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WO2012006181A2 - Compositions et procédés pour l'inhibition de micro-arn oncogènes et le traitement du cancer - Google Patents

Compositions et procédés pour l'inhibition de micro-arn oncogènes et le traitement du cancer Download PDF

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WO2012006181A2
WO2012006181A2 PCT/US2011/042436 US2011042436W WO2012006181A2 WO 2012006181 A2 WO2012006181 A2 WO 2012006181A2 US 2011042436 W US2011042436 W US 2011042436W WO 2012006181 A2 WO2012006181 A2 WO 2012006181A2
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mir
mirna
cell
inhibitor
cancer
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WO2012006181A3 (fr
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Josep M. Llovet
Sara Toffanin
Radoslav Savic
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Icahn School of Medicine at Mount Sinai
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Mount Sinai School of Medicine
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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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs

Definitions

  • This invention relates to microRNA inhibitors and methods of use thereof.
  • Hepatocellular carcinoma is the third leading cause of cancer- related death worldwide and the principal cause of mortality among cirrhotic patients. (El- Docket No. 27527-0076WO1
  • HCC mortality has significantly increased in the 1990-2005 period (Jemal et al., 2009), probably due to the large number of patients with long standing hepatitis (Llovet et al, 2003). Only one third of patients are eligible for curative treatments (resection, liver transplantation), and the multikinase inhibitor sorafenib represents the only approved systemic agent for advanced cases (Lang, 2008; Llovet et al, 2003; Llovet et al, 2008). Thus, treatment options for HCC are currently very limited, and new and improved methods for diagnosing and treating HCC are needed.
  • HCC is a highly heterogeneous tumor.
  • Increasing evidence suggests that the complexity of this disease and its clinical variability mainly depend on different molecular alterations that occur during the development and progression of HCC (Hoshida et al, 2010; Villanueva et al, 2007; Zucman-Rossi and Laurent-Puig, 2007).
  • a variety of molecular alterations leading to aberrant activation of different cellular pathways, including IGF, Ras, Akt/mTOR and Wnt signaling, have been reported in HCC (Newell et al, 2009; Tovar et al, 2010; Villanueva et al, 2008).
  • a major clinical challenge is to identify the predominant molecular mechanisms in distinct HCC molecular classes in order to select the best therapeutic option for each individual on the basis of the genomic background of their tumor.
  • a new area of research has focused on microRNA (“miRNA”) expression in cancers, such as HCC.
  • MicroRNAs are an abundant class of short (about 19-25 nucleotides), non-coding endogenous RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mRNAs. miRNA genes are usually transcribed by RNA polymerase II (Pol II). The polymerase binds to a promoter found near the DNA sequence encoding what will become the hairpin loop of a pre-miRNA, which is a longer (ca 70-80 nt) hairpin- like precursors termed pre-miRNAs.
  • the pre- miRNA transcript is capped with a specially-modified nucleotide at the 5' end, polyadenylated with multiple adenosines (a poly(A) tail), and spliced.
  • the product called a primary miRNA (“pri-miRNA”), may be hundreds or thousands of nucleotides in length and can contain one or more miRNA stems. Docket No. 27527-0076WO1
  • a single pri-miRNA may contain from one to six miRNA precursors. These hairpin loop structures are composed of about 70 nucleotides each. Each hairpin is flanked by sequences necessary for efficient processing.
  • the double-stranded RNA structure of the hairpins in a pri-miRNA is recognized by a nuclear protein known as DiGeorge Syndrome Critical Region 8 (DGCR8), named for its association with DiGeorge Syndrome. DGCR8 associates with the enzyme Drosha, a protein that cuts RNA, to form the "Microprocessor" complex.
  • DGCR8 DiGeorge Syndrome Critical Region 8
  • DGCR8 orients the catalytic RNase III domain of Drosha to liberate hairpins from pri-miRNAs by cleaving RNA about eleven nucleotides from the hairpin base (two helical RNA turns into the stem).
  • the resulting hairpin known as a pre-miRNA, has a two-nucleotide overhang at its 3' end; it has 3' hydroxyl and 5' phosphate groups.
  • the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer. This endoribonuclease interacts with the 3' end of the hairpin and cuts away the loop joining the 3' and 5' arms, yielding an imperfect miRNA:miRNA duplex about 22 nucleotides in length. Although either strand of the duplex may potentially act as a functional miRNA, only one strand is usually incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact.
  • RISC RNA-induced silencing complex
  • MicroRNAs assemble in ribonucleoprotein complexes termed miRNPs and recognize their target sites by antisense complementarity, thereby mediating down-regulation of their target genes. Near-perfect or perfect complementarity between the miRNA and its target site results in target mRNA cleavage, whereas limited complementarity between the microRNA and the target site results in translational inhibition of the target gene.
  • miRNAs can regulate the expression of hundreds of genes, thereby simultaneously affecting a variety of molecular pathways (Farh et al., 2005; Lewis et al, 2003).
  • miRNAs regulate target niRNAs via a 7 bp "seed" sequence (i.e., sequence at 5' positions 1-7 or 2-8 of miRNA). Complementarity of an mRNA sequence to the "seed" is normally found in the 3' untranslated region (3' UTR). Docket No. 27527-0076WO1
  • Dysregulation of miRNA expression has been observed in cancer and can be related to different mechanisms, such as amplification/deletion, chromosomal rearrangement and epigenetic regulation (Calin et al., 2004; Chen, 2005; Esquela-Kerscher and Slack, 2006).
  • the C19MC locus is a poorly characterized primate-specific cluster of miRNAs with similar nucleotide sequence, which are expressed in undifferentiated tissues and cell type including placenta, human embryonic and hematopoietic stem cells (Bar et al., 2008; Bentwich et al., 2005; Laurent et al., 2008; Ren et al., 2009). Bar et al.
  • compositions comprising miRNAs and methods of use thereof.
  • the present invention also provides compositions and methods for treating HCC and other cancers by regulating miRNA expression level and/or activity. Further the invention provides anti-neoplastic compositions and methods for inhibiting growth, proliferation and/or metastasis of cancer cells. Such cancer cells can include but are not limited to tumors.
  • the present invention provides novel methods for decreasing cell viability, e.g., growth and/or proliferation, and/or migration, as well as methods for treating cancer (e.g., HCC) and/or for inhibiting metastasis of a cancer cell using miRNA inhibitors of the invention.
  • the present invention provides compositions and methods for increasing expression of LTBP-4, a protein presently discovered to be a target of miR-517a.
  • the invention provides novel methods for identifying a candidate subject or patient in need of treatment according to a method of the present invention.
  • the invention provides methods for determining a subject's HCC prognosis (i.e. likeliness of metastasis of HCC tumor) based on the expression levels of C19MC miRNAs in tumor cells of the subject. Docket No. 27527-0076WO1
  • the present invention provides a method for decreasing cell viability, the method comprising introducing into the cell an inhibitor of an RNA molecule selected from the group consisting of microRNA (miR)-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR- 524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR-520f, miR-518f* and precursors thereof, in an amount effective for decreasing cell viability.
  • an RNA molecule selected from the group consisting of microRNA (miR)-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, mi
  • the method for decreasing cell viability comprises decreasing cell proliferation. In another aspect, the method for decreasing cell viability comprises increasing apoptosis. In another aspect, the cell is a cancer cell, such as a hepatocellular carcinoma cell.
  • the inhibitor for decreasing cell viability is a nucleic acid molecule, wherein the nucleic acid molecule (i) hybridizes under stringent conditions to the RNA molecule, or (ii) comprises a sequence having at least 70% identity with one of SEQ ID NOs: 46 - 91.
  • the inhibitor is selected from the group consisting of an antisense oligonucleotide, an siRNA, an shRNA and a ribozyme.
  • the inhibitor is an siRNA molecule having a core nucleic acid sequence selected from SEQ ID NOs: 69 - 91.
  • the RNA molecule is miR-517a or a precursor thereof. In another aspect, the RNA molecule is miR-520c or a precursor thereof.
  • the method for decreasing cell viability is carried out in vivo. In another embodiment, the method is carried out in vitro.
  • the invention provides a method for treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of an RNA molecule, wherein the RNA molecule is selected from the group consisting of microRNA (miR)-517a, miR-517c, miR- 520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, Docket No.
  • miR microRNA
  • the subject having cancer has a cancer cell that overexpresses at least one microRNA selected from the group consisting of microRNA (miR)-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR- 520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR-520f, miR-518f*, and precursors thereof.
  • microRNA microRNA
  • the method for treating cancer comprises inhibiting metastasis of a cancer cell.
  • the cancer is hepatocellular carcinoma.
  • the inhibitor is a nucleic acid molecule, wherein the nucleic acid molecule (i) hybridizes under stringent conditions to the RNA molecule, or (ii) comprises a sequence having at least 70% identity with one of SEQ ID NOs: 46 - 91.
  • the inhibitor for treating cancer is selected from the group consisting of an antisense oligonucleotide, an siRNA, a micromir, and a ribozyme.
  • the inhibitor is an siRNA molecule having a core nucleic acid sequence selected from SEQ ID NOs: 69 - 91.
  • the antisense oligonucleotide has a nucleic acid sequence selected from SEQ ID NOs: 46 - 91.
  • the RNA molecule is miR-517a or a precursor thereof. In other aspects, the RNA molecule is miR-520c or a precursor thereof. In some embodiments, the method for treating cancer, comprises coadministering the inhibitor with a chemotherapeutic agent or small molecule.
  • the cancer is hepatocellular carcinoma and the miRNA is miR-517a or miR-520c.
  • the invention provides a method for determining whether a hepatocellular carcinoma (HCC) in a subject is likely to become Docket No. 27527-0076WO1 metastatic, the method comprising: (a) determining the expression level of one or more targets genes in the C19MC locus in a sample from the subject; and (b) comparing the expression level of (a) to the expression level of the one or more targets from subjects having known HCC outcomes; whereby the likeliness of HCC metastasis in the subject is determined from step (b); wherein the one or more target genes is selected from the group consisting of microRNA (miR)-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR- 516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR
  • miR microRNA
  • the sample is an RNA- containing sample.
  • the invention provides a method for increasing expression of latent TGF- ⁇ Binding Protein 4 (LTBP-4) in a cell, the method comprising introducing into the cell an inhibitor of microRNA (miR)-517a in an amount effective for decreasing the expression level or LTBP-4 inhibiting activity of mi-517a.
  • LTBP-4 latent TGF- ⁇ Binding Protein 4
  • the invention provides a composition comprising an anti-miR-517a oligonucleotide having a core nucleic acid sequence ACACTCTAAAGGGATGCACGAT (SEQ ID NO: 46) or
  • the composition further comprises a synthetic polymer.
  • the synthetic polymer is poly(ethylene glycol) (PEG)-polycation diblock copolymer.
  • the invention provides a pharmaceutical composition comprising a C19MC miRNA oligonucleotide inhibitor and a pharmaceutically acceptable diluent or carrier.
  • the oligonucleotide inhibitor comprises a nucleic acid sequence selected from SEQ ID NOs: 46 - 91.
  • Figure 1A is a graph quantifying the expression level of miR-517a following treatment with 5-aza-2'deoxycytidine 3 ⁇ and 4-phenylbutyric acid 3mM in Huh7 cells compared with untreated cells. Results are expressed as fold changes ⁇ standard error of the mean (SEM); *p ⁇ 0.001.
  • Figure IB is an image of an agarose gel showing PCR products following methylation-specific PCR using primers specific for unmethylated (“U”) or methylated (“M”) sequences of the CpG located upstream of the C19MC locus.
  • Untreated DNA DNA not treated with sodium bisulfate
  • Ctrl sodium bisulfate-treated DNA following incubation with Sssl methylase.
  • Figure 2A is a graph quantifying cell proliferation at 24, 48 and 72 hours following transfection with control oligonucleotide, miR-517a or miR-520c ( ⁇ ) in Huh7 cells. Results are expressed as percentage of 3 H-thymidine incorporation (counts per million) ⁇ SEM compared with control; *p-value ⁇ 0.05.
  • Figure 2B is a graph quantifying cell migration (# migrated cells per field) in each group. Data represent the mean of 10 different fields; *p-value ⁇ 0.001.
  • Figure 2C is a graph quantifying the number of invaded cells per field; *p-value ⁇ 0.05.
  • Figure 2D is a graph quantifying cell migration in a wound healing assay. Results are expressed as distance (in ⁇ ) between the two edges ⁇ SEM; *p ⁇ 0.002, **p ⁇ 0.001.
  • Figure 3 A is flow chart representing the strategy used to identify potential target genes of an miRNA. Docket No. 27527-0076WO1
  • Figure 3B is a graph quantifying expression of LTBP-4 in Huh7 stably expressing miR-517a determined by qRT-PCR. Results are expressed as relative expression levels compared with control (cells stably transduced with empty vector); *p ⁇ 0.05.
  • Figure 3C is a graph quantifying luciferase expression in Huh7 cells after co-transfection with a reporter vector harboring the 3'UTR target sequence of LTBP-4 downstream from the luciferase gene, and synthetic miR-517a. Results are expressed as percentage of luciferase activity normalized to renilla expression; *p ⁇ 0.05.
  • Figure 3D is a graph quantifying luciferase expression in Huh7 cells after co-transfection with a reporter vector engineered with a mutated 3'UTR LTBP4 sequence downstream from the luciferase gene, and synthetic miR-517a. Results are expressed as percentage of luciferase activity normalized to renilla expression; *p ⁇ 0.05.
