease and related diseases. Thakral et al. 2015 Curr. Gene Ther. 15:142-150 and references cited therein. miR-122 has a key role in some viral infections, facilitating replication of infectious viruses in hepatic cells. Jopling et al. 2005 Science 309: 1577-81 . Santaris Pharma has developed miravirsen, an anti-miR-122 drug that showed promising results in chronic HCV type 1 infected patients. Janssen et al. 2013 N. Eng. J. Med. 368:1685-94. Thus, anti-miR- 122 molecules may be useful in treating viral infections in the liver, including Hepatitis C virus. Lanford et al. 2010 Science 327: 198-201 . miRNA-122 expression or over-expression is thus associated with viral infections, including HCV infection, and dysregulated
  • a miR-122 inhibitor can thus be used to treat any of these diseases or any other disease known in the art related to expression or over-expression of miR-122.
  • an AMO known in the art can be shortened on its 5' end (the complement to the 3' end of the miRNA antisense strand), which is the end furthest from the seed region.
  • the seed region positions 2-8 of the miRNA antisense strand
  • Efficacious tiny AMOs have been constructed which are only 8 nt long and only bind to the seed region. As these are constructed from LNA, these are also known as "tiny LNAs". Obad et al. 201 1 Nat. Genet. 43: 371 -378.
  • an AMO of the invention need not recognize or anneal to the seed region of the AMO.
  • an anti-miRNA oligo can, in fact, not anneal to the miRNA at all, but rather to a sequence adjacent to the miRNA, such as a Dicer-excised region.
  • an anti-miRNA oligo can anneal to a portion of a miRNA and also a portion of an adjacent sequence, such as a Dicer-excised region or Drosha- excised region.
  • Many efficacious AMOs are disclosed herein, wherein the AMO does not anneal to the seed region of the miRNA.
  • a miRNA inhibitor of the invention can be, for example, about 6 to about 21 mers long; about 6 to 21 ; 6 to about 21 ; 6 to 21 ; or 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 mers long; or longer than any of these lengths.
  • miRNA inhibitors of the invention can be constructed from mers (subunits), wherein the mers are nucleotides (RNA or DNA), modified nucleotides and/or nucleotide analogues. Multiple types of mers (e.g., DNA and LNA, modified RNA and non-modified RNA, etc.) can be combined in the same AMO. Some of these molecules may be designated as mixmers.
  • NUCLEOTIDES MODIFIED NUCLEOTIDES AND NUCLEOTIDE ANALOGS FOR USE IN AMOs
  • modifications confer greater stability against nucleases; increase binding affinity to the target miRNA; aid in cellular uptake and/or reduce the triggering of an immune response.
  • modifications at the phosphate include replacement of one or more phosphate with any of: phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker, C 1-6 alkyl, and a compound of formula (I):
  • R 3 is selected from O “ , S “ , NH 2 , BH 3 , CH 3 , C 1-6 alkyl, C 6 . 10 aryl, C 1-6
  • alkoxy and C 6 . 10 aryl-oxy wherein C 1-6 alkyl and C 6 . 10 aryl are unsubstituted or optionally independently substituted with 1 to 3 groups independently selected from halo, hydroxyl and NH 2 ; and R 4 is selected from O, S, NH, or CH 2 .
  • one or more phosphate of the modified nucleotide or polynucleotide is replaced by phosphorothioate (PS).
  • PS phosphorothioate
  • Both endo- and exonucleses degrade nucleic acids by cleaving the phosphate bonds between nucleotides.
  • the phosphorothioate modification substitutes a sulfur atom for a non-bridging oxygen in the phosphate bond, which reduces the ability of nucleases to degrade this bond.
  • AMOs have been constructed which comprise a phosphorothioate. AMOs comprising a PS have been described in: Krutzfeldt et al. 2005 Nature 438: 685-689; Krutzfeldt et al.
  • Non-limiting examples of modified nucleotides which can be used to generate the miRNA inhibitor include 2-aminopurine, (S)-1 -[3-hydroxy-2-
  • Non-limiting examples of the sugar include, for example, a modification at the 2' carbon.
  • at least one modified nucleotide is selected from among 2' alkoxyribonucleotide, 2' alkoxyalkoxy ribonucleotide, or 2'-fluoro ribonucleotide.
  • said at least one modified nucleotide is selected from 2'-OMe, 2'- MOE and 2'-H.
  • the nucleotide subunit is chemically modified at the 2' position of the sugar.
  • the 2' chemical modification is selected from a halo, a C1 -10 alkyl, a C1 -10 alkoxy, and the like.
  • the 2' chemical modification is a C1 -10 alkoxy selected from -OCH 3 (i.e., "OMe"), -OCH 2 CH 3 (i.e., "OEt") or -CH 2 OCH 2 CH 3 (i.e., methoxyethyl or "MOE"); or is a halo selected from F.
  • a 2'-OMe can improve binding affinity; the melting temperature of a 2'-OMe:RNA duplex is about 1 .6 degrees C higher than that of a corresponding DNA:RNA duplex of the same sequence.
  • AMOs comprising a 2'-MOE, 2'-OMe, or 2'F have described in, inter alia: Meister et al. 2004 RNA 10: 544-550; Hutvagner et al. 2004 PLoS 2: E98; Davis et al. 2006 Nucl. Acids Res. 34: 2294-2304; and Davis et al. 2009 Nucl. Acids Res. 37: 70-77.
  • An additional modification, napthyl-based chemical modifying group has been successfully used in AMOs, particularly near each end of the antisense strand. Lennox et al. 2008 Oligo. 16: 26-42; and Melkman-Zehavi et al. 201 1 EMBO J. 30: 835-845.
  • An AMO can thus comprise any of: a nucleotide, a modified nucleotide, and/or a nucleotide analogue.
  • the nucleotide analogue useful in this invention is any nucleotide analogue known in the art, including, but not limited to, peptide nucleic acid (PNA), PNA carrying an alternating ⁇ / ⁇ amino acid backbone consisting of (2'R,4'R)-nucleobase- substituted proline and (1 S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), and/or unlocked nucleic acid (UNA), or xeno nucleic acid (XNA), or
  • PNA peptide nucleic acid
  • nucleotide equivalents have advantages over nucleotides in some cases.
  • PNAs for example, have been used to create inhibitors to specific miRNAs; these have exceptional biological and chemical stability. Because the intra-molecular distances and the configuration of the bases are similar to those found in naturally occurring DNA molecules, hybridization occurs specifically between the PNA and its complementary RNA sequences. Compared with DNA-RNA duplexes, PNA-RNA duplexes exhibit increased thermal stability. Oh et al. 2009 Mol. Cells 28: 341 -345.
  • AMO comprising both nucleotides and nucleotide analogs
  • AMOs comprising a nucleotide analog such as LNA, PNA or morpholino, or a mixture of nucleotides and nucleotide analogues have been described in, for example: Lennox et al. 2010 Pharm. Res. 27: 1788-1799; Chan et al. 2005 Cancer Res. 65: 6029-6033; Flynt et al. 2007 Nat. Genet. 39: 259-263; Obad et al. 201 1 Nat. Genet. 43: 371 -378; Fabani et al. 2008 RNA 14: 336-346; Oram et al.
  • an AMO can comprise nucleotides, modified nucleotides and/or nucleotide analogues, or a mixture thereof.
  • a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.
  • a therapeutically effective amount of a miRNA inhibitor targeting a miRNA can reduce miRNA levels by at least 10%.
  • a given clinical treatment is considered effective where there is at least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction in a measurable parameter associated with a disease or disorder, and the therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction, respectively, in that parameter.
  • miRNA inhibitors can also be modified at the 5' and/or 3' end. Such modifications may increase performance, and/or may decrease degradation or increase stability. Any modification at the 5' and/or 3' end of a miRNA inhibitor can be used with any miRNA comprising any combination of nucleotides, modified nucleotides, and/or nucleotide analogues.
  • miRNA inhibitors can also be modified on the 5' end.
  • any of the following can be conjugated to the AMO at the 5' end:
  • CPP cell-penetrating peptides
  • RNA loop binders which bind to the loop of a pre-miRNA or pri-miRNA; these include, without limitation, dimers of deoxystreptamine, Thomas et al. 2005 J. Am. Chem. Soc. 127: 12434-12435; and molecules that bind to TAR, U1A, and GNRA, Thomas et al. 2008 Chem. Rev. 108: 1 171 ;
  • miRNA inhibitors can also be modified on the 3' end.
  • any of the following can be conjugated to the AMO at the 3' end:
  • Any minor groove binder known in the art can be used in combination with a miRNA inhibitor described herein (e.g., the miRNA inhibitor can have a 3' end modification that is a minor grove binder).
  • minor groove binders have a molecular weight of approximately 150 to approximately 2000 Daltons.
  • a minor groove binder generally binds in a non-intercalating manner into the minor groove of double stranded (or higher order aggregation) DNA, RNA or hybrids thereof, preferably, with an association constant greater than approximately 10 3 M "1 .
  • Minor groove binding compounds have widely varying chemical structures, but many minor groove binders have a crescent shape three-dimensional structure.
  • Minor groove binders are known in the art. And of these can be conjugated to an anti-miRNA molecule of the invention. As a non-limiting example, the minor groove binder can be present at the 3' or 5' end of a molecule comprising an anti-miRNA molecule.
  • Minor groove binders useful for the invention include any of those known in the art. These include, as non-limiting examples:
  • Naturally-occurring compounds and antibiotics such as netropsin, distamycin, and lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin; Derivatives or analogues of naturally-occurring compounds and antibiotics, such as derivatives or analogues of netropsin, distamycin, lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin [Sondhi et al 1997 Curr. Med. Chem. 4: 313; Reddy et al. 1999 Pharm. Therap.
  • a multimer (e.g., a dimer, trimer, tetramer, pentamer, hexamer or longer) of 1 ,2- dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI);
  • a multimer (e.g., a dimer, trimer, tetramer, pentamer, hexamer or longer) of N- methylpyrrole-4-carbox-2-amide (MPC);
  • Bisquarternary ammonium heterocyclic compounds diarylamidines, pentamidine, stilbamidine and berenil, CC-1065 and related pyrroloindole and indole polypeptides, Hoechst 33258, 4'-6-diamidino-2-phenylindole (DAPI);Synthetic minor groove binding molecule, USPN 8,012,967;
  • oligopeptides comprising naturally-occurring or synthetic amino acids are minor groove binder compounds.
  • exemplary minor groove binders are described in U.S. Pat. Nos. 6,084,102 and 7,700,765; or
  • Any minor groove binder known in the art can be used in combination with a miRNA inhibitor described herein (e.g., the miRNA inhibitor can have a 3' end modification that is a minor grove binder).
  • miRNA INHIBITORS WHICH INVADE THE pri-miRNA AND/OR pre-miRNA HAIRPIN, BIND TO THE LOOP AND/OR STRADDLE A CLEAVAGE SITE
  • Inhibitors of miRNA known in the art generally act by annealing to the mature miRNA, especially the seed region, and blocking the recognition of the target by the miRNA.
  • the invention provides miRNA inhibitors which anneal to a sequence outside the miRNA (e.g., in a Dicer-excised region or Drosha-excised region), and - in a forty-fifth embodiment - also anneal to at least a portion of the mature miRNA sequence.
  • these miRNA inhibitors function by preventing or reducing the formation of the mature miRNA from a pre-miRNA or pri- miRNA, e.g., by preventing or reducing cleavage by Dicer and/or Drosha.
  • mature miRNA molecules comprise two at-least-partially complementary strands, each strand about 18-25 nt long, often with a 3' overhang (single- stranded) region on either side. These molecules are cleaved from a longer molecule, a pre- miRNA, which forms a stem loop hairpin structure. Cleavage is performed by two enzymes, Dicer and Drosha. The portions deleted from the molecule to form a mature miRNA are designated the Dicer-excised region and Drosha-excised region.
  • the miRNA inhibitor comprises a sequence such that it invades the hairpin and/or the terminal loop (e.g., a part of the Dicer-excised region) of a pri-miRNA and/or pre-miRNA. Annealing to one strand, the terminal loop, Dicer-excised region or Drosha-excised region, and/or the other strand disturbs the structure of the pri- miRNA and/or pre-miRNA, thus preventing processing (e.g., cleavage) of the pri-miRNA and/or pre-miRNA to produce a miRNA.
  • the miRNA inhibitor comprises a sequence (or its complement) which straddles at least one cleavage site of the pri-miRNA and/or pre-miRNA and/or anneals to the terminal loop of the pri-miRNA and/or pre-miRNA.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region (or a portion thereof) of a pri-miRNA hairpin or pre-miRNA hairpin, wherein (a) the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the first to fifth nt counting from the 5' end of the Dicer-excised region; or (b) the region of complementarity is at the 5' end of the miRNA inhibitor, and the mer at the 5' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • the entire miRNA inhibitor is complementary to the Dicer-excised region (or a portion thereof); thus, the miRNA inhibitor consists of a region of complementarity.
  • the region of complementarity has 1 or 2 or 3 mismatches to the Dicer-excised region.
  • Position 1 is the 5' end; position 64 is the 3' end. This represents a portion of the molecule; the complete molecule would extend 5' to nt 1 , and 3' to nt 64.
  • the miRNA has an antisense strand (or 5p strand) of nt 6 to 26 (bold, underlined), and a sense strand (or 3p strand) of 41 to 61 (also bold, underlined).
  • the loop (e.g., terminal loop) is represented by nucleotides 27 to 38.
  • the stem shown is represented by nt 1 to 26 and 39 to 64.