  • Figure 4 shows graphs plotting the amount of Relative Luciferase Light Unit (RLU) in the liver area over time, as an indicator of tumor growth in individual BALB/c nude mice at the indicated time points following injection into the left lobe of the liver with luciferase-tagged Huh7 cells stably transduced with miR-517a (upper) or control vector (lower panel).
  • RLU Relative Luciferase Light Unit
  • Figure 5 is a graph quantifying miR-517a expression determined by qRT-PCR in Huh7 cells stably transfected with control (pCDH vector) or miR-517a vector, following cell sorting of GFP positive cells.
  • Figure 6 is a Kaplan-Meier survival curve showing survival of mice injected with miR-517a-transduced Huh7 or control Huh7 cells at the indicated time points following injection.
  • Figure 7A is a graph quantifying the expression of miR-517a in SNU- 423 cells and in Huh7 cells that were stably transduced with miR-517a ("miR-517a- Huh7"), as determined by qRT-PCR. Ct values are displayed as an indicator of miR-517a expression. Docket No. 27527-0076WO1
  • Figure 7B is a graph showing the fold change in miR-517a expression in SNU-423 cells, as determined by qRT-PCR, 72 hours post-transfection with lOOnM of control ("Ctrl”) anti-miR or lOOnM anti-miR-517a. Results are expressed as fold change compared to control.
  • Figure 7C is a graph showing the percentage of viable SNU-423 cells following transfection with lOOnM or 200nM control ("ctrl") anti-miR or anti-miR-517a, as determined in an MTT assay. Results are expressed as percentage of viable cells compared to the control. "P ⁇ 0.05" indicates statistical significance.
  • Figure 7D is a graph showing the percentage of invaded SNU-423 cells per field 24 hours after transfection with either lOOnM or 200nM control anti-miR or anti- miR-517a. Results are expressed as percentage of invaded cells/field, compared to control.
  • Figure 7E left panel, shows representative images of SNU-423 cells in a migration assay 24 hours after transfection with lOOnM or 200nM control anti-miR ("Ctrl anti-miR") or anti-miR-517a.
  • Figure 7E, right panel, is a graph quantifying the percentage of migrated SNU-423 cells per field 24 hours after transfection with either lOOnM or 200nM control anti-miR or anti-miR-517a. Results are expressed as percentage of migrated cells/field, compared to control.
  • Figure 7F is a Western blot image showing expression of total ER , phosho ERK ("pERK”), or Tublin (control) in SNU-423 cells transfected with 300 nM anti- miR-517a or 300 nM control (“Ctrl”) anti- miR.
  • the phrase "decreasing cell viability" with respect to a microRNAs inhibitor of the invention means the inhibitor causes a cell to stop actively growing and/or dividing.
  • the decrease in cell viability can also be associated with apoptosis of the cell.
  • An increase in apoptosis in a cell population is relative to the Docket No. 27527-0076WO1 incidence of cell death or apoptosis observed in a population of the cell in the absence of an miRNA inhibitor of the invention (e.g., more of the cells are induced into the death process as compared to non-exposure to (contact with) the miRNA inhibitor.
  • an effective amount of the miRNA inhibitor is introduced into the cell to result in a decrease in cell viability, such as an increase in cell death or apoptosis.
  • Apoptosis is generally considered to be a form of programmed cell death in which a controlled sequence of events (or program) leads to the elimination of the cell.
  • the phrase "decreasing cell proliferation” means that the proliferation and/or ability of a cell to proliferate is preferably decreased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, more preferably by at least about 50%, at least about 60%, at least about 70%, and most preferably by at least about 80%, at least about 90%, or at least about 100%. Proliferation is determined by any suitable method, such as 3 H-Thymidine incorporation.
  • the phrase "inhibiting metastasis of a cancer cell” means full or partial inhibition of a metastasis.
  • the inhibition of metastasis is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, more preferably at least about 70%, at least about 80%, and most preferably at least about 90%, or at least about 100%.
  • microRNAs refer to a small 19-25 bp endogenous single stranded RNA that regulates the expression of target mRNAs via a 7 bp "seed" sequence (i.e., sequence at 5' positions 1-7 or 2-8 of miRNA). Complementarity of an mRNA sequence to the "seed" is normally found in the 3' untranslated region (3' UTR).
  • C19MC miRNA(s) refers to one or more of the miRNA molecules in the C19MC locus, such as e.g., any one or more of miR-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-
  • miRNA-524* miRNA-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR-526a, miR-518b, and miR-525, miR-517b, miR-520h, miR-520b, miR-520f, miR-518f*.
  • An asterisk (*) next to an miRNA means that the miRNA derives from the Docket No. 27527-0076WO1 other arm of the precursor from which the most predominant form derives (in other words, a precursor miRNA can give rise to two excised forms, one from each arm. The asterisk is used to denote the less predominant form).
  • the term "miRNA precursor,” or “precursor thereof in reference to a particular miRNA refers broadly to any precursor which through processing in a cell results in the specified miRNA.
  • the term thus includes the corresponding pri- miRNA, pre -miRNA or variant thereof.
  • the precursor is the corresponding pri-miRNA or pre-miRNA.
  • the pre-miRNA sequence may include, for example, from 45-90, 60-80 or 60-70 nucleotides.
  • the sequence of the pre-miRNA may include the entire miRNA sequence, or be that of a pri-miRNA excluding from 0-160 nucleotides from the 5' and 3' ends of the pri-miRNA.
  • the sequence of the pre-miRNA may comprise the sequence of a hairpin loop.
  • the pri-miRNA sequence may comprise, for example, from 45-250, 55-200, 70-150 or 80-100 nucleotides.
  • the sequence of the pri- miRNA may include the pre-miRNA or miRNA.
  • the pri-miRNA may also include a hairpin structure (e.g., from 37-50 nucleotides).
  • complementarity means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interactions such as Wobble-base pairing which permits binding of guanine and uracil.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid sequence.
  • the term "therapeutically effective” applied to dose or amount refers to that quantity of a compound or composition (e.g., pharmaceutical composition) that is sufficient to result in a desired activity upon administration to an animal in need thereof.
  • the term “therapeutically effective amount” refers to that quantity of a compound or composition that is sufficient to treat at least one symptom of a cancer, such as but not limited to cancer cell proliferation, tumor growth, resistance to apoptosis, and angiogenesis, and/or to inhibit metastasis of a cancer cell.
  • 27527-0076WO1 effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
  • a "prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of cancer, or reducing the likelihood of the onset (or recurrence) of cancer or cancer symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations.
  • the term “inhibit” and its grammatical variations are used to refer to any level of reduction in a function or amount.
  • the term “inhibit” may mean without limitation reduction in the size of individual metastases and/or a reduction or complete elimination in the total number of distinct metastatic foci in a subject.
  • the term “inhibit” or “inhibitor” may mean without limitation reduction in the expression level or activity of the targeted miRNA.
  • an miRNA inhibitor may function by reducing the expression of an miRNA and/or by reducing the ability of miRNA to interact with its downstream target(s).
  • miRNA inhibitors of the present invention include without limitation antisense nucleic acids (oligonucleotides), ribozymes, triple helix forming oligonucleotides (TFOs), RNAi oligonucleotides, morpholino-oligos (see, U.S. Pat. No. 5,034,506), Locked Acid Nucleic (LNA) (reviewed in, e.g., Jepsen and Wengel, Curr. Opin. Drug Discov. Devel. 2004; 7:188-194; Crinelli et al, Curr.
  • antisense nucleic acids oligonucleotides
  • ribozymes triple helix forming oligonucleotides (TFOs)
  • TFOs triple helix forming oligonucleotides
  • RNAi oligonucleotides morpholino-oligos
  • LNA Locked Acid Nucleic
  • microRNA sponges any chemically modified or stabilized forms thereof including, without limitation, nano- and micro-sized polymeric or lipid carriers of the same.
  • Ebert MS Nat Methods 2007;4:721; Mattes et al, Curr Opin Mol Ther 2008;10: 150; Brown BD Nat Rev Genet 2009;10:578.
  • phrases "pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory Docket No. 27527-0076WO1 agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which a compound (e.g., a C19MC miRNA inhibitor) is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution, saline solutions, and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin, 18th Edition.
  • polynucleotide or “nucleotide sequence” mean a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and mean any chain of two or more nucleotides.
  • a nucleotide sequence can carry genetic information, including the information used by cellular machinery to make proteins and enzymes or can be a microRNA. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids.
  • the polynucleotides herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
  • the nucleic acids may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include methylation, "caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • Polynucleotides may contain one or more additional covalently Docket No.
  • 27527-0076WO1 linked moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • proteins e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • intercalators e.g., acridine, psoralen, etc.
  • chelators e.g., metals, radioactive metals, iron, oxidative metals, etc.
  • alkylators e.g., metals, radioactive metals, iron, oxidative metals, etc.
  • the polynucleotides may be derivatized by formation of a methyl or
  • express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example, producing an non-coding (untranslated) RNA or a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product” such as RNA or a protein.
  • the expression product itself, e.g. the resulting RNA or protein, may also be said to be “expressed” by the cell.
  • vector means the vehicle by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and clone the vector or promote expression of the introduced sequence.
  • Vectors include plasmids, cosmids, phages, viruses, etc. Vectors may further comprise selectable markers.
  • host cell means any cell of any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example, the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme.
  • host cells of the invention include without limitation Huh7, Hep3B, HepG2, PLC/PRF/5, SNU-387, SNU-398, SNU-423, SNU-449, SNU-475 cells.
  • oligonucleotide refers to a nucleic acid, generally of at least 8, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an RNA molecule such as an miRNA.
  • Oligonucleotides can be labeled, e.g., with 32 P -nucleotides or nucleotides to which a label, Docket No. 27527-0076WO1 such as biotin, has been covalently conjugated.
  • an oligonucleotide of the invention can be used as antisense oligonucleotides to inhibit the expression level or activity of an miRNA molecule (as described below).
  • oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
  • a sequence "encoding" an expression product such as an RNA (e.g., miRNA), polypeptide, protein, or enzyme, is a minimum nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme.
  • nucleic acid is “antisense” to an miRNA or miRNA precursor (i.e., is an “antisense oligonucleotide”) when, written in the 5' to 3' direction, it comprises the reverse complement of the corresponding region of the target nucleic acid.
  • Antisense compounds are also often defined in the art to comprise the further limitation of, once hybridized to a target, being able to modulate expression levels, or function of the target compound.
  • antisense nucleic acid molecule or oligonucleotide is used in the present disclosure to refer to a single stranded (ss) or double stranded (ds) nucleic acid molecule, which may be DNA, RNA, a DNA-RNA chimera, or a derivative thereof, which, upon hybridizing under physiological conditions with complementary bases in an RNA or DNA molecule of interest, inhibits the expression level or activity of the target miRNA molecule.
  • antisense broadly includes RNA-RNA interactions, RNA- DNA interactions, and RNase-H mediated arrest.
  • RNA interference refers to the ability of double stranded RNA (dsRNA) to suppress the expression level or activity of a specific target miRNA molecule of interest in a homology-dependent manner. While not intended to be bound by a particular theory or mechanism, it is currently believed that RNA interference acts post-transcriptionally by targeting RNA molecules for degradation. RNA interference commonly involves the use of dsRNAs that are greater than 500 bp; however, it can also be mediated through small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs), which can be 10 or more nucleotides in length and are typically 18 or more nucleotides in Docket No. 27527-0076WO1 length. For reviews, see Bosner and Labouesse, Nature Cell Biol. 2000; 2:E31-E36 and Sharp and Zamore, Science 2000; 287:2431-2433.
  • siRNAs small interfering RNAs
  • shRNAs small hairpin RNAs
  • TFO triple helix forming oligonucleotide
  • TFOs bind to the purine-rich strand of the duplex through Hoogsteen or reverse Hoogsteen hydrogen bonding. They exist in two sequence motifs, either pyrimidine or purine. According to the present invention, TFOs can be employed as an alternative to antisense oligonucleotides to inhibit miRNA expression level TFOs have also been shown to produce mutagenic events, even in the absence of tethered mutagens.
  • TFOs can increase rates of recombination between homologous sequences in close proximity.
  • TFOs of the present invention may be conjugated to active molecules (for a review, see Casey and Glazer, Prog. Nucleic Acid. Res. Mol. Biol. 2001; 67: 163-92).
  • ribozyme is used herein to refer to a catalytic RNA molecule capable of mediating catalytic reactions on (e.g., cleaving) RNA substrates (e.g., miRNA molecules). Ribozyme specificity is dependent on complementary RNA-RNA interactions (for a review, see Cech and Bass, Annu. Rev. Biochem. 1986; 55:599-629). Two types of ribozymes, hammerhead and hairpin, have been described. Each has a structurally distinct catalytic center.
  • the present invention contemplates the use of ribozymes designed on the basis of the miRNA or miRNA-encoding nucleic acid molecules of the invention to induce catalytic reaction (e.g., cleavage) of the miRNA, thereby modulating (e.g., inhibiting) a function or expression of the miRNAs of the invention.
  • catalytic reaction e.g., cleavage
  • Ribozyme technology is described further in Intracellular Ribozyme Applications: Principals and Protocols, Rossi and Couture ed., Horizon Scientific Press, 1999.