  • a hairpin comprises a stem and a loop.
  • the staggered Dicer cleavage site is close to the loop; Dicer cleaves between nt 26 and 27, and between nt 40 and 41 ; Drosha cleaves between nt 5 and 6, and between nt 61 and 62.
  • the Dicer-excised region is nt 27 to 40 (inclusive).
  • the Dicer-excised region can comprise a single large terminal loop (e.g., a loop at the end) and an additional sequence, as in this example.
  • Other example Dicer-excised regions are nt 39 to 53 (inclusive) of miR-145 (see Fig. 1 A), and nt 37 to 50 (inclusive) of miR-122 (Fig. 7B).
  • the Dicer-excised region can comprise a small terminal loop and/or one or more loops which are not terminal loops, along with additional sequences; examples of such a Dicer-excised region is nt 47 to 58 (inclusive) of miRNA-137 (Fig. 6B), and nt 31 to 43 (inclusive) of miR-208a (Fig. 4D).
  • the Drosha-excised region is positions 1 to 5 (and positions 5' of 1 , but not shown here) and positions 62 to 64 (and positions 3' of 64, but also not shown here).
  • the nt 27-38 in the pre-miRNA diagram above indicate the terminal loop. Nucleotides 6-7, 15, 50, and 58-59, while forming single-stranded regions, would not represent a terminal loop.
  • A. C (nt 14), UC (17-18), C (28), U (67), and UU (72-73) are not terminal loops, but UAGAUGCUAAGAU (39-51) (SEQ ID NO: 324) is a terminal loop.
  • D. G (nt 5), G (7), GG (23-24), C (31), GA (48-49), A (52), A (55), and G (67) are not terminal loops, but UGC (35-37) is a terminal loop.
  • the nt 1 -26 and 39-64 form the stem of a pri- miRNA in the diagram above.
  • the nt 1 -38 and 52-88 form the stem of a pre-miRNA.
  • nt 1 -34 and 38-71 form the stem of a pri-miRNA.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region of a pri-miRNA hairpin or pre-miRNA hairpin, wherein (a) the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the first to fifth nt counting from the 5' end of the Dicer-excised region.
  • the first to fifth nt counting from the 5' end of the Dicer-excised region are positions 27, 28, 29, 30 and 31 .
  • Examples of such a miRNA inhibitor include, without limitation, those with the sequences, in 3' to 5' order: 27, 28, 29, 30, 31 , etc.; 28, 29, 30, 31 , 32, etc.; 29, 30, 31 , 32, 33, etc.; 30, 31 , 32, 33, 34, etc.; 31 , 32, 33, 34, 35, etc.; where "etc.” indicates one or more addition mers at the 5' end.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region of a pri-miRNA hairpin or pre-miRNA hairpin, wherein the region of complementarity is at the 5' end of the miRNA inhibitor, and the mer at the 5' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • the third to seventh nt counting from the 3' end of the Dicer-excised region are represented by positions 38, 37, 36, 35, and 34.
  • the sequence of the miRNA inhibitor can consist of a sequence that is the entire Dicer-excised region or a portion thereof. In such a case, the sequence of the miRNA does not actually comprise a sequence of the miRNA itself. In this example, such a miRNA can comprise a sequence, as non-limiting examples, of: 27-40, 27-
  • a miRNA inhibitor comprises at least 6 mers.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region of a pri-miRNA hairpin or pre-miRNA hairpin, wherein the 3' end of the region of complementarity anneals to any of the first to fifth nt counting from the 5' end of the Dicer-excised region, and the mer at the 5' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • Non-limiting examples of such miRNAs comprise the sequences of positions: 27-38, 27-37, 27-36, 27-35, 27-34, 28-38, 28-37, 28-36, 28-35, 28-34, 29-38, 29-37, 29-36, 29-35, 29-34, 30-38, 30-37, 30-36, 30-35, 30-34, 31 -38, 31 -37, 31 -36, 31 -35, or 31 -34.
  • the miRNA inhibitor comprises at least 6 mers.
  • the miRNA inhibitor of the invention can comprise a sequence which anneals (or is complementary or substantially complementary) to a sequence in the stem and/or the terminal loop of a pre-miRNA or pri-miRNA, such a Dicer-excised region.
  • the loop (or terminal loop) sequence is the sequence that is between the Dicer cleavage positions in a pre-miRNA hairpin, specifically the sequence extending from the 5'cleavage Dicer cleavage site to the 3'-Dicer cleavage site of a pre- miRNA.
  • the miRNA inhibitor comprises a sequence (or its complement) which straddles at least one pri-miRNA and/or pre-miRNA cleavage site.
  • the miRNA inhibitor is a polymer (e.g., a polymer comprising nucleotides or functionally equivalent molecules) comprising a sequence which straddles at least one cleavage site of a pri-miRNA and/or pre-miRNA, therefore interfering with cleavage and production of the miRNA.
  • a miRNA inhibitor comprising a sequence which straddles the cleavage site between nt 5 and 6 can comprise nt 5 and 6 or the complementary sequence (nt 59 and 60).
  • a miRNA comprising a sequence which straddles the cleavage site between nt 61 and 62 can comprise nt 61 and 62 or the complementary sequence (nt 3 and 4).
  • Cleavage of a pre-miRNA generally produces staggered ends. Therefore, the cleavage site on one strand is not generally aligned with the cleavage site on the other strand. If the two cleavage sites were aligned, two blunt-ended molecules would result from cleavage. In the example miRNA directly above, one cleavage site is between nt 5 and 6, but this is not directly aligned with the cleavage site between nt 61 and 62.
  • a miRNA inhibitor comprising a sequence which straddles a cleavage site on one strand may or may not straddle the cleavage site on the other strand.
  • a miRNA with a sequence with a 5' end beginning at nt 4 or 5 e.g., 4-22, 4-23, 4-24, 4-25, 4-26, or 5-23, 5-24, 5-25, 5-26, etc.
  • Various miRNA inhibitors with sequences complementary to sequences of 4-22, 4-23, 4-24, 4-25, 4- 26, or 5-23, 5-24, 5-25, 5-26, etc. would also be termed to straddle a cleavage site.
  • a miRNA inhibitor can comprise a sequence which straddles a cleavage site by at least 1 nt (e.g., it comprises a sequence comprising at least 1 nt on either side of a cleavage site on one strand or the other).
  • a miRNA inhibitor can comprise a sequence which straddles a cleavage site by at least 2 nt (e.g., it comprises a sequence comprising at least 2 nt on either side of a cleavage site on one strand or the other).
  • a miRNA inhibitor can comprise a sequence which straddles a cleavage site by at least 3 nt (e.g., it comprises a sequence comprising at least 3 nt on either side of a cleavage site on one strand or the other).
  • a miRNA inhibitor can comprise a sequence which straddles a cleavage site by 4 at least nt (e.g., it comprises a sequence comprising at least 4 nt on either side of a cleavage site on one strand or the other).