  • nucleic acid hybridization refers to the pairing of complementary strands of nucleic acids, including triple-stranded nucleic acid hybridization.
  • the mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of nucleic Docket No. 27527-0076WO1 acids.
  • adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
  • Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions.
  • Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters.
  • Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated.
  • hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length.
  • high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, followed by washing in O.
  • lx SSC/0.1% SDS (where lx SSC is 0.15 M NaCl, 0.15 M Na citrate) at 68°C or for oligonucleotide inhibitors washing in 6xSSC/0.5% sodium pyrophosphate at about 37°C (for 14 nucleotide- long oligos), at about 48°C (for about 17 nucleotide-long oligos), at about 55°C (for 20 nucleotide-long oligos), and at about 60°C (for 23 nucleotide-long oligos).
  • Conditions of intermediate or moderate stringency such as, for example, an aqueous solution of 2xSSC at 65°C; alternatively, for example, hybridization to filter- bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C followed by washing in 0.2 x SSC/0.1% SDS at 42°C
  • low stringency such as, for example, an aqueous solution of 2xSSC at 55°C
  • Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA or RNA molecule and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol. Docket No. 27527-0076WO1
  • standard hybridization conditions refers to hybridization conditions that allow hybridization of two nucleotide molecules having at least 50% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 75% sequence identity, at least 80%> sequence identity, at least 90%> sequence identity, at least 95% sequence identity, or at least 99%> sequence identity.
  • the phrases “specifically hybridizable”, “hybridizes specifically to”, “specifically targets”, and other similar phrases refer to the association of a nucleic acid with an miRNA, or miRNA precursor, resulting in interference with the normal function of the miRNA, or miRNA precursor (e.g. by altering the activity, disrupting the function, or modulating the expression level of the miRNA or miRNA precursor).
  • a nucleic acid is “specifically hybridizable,” to an miRNA or miRNA precursor, there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to nucleic acid sequences other than the intended miRNA or miRNA precursor under conditions in which specific hybridization is desired (e.g.
  • the sequence of the nucleic acid need not be 100% complementary to that of its target miRNA or miRNA precursor to be specifically hybridizable. Moreover, the nucleic acid may hybridize over one or more segments of the miRNA or miRNA precursor such that intervening or adjacent segments are not involved in the hybridization (e.g., a bulge, a loop structure or a hairpin structure).
  • Nucleic acid molecules that "hybridize” to or “target” any of the miRNAs of the present invention may be of any length. In one embodiment, such nucleic acid molecules are at least 10, at least 15, at least 20, or at least 25 nucleotides in length. In another embodiment, nucleic acid molecules that hybridize are of about the same length as Docket No. 27527-0076WO1 the particular miRNA targeted by the nucleic acid molecule. In another embodiment, more than one miRNA inhibitor can be assembled into one vector of at least 25 nucleotides.
  • homologous as used in the art commonly refers to the relationship between nucleic acid molecules or proteins that possess a “common evolutionary origin,” including nucleic acid molecules or proteins within superfamilies (e.g., the immunoglobulin superfamily) and nucleic acid molecules or proteins from different species (Reeck et al, Cell 1987;50:667). Such nucleic acid molecules or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of substantial percent similarity or the presence of specific residues or motifs at conserved positions.
  • sequence similarity generally refers to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.
  • the sequences are aligned for optimal comparison purposes.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. In one embodiment, the two sequences are, or are about, of the same length.
  • the percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and
  • Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 1997;25:3389.
  • PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules.
  • detectable change as used herein in relation to a function or expression level of a gene or miRNA (e.g., miR-517a) means any measurable change, preferably a statistically significant change, and preferably at least a 1.5 -fold change as measured by any available technique such as electrophoretic gel mobility shift assays (EMSA), denaturing (8M urea) PAGE analysis, SDS-PAGE, Western blotting, nucleic acid hybridization, quantitative PCR, etc.
  • ESA electrophoretic gel mobility shift assays
  • 8M urea denaturing
  • SDS-PAGE SDS-PAGE
  • Western blotting nucleic acid hybridization
  • quantitative PCR quantitative PCR
  • the term "expression level" of an miRNA means the amount of miRNA in a cell or sample, which can be quantified by any suitable method known in the art, such as, e.g., quantitative reverse transcriptase polymerase chain reaction ("qRT-PCR").
  • An miRNA inhibitor can inhibit the expression level of an miRNA.
  • An miRNA inhibitor can also inhibit the "activity” or "function” of an miRNA.
  • the terms "activity” and “function” with respect to an miRNA molecule are used interchangeably, and mean any property of the miRNA molecule that is directly or Docket No. 27527-0076WO1 indirectly related to causing a cancer phenotype of a cell or tissue.
  • An miRNA inhibitor can inhibit one or more functions of an miRNA molecule. As discussed, supra, a single miRNA molecule can have hundreds of targets. Thus, for example, miR-517a is presently discovered to modulate the expression of the protein, LTBP-4.
  • An miR-517a inhibitor can inhibit miR-517a activity, e.g., by decreasing or abolishing the ability of miR-517a to modulate the expression of LTBP-4.
  • the miR-517a inhibitor may or may not also inhibit other functions of miR-517a, e.g., the inhibitor may or may not inhibit the ability of miR- 517a to modulate the expression of other target genes.
  • the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
  • the term “treat” may mean to relieve or alleviate at least one symptom selected from the group consisting of tumor growth, metastasis, sensitivity of tumor cells to treatments such as chemotherapy, radiation therapy, thermotherapy, etc.
  • the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
  • treating cancer comprises inhibiting metastasis.
  • cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemia, carcinomas and sarcomas.
  • exemplary cancers include cancer of the breast, brain, cervix, colon, head & neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
  • Additional examples include, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine and exocrine pancreas, and prostate cancer. Docket No. 27527-0076WO1
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere
  • anti-plastic means inhibiting or preventing the growth of cancer.
  • the miRNA inhibitors of the invention e.g., oligonucleotide inhibitors
  • the miRNA inhibitors of the invention are antineoplastic nucleic acids.
  • Combination therapy means that the patient in need of the drug is treated or given another drug for the disease in conjunction with the nucleic acid.
  • Combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These drugs are said to be “coadministered”.
  • HCC Hepatocellular carcinoma
  • the present invention provides compositions and methods for treating HCC and other cancers by regulating miRNA expression level and/or activity. Further the invention provides antineoplastic compositions and methods for inhibiting growth, proliferation and/or metastasis of cancer cells.
  • cancer cells can include but are not limited to tumors.
  • a subclass of HCC patients were defined by the overexpression of a large cluster of miRNAs located on chromosome 19ql3.41 ("the C19MC locus"). These microRNAs include miR-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512- 3p, miR-522, miR-526a, miR-518b, and miR-525, miR-517b, miR-520h, miR-520b, miR- 520f, miR-518f*.
  • miRNAs were shown to play a significant role in promoting cancer.
  • these miRNAs promote HCC tumor growth and metastasis.
  • miR-517a and miR-520c were demonstrated to promote proliferation, survival and migration of cells in experimental models of HCC.
  • miR-517a was demonstrated to promote aggressive metastasis of HCC tumor cells and to decrease the expression of Latent TGF- ⁇ Binding Protein 4 (LTBP-4). Docket No. 27527-0076WO1
  • LTBP-4 is an extracellular protein potentially involved in cell adhesion due to the presence of heparin binding sites into its molecular structure (Kantola et al., 2008). Loss of LTBP-4 has been reported to promote abnormal lung development and colorectal cancer through deregulation of TGF- ⁇ signaling (Sterner-Kock et al., 2002). TGF- ⁇ signaling pathway plays an important role in invasiveness and epithelial- mesenchymal transition of tumor cells (Coulouarn et al., 2008; Thiery and Sleeman, 2006).
  • the nucleic acid and amino acid sequences of LTBP-4 are known and have been described.
  • the human LTBP-4 nucleic acid sequence has GenBank® Accession No. NM_001042544.1, which has the nucleic acid sequence:
  • Transcript variants of LTBP-4 are also known, and have GenBank® Accession Nos. NM OO 1042545.1 (mRNA) and NP OO 1036010.1 (protein); and Docket No. 27527-0076WO1
  • NM_003573.2 mRNA
  • NP_003564.2 protein
  • the nucleic acid sequence of NM_001042545.1 is:
  • amino acid sequence of NP OO 1036010.1 is:
  • NP 003564.2 The amino acid sequence of NP 003564.2 is:
  • the present invention is based on the discovery that certain C19MC miRNAs are overexpressed and play a role in promoting cancer, such as promoting cell proliferation, migration, survival (e.g., resistance to apoptosis), tumor formation, angiogenesis and metastasis.
  • C19MC miRNA molecules include, e.g., miR-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR- 526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR-520f, miR-518f* and precursors thereof.
  • miR-517a is overexpressed in HCC, and further that miR-517a regulates, for example and without limitation, the expression of LTBP-4. miR-517a may also have other targets. Further, miR- 520c is discovered to be upregulated in HCC. Mature forms of miR-517a and miR-520c are each 22 nucleotides in length. miR-517a and miR-520c share 77% homology in their mature nucleic acid sequences and 57% homology in their seed sequences. Using computational prediction approaches, miR-517a and miR-520c have very few target genes in common.
  • the present invention also contemplates inhibitors that can target miRNA molecule mutants.
  • the invention provides miRNAs precursor sequences, which are also contemplated as targets of the miRNA inhibitors of the present invention.
  • Precursor sequences for miRNAs are given in Table II, below: Docket No. 27527-0076WO1
  • an miRNA inhibitor inhibits (i.e., Docket No. 27527-0076WO1 targets) an miRNA selected from the group consisting of miR-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR- 516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR-526a, miR-518b, miR-525, miR- 517b, miR-520h, miR-520b, miR-520f, miR-518f* and precursors thereof.
  • an miRNA inhibitor specifically targets miR-520c, and most preferably, a miRNA inhibitor specifically targets miR-517a.
  • the invention provides methods for increasing the concentration of LTBP-4 protein within a cell including introducing into the cell a nucleic acid hybridizable to miR-517a or a precursor thereof. In other embodiments, the invention provides methods for decreasing viability of a cell, such as a cancer cell, and/or for treating cancer or inhibiting metastasis of a cancer cell using one or more miRNA inhibitors of the invention.
  • inhibitors that target nucleic acid sequences, such as miRNAs, are well known in the art, and can be readily designed and synthesized by the skilled artisan, once the target nucleic acid sequence has been identified (i.e., the miRNA sequence).
  • inhibitors can be obtained from commercial sources, such as Dharmacon, Inc. (Lafayette, CO), Exiquon (Woburn, MA), Ambion® (Austin, TX).
  • Such inhibitors are designed to be highly specific for the target miRNA; thus, miRNA sequences that vary even by only several nucleic acids can be specifically targeted by inhibitors, such that the inhibitor of one miRNA will not cross react with another miRNA having a highly homologous sequence.
  • an miRNA-517a inhibitor does not inhibit expression level or activity of miR-520c.
  • an miR-520c inhibitor does not inhibit the expression level or activity of miR-517a.
  • an miRNA inhibitor may cross react with one or more other miRNA molecules.
  • Non-limiting examples of miRNA inhibitors include oligonucleotides, such as antisense oligonucleotides, short interfering (siRNA) or small hairpin (shRNA)
  • RNA molecules triple helix forming oligonucleotides (TFOs), ribozymes, antagomirs, morpholino-oligos, Locked Acid Nucleic (LNA), microRNA sponges and decoys capable Docket No. 27527-0076WO1 of inhibiting the activity or expression level of the miRNA molecules of the invention.
  • Other inhibitors include but are not limited to peptides, small molecules, and any other inhibitor now known and to be discovered that are capable of inhibiting the expression level or function of an miRNA of the invention. Non-limiting examples of miRNA inhibitors are described in detail, infra.
  • the sequence of an oligonucleotide inhibitor may be designed such that it will hybridize to a particular miRNA or miRNA precursor or a region or segment thereof.
  • “Targeting” thus includes determination of at least one target region, segment, or site within the target miRNA or miRNA precursor for the interaction to occur such that the desired effect, e.g., inhibition of expression level or function (activity), will result.
  • region or “target region” is defined as a portion of the target miRNA or miRNA precursor having at least one identifiable sequence, structure, function, or characteristic.
  • Oligonucleotide-based inhibitors of miRNAs are also known as antagomirs and micromirs. See, for example, U.S. Patent Application Publication No. 2010/0069471 and U.S. Patent Application Publication No: 2009/0143326.
  • an oligonucleotide inhibitor is designed to hybridize to a single continuous region of the target miRNA or miRNA precursor.
  • a single nucleic acid is designed to bind to two different regions of a target miRNA or miRNA precursor.
  • an oligonucleotide inhibitor may be designed to block the processing of pre-miRNAs by Dicer by targeting part of the loop and part of the stem of a pre -miRNA.
  • nucleic acids are designed to block the processing of pri-miRNAs by Drosha by targeting part of the stem and part of either part of the single stranded RNA at the base of the stem.
  • the export of pre-miRNA to the cytoplasm by Exportin may be blocked by targeting pre -miRNA.
  • 27527-0076WO1 may be designed to block Drosha processing by targeting two discontinuous extensions of the base of the stem in a pri-miRNA sequence.
  • a nucleic acid may be designed to target the stem portion of an miRNA precursor.
  • any portions of the miRNA participating in mRNA binding may be targeted.