  • a miRNA inhibitor can comprise a sequence which straddles a cleavage site by at least 5 nt (e.g., it comprises a sequence comprising at least 5 nt on either side of a cleavage site on one strand or the other).
  • the miRNA inhibitor comprises a sequence complementary to all or a portion of the Dicer-excised region.
  • the miRNA inhibitor might anneal to a portion of the Dicer-excised region (e.g., annealing to at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more nt of the Dicer-excised region).
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 1 nt of the Dicer- excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 2 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 3 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 4 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 5 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 6 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 7 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 8 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 9 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 10 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 1 1 nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which is
  • a miRNA inhibitor can comprise a sequence which is complementary to a sequence of at least 12 or more nt of the Dicer-excised region.
  • a miRNA inhibitor can comprise a sequence which both straddles a cleavage site (by any measure indicated, e.g., 1 , 2, 3, 4, 5, 5 or more nt) and be complementary to a sequence of the Dicer-excised region of any length indicated (at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 12 or more nt) of the Dicer-excised region, or any combination thereof (e.g., straddling a cleavage site by 1 nt, and complementary to 5 nt of the Dicer-excised region; straddling a cleavage site by 3 nt, and complementary to 2 nt of the Dicer-excised region; straddling the cleavage site by 5 nt, and complementary to 5 nt of the Dicer-excised region, etc.).
  • the present invention provides, inter alia, miRNA inhibitors and methods of their use.
  • miRNA inhibitors are disclosed in Tables 1 to 10 and the Figures, in addition to elsewhere in the specification.
  • Various anti-miR-145 oligos are described and illustrated herein, e.g., in Figures 1 to 3 and Tables 1 to 4. These can be used to treat any disease related to expression or over-expression of miR-145.
  • Various anti-miR-208a oligos are described and illustrated herein, e.g., in Figures 4 and 5 and Table 5. These can be used to treat any disease related to expression or over-expression of miR-208a.
  • Various anti-miR-137 oligos are described and illustrated herein, e.g., in Figure 6 and Table 6. These can be used to treat any disease associated with miR-137 expression or over-expression.
  • Various anti-miR-122 oligos are described and illustrated herein, e.g., in Figure 7 and Table 7. These can be used to treat any disease associated with miR-122 expression or over-expression.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 381 , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 382, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 383, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 384, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 385, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 386, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 387, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 388, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 389, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 390, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 391 , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 392, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 393, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 394, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 395, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 396, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 397, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 398, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 399, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 400, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 401 , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 402, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 403, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 404, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 405, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 406, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 407, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of SEQ ID NO: 408, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 381 , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 382, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 383, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 384, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 385, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 386, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 387, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 388, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 389, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 390, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 391 , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 392, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 393, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 394, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 395, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 396, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 397, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 398, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 399, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 400, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 401 , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 402, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 403, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 404, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: , wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 405, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 406, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 407, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor consists of the sequence of SEQ ID NO: 408, wherein the miRNA inhibitor comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a method of treating or ameliorating a disease associated with the expression or over-expression of miR-145 in an individual comprising the step of administering to the individual a therapeutically effective amount of any one or more of:
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 381 , wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 382, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 383, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 384, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 385, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 386, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 387, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 388, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 389, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues, and/or
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 390, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a method of treating or ameliorating a disease associated with the expression or over-expression of miR-208a in an individual comprising the step of administering to the individual a therapeutically effective amount of any one or more of:
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 391 , wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 392, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 393, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 394, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 395, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 396, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 397, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 398, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 399, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 400, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues, and/or
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 401 , wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a method of treating or ameliorating a disease associated with the expression or over-expression of miR-137 in an individual comprising the step of administering to the individual a therapeutically effective amount of any one or more of:
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 402, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 403, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues, and/or
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 404, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • a method of treating or ameliorating a disease associated with the expression or over-expression of miR-122 in an individual comprising the step of administering to the individual a therapeutically effective amount of any one or more of:
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 405, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 406, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues,
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 407, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues, and/or
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises or consists of the sequence of SEQ ID NO: 408, wherein the miRNA comprises one or more nucleotides, modified nucleotides and/or nucleotide analogues.
  • the miRNA inhibitors of this invention may comprise only a first strand.
  • the miRNA inhibitors can comprise a first and a second strand.
  • the miRNA inhibitors can comprise any combination of one or more nucleotides, modified nucleotides and/or nucleotide analogues, and can further optionally comprise any 5' or 3' end modification disclosed herein or known in the art, and can be in combination with any delivery vehicle disclosed herein or known in the art, and can be used for any method of treatment or reducing the level, activity or efficacy of any cognate miRNA.
  • compositions for delivery of the miRNA inhibitors of the invention are described herein, and below.
  • the miRNA inhibitors of the invention can be delivered using any method or composition described herein, or known in the art to be used for delivering miRNA inhibitors or small nucleic acids.
  • miRNA inhibitor can optionally be attached to a ligand selected to improve one or more characteristic, such as, e.g., stability, distribution and/or cellular uptake of the agent.
  • the miRNA inhibitor(s) can be isolated or be part of a pharmaceutical composition used for the methods described herein.
  • the pharmaceutical composition can be formulated for delivery to specific tissues (e.g., those afflicted with a miRNA-related disease) or formulated for parenteral administration.
  • the pharmaceutical composition can optionally comprise two or more miRNA inhibitors, each one directed to the same, overlapping or a different segment of the pre-miRNA or pri- miRNA.
  • the pharmaceutical composition can further comprise or be used in conjunction with any known treatment for the particular miRNA-related disease.
  • composition comprising a miRNA Inhibitor
  • Additional components of a pharmaceutical composition comprising a miRNA inhibitor are contemplated to aid in delivery, stability, efficacy, or reduction of
  • Liposomes have been used previously for drug delivery (e.g., delivery of a chemotherapeutic).
  • Liposomes e.g., cationic liposomes
  • a process of making liposomes is also described in WO04/002453A1 .
  • neutral lipids have been incorporated into cationic liposomes (e.g., Farhood et al. 1995), as well as PEGylated lipids.
  • Cationic liposomes have been used to deliver drugs to various cell types (Sioud and Sorensen 2003; U.S. Patent Application 2004/0204377; Duxbury et al., 2004; Donze and Picard, 2002).
  • a variety of molecules have been used for cell-specific nucleic acid delivery. See, for example, WO/201 1/076807.
  • the nucleic acid-condensing property of protamine has been combined with specific antibodies to deliver small nucleic acids. Song et al. 2005 Nat Biotech. 23: 709-717. The self-assembly PEGylated polycation
  • PEI polyethylenimine
  • the miRNA inhibitors of the invention can be delivered via, for example, Lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles; or via modification of the miRNA inhibitor (e.g., covalent attachment), or by any method known in the art for delivery of a miRNA inhibitor.