  • the first 6, 7, or 8 nucleotides from the 5' end of the miRNA may be targeted.
  • Such locations on the target miRNA or precursor thereof to which nucleic acid hybridizes may be referred to as a "suitable target segment.”
  • the term "suitable target segment" is defined as at least a 6, 7 or 8-nucleotide portion of a target region to which an oligonucleotide inhibitor is targeted.
  • an oligonucleotide inhibitor is designed to be sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect (e.g. anti-cancer effect, antineoplastic effect, increasing apoptosis, and or increasing intracellular and/or extracellular concentrations of LTBP-4 protein).
  • an oligonucleotide inhibitor is designed to target, at least in part, the seed region of the miRNA.
  • the target region includes at least a portion or the entire seed region of the miRNA.
  • seed region refers to nucleotides at the 5' end of the miRNA sequence that are typically common to an miRNA family.
  • the seed region includes 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides within the miRNA sequence.
  • the seed region of the miRNA is 6, 7, or 8 consecutive nucleotides within the miRNA sequence.
  • the seed region of the miRNA sequence may be nucleotides 1 through 7, 1 through 8, 2 through 7, 2 through 8, 1 through 9, 1 through 10, 2 through 9, 2 through 10, 3 through 10, or 4 through 12 from the 5' end of the miRNA sequence.
  • the seed region of the miRNA sequence may advantageously be inclusively defined as nucleotides 1 through 7, 1 through 8, 2 through 7, or 2 through 8 from the 5' end of the miRNA sequence.
  • the methods described herein include the use of an oligonucleotide inhibitor that is hybridizable to an RNA molecule, is antisense to an RNA molecule, and/or is substantially complementary to an RNA molecule, and/or Docket No. 27527-0076WO1 has at least 70% sequence identity to an miRNA.
  • RNA molecule in these methods is an miRNA selected from miR-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516- 5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR- 512-3p, miR-522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR- 520f, miR-518f* (the nucleic acid sequences of which are disclosed in Table I, supra), and precursors thereof (the nucleic acid sequences of which are disclosed in Table II, supra).
  • the oligonucleotide inhibitor comprises or consists of a sequence having at least 70%> sequence identity to a 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleobase sequence that is complementary to a nucleobase sequence that is of or within one of SEQ ID NOs: 1 - 45.
  • the "nucleobase sequence” refers to a consecutive nucleobases within the relevant SEQ ID NO.
  • the oligonucleotide inhibitor may comprise or consist of a nucleotbase sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%o, at least 99%, or 100% sequence identity to a sequence that is complementary to any one of SEQ ID NOs: 1 - 45, or to a 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobase sequence that is complementary to a nucleobase sequence within any one of SEQ ID NOs: 1 - 45.
  • the oligonucleotide inhibitor comprises or consists of a sequence having at least 75% or 80%> sequence identity to a sequence that is complementary to one of SEQ ID NOs: 1 - 45. In other instances, the oligonucleotide inhibitor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%o, at least 99% or 100% sequence identity with a sequence that is complementary to one of SEQ ID NOs: 1 - 45. In certain embodiments, the oligonucleotide inhibitor comprises or consists of a sequence having 100%) sequence identity with a sequence that is complementary to one of SEQ ID NOs: 1 - 45.
  • the oligonucleotide inhibitor is at least 12 nucleobases in length. In certain embodiments, the oligonucleotide inhibitor is at least 15 nucleobases in length. The oligonucleotide inhibitor may also be less than 22 nucleobases in length. Thus, in certain embodiments, the oligonucleotide inhibitor is from 7 to 21 nucleobases in length. In other embodiments, the oligonucleotide inhibitor is from 8 to 21, 9 to 21, to 21, 11 to 21, 12 to 21, 13 to 21, 14 to 21, 15 to 21, 16 to 21, 17 to 21, or 18 to 21 Docket No. 27527-0076WO1 nucleobases in length. In some instances, the oligonucleotide inhibitor is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleobases in length.
  • the oligonucleotide inhibitor may include a complementary sequence that differs by no more than 8 nucleobases (or nucleotides) from any one of the miRNA sequences identified in Table I or Table II (SEQ ID NOs: 1 - 45). In other embodiments, the oligonucleotide inhibitor may include a sequence that differs by no more than 5, 6, or 7 nucleobases (or nucleotides) from any one of any one of the miRNA sequences identified in Table I or Table II (SEQ ID NOs: 1 - 45).
  • the oligonucleotide inhibitor may include a sequence that differs by no more than 1, 2, 3 or 4 nucleobases (or nucleotides) from a complementary sequence to any one of the miRNA sequences identified in Table I and Table II (SEQ ID NOs: 1 - 45).
  • An oligonucleotide inhibitor may hybridize under stringent conditions to the RNA molecule. In certain embodiments, an oligonucleotide inhibitor hybridizes under low stringency hybridization conditions to the RNA molecule. In other embodiments, the oligonucleotide inhibitor hybridizes under moderately stringent hybridization conditions to the RNA molecule. In other embodiments, the oligonucleotide inhibitor hybridizes under highly stringent hybridization conditions to the RNA molecule.
  • the oligonucleotide inhibitor is substantially complementary to the miRNA or miRNA precursor.
  • the oligonucleotide inhibitor may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% sequence complementarity to a target region (e.g. seed region) within the miRNA or miRNA precursor.
  • the oligonucleotide inhibitor includes at least 90% sequence complementarity to a target region (e.g. seed region) within the miRNA or miRNA precursor.
  • the oligonucleotide inhibitor includes at least 95%), at least 96%>, at least 97%, at least 98%>, or at least 99% sequence complementarity to a target region (e.g. seed region) within the miRNA or miRNA precursor.
  • a target region e.g. seed region
  • an oligonucleotide inhibitor in which 18 of 20 of its nucleobases are complementary to a target sequence would represent 90 percent complementarity.
  • an oligonucleotide inhibitor is substantially complementary to a miRNA or precursor, the Docket No.
  • 27527-0076WO1 remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • an oligonucleotide inhibitor which is 22 nucleobases in length having 6 (six) non-complementary nucleobases which are flanked by two regions of complete complementarity with the target miR A or miR A precursor would have 72.7% overall complementarity with the miRNA or miRNA precursor.
  • Percent complementarity of an oligonucleotide inhibitor with a region of an miRNA or miRNA precursor can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al, J. Mol. Biol, 1990, 215, 403- 410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • the oligonucleotide inhibitor is perfectly complementary to the miRNA or miRNA precursor.
  • Any appropriate method for introducing an oligonucleotide inhibitor into a cell may be employed.
  • suitable methods include, for example and without limitation, cell transfection methods such as chemical, biological or mechanical means. Such methods include electroporation, use of a virus vector, lipofection, gene guns, and microinjection.
  • the method may be practiced with any appropriate cell, such as, e.g., a cell from a mammal, such as a human.
  • the cell may also be a cancer cell, such as a human HCC cell.
  • the methods of introducing the oligonucleotide inhibitor into the cell are performed in vitro. Once introduced into the cell, the oligonucleotide inhibitor performs a function of an miRNA inhibitor, such as, e.g., decreasing cell viability, proliferation, and/or migration, and/or inhibiting metastasis of a cancer cell.
  • an oligonucleotide inhibitor may act by one or more of a number of mechanisms, including a cleavage-dependent or cleavage-independent mechanism.
  • a cleavage-based mechanism can be RNAse H dependent and/or can include RISC complex function.
  • Cleavage-independent mechanisms include occupancy-based translational arrest, such as can be mediated by miRNAs, or binding of the oligonucleotide inhibitor to a protein, as do aptamers.
  • Oligonucleotide Docket No. 27527-0076WO1 inhibitors may also be used to alter the expression of genes by changing the choice of splice site in a pre-mRNA. Inhibition of splicing can also result in degradation of the improperly processed message, thus down-regulating gene expression.
  • oligonucleotide inhibitors are described in more detailed, infra. i. Antisense Nucleic Acids
  • Antisense oligonucleotides can be used to achieve inhibition of the expression level or activity of an miRNA molecule.
  • Antisense oligonucleotides typically comprise from about 5 nucleotides to about 30 nucleotides in length, preferably from about 10 to about 25 nucleotides in length, and more preferably from about 20 to about 25 nucleotides in length.
  • an antisense oligonucleotide e.g., SEQ ID NO: 46, or SEQ ID NO: 69, see Tables III and IV, respectively
  • an antisense oligonucleotide inhibits expression of miR-517a and re-activates the expression of LTBP-4.
  • an example of an antisense oligonucleotide is an antagomir.
  • Antagomirs harbor optimized phosphorothioate modifications and require >19- nt length for highest efficiency. Such antagomirs can discriminate between single nucleotide mismatches of the targeted miRNA (See, Krutzfeldt et al. Nucleics Acid Research 2007;35(9):2885-2892). Antagomir function can be abolished when the length of the antagomir is reduced to 19 nucleotides, whereas a 25 -nucleotide antagomir shows optimal functional activity.
  • an antisense nucleic acid molecule has for example but not limited to a sequence disclosed in Table III, below.
  • the antisense oligonucleotides can be DNA or R A or chimeric mixtures, or derivatives or modified versions thereof, and can be single-stranded or double- stranded.
  • the antisense oligonucleotides set forth in herein when a sequence includes thymidine residues, one or more of the thymidine residues may be replaced by uracil residues and, conversely, when a sequence includes uracil residues, one Docket No. 27527-0076WO1 or more of the uracil residues may be replaced by thymidine residues.
  • Non examples of oligonucleotide miR A inhibitors are given in Table IV, below.
  • Antisense oligonucleotides comprise sequences complementary to at least a portion of the corresponding miRNA molecule. However, 100% sequence complementarity is not required so long as formation of a stable duplex (for single stranded antisense oligonucleotides) or triplex (for double stranded antisense oligonucleotides) can be achieved. The ability to hybridize will depend on both the degree of complementarity Docket No. 27527-0076WO1 and the length of the antisense oligonucleotides. Generally, the longer the antisense oligonucleotide, the more base mismatches with the corresponding nucleic acid target can be tolerated. One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (see, e.g., U.S. Patent Nos. 5,814,500 and 5,811,234), or alternatively they can be prepared synthetically (see, e.g., U.S. Patent No. 5,780,607).
  • the function(s) of a C19MC microRNA (e.g., miR-517a, miR-517c, etc.) in a cancer cell may be inhibited and studied using antisense nucleic acids derived on the basis of miRNA nucleic acid molecules described herein.
  • miRNA-specific antisense oligonucleotides may be useful without limitation as therapeutics to decrease cell viability and/or proliferation, to treat cancer in a subject, and to inhibit metastasis of cancer cells.
  • the antisense oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, or a combination thereof.
  • the miRNA- specific antisense oligonucleotide comprises at least one modified sugar moiety, e.g., a sugar moiety selected from arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the miRNA-specific antisense oligonucleotide comprises at least one modified phosphate backbone selected from a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • Examples include, without limitation, phosphorothioate antisense oligonucleotides (e.g., an antisense oligonucleotide phosphothioate modified at 3' and 5' ends to increase its stability) and chimeras between methylphosphonate and phosphodiester oligonucleotides. These oligonucleotides provide good in vivo activity due to solubility, nuclease resistance, good cellular uptake, ability to activate RNase H, and high sequence selectivity.
  • oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, Docket No. 27527-0076WO1 short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH2-NH-0-CH2, CH2- N(CH3)-0-CH2, CH2-0-N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and 0-N(CH3)-CH2- CH2 backbones (where phosphodiester is 0-P02-0-CH2).
  • 5,677,437 describes heteroaromatic oligonucleoside linkages. Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U.S. Patent Nos. 5,792,844 and 5,783,682). U.S. Patent No. 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds.
  • the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al., Science 1991;254: 1497).
  • oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, 0(CH 2 ) felicitNH 2 or 0(CH 2 ) n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; 0-; S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group.
  • Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine may be used, such as inosine.
  • locked nucleic acids LNA can be used (reviewed in, e.g., Jepsen and Wengel, Curr. Opin. Drug Discov. Devel. 2004; 7: 188-194; Crinelli et al, Curr. Drug Targets 2004; 5:745-752).
  • LNA are nucleic acid analog(s) with a 2'-0, 4'-C methylene bridge. This bridge restricts the flexibility of the ribofuranose ring and locks the structure into a rigid C3-endo conformation, conferring enhanced hybridization Docket No. 27527-0076WO1 performance and exceptional biostability. LNA allows the use of very short oligonucleotides (less than 10 bp) for efficient hybridization in vivo.
  • an miRNA-specific antisense oligonucleotide can comprise at least one modified base moiety selected from a group including but not limited to 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- methylgu
  • the antisense oligonucleotide can include a- anomeric oligonucleotides.
  • An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, Nucl. Acids Res. 1987; 15:6625-6641).
  • Oligonucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
  • the antisense oligonucleotide can be a morpholino antisense oligonucleotide (i.e., an oligonucleotide in which the bases are linked to 6-membered morpholine rings, which are connected to other morpholine-linked bases via non-ionic phosphorodiamidate intersubunit linkages).
  • Morpholino oligonucleotides are highly resistant to nucleases and have good targeting predictability, high in-cell efficacy and high sequence specificity (U.S. Patent No.
  • Antisense oligonucleotides may be chemically synthesized, for example using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Antisense nucleic acid oligonucleotides can also be produced intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell within which the vector or a portion thereof is transcribed to produce an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, so long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • "naked" antisense nucleic acids can be delivered to adherent cells via "scrape delivery", whereby the antisense oligonucleotide is added to a culture of adherent cells in a culture vessel, the cells are scraped from the walls of the culture vessel, and the scraped cells are transferred to another plate where they are allowed to re-adhere. Scraping the cells from the culture vessel walls serves to pull adhesion plaques from the cell membrane, generating small holes that allow the antisense oligonucleotides to enter the cytosol.
  • RNAi RNA Interference
  • RNA interference is a process of sequence-specific post- transcriptional gene silencing by which double stranded RNA (dsRNA) homologous to a target locus can specifically inactivate gene function in plants, fungi, invertebrates, and vertebrates, including mammals (Hammond et al., Nature Genet. 2001;2: 110-119; Sharp, Genes Dev. 1999;13: 139-141).
  • dsRNA double stranded RNA
  • RNAi-mediated gene silencing is thought to occur via sequence-specific RNA degradation, where sequence specificity is determined by the interaction of an siRNA with its complementary sequence within a target RNA (see, e.g., Tuschl, Chem. Biochem. 2001; 2:239-245). Docket No. 27527-0076WO1
  • RNAi commonly involves the use of dsRNAs that are greater than 500 bp; however, it can also be activated by introduction of either siRNAs (Elbashir, et al, Nature 2001; 411 : 494-498) or short hairpin RNAs (shRNAs) bearing a fold back stem-loop structure (Paddison et al, Genes Dev. 2002; 16: 948-958; Sui et al, Proc. Natl. Acad. Sci. USA 2002; 99:5515-5520; Brummelkamp et al, Science 2002; 296:550-553; Paul et al, Nature Biotechnol. 2002; 20:505-508).
  • siRNAs Elbashir, et al, Nature 2001; 411 : 494-4908
  • shRNAs short hairpin RNAs bearing a fold back stem-loop structure
  • the siRNAs are preferably short double stranded nucleic acid duplexes comprising annealed complementary single stranded nucleic acid molecules.
  • the siRNAs are short dsRNAs comprising annealed complementary single strand RNAs.
  • siRNAs may also comprise an annealed RNA:DNA duplex, wherein the sense strand of the duplex is a DNA molecule and the antisense strand of the duplex is a RNA molecule.
  • each single stranded nucleic acid molecule of the siRNA duplex is of from about 19 nucleotides to about 27 nucleotides in length.
  • duplexed siRNAs have a 2 or 3 nucleotide 3 ' overhang on each strand of the duplex.
  • siRNAs have 5 '-phosphate and 3'-hydroxyl groups.
  • RNAi molecules comprise nucleic acid sequences that are complementary to the nucleic acid sequence of a portion of a target miRNA molecule.
  • the portion of the target nucleic acid to which the RNAi probe is complementary is at least about 15 nucleotides in length.
  • the target locus to which an RNAi probe is complementary may represent a transcribed portion of the miRNA gene or an untranscribed portion of the miRNA gene (e.g., intergenic regions, repeat elements, etc.)
  • the invention thus provides miRNA-directed siRNAs.
  • the core sequences of siRNA inhibitors can be the same as the core sequences of the antisense oligonucleotide inhibitors described, supra (SEQ ID NOs: 69-91).
  • an siRNA targeting miR-517a has a core sequence ACACUCUAAAGGGAUGCACGAU (SEQ ID NO: 69) and further includes necessary modifications, such as those described below.
  • the core sequence of the siRNA targeting miR-520c is ACCCUCUAAAAGGAAGCACUUU (SEQ ID NO: 78). Docket No. 27527-0076WO1
  • RNAi molecules may include one or more modifications, either to the phosphate-sugar backbone or to the nucleoside.
  • the phosphodiester linkages of natural RNA may be modified to include at least one heteroatom other than oxygen, such as nitrogen or sulfur.
  • the phosphodiester linkage may be replaced by a phosphothioester linkage.
  • bases may be modified to block the activity of adenosine deaminase.
  • a modified ribonucleoside may be introduced during synthesis or transcription.
  • siRNAs may be introduced to a target cell as an annealed duplex siRNA, or as single stranded sense and antisense nucleic acid sequences that, once within the target cell, anneal to form the siRNA duplex.
  • the sense and antisense strands of the siRNA may be encoded on an expression construct that is introduced to the target cell. Upon expression within the target cell, the transcribed sense and antisense strands may anneal to reconstitute the siRNA.
  • shRNAs typically comprise a single stranded "loop" region connecting complementary inverted repeat sequences that anneal to form a double stranded "stem” region. Structural considerations for shRNA design are discussed, for example, in McManus et al, RNA 2002;8:842-850. In certain embodiments the shRNA may be a portion of a larger RNA molecule, e.g., as part of a larger RNA that also contains U6 RNA sequences (Paul et al, supra).
  • the loop of the shRNA is from about 1 to about 9 nucleotides in length.
  • the double stranded stem of the shRNA is from about 19 to about 33 base pairs in length.
  • the 3' end of the shRNA stem has a 3' overhang.
  • the 3' overhang of the shRNA stem is from 1 to about 4 nucleotides in length.
  • shRNAs have 5 '-phosphate and 3'-hydroxyl groups.
  • RNAi molecules preferably contain nucleotide sequences that are fully complementary to a portion of the target nucleic acid, 100% sequence complementarity between the RNAi probe and the target nucleic acid is not required. Docket No. 27527-0076WO1
  • RNAi molecules can be synthesized by standard methods known in the art, e.g., by use of an automated synthesizer. RNAs produced by such methodologies tend to be highly pure and to anneal efficiently to form siRNA duplexes or shRNA hairpin stem-loop structures. Following chemical synthesis, single stranded RNA molecules are deprotected, annealed to form siRNAs or shRNAs, and purified (e.g., by gel electrophoresis or HPLC).
  • RNA polymerase promoter sequences e.g., T7 or SP6 RNA polymerase promoter sequences.
  • Efficient in vitro protocols for preparation of siRNAs using T7 RNA polymerase have been described (Donze and Picard, Nucleic Acids Res. 2002; 30:e46; and Yu et al, Proc. Natl. Acad. Sci. USA 2002; 99:6047-6052).
  • an efficient in vitro protocol for preparation of shRNAs using T7 RNA polymerase has been described (Yu et al, supra).
  • the sense and antisense transcripts may be synthesized in two independent reactions and annealed later, or may be synthesized simultaneously in a single reaction.
  • RNAi molecules may be formed within a cell by transcription of RNA from an expression construct introduced into the cell.
  • siRNAs are described in Yu et al, supra.
  • protocols and expression constructs for in vivo expression of shRNAs have been described (Brummelkamp et al, supra; Sui et al, supra; Yu et al, supra; McManus et al, supra; Paul et al, supra).
  • RNAi expression constructs for in vivo production of RNAi molecules comprise RNAi encoding sequences operably linked to elements necessary for the proper transcription of the RNAi encoding sequence(s), including promoter elements and transcription termination signals.
  • Preferred promoters for use in such expression constructs include the polymerase-III HI-RNA promoter (see, e.g., Brummelkamp et al., supra) and the U6 polymerase-III promoter (see, e.g., Sui et al, supra; Paul, et al. supra; and Yu et al, supra).
  • the RNAi expression constructs can further comprise vector sequences that facilitate the cloning of the expression constructs. Standard vectors are known in the art (e.g., pSilencer 2.0-U6 vector, Ambion Inc., Austin, TX). Docket No. 27527-0076WO1 iii. Ribozyme Inhibition
  • a function of or expression levels of miRNA molecules can be modulated by ribozymes designed based on the nucleotide sequence thereof.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the sequence-specific cleavage of RNA (for a review, see Rossi, Current Biology 1994;4:469- 471).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event.
  • the composition of ribozyme molecules must include: (i) one or more sequences complementary to the target RNA; and (ii) a catalytic sequence responsible for RNA cleavage (see, e.g., U.S. Patent No. 5,093,246).
  • hammerhead ribozymes cleave RNAs at locations dictated by flanking regions that form complementary base pairs with the target RNA. The sole requirement is that the target RNA has the following sequence of two bases: 5'-UG-3'.
  • the construction of hammerhead ribozymes is known in the art, and described more fully in Myers, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, 1995 (see especially Figure 4, page 833) and in Haseloff and Gerlach, Nature 1988; 334:585-591.
  • Ribozymes preferably are engineered so that the cleavage recognition site is located near the 5' end of the miRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non- functional miRNA molecules.
  • ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.). These can be delivered to cells which express the target miRNA molecule in vivo.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to catalyze miRNA cleavage.
  • ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration may be required to achieve an adequate level of efficacy. Docket No. 27527-0076WO1
  • Ribozymes can be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. Ribozyme technology is described further in Intracellular Ribozyme Applications: Principals and Protocols, Rossi and Couture eds., Horizon Scientific Press, 1999. iv. Triple Helix Forming Oligonucleotides (TFOs)
  • Nucleic acid molecules useful to inhibit activity or expression level of an miRNA molecule via triple helix formation are preferably composed of deoxynucleotides.
  • the base composition of these oligonucleotides is typically designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, resulting in TAT and CGC triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine -rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, e.g., those containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • sequences can be targeted for triple helix formation by creating a so-called "switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • triple helix molecules can be prepared by any method known in the art. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides such as, e.g., solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences "encoding" the particular RNA molecule. Docket No. 27527-0076WO1
  • RNA polymerase promoters such as the T7 or SP6 polymerase promoters. See, Nielsen, P.E. "Triple Helix: Designing a New Molecule of Life", Scientific American, December, 2008; Egholm, M., et al. "PNA Hybridizes to Complementary Oligonucleotides Obeying the Watson-Crick Hydrogen Bonding Rules.” (1993) Nature, 365, 566-568; Nielsen, P.E. 'PNA Technology'. Mol Biotechnol. 2004;26:233-48]
  • Oligonucleotide inhibitors can be modified to contain delivery or targeting agents that facilitate entry into a cell and/or nucleus, and/or that target a specific cell type, such as a cancer cell.
  • an oligonucleotide may comprise a synthetic polymer.
  • the synthetic polymer can be poly(ethylene glycol) (PEG)-polycation diblock copolymer for encapsulation of an miRNA of the invention (e.g., miR-517a) into polyp lex micelles with endosomal escaping capabilities.
  • Polyplex micelles are formed through electrostatic interaction between the miRNA of the invention (e.g., miR-517a) and PEG-polycation block copolymers providing a nano-sized delivery vehicle with excellent biocompatibility. Any existing copolymer or analogues thereof are suitable for the methods and compositions described herein.
  • targeting molecules include cell targeting peptides (e.g. TAT sequences, antennapedia domain), proteins, antibodies, lipids, and cancer-cell specific or selective cell surface markers.
  • the antisense oligonucleotide can include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl. Acad. Sci. USA 1989; 86:6553-6556; Lemaitre et al, Proc. Natl. Acad. Sci. USA 1987; 84:648-652; PCT Publication No. WO 88/09810) or blood-brain barrier (see, e.g., PCT Publication No.
  • an oligonucleotide miRNA inhibitor to which is conjugated a lipophilic moiety e.g., cholesterol, e.g., by conjugation by a linker to the oligonucleotide
  • a lipophilic moiety e.g., cholesterol
  • linkers are described in detail in U.S. Patent Application Publication No. 2010/0069471.
  • lipophilic moieties can enhance entry of the oligonucleotide inhibitor into a cell.
  • the cell is part of an organism, tissue, or cell line, e.g., a primary cell line, immortalized cell line, or any type of cell line disclosed herein.
  • the conjugated oligonucleotide inhibitor can be used to inhibit expression level or function of a target miRNA in an organism, e.g., a mammal, e.g., a human, or to increase expression level or function of a target miRNA in a cell line or in cells which are outside an organism.
  • An oligonucleotide inhibitor may further be conjugatedto an antibody that is specific for the target cell or tissue.
  • an antibody is specific for a HCC-specific marker, such as glypican 3, which allows the oligonucleotide inhibitor to be specifically targeted to the cancer cell.
  • a HCC-specific marker such as glypican 3
  • Such an antibody-conjugated oligonucleotide inhibitor may also be further modified with any of the cell or nuclear targeting agents and/or any other modifications described herein.
  • an miRNA inhibitor may be conjugated to a ligand which targets the inhibitor to a specific cell-type, e.g. a cancer cell, expressing a cell surface receptor for the ligand.
  • ligands include transferrin, folate, and lectin.
  • compositions preferably comprise one or more miRNA inhibitors as described herein (e.g., an inhibitor comprising a sequence identified by one of SEQ ID NOs: 46 - 91).
  • the one or more miRNA inhibitors may be targeted Docket No. 27527-0076WO1 to a first miRNA target and one or more additional miRNA inhibitors targeted to a second miRNA target.
  • compositions may contain two or more miRNA inhibitors targeted to different regions, segments or sites of the same miRNA target. Two or more combined miRNA inhibitors may be used together or sequentially. Further, two or more compositions may be used in combination according to the method of the present invention.
  • compositions described herein are useful, for example and without limitation, for decreasing cell viability and/or cell proliferation and/or migration, for treating cancer and/or at least one symptom associated with cancer, and/or for inhibiting metastasis.
  • compositions include an active agent and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the pharmaceutical compositions including each of the active agents, will be prepared in accordance with good manufacturing process (GMP) standards, as set by the Food & Drug Administration (FDA).