  • LNP Lipid nanoparticles
  • NL neutral liposomes
  • polymer nanoparticles or via modification of the miRNA inhibitor (e.g., covalent attachment), or by any method known in the art for delivery of a miRNA inhibitor.
  • Lipid nanoparticles are self-assembling cationic lipid based systems. These can comprise, for example, a neutral lipid (the liposome base); a cationic lipid (for small nucleic acid loading); cholesterol (for stabilizing the liposomes); and PEG-lipid (for stabilizing the formulation, charge shielding and extended circulation in the bloodstream).
  • a neutral lipid the liposome base
  • a cationic lipid for small nucleic acid loading
  • cholesterol for stabilizing the liposomes
  • PEG-lipid for stabilizing the formulation, charge shielding and extended circulation in the bloodstream.
  • the cationic lipid can comprise, for example, a headgroup, a linker, a tail and a cholesterol tail.
  • the LNP can have, for example, good delivery to the diseased area, extended circulation in the blood, small particles (e.g., less than 100 nm), and stability in the microenvironment of the diseased area (which may have low pH and/or be hypoxic).
  • Neutral liposomes are non-cationic lipid based particles.
  • Polymer nanoparticles are self-assembling polymer-based particles.
  • Neutral liposomes known in the art can be used to deliver miRNA inhibitors of the invention.
  • miRNA inhibitor compositions in a Lipid nanoparticles (LNP) comprising a neutral lipid; a cationic lipid; cholesterol; and PEG-lipid
  • Lipid nanoparticles are self-assembling cationic lipid based systems. These can comprise, for example, a neutral lipid (the liposome base); a cationic lipid (for nucleic acid loading); cholesterol (for stabilizing the liposomes); and PEG-lipid (for stabilizing the formulation, charge shielding and extended circulation in the bloodstream).
  • a neutral lipid is, for example, the liposome base.
  • a cationic lipid is, for example, for nucleic acid loading.
  • Cholesterol is, for example, for stabilizing the liposomes.
  • PEG-lipid is, for example, for stabilizing the formulation, charge shielding and extended circulation in the bloodstream.
  • Lipid nanoparticles known in the art can be used to deliver miRNA inhibitors of the invention.
  • the miRNA inhibitor is packaged as a monotherapy into a delivery vehicle, or may be further ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptid
  • the miRNA inhibitors of the invention can be prepared in a pharmaceutical composition comprising various components appropriate for the particular method of administration of the miRNA inhibitor.
  • the invention further provides methods for reducing the level of mature miRNA in a cell, particularly in the case of a disease characterized by expression, over-expression or hyper-activity of a miRNA.
  • the present methods utilize the cellular mechanisms involved in RNA interference to selectively degrade the target RNA in a cell and are comprised of the step of contacting a cell with one of the miRNA inhibitors of the invention.
  • the invention also encompasses a method of treating a human subject having a pathological state mediated at least in part by miRNA expression, over-expression or hyperactivity, the method comprising the step of administering to the subject a therapeutically effective amount of a miRNA inhibitor. Additional methods involve preventing, treating, modulating and/or ameliorating a pathological state wherein disease progression requires miRNA, although miRNA is not amplified or over-expressed. Such methods comprise the step of administering one of the miRNA inhibitors of the invention to a subject, as further described below. Such methods can be performed directly on a cell or can be performed on a mammalian subject by administering to a subject one of the miRNA
  • inhibitors/pharmaceutical compositions of the invention Reduction of target mature miRNA in a cell results in a reduction in the amount of mature miRNA produced. In an organism, this can result in restoration of balance in a pathway involving miRNA, and/or prevention of miRNA accumulation, and/or a reduction in miRNA activity and/or expression, and/or prevention of miRNA-mediated activation of other genes, and/or amelioration, treatment and/or prevention of a miRNA-related disease.
  • a reduction in miRNA expression, level or activity can limit disease growth.
  • compositions of the invention can be used in any appropriate dosage and/or formulation described herein or known in the art, as well as with any suitable route of administration described herein or known in the art.
  • a method of treatment using a composition comprising a miRNA inhibitor described herein is a method of treatment using a composition comprising a miRNA inhibitor described herein.
  • the invention relates to a method of treating a miRNA-related disease in an individual, comprising the step of administering to the individual a
  • the miRNA inhibitor comprises an antisense strand duplexed with a sense strand, wherein the sense and antisense strands are selected from one or more of the sequences provided in any of Tables 1 to 10.
  • the invention relates to such a method, wherein the composition comprising a miRNA inhibitor further comprises a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by 0, 1 , 2, or 3 nucleotides from the sense strand of a miRNA inhibitor selected from the specific duplexes provided herein and as listed, e.g., in any Table herein.
  • the miRNA inhibitor comprises at least an anti- sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti-sense strand is the sequence of the sense and/or the anti-sense strand of a miRNA inhibitor selected from those specific duplex provided herein and as listed, e.g., in Table 1 , wherein the composition further comprises a pharmaceutically effective formulation.
  • the miRNA inhibitor comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti-sense strand comprises the sequence of the sense and/or the anti-sense strand of a miRNA inhibitor selected from those specific duplex provided herein and as listed, e.g., in any of Tables 1 to 10, wherein the composition further comprises a pharmaceutically effective formulation.
  • the invention provides a method of inhibiting expression of miRNA in a subject, wherein the method comprises the step of administering to the subject an effective amount of a composition comprising a miRNA inhibitor (e.g., any described herein).
  • a composition comprising a miRNA inhibitor (e.g., any described herein).
  • the subject is human.
  • the subject has a miRNA-related disease.
  • the method further comprises the step of administering an additional treatment.
  • the additional treatment is a method (or procedure). In one aspect, the additional treatment is a therapeutically effective dose of a composition.
  • the additional treatment and the miRNA inhibitor can be administered in any order, or can be administered simultaneously.
  • the method further comprises the step of administering an additional treatment for a miRNA-related disease.
  • the method further comprises the step of administering an additional treatment.
  • a miRNA inhibitor can be used in conjunction with any additional treatment disclosed herein, as appropriate for the disease, optionally, in further conjunction with one or more additional miRNA inhibitors to miRNA.
  • the composition comprises a second miRNA inhibitor.
  • the second miRNA inhibitor is physically distinct from the first, or the two are physically connected (e.g., linked or conjugated).
  • the first and second miRNA inhibitors are combined within the same composition (e.g., both in the same lipid nanoparticle).
  • the invention provides a method of inhibiting the expression of miRNA in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition comprising a miRNA inhibitor of the invention.
  • the miRNA inhibitor comprises a strand, wherein the strand comprises at least 15 contiguous mers differing by 0, 1 , 2, or 3 mers from the sequence of a miRNA inhibitor listed in any of Tables 1 to 10, wherein the composition is in a pharmaceutically effective formulation.