  • GMP Manufacturing process
  • FDA Food & Drug Administration
  • Quality assurance (QA) and quality control (QC) standards will include testing for purity and function and other standard measures.
  • a preferred delivery vehicle is any chemical entity that ensures delivery of an inhibitor to a cancer cell in a selective manner, achieves sufficient concentration of active inhibitor in the cancer cell, and is preferably orally bioavailable.
  • This can include, without limitation, standard pharmaceutical dosage forms (e.g., tablets, capsules, powders, solutions, suspensions, emulsions) with or without controlled release. Docket No. 27527-0076WO1
  • the invention provides methods for inhibiting the function or expression level of a C19MC miRNA of the invention in a cell using the miRNA inhibitors of the invention.
  • the invention provides methods for decreasing cell viability, wherein the method comprises introducing into the cell an inhibitor of an RNA molecule, wherein: (a) the RNA molecule is selected from the group consisting of microRNA (miR)-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR- 519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR-520f and miR-518f*, and precursors thereof; and (b) said RNA molecule inhibitor is introduced in an amount effective for decreasing cell viability or proliferation.
  • the RNA molecule is selected from the group consisting of microRNA (miR)-517a, miR-517c
  • the method for decreasing cell viability comprises introducing into the cell an inhibitor of miR-517a or miR-520c in an amount effective for decreasing cell viability.
  • a combination of inhibitors may be introduced to the cell in order to inhibit two or more different C19MC microRNAs.
  • Such methods are useful for treating a cell having a proliferative disorder, such as, but not limited to cancer. Treatment can be in vitro, ex vivo, and/or in vivo.
  • the miRNA inhibitor(s) can be any one or more of those described above.
  • Examples of cells for use in the methods described herein include, but are not limited to SNU-387, SNU-398, SNU-423, SNU-449, SNU-475, Hep3B, HepG2, PLC/PRF/5, and Huh7 cells (HCC cancer cells), primary cancer cells obtained from mammalian subjects, or any cell expressing one or more C19MC miRNAs.
  • Such cells are available from ATCC (SNU-387 ATCC NUMBER: CRL-2237TM; SNU-398 ATCC NUMBER:CRL-2233TM; SNU-423 ATCC NUMBER:CRL-2238TM, SNU-449 ATCC NUMBER:CRL-2234TM, SNU-475 ATCC NUMBER: CRL-2236TM, Hep3B ATCC NUMBER: HB-8064TM; HepG2 ATCC NUMBER: CRL- 10741TM; PLC/PRF/5 ATCC Docket No. 27527-0076WO1
  • Cells can be transfected with one or more miRNAs, e.g., as described in the Examples, infra, in order to facilitate study and testing of overexpressed miRNAs. Treatment of the cells can also occur in vivo, wherein the cell is part of a tissue, organ, or circulatory system, or any other location in the subject to be treated.
  • the invention thus provides methods for treating cancer and/or for treating at least one symptom associated with cancer, comprising inhibiting the expression or function of one or more C19MC miRNA molecules using anti-cancer agents, as described in more detail below.
  • the methods of the invention are useful for treating a subset of HCC patients that have cancer cells that overexpress a subset of C19MC miRNAs.
  • the invention is useful for treating a subject or patient having cancer, wherein the subject having cancer has a cancer cell that overexpresses at least one microRNA selected from the group consisting of microRNA (miR)-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR- 520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR-520f, miR-518f*, and precursors thereof.
  • the invention provides novel anti-cancer agents based on the miRNA-specific antisense oligonucleotides, RNA interference (RNAi) molecules, ribozymes, and triple helix forming oligonucleotides (TFOs) described herein, as well as methods for using such agents to treat cancer.
  • RNA interference RNA interference
  • TFOs triple helix forming oligonucleotides
  • the anti-cancer agents described herein can be used in conjunction with any existing treatments such as thermo-, chemo-, and Docket No. 27527-0076WO1 radiotherapeutic treatments, transarterial embolization (TACE), and molecular targeted therapies.
  • the miRNA-specific antisense oligonucleotides described herein are capable of inhibiting miRNA function and/or expression in vivo when transfected in HCC cells.
  • Such treatment can promote pro-apoptotic processes and/or decrease cell viability and/or proliferation, processes that are otherwise blocked by increased C19MC miRNA expression.
  • Anti-cancer agents may be based on the miRNA-specific antisense oligonucleotides, RNA interference (RNAi) molecules, ribozymes, and triple helix forming oligonucleotides (TFOs), candidate molecules can be introduced in a cancer cell line (e.g., SNU-387, SNU-398, SNU-423, SNU-449, SNU-475, Hep3B, HepG2, PLC/PRF/5, and/or Huh7 cells) followed by evaluating the miRNA expression levels and /or survival, growth, migration, and/or proliferation of cells in comparison to that of untransfected cells.
  • a cancer cell line e.g., SNU-387, SNU-398, SNU-423, SNU-449, SNU-475, Hep3B, HepG2, PLC/PRF/5, and/or Huh7 cells
  • miRNA-specific antisense oligonucleotides RNA interference (RNAi) molecules, ribozymes, and triple helix forming oligonucleotides (TFOs) which show the strongest effect on cell survival, growth, migration, and/or proliferation and miRNA expression can be further optimized (e.g., by increasing their resistance to nucleases, increasing the efficiency of their targeting to cancer cells, increasing their sequence specificity [e.g., by introducing phosphothioate or morpholino modifications or using LNA], and reducing the size) making them even more potent in inhibition of cell survival and inhibition of the target miRNA expression and/or function.
  • the most potent anti-cancer molecules selected in tissue culture experiments can be further tested for their ability to affect the growth of tumors induced in nude mice (e.g., by injection of HCC cells) and subjected to chemo- or radiation therapy.
  • the therapeutics described herein may be used in a method for treating cancer in a mammal Docket No. 27527-0076WO1 comprising administering said therapeutics to the mammal.
  • the mammal is human.
  • the mammal has a cancer cell that overexpresses one or more C19MC microRNAs.
  • the method further comprises subjecting the mammal to a treatment selected from the group consisting of radiation therapy, chemotherapy, thermotherapy, transarterial embolization (TACE), molecular targeted therapy and any combination thereof.
  • a method for treating cancer and/or for inhibiting metastasis of a cancer cell in a subject may comprise administering to a subject in need thereof a therapeutically effective amount of an inhibitor of an RNA molecule, wherein said RNA molecule is selected from the group consisting of microRNA (miR)- 517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR- 526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR-520f, miR-518f* and precursors thereof.
  • such inhibitor is conjointly administered with another anti-cancer treatment, such as e.g.,
  • the relative timing of administration of the miRNA inhibitor (e.g., antisense/RNAi/ribozyme/TFO administration) and anti-cancer treatment depends on the delivery mechanism for the specific miRNA inhibitor and on the type of the specific anticancer treatment used. Generally, cells may become more sensitive to an anti-cancer treatment as soon as they come in contact with antisense/RNAi/ribozyme/TFO administration.
  • a preferred cancer to be treated is HCC and a preferred inhibitor is an anti-miR-517a oligonucleotide or a precursor thereof.
  • a preferred inhibitor is an anti-miR-517a oligonucleotide or a precursor thereof.
  • any cancer that is associated with upregulated expression levels of one or more miRNA molecules in the C19MC locus is a candidate for treatment according to the methods of the present invention.
  • patients with other cancers such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, Docket No.
  • endotheliosarcoma lymphangiosarcoma, lymphangioendothelio-sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
  • Anti-cancer compositions can be advantageously used in combination with other treatment modalities, including without limitation radiation, chemotherapy, thermotherapy and molecular targeted therapies.
  • Chemotherapeutic agents used in the methods described herein include without limitation taxol, taxotere and other taxoids (e.g., as disclosed in U.S. Patent Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072; 5,587,493; European Patent No. EP 253 738; and PCT Publication Nos.
  • WO 91/17976, WO 93/00928, WO 93/00929, and WO 96/01815) cisplatin, carboplatin, (and other platinum intercalating compounds), etoposide and etoposide phosphate, bleomycin, mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, methotrexate, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, nitrosoureas, mitomycin, dacarbazine, procarbizine, campathecins, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, calicheamicin, and the like.
  • Typical radiation therapy includes without limitation radiation at 1-2 Gy.
  • Examples of radiation therapy include without limitation ⁇ -radiation, neutron beam Docket No. 27527-0076WO1 radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes.
  • Radioconjugates and chemotherapeutics may also be used in the methods described herein.
  • Directing the cytotoxic exposure directly to the tumor itself is a commonly used approach to deliver a cytotoxic drug while minimizing the cytotoxic exposure of normal tissues.
  • one of the factors which limit the effectiveness of such an approach is incomplete induction of tumor cell death because of limited dose delivery.
  • Tumor-specific delivery is commonly achieved by conjugating a cytotoxic agent (e.g., a toxins (such as ricin) or a radioisotope) to an antibody that preferentially targets the tumor (e.g., glypican-3 in HCC), anti-CD2 in neuroblastoma or anti-Her2-neu in certain breast carcinomas).
  • a cytotoxic agent e.g., a toxins (such as ricin) or a radioisotope
  • an antibody that preferentially targets the tumor e.g., glypican-3 in HCC
  • anti-CD2 in neuroblastoma
  • anti-Her2-neu in certain breast carcinomas
  • the targeting may be also done with natural targeting (i.e., with radioactive iodine in the treatment of thyroid carcinoma), physical targeting (i.e., administration of a radioisotope to a particular body cavity), or other targeting protein (e.g., ferritin in hepatocellular carcinoma).
  • C19MC miRNA-targeted therapy of a tumor or cancer cell can be combined with other anti-cancer therapies, including but by no means limited to small tyrosine kinase inhibitors (e.g., sorafenib, erlotinib, everolimus, gefitinib, brivanib, sunitinib, lapatinib, cediranib, vatalanib), monoclonal antibodies (e.g.
  • cetuximab bevacizumab, IMC-A12, IMC1121B, panitumumab, trastuzumab
  • suicide gene therapy i.e., introduction of genes that encode enzymes capable of conferring to tumor cells sensitivity to chemotherapeutic agents such as thymidine kinase of herpes simplex virus or varicella zoster virus and bacterial cytosine deaminase
  • anti-oncogene or tumor suppressor gene therapy e.g., using anti-oncogene molecules including monoclonal antibodies, single chain antibody vectors, antisense oligonucleotide constructs, ribozymes, immunogenic peptides, etc.
  • administration of tumor growth inhibitors e.g., interferon (IFN)-y, tumor Docket No.
  • IFN interferon
  • angiogenesis inhibitors e.g., fragments of angiogenic proteins that are inhibitory [such as the ATF of urokinase], angiogenesis inhibitory factors [such as angiostatin and endostatin], tissue inhibitors of metalloproteinase, soluble receptors of angiogenic factors [such as the urokinase receptor or FGF/VEGF receptor], molecules which block endothelial cell growth factor receptors, and Tie-1 or Tie-2 inhibitors), vasoconstrictive agents (e.g., nitric oxide inhibitors), immune therapies with an immunologically active polypeptide (including immunostimulation, e.g., in which the active polypeptide is a cytokine, lymphokine, or chemokine [e.g., IL-2
  • mapatumumab mapsatumumab
  • proteosome inhibitors e.g. bortezomib
  • cell cycle inhibitors e.g. flavopiridol
  • DNA methylation inhibitors e.g. 5-Aza-cytidine
  • Methods for treating cancer include gene therapy, such as, e.g., gene replacement therapy wherein a gene encoding an miRNA of the invention is replaced with, e.g., a gene encoding a non- functional miRNA molecule or a non-expressed miRNA gene.
  • gene therapy such as, e.g., gene replacement therapy wherein a gene encoding an miRNA of the invention is replaced with, e.g., a gene encoding a non- functional miRNA molecule or a non-expressed miRNA gene.
  • Nucleic acid constructs which include the exogenous gene and, optionally, nucleic acids encoding a selectable marker, along with additional sequences necessary for expression of the exogenous gene in recipient primary or secondary cells, are used to transfect primary or secondary cells in which the encoded product is to be produced.
  • Such constructs include but are not limited to infectious vectors, such as retroviral, herpes, adenovirus, adenovirus-associated, mumps and poliovirus vectors, can be used for this purpose.
  • an overexpressed C19MC miRNA in a cancer cell is replaced using gene therapy with a C19MC miRNA gene that has been inactivated (e.g., via mutation), such that it is not transcribed, or such that it encodes a mutant miRNA that cannot carry out one or more oncogenic functions of the wild type miRNA (e.g,. that promote cell proliferation, growth, resistance to apoptosis of the cell, and/or metastasis).
  • This achieves the effect of inhibiting the expression or oncogenic activity of the C19MC miRNA, thereby treating the cancer.
  • Such effect may be achieved, e.g., by decreasing the proliferation of the cell, decreasing the resistance to apoptosis of the cell, and/or by inhibiting metastasis of the cell.
  • Subjects amenable to treatment may be identified prior to the administration of an miRNA inhibitor. Accordingly, methods provided herein may be used as a targeted therapy, wherein treatment is tailored to a subject based on the particular characteristics of the cancer cells in the subject. For instance, as described in the Examples, infra, C19MC miRNAs, such as miR-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR- 519e, miR-512-3p, miR-522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR- 520b, miR-520f, miR-518f* and precursors thereof, are overexpressed in a subset of HCC patients.