  • the miRNA inhibitor comprises a strand, wherein the strand comprises at least 15 contiguous mers from the sequence of a miRNA inhibitor listed in any of Tables 1 to 10, wherein the composition is in a pharmaceutically effective formulation.
  • the miRNA inhibitor comprises a strand, wherein the strand comprises the sequence of a miRNA inhibitor listed in any of Tables 1 to 10, wherein the composition is in a pharmaceutically effective formulation.
  • the miRNA inhibitor comprises a strand, wherein the strand consists of the sequence of a miRNA inhibitor listed in any of Tables 1 to 10, wherein the composition is in a pharmaceutically effective formulation.
  • the miRNA inhibitor comprises a first strand and a second strand.
  • the individual is afflicted with or susceptible to a miRNA-related disease.
  • the method further comprises the step of administering an additional treatment.
  • the additional treatment and the miRNA inhibitor can be administered in any order or can be administered simultaneously.
  • the composition comprises a second miRNA inhibitor.
  • the second miRNA inhibitor is physically separate from the first, or the two are physically connected (e.g., covalently linked or otherwise conjugated).
  • the first and second miRNA inhibitors are combined within the same composition (e.g., both in the same lipid nanoparticle).
  • the method further comprises the step of administering an additional miRNA inhibitor.
  • miRNA inhibitor for use in treating various miRNA-related diseases
  • the miRNA inhibitor of the invention comprises a sequence disclosed herein and is administered to a patient in need thereof (e.g., a patient suffering from a miRNA-related disease disclosed herein or known in the literature).
  • the miRNA inhibitor of the invention is administered to a patient in need thereof, along with one or more additional pharmaceutical agent appropriate for that disease.
  • a patient suffering from a miRNA-related disease can be administered a pharmacologically effective amount of one or more miRNA inhibitor along with a
  • a patient suffering from a miRNA-related disease can be administered one or more miRNA inhibitor and one or more additional miRNA-related disease treatment.
  • This additional treatment can be selected from the list of any disease treatment listed herein, and/or any anti- miRNA-related disease treatment known in the art.
  • the miRNA inhibitors of the invention can be administered along with (as part of the same therapeutic treatment regimen, prior to, simultaneously with, or after) one or more additional therapeutics to treat a miRNA-related disease.
  • the patient can also be administered as more than one miRNA inhibitor.
  • the miRNA inhibitor(s) and additional disease treatment(s) can be administered in any order, simultaneously or sequentially, or in multiple doses over time.
  • Administration of the miRNA inhibitor and the additional treatment can be, for example, simultaneous, concurrent, separate or sequential.
  • Simultaneous administration may, e.g., take place in the form of one fixed combination with two or more active ingredients, or by simultaneously administering two or more active ingredients that are formulated independently.
  • Sequential use (administration) preferably means administration of one (or more) components of a combination at one time point, other components at a different time point, that is, in a chronically staggered manner, preferably such that the combination shows more efficiency than the single compounds administered independently (especially showing synergism).
  • Separate use (administration) preferably means administration of the components of the combination independently of each other at different time points, preferably meaning that the components (a) and (b) are administered such that no overlap of measurable blood levels of both compounds are present in an overlapping manner (at the same time).
  • combination component-drugs show a joint therapeutic effect that exceeds the effect found when the combination component-drugs are used independently at time intervals so large that no mutual effect on their therapeutic efficiency can be found, a synergistic effect being especially preferred.
  • the miRNA inhibitor inhibits miR-145, miR-208a, miR-137 or miR-122.
  • the invention also pertains to methods of using these miRNA inhibitors to inhibit the miRNA, and/or to treat, prevent or ameliorate a disease related to expression or over-expression of the miRNA.
  • the invention provides for the use of the miR-145 inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miR-145, including Marfan syndrome, Loeys-Dietz syndrome, Takayasu's arteritis, giant cell arteritis, liver fibrosis, kidney fibrosis, heart fibrosis, respiratory disease, pulmonary arterial hypertension (PAH), idiopathic pulmonary fibrosis (IPF), Asthma, Chronic Obstructive Pulmonary Disease (COPD), chronic/scarless wound healing, and primary open angle glaucoma (POAG).
  • diseases related to expression or over-expression of miR-145 including Marfan syndrome, Loeys-Dietz syndrome, Takayasu's arteritis, giant cell arteritis, liver fibrosis, kidney fibrosis, heart fibrosis, respiratory disease, pulmonary arterial hypertension (PAH), idiopathic pulmonary fibrosis (IPF), Asthma, Chronic Obstruct
  • the invention provides for the use of the miR-208a inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miR-208a, including Nonalcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, heart disease, heart failure, acute coronary syndrome (ACS), hypertrophic heart dysfunction, acute myocardial infarction, obesity, fatty liver, metabolic syndrome, and Type II diabetes mellitus (T2DM).
  • NAFLD Nonalcoholic fatty liver disease
  • NASH Non-alcoholic steatohepatitis
  • liver fibrosis liver fibrosis
  • cirrhosis cirrhosis
  • heart disease heart failure
  • ACS acute coronary syndrome
  • T2DM Type II diabetes mellitus
  • the invention provides for the use of the miRNA inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miRNA-137, or miRNA-137 variants with a single nucleotide polymorphism, including schizophrenia.
  • the invention provides for the use of the miRNA-122 inhibitor in the treatment or amelioration of various diseases related to expression or over-expression of miRNA-122, including viral infections, including HCV infection, and dysregulated homeostasis, including high cholesterol and fatty liver disease.
  • compositions for delivery of a miRNA inhibitor can be used with any of the miRNA inhibitors described herein.
  • methods of use described herein and known in the art can be used with any miRNA inhibitor described herein.
  • This invention presents various novel miRNA inhibitors, including anti-miRNA oligonucleotides. Sequences of these various novel miRNA inhibitors are presented in the Tables below.
  • Table 1 Human microRNA stem loop hairpin ligands for SPR binding assays.
  • Table 9 A list of some miRNA inhibitors to miR-145, miR-208a, miR-137 and miR-122.
  • Table 1 shows miRNA stem loop hairpin ligands used in SPR binding assays, including those described in Example 1 . "hsa” indicates Homo sapiens.
  • Table 2 shows human pre-microRNA hairpin substrates for in vitro Dicer processing assays, as detailed in Example 1 .
  • "005" indicates a 2'-MOE modification.
  • C005" represent a modified nucleotide wherein the base is, respectively, A, G, or C, respectively, and the sugar has a 2'-MOE modification.
  • 006 is also MOE. It refers to a MOE-C without the 5-Me modification on the nucleobase. Below, C006 is on the left and C005 is on the right. Both are MOE-C but C005 has the 5-Me group.
  • R1 and R2 are where the molecule attaches to the rest of the oligonucleotide via the PO (phosphate) or PS (phosphorothioate) linkages.
  • "007” indicates a 2'-F.