  • C19MC miRNAs such as miR
  • methods provided herein may include identifying a subset of patients with HCC and any other cancer determined to be associated with increased
  • C19MC miRNA expression e.g. increased expression of one or more miRNAs selected Docket No. 27527-0076WO1 from miR-517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR- 522, miR-526a, miR-518b, miR-525, miR-517b, miR-520h, miR-520b, miR-520f and miR- 518f* and precursors thereof) leading to the pathological state, and treating such patients with an miRNA inhibitor that specifically targets the overexpressed C19MC miRNA sequence(s).
  • An exemplary inhibitor includes but is not limited to an oligonucleotide that comprises the sequence set forth in one of SEQ ID NOs: 46 - 91
  • the methods described herein are also useful for determining whether a hepatocellular carcinoma (HCC) in a subject is likely to become metastatic. These methods include the steps of: (a) determining the expression level of one or more miRNA molecules in the C19MC locus in a sample from the subject; and (b) comparing the expression level of the miRNA molecules in (a) to the expression level of the one or more miRNA molecules from subjects having known HCC outcomes; whereby the likeliness of metastasis of HCC is determined from step (b); and wherein the one or more C19MC miRNA molecules is selected from the group consisting of miR-517a, miR- 517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, mi
  • a patient with a known clinical outcome of metastatic HCC will have a specific C19MC miRNA expression profile.
  • a patient having a similar expression profile i.e., upregulated expression of one or more C19miRNA molecules, and preferably upregulated expression of miR-517a and/or miR-520c), to that of the patient known to have metastatic HCC, is also likely to have metastatic HCC.
  • Tumor load is assessed prior to therapy by means of objective scans of the tumor such as with x-ray radiographs, computerized tomography (CAT scans), nuclear magnetic resonance (NMR) scans or direct physical palpation of the tumor mass.
  • objective scans of the tumor such as with x-ray radiographs, computerized tomography (CAT scans), nuclear magnetic resonance (NMR) scans or direct physical palpation of the tumor mass.
  • CAT scans computerized tomography
  • NMR nuclear magnetic resonance
  • the tumor may secrete a marker substance such as alphafetoprotein from Docket No. 27527-0076WO1 colon cancer, CA 125 antigen from ovarian cancer, or serum myeloma "M" protein from multiple myeloma, or AFP for HCC.
  • a marker substance such as alphafetoprotein from Docket No. 27527-0076WO1 colon cancer, CA 125 antigen from ovarian cancer, or serum myeloma "M" protein from multiple myeloma, or AFP for HCC.
  • the levels of these secreted products then allow for an estimate of tumor burden to be calculated.
  • These direct and indirect measures of the tumor load are done pretherapy, and are then repeated at intervals following the administration of the drug in order to gauge whether or not an objective response has been obtained.
  • An objective response in cancer therapy generally indicates >50% shrinkage of the measurable tumor disease (a partial response), or complete disappearance of all measurable disease (a complete response).
  • these responses must be maintained for a certain time period, usually one month, to be classified as a true partial or complete response.
  • time period usually one month
  • tumor shrinkage that is ⁇ 50%
  • increased survival is associated with obtaining a complete response to therapy and, in some cases, a partial response if maintained for prolonged periods can also contribute to enhanced survival in the patient.
  • Patients receiving chemotherapy are also typically "staged” as to the extent of their disease before and following chemotherapy are then restaged to see if this disease extent has changed. In some situations the tumor may shrink sufficiently and if no metastases are present, then surgical excision may be possible after chemotherapy treatment where it was not possible beforehand due to the widespread disease. In this case the chemotherapy treatment with the novel pharmaceutical compositions of the invention is being used as an adjuvant to potentially curative surgery.
  • patients may have individual lesions in the spine or elsewhere that produce symptomatic problems such as pain and these may need to have local radiotherapy applied. This may be done in addition to the continued use of the systemic pharmaceutical compositions.
  • Patients are assessed for toxicity with each course of miRNA inhibitor administration typically looking at effects on liver function enzymes and renal function enzymes such as creatinine clearance or BUN as well as effects on the bone marrow, typically a suppression of granulocytes important for fighting infection and/or a suppression of platelets important for hemostasis or stopping blood flow.
  • liver function enzymes and renal function enzymes such as creatinine clearance or BUN as well as effects on the bone marrow
  • effects on the bone marrow typically a suppression of granulocytes important for fighting infection and/or a suppression of platelets important for hemostasis or stopping blood flow.
  • normal blood counts may be reached between 1-3 weeks after therapy. Recovery then ensues over the next 1-2 weeks. Based on the recovery of normal white blood counts, treatments may then be resumed.
  • complete and partial responses are associated with at least a 1- 2 log reduction in the number of tumor cells (a 90-99% effective therapy).
  • Patients with advanced cancer will typically have >10 9 tumor cells at diagnosis, multiple treatments will be required in order to reduce tumor burden to a very low state and potentially obtain a cure of the disease.
  • a pharmaceutical formulation of the invention which could comprise several weeks of continuous drug dosing
  • patients can be evaluated for response to therapy (complete and partial remissions), toxicity measured by blood work and general well-being classified performance status or quality of life analysis.
  • the latter includes the general activity level of the patient and their ability to do normal daily functions. It has been found to be a strong predictor of response and some anticancer drugs may actually improve performance status and a general sense of well-being without causing significant tumor shrinkage.
  • the pharmaceutical formulations may similarly provide a significant benefit, well-being performance status, etc. without affecting true complete or partial remission of the disease.
  • a number of biological assays are available to evaluate and to optimize the choice of nucleic acids for optimal antitumor/anticancer activity. These assays can be roughly split into two groups; those involving in vitro exposure of miRNA inhibitors to tumor/cancer cells and in vivo antitumor/anticancer assays in rodent models and rarely, in larger animals.
  • Cytotoxic assays in vitro for miRNA inhibitors generally involve the use of established tumor/cancer cell lines both of animal and, especially of human origin. These cell lines can be obtained from commercial sources such as the American Type Tissue Docket No. 27527-0076WO1
  • the enumeration of the total number of cells is one approach to in vitro testing with either cell lines or fresh tumor biopsies.
  • clumps of cells are typically disaggregated into single units which can then be counted either manually on a microscopic grid or using an automated flow system such as either flow cytometry or a CoulterTM counter.
  • Control (untreated) cell growth rates are then compared to the treated (with a nucleic acid) cell growth rates.
  • Vital dye staining is another one of the older hallmarks of antitumor assays.
  • cells either untreated or treated with a cancer drug are subsequently exposed to a dye such as methylene blue, which is normally excluded from intact (viable) cells.
  • the number of cells taking up the dye is the numerator with a denominator being the number of cells which exclude the dye.
  • viability can be assessed using the incorporation of radiolabeled nutrients and/or nucleotides.
  • a typical Docket No. 27527-0076WO1 experiment involves the incorporation of either ( 3 H) tritium- or 14 C-labeled nucleotides such as thymidine.
  • Control (untreated) cells are shown to take up a substantial amount of this normal DNA building block per unit time, and the rate of incorporation is compared to that in the drug treated cells. This is a rapid and easily quantifiable assay that has the additional advantage of working well for cells that may not form large (countable) colonies.
  • Drawbacks include the use of radioisotopes which present handling and disposal concerns.
  • Suitable cell lines include but are not limited to Huh7, Hep3B, SNU-387, SNU-398, SNU-423, SNU-449, and SNU-475.
  • the current test system used by the National Cancer Institute uses a bank of over 60 established sensitive and multidrug-resistant human cells lines of a variety of cell subtypes. This typically involves 5-6 established and well-characterized human tumor/cancer cells of a particular subtype, such as non-small cell or small cell lung cancer, for testing new agents.
  • CompareTM the overall sensitivity in terms of dye uptake (either sulforhodamine B or MTT tetrazolium dye) is determined.
  • the specific goal of this approach is to identify nucleic acids that are uniquely active in a single histologic subtype of human cancer.
  • the endpoint for certain assays is the incorporation of a protein dye called sulforhodamine B (for adherent tumor cells) and the reduction of a tetrazolium (blue) dye in active mitochondrial enzymes (for non-adherent, freely-floating types of cells).
  • sulforhodamine B for adherent tumor cells
  • tetrazolium blue
  • miR A inhibitors are typically then injected at some later time point(s), either by intraperitoneal or intravenous administration, or administered by the oral routes.
  • Tumor growth rates and/or survival are determined and compared to untreated controls.
  • growth rates are typically measured for tumors growing in the front flank of the animal, wherein perpendicular diameters of tumor width are translated into an estimate of total tumor mass or volume.
  • the time to reach a predetermined mass is then compared to the time required for equal tumor growth in the untreated control animals.
  • significant findings generally involve a >25% increase in the time to reach the predetermined mass in the treated animals compared to the controls.
  • significant findings involve a >42% increase in the time to reach the predetermined mass in the treated animals compared to the controls.
  • the significant findings are termed tumor growth inhibition.
  • survival can be used as an endpoint and a comparison is made between the treated animals and the untreated or solvent treated controls.
  • a significant increase in life span for a positive new agent is again >20-42% longer life span due to the treatment.
  • anticancer miRNA inhibitors are generally tested at doses very near the lethal dose and 10% (LDio) and/or at the determined maximally- tolerated dose, that dose which produces significant toxicity, but no lethality in the same strain of animals and using the same route of administration and schedule of dosing. Similar studies can also be performed in rat tumor models.
  • nude mice which are typically hairless and lack a functional thymus gland
  • human tumors millions of cells
  • take This visible development of a palpable tumor mass is called a "take”.
  • Anticancer drugs such as the miRNA inhibitors disclosed herein are then injected by some route (intravenous, intramuscular, subcutaneous, per os) distal to the tumor implant site, and growth rates are calculated by perpendicular measures of the widest tumor widths as described earlier.
  • SCID mice A number of human tumors are known to successfully "take” in the nude mouse model.
  • An alternative mouse model for this work involves mice with a severe combined immunodeficiency disease (SCID), in which there is a defect in maturation of lymphocytes. Because of this, SCID mice do not produce functional B- and T-lymphocytes. However, these animals do have normal cytotoxic T-killer cell activity. Nonetheless, SCID mice will "take” a large number of human tumors. Tumor measurements and drug dosing are generally performed as above. Again, positive compounds in the SCID mouse model are those that inhibit tumor growth rate by >20-42% compared to the untreated control.
  • Anti-miR A inhibitors can be administered to individuals through injection (for example, intravenous, epidural, intrathecal, intramuscular, intraluminal, intratracheal or subcutaneous), orally, transdermally, or other methods known in the art.
  • An individual in need thereof is, for example, a human or other mammal that would benefit by the administration of an anti-miRNA inhibitor, such as a human or other mammal with cancer.
  • the pharmaceutical composition of this disclosure can be introduced parenterally, transmucosally, e.g., orally (per os), nasally, or rectally, or transdermally.
  • Parental routes include intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • Specific organs may be targeted, e.g., by direct administration to the targeted organ.
  • direct administration to the liver may be advantageous for the treatment of HCC.
  • a preferred route of administration of the compositions of the invention is oral.
  • the pharmaceutical compositions may take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch or sodium starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); Docket No. 27527-0076WO1 emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the chaperones for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing
  • compositions may be added to a retained physiological fluid such as blood or synovial fluid.
  • the active ingredient can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • a liposome see Langer, Science 249: 1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • miPvNA inhibitor-containing compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Docket No. 27527-0076WO1 the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • miRNA inhibitor- containing compounds and compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds and compositions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the therapeutic compound can be delivered in a controlled release system.
  • an miRNA inhibitor may be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly-lactic/glutamic acid (PLGA), a pellet containing a mixture of cholesterol and the active ingredient (SilasticR.TM.; Dow Corning, Midland, Mich.; see U.S. Pat. No. 5,554,601) implanted subcutaneously, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • PLGA poly-lactic/glutamic acid
  • SilasticR.TM. Dow Corning, Midland, Mich.
  • U.S. Pat. No. 5,554,601 implanted subcutaneously, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • DNA may be injected directly into a tumor.
  • Administration of miRNA inhibitors may be once a day, twice a day, or more often, but frequency may be decreased during a maintenance phase of the disease or disorder, e.g., once every second or third day instead of every day or twice a day.
  • the dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in Docket No. 27527-0076WO1 the art. More generally, dose and frequency will depend in part on recession of pathological signs and clinical and subclinical symptoms of a disease condition or disorder contemplated for treatment with the present compounds.
  • a median effective dose (ED 50 ) of an miR A inhibitor can be about 10 mg/kg/day (dose range: 1 to 200 mg/kg/day) (See, Elmen et al, Nature 2008;452, 896-899).
  • Dosages and administration regimen can be adjusted depending on the age, sex and physical condition of the subject or patient as well as the benefit of the treatment and side effects in the patient or mammalian subject to be treated and the judgment of the physician, as is appreciated by those skilled in the art.
  • FFPE Formalin-fixed paraffin-embedded
  • BCLC Barcelona Clinic Liver Cancer.
  • RNA from frozen tissues and Huh7 cells was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions.
  • RNA from FFPE sections was extracted as previously described (Hoshida et al, 2009).
  • RNA was purified using Clean-Up Purification kit (Qiagen, Germantown, MD) and quantified using the NanoDrop ND-1000 spectrometer (NanoDrop, Wilmington, DE). Quality and integrity were measured with a bioanalyzer (Agilent, Palo Alto, CA).