  • “A007”, “G007”, or “C007”, etc. represent a modified nucleotide wherein the base is, respectively, A, G, or C, respectively, and the sugar has a 2'-F modification.
  • p indicates a phosphate (a component of a nucleotide).
  • X003 indicates a C6 3' end cap, which is a C6 modification at the 3' carbon of the sugar.
  • ALL MOE indicates that all or almost all the mers are 2'MOE; "Alternating” indicates that, in general, two different modifications alternate (e.g., 007 and 006).
  • Table 3 shows Human miR-145 AMO hairpin walk biochemical screening data.
  • those molecules with a higher SPR and lower miR-145 processing can be considered more efficacious. Processing can be considered more efficacious, as the masses are roughly equivalent/comparable.
  • "1 .17" indicates 1 .17% cleavage of the miR-145 pre-miRNA by Dicer in the presence of 36XD compared to the negative control treatment (100% cleavage).
  • Man is short for Manual, meaning the modifications are assigned by hand (manually) rather than according to a predetermined pattern.
  • LXH874 is a Human miR-145 PNA used in Figure 3.
  • Table 5 shows Human miR-208a AMO hairpin walk biochemical screening data. The final column shows the amount of pre-miRNA remaining uncleaved by Dicer after addition of the miRNA inhibitor.
  • Table 6 shows Human miR-137 AMO hairpin walk biochemical screening data.
  • the final column shows the amount of pre-miRNA remaining uncleaved by Dicer after addition of the miRNA inhibitor. For example, in the first row, 0.90 or 90% of the test miRNA pre-miRNA remained uncleaved in the presence of miRNA inhibitor 58VE.
  • Table 7 shows the results of the human miR-122 AMO hairpin walk biochemical screening data.
  • a more negative number in the last column indicates a more active compound.
  • the processing here is listed as the % of guide strand that is produced relative to having no inhibitor. So, the more negative the number, the more inhibition. "-100.00" would indicate no guide strand produced at all, or complete inhibition.
  • the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region (or a portion thereof) of a pri-miRNA hairpin or pre-miRNA hairpin, wherein (a) the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the first to fifth nt counting from the 5' end of the Dicer-excised region; or (b) the region of complementarity is at the 5' end of the miRNA inhibitor, and the mer at the 5' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • the invention provides a miRNA inhibitor, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region (or a portion thereof) of a pri- miRNA hairpin or pre-miRNA hairpin, wherein the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • Table 9 represents some, but not necessarily all, of the particularly efficacious miRNA inhibitors disclosed herein.
  • Report Point is a snapshot quantifier for the surface plasmon resonance (SPR) assay.
  • the Report Point is proportional to ligand binding measured in a small window in time in the assay, just toward the end of the injection. The higher the Report Point, the more mass (or ligand) is bound to the surface of the chip that has the RNA on its surface.
  • AMOs that bind to the target RNA e.g., miR-145) will bind to the RNA, then the mass increases at the surface of the chip, and the Report Point will go up. Less binding of the AMO indicates a lower Report Point.
  • RP/Length is the ratio of "Report Point/Length” and is a way to normalize the data to account for different masses of the ligands (AMOs). Smaller (in this case, shorter) AMOs will have less effect on the mass change on the surface of the SPR chip when they bind, relative to larger (e.g., longer) ones. Thus, this is a computational way to correct for the difference using the AMO length.
  • the final column presents RP/Mwt*1000.
  • RP/Mwt is multiplied by 1000 to change the denominator units to kDa, rather than the Da that the molecular weights are recorded in.
  • SPR assays were run on the ProteOn XPR36 system (BioRad) using a neutravidin-coated NLC Sensor Chip (BioRad, #176-5021 ).
  • Human microRNA stem loop hairpin ligands (5' biotinylated, RNAse-free HPLC purification; Table 1) were synthesized by Integrated DNA Technologies (IDT) or TriLink BioTechnologies and diluted to 800 nM in Buffer A (20 mM Tris-HCI, pH 7.5, 140 mM NaCI, 5 mM MgCI 2 , 2 mM KCI, 2 mM CaCI 2 and 0.05% P20).
  • Ligands were heated for 5 min at 90°C and then cooled to 37°C at a rate of 3.5°C/min to ensure proper folding. Ligands (25 ⁇ total) were bound to the NLC chip by injection at a rate of 50 ⁇ /min.
  • the coupled surface was washed sequentially with 1 M NaCI, Buffer A and Regeneration Buffer.
  • Regeneration Buffer was composed of a 1 :1 :1 mix of water, acid solution (37.5 mM oxalic acid, 37.5 mM phosphoric acid, 37.5 mM formic acid and 37.5 mM malonic acid), and ionic solution (0.4 M KCI, 1 .8 M MgCI 2 , 0.9 M urea, 1 .8M guanidine HCI).
  • Anti-miR-oligonucleotide (AMO; Novartis Biologies Center) or peptide nucleic acid (PNA; PNA Bio) analytes were prepared as a 1 ⁇ working stock in Buffer A. Analytes were heated to 85°C for 5 min, cooled to 37°C at a rate of 3.2°C/min and diluted to a final concentration of 100 nM in Buffer A. Sample injections (60 ⁇ total) were performed for 6 analytes/round at a rate of 50 ⁇ /min at 37°C. The surface was regenerated between injections by washing with Regeneration Buffer (30 ⁇ total) at a rate of 100 ⁇ /min.
  • Regeneration Buffer (30 ⁇ total) at a rate of 100 ⁇ /min.
  • In vitro processing assays were performed using the Recombinant Dicer Enzyme Kit (Genlantis, #T510008) according to the manufacturer's protocol. AMOs and substrates were pre-incubated for 20 min at a molar stoichiometry of 1 :1 or 2:1 . Reaction samples were prepared by addition of 12 ⁇ of 2X Loading Buffer (9.7 ml deionized formamide, 0.2 ml 0.5 M EDTA, 0.1 ml 100 mg/ml tartrazine) and subjected to
  • HPLC purification were synthesized by Integrated DNA Technologies (IDT) or TriLink BioTechnologies. Substrates were diluted to a final concentration of 25 ⁇ in RNase free water. AMOs were suspended to a final concentration of 80 ⁇ in 1 X PBS. In vitro processing assays were performed using the Recombinant Dicer Enzyme Kit (Genlantis, #T510008). An assay mixture was prepared by mixing of 25 of hairpin solution, 10 of 10 mM ATP, 5 of 50 mM MgCI 2 , and 40 of reaction buffer. Each assay mixture was made by mixing 8 of the assay mixture, 2 ⁇ L ⁇ of the AMO solution, and 1 ⁇ L ⁇ of Dicer solution.
  • a control wells used 2 ⁇ of 1 X PBS in place of the AMO solution and another control well used RNase free water in place of the Dicer solution and 1X PBS in place of the AMO solution.
  • Samples were incubated at 37°C for 24 h and then 2 of each sample was loaded onto a 1 .2% agarose SybrSafe gel. Gels were imaged using a ChemiDoc MP imager. The bands were quantitated using the accompanying software (version 4.1 ). The percent hairpin remaining was calculated by comparing band intensities to the sample from the control well which did not include AMO or Dicer. Experiments were performed in triplicate and average results are reported.
  • transfection master mix was prepared by mixing 0.05 L l/well Lipofectamine RNAiMAX (Life Technologies, #13778150) in 8.95 ⁇ /well Opti-MEM Reduced Serum Medium (Life Technologies, #31985070) followed by incubation for 5 min at room temperature. Transfection master mix (9 ⁇ /well) was transferred to a black, clear-bottom collagen-coated 384-well destination plate (Greiner BioOne, #781956) using a Matrix WellMate (Thermo Scientific). Plates were centrifuged for 1 min at 300xg.
  • AMOs (1 .5 ⁇ at 1 ⁇ ) in 1 X siRNA Buffer were transferred from a 384-well clear, v-bottom source plate (Greiner BioOne, #781271) to the destination plate using a Matrix Platemate 2x2 (Thermo Scientific) and incubated for 20 min at room temperature to allow complex formation (50 nM final AMO concentration).
  • Pulmonary arterial smooth muscle cell (PASMCs; Lonza, #CC-2581) were trypsinized (ReagentPack; Lonza, CC-5034), centrifuged at 300xg for 5 min at room temperature and resuspended in complete growth medium (SmGM-2; Lonza, #CC-3182) without antibiotics at 6.0 x 10 4 viable cells/ml. Reverse transfection was performed by transferring 20 ⁇ of PASMC cell suspension (1200 cells/well) to the 384-well destination plate using a Matrix WellMate. The destination plates were centrifuged for 1 min at 300xg at room temperature and incubated at 37°C + 5% C0 2 overnight.
  • a miRNA inhibitor comprising a polymer, wherein the sequence of the miRNA inhibitor comprises a region of complementarity complementary to the Dicer-excised region or a portion thereof of a pri-miRNA hairpin or pre-miRNA hairpin, wherein (a) the region of complementarity is at the 3' end of the miRNA inhibitor and the mer at the 3' end of the region of complementarity anneals to any of the first to fifth nt counting from the 5' end of the Dicer-excised region; or (b) the region of complementarity is at the 5' end of the miRNA inhibitor, and the mer at the 5' end of the region of complementarity anneals to any of the third to seventh nt counting from the 3' end of the Dicer-excised region.
  • R 3 is selected from O “ , S “ , NH 2 , BH 3 , CH 3 , C 1-6 alkyl, C 6 _ 10 aryl, C 1-6
  • the miRNA inhibitor of embodiment 1 wherein the modified nucleotide is selected from: 2-aminopurine, (S)-1 -[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine, 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylamin
  • nucleotide analogue is: peptide nucleic acid (PNA), PNA carrying an alternating a/b amino acid backbone consisting of (2'R,4'R)-nucleobase-substituted proline and (1 S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), and/or unlocked nucleic acid (UNA), or xeno nucleic acid (XNA), or xylo nucleic acid (XNA).
  • PNA peptide nucleic acid
  • PNA carrying an alternating a
  • the miRNA inhibitor of claim 1 wherein the sequence of the miRNA inhibitor is the sequence of any miRNA inhibitor sequence disclosed herein.
  • a miRNA inhibitor comprising a polymer which can anneal to a pri-miRNA and/or pre-miRNA in a sequence-dependent manner and comprises a sequence which can straddle a cleavage site by at least 3 to 7 nucleotides, and/or anneal to a Dicer-excised region or Drosha-excised region in the pri-miRNA and/or pre-miRNA.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises 15 contiguous positions of the sequence of any miRNA inhibitor sequence disclosed herein.
  • a miRNA inhibitor wherein the sequence of the miRNA inhibitor comprises the sequence of any miRNA inhibitor sequence disclosed herein.
  • a miRNA inhibitor to miR-145, miR-137, miR-208a, or miR-122, wherein the sequence of the miRNA inhibitor comprises at least 15 contiguous positions of, comprises, or is the sequence of any miRNA inhibitor sequence disclosed herein.
  • a method of reducing the level, expression and/or activity of miR-145, miR-137, miR-208a, or miR-122 in a patient comprising the step of
  • a method of treating or ameliorating a disease or condition related to expression or over-expression of miR-145, miR-137, miR-208a, or miR-122 comprising the step of administering a therapeutically effective amount of a miRNA inhibitor of claim 17.
  • PHO pulmonary arterial hypertension
  • IPF idiopathic pulmonary fibrosis
  • COPD Chronic Obstructive Pulmonary Disease
  • POAG primary open angle glaucoma
  • NASH Non-alcoholic fatty liver disease
  • NASH Non-alcoholic steatohepatitis
  • cirrhosis cirrhosis
  • heart disease heart failure
  • ACS acute coronary syndrome
  • T2DM Type II diabetes mellitus
  • a method of treating a patient afflicted with schizophrenia comprising the step of administering a therapeutically effective amount of an inhibitor to miR-137 disclosed herein.
  • a method of treating a patient afflicted with a viral infection, HCV infection, and/or dysregulated homeostasis, including high cholesterol and fatty liver disease comprising the step of administering a therapeutically effective amount of an inhibitor to miR-122 disclosed herein.
  • the technical and scientific terms used herein have the same meaning as that usually understood by a specialist familiar with the field to which the invention belongs.

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Abstract

L'invention concerne un inhibiteur de miARN, la séquence de l'inhibiteur de miARN comprenant une région de complémentarité complémentaire de la région excisée par Dicer (ou une partie de celle-ci) d'une épingle à cheveux de pri-miARN ou d'une épingle à cheveux de pré-miARN, (a) la région de complémentarité se trouvant à l'extrémité 3' de l'inhibiteur de miARN et le motif monomère à l'extrémité 3' de la région de complémentarité s'hybridant à l'un quelconque des premier à cinquième nucléotides à partir de l'extrémité 5' de la région excisée par Dicer; ou (b) la région de complémentarité se trouvant à l'extrémité 5' de l'inhibiteur de miARN, et le motif monomère à l'extrémité 5' de la région de complémentarité s'hybridant à l'un quelconque des troisième à septième nucléotides à partir de l'extrémité 3' de la région excisée par Dicer. L'invention concerne également un procédé de réduction de la formation d'un miARN à partir d'un pri-miARN et/ou d'un pré-miARN, par mise en contact du pri-miARN et/ou du pré-miARN avec un inhibiteur de miARN. L'invention concerne en outre une méthode de traitement d'une maladie liée à l'expression ou à la surexpression d'un miARN chez un patient, par administration d'un inhibiteur de miARN.
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WO2023060238A3 (fr) * 2021-10-08 2023-05-11 Regulus Therapeutics Inc. Procédés et compositions pour éviter des effets hors cible

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