  • the expression levels of 358 miRNAs were determined using a bead-based amplification method as previously described (Lu et al, 2005; Mi et al, 2007).
  • Genome -wide mRNA and copy number profiling of the training set were performed as previously described (Chiang et al, 2008). mRNA profiling of the validation set was performed using whole-genome DNA-mediated Annealing, Selection, extension, and Ligation (DASL) Assay (April et al, 2009; Fan et al, 2004). Human Gene 1.0 ST Array (Affymetrix®, Santa Clara, CA) was used for mRNA profiling of Huh7 cells. Dataset is available from NCBI's Gene Expression Omnibus: accession number GSE20596. Docket No. 27527-0076WO1
  • miRNA expression analysis cDNA was synthesized using miRNA-specific primers using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Austin, TX) according to manufacturer's instruction. miR-23 (constant expression among the samples of the training set) was used to normalize the expression levels of miR-517a, miR-520g and miR-516-5p in the validation set (FFPE tissues). The ddCt method (Llovet et al, 2006) was used to calculate miRNA expression levels. Median relative expression levels> 100 for all the 3 miRNAs (miR-517a, miR-520g and miR-516- 5p) in HCC compared with cirrhotic tissues were used to define subclass C2 in the validation set.
  • LTBP-4 expression analysis Sprint RT-Complete-Random Hexamer kit (Clontech) was used to synthesize cDNA from total RNA.
  • Taqman gene expression assay Hs00186025_ml was used to analyze the expression levels of LTBP4 normalized to 18S expression (Hs99999901_sl).
  • Huh7 cells were treated with the demethylated agent 5-aza- 2'deoxycytidine 3 ⁇ (5-Aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid 3mM (PBA) (Sigma-Aldrich, St. Louis, MO, USA). 5-Aza-CdR was removed after 24h whereas PBA was daily administered for 6 days.
  • PBA histone deacetylase inhibitor 4-phenylbutyric acid 3mM
  • Genomic DNA was treated with sodium bisulfite using the EpiTect Bisulfite Kit (Quiagen, Valencia, CA) according to manufacturer's instructions.
  • PCR conditions for methylation-specific PCR reactions were: 95°C 10 min, 40 cycles at 94°C 15 Docket No. 27527-0076WO1 sec, 50°C 30 sec, 72°C 30 sec followed by 1 cycle at 72°C 10 min.
  • DNA extracted from Huh7 treated with M.SssI-methylase 4 ⁇ / ⁇ 1 New England Biolabs, Beverly, MA
  • DNA from Huh7 not treated with sodium bisulfite were used as positive and negative controls respectively.
  • PCR products were separated by 2% agarose gel electrophoresis. The PCR primer sequences are described in the following Table VI:
  • Pre-miRNA Precursor Molecule (Ambion, Austin, TX) mimicking miR- 517a, miR-520c or non-specific control miRNA (Pre-miR Negative Control #1, Ambion, Austin, TX) was transfected into Huh7 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 24 hours, the transfection medium was removed and complete medium was added.
  • Huh7 cells transfected with control oligonucleotide, miR-517a or miR- 520c were starved for 24 hours, suspended in serum- free medium and added (2 x 10 5 cell/ml, 250 ⁇ ) to the upper chamber of the Transwell chambers. Complete medium was Docket No. 27527-0076WO1 added to the bottom well of the chamber. After 16h, the cells on the upper surface of the filter were removed with a cotton swab, and the cells that had migrated to the lower surface of the filter were stained with crystal violet and counted using an inverted microscope by randomly selecting ten fields per filter (200X magnification). Matrigel-coated filters were used to assess invasive potential of miRNA-transfected cells.
  • SNU-423 cells were grown in RPMI supplemented with 10% heat- inactivated fetal bovine serum ("complete medium”) and maintained at 37°C in a 5% C02 atmosphere.
  • the antomir anti-miR-517a and control anti-miR were obtained from Regulus TherapeuticsTM (San Diego, CA).
  • SNU-423 cells were transfected with Lipofectin (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. After 24 hours, the transfection medium was removed and complete medium was added.
  • Transfected cells were grown to confluence and serum- starved for 24h. After pre-treatment with mytomycin C for 3h to inhibit proliferation, cell monolayers were wounded with a sterile tip and maintained in serum-free medium for 24h. Images were taken after 0 and 24h post- wounding on an inverted microscope (100X). Distance measurements between the two edges were performed using Image J software. miRNA Stable Transfection
  • Recombinant lentivirus were produced by co-transfecting human embryonic kidney 293T cells with GFP-miR-517a or GFP-only vectors (ViraPowerTM Lentiviral Packaging Mix, Invitrogen, Carlsbad, CA) using Lipofectamine 2000. Supernatant containing lentivirus was collected 48h after transfection and added to Huh7 cells. GFP -positive Huh7 were sorted by fluorescence-activated cell sorting (FACS) analysis (Becton Dickinson Instrument, San Jose, CA).
  • FACS fluorescence-activated cell sorting
  • GFP-sorted Huh7 cells were transfected with firefly luciferase plasmid (Addgene plasmid 18964) (Safran et al., 2006) and selected in medium containing G148 for 2 weeks. Luciferase expression was confirmed using a bioluminescent system. Docket No. 27527-0076WO1
  • the 3'UTR LTBP4 target sequence of miR-517a was cloned into the miRGLO vector (Promega) according to manufacturer's instructions.
  • a mutated sequence of the target 3'UTR LTBP4 sequence was designed and cloned into the miRGLO vector as control.
  • the correct insertion of both LTBP4 sequences into the vector was confirmed by DNA sequencing.
  • Luciferase-reporter assays were performed 24 hours after co-transfection with plasmids harboring wild-type or mutated 3'UTR sequences (100 ng) and synthetic miR-517a ( ⁇ ) (Ambion) using Lipofectamine 2000. Renilla expression was used to normalize luciferase signal.
  • Luciferase-tagged Huh7 cells expressing with miR-517a or control vector were injected directly into the left liver lobe of nude BALB/C mice (6-8 weeks of age). This study was performed in accordance with the guidelines and regulation from the Institutional Animal Care and Use Committee (IACUC). BALB/c nude mice (6-8 weeks of age) were anesthetized intraperitoneally with a solution of 80 mg/kg Ketamine and 100 mg/kg Xylazine. A small transverse incision below the sternum was made to expose the liver.
  • IACUC Institutional Animal Care and Use Committee
  • mice were anesthetized with isoflurane/oxygen, injected intraperitoneally with the substrate luciferin at a dose of 150 mg/kg body weight and placed on the imaging stage. Mice were imaged 1 day after surgery and once a week for 8 consecutive weeks. Ventral images were collected for 30 seconds to 1 minute using the I VIS system (Xenogen Corp., Alameda, CA). Photons emitted from luciferase-expressing cells were quantified using Living Image software c (Xenogen Corp., Alameda, CA) and used as indicator of Docket No. 27527-0076WO1 tumor growth. At week 9 mice were sacrificed and ex-vivo imaging of collected organs were performed.
  • liver tumors and secondary organs (spleen, stomach, lung, colon, kidneys) from mice included in the study were excised, fixed in 4% paraformaldehyde solution and embedded in paraffin. Five- ⁇ slides were stained with hematoxylin and eosin (H&E) for histological evaluation.
  • H&E hematoxylin and eosin
  • Unsupervised hierarchical clustering was performed using dChip software (http://biosunl .harvard.edu/complab/dchip/). Subclass mapping algorithm was used to evaluate similarity in global gene-expression profile between the training and validation sets (Hoshida et al., 2007).
  • the UCSC Universality of California Santa Cruz Genome Bioinformatics
  • Genome Browser database www.genome.ucsc.edu
  • MethylPrimer software http://www.urogene.Org//methprimer was used to design primers matching methylated and unmethylated sequences for methylation-specific PCR.
  • GSEA Gene Set Enrichment Analysis
  • Predicted targets for miR-517a and miR-520c were analyzed using miRBase Targets Release Version v5 (www.mirbase.org/) and TargetScan (www.targetscan.org) (Griffiths- Jones et al, 2008; Lewis et al, 2005).
  • the human 3'UTR sequence of LTBP-4 was extracted from Ensembl genome browser release 56. Docket No. 27527-0076WO1
  • SNU-423 cells (2000 cells/well) were plated in a 96-well plate and transfected with anti-miR-517a or control anti-miR as described above. Cells proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI), according to manufacturer's instruction. Absorbance was measured at 490nm using a spectrophotometer.
  • Example 1 miRNA-based Molecular Classification of
  • HCV hepatitis-C
  • Subclass C2 specifically overexpressed 18 miRNAs, including miR- 517a, miR-517c, miR-520g, miR-519b, miR-519d, miR-516-5p, miR-519a, miR-520c, miR-526b*, miR-524*, miR-516-1, miR-526b, miR-519e, miR-512-3p, miR-522, miR- 526a, miR-518b, and miR-525, miR-517b, miR-520h, miR-520b, miR-520f and miR-518f* (median fold change: 8.8, FDR ⁇ 0.01) belonging to the largest miRNA cluster ever reported (-100 Kb) located on chrl9ql3.41 (also referred to as chromosome 19 miRNA cluster, C19MC) (Bentwich et al, 2005) (Table VII). Docket No. 27527-0076WO1
  • JLA12 hsa-miR-362 (j-mir-48) 0.01 1 .66
  • the chrl9ql3.41 miR A cluster (up-regulated in Subclass C2) constitutes a primate-specific and poorly characterized family of miRNAs, whose expression has never been previously reported in HCC.
  • Example 4 miR-517a and miR-520c, Members of C19MC Polycistron,
  • a wound-healing assay was performed to evaluate migratory capability of Huh7 cells transfected with scramble oligonucleotide, miR-517a or miR-520c. Cell monolayers were scratched and images were taken after 0 and 24h from wounding. Consistently, expression of both miRNAs determined an increase in the migratory capability of Huh7 in a wound-healing assay (p ⁇ 0.002) ( Figure 2D).
  • miR-517a was selected for further investigations due to its overexpression in HCC.
  • a stably miR-517a-transduced Huh7 cell line was generated using a lentiviral vector containing both miR-517a precursor and the gene of green fluorescent protein (GFP) to allow for selection of efficiently transduced cells by flow cytometry.
  • GFP green fluorescent protein
  • the expression of miR-517a-transduced Huh7 cells was confirmed by qRT-PCR ( Figure 5). Cells stably transduced with GFP vector lacking miR-517a gene were also generated and used as a control.
  • mRNA expression profiling of Huh7 stably transduced with miR-517a was performed to investigate the effects of miR-517 overexpression on global molecular status of HCC cells.
  • gene sets positively involved in cell cycle e.g. CCNA, CDK2, CDC6, CDC7
  • DNA replication e.g., POLB, POLD2, POLE2, MCM3, MCM7
  • POLB, POLD2, POLE2, MCM3, MCM7 were found to be significantly induced in miR-517a-transduced cells (p ⁇ 0.001).
  • enrichment of genes involved in the Angiotensin II pathway e.g.
  • TIMP4 TIMP metallopeptidase inhibitor 4 0.000 -1.17 0.994
  • KIR2DS4 killer cell immunoglobulin-like receptor two domains, short
  • RXFP4 relaxin/insulin-like family peptide receptor 4 0.000 -1.23 0.988
  • PTPRG protein tyrosine phosphatase, receptor type G 0.000 -1.25 0.981
  • SLC7A3 solute carrier family 7 (cationic amino acid transporter, y+ system)
  • KCNA5 potassium voltage-gated channel, shaker-related subfamily, member 5 0.000 -1.35 0.975
  • FRG2B FSHD region gene 2 family member B 0.000 -1.35 0.973
  • CSF2 colony stimulating factor 2 (granulocyte-macrophage)
  • FLJ40235 hypothetical protein FLJ40235
  • ARAP3 ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 3
  • GALNT9 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 9 (GalNAc-T9)
  • ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 motif, 2
  • OR4X1 olfactory receptor, family 4, subfamily X, member 1
  • MS4A6A membrane-spanning 4-domains, subfamily A, member 6A
  • FAM75C1 // FAM75C1 // FAM75C1 // FAM75C1 // FAM75C1 // FAM75C1 // FAM75C1 // FAM75C1 // FAM75C1 // FAM75C1
  • CDH5 cadherin 5 type 2 (vascular endothelium)
  • NEGRI neuronal growth regulator 1 NEGRI neuronal growth regulator 1
  • SNORD1 14-6 small nucleolar RNA, C/D box 1 14-6
  • GJC2 gap junction protein gamma 2, 47kDa
  • TNFSF18 tumor necrosis factor (ligand) superfamily member 18
  • COLEC1 1 collectin sub-family member 1 1
  • SH3TC1 SH3 domain and tetratricopeptide repeats 1
  • SLC7A10 solute carrier family 7, neutral amino acid transporter, y+ system
  • CD164L2 CD164 sialomucin-like 2
  • SLC6A3 solute carrier family 6 neurotransmitter transporter, dopamine
  • CTRB1 chymotrypsinogen B1
  • SLC18A3 solute carrier family 18 vesicular acetylcholine
  • TFAP2A transcription factor AP-2 alpha activating enhancer binding protein 2
  • KRTAP4-5 keratin associated protein 4-5
  • FUT9 fucosyltransferase 9 (alpha (1 ,3) fucosyltransferase)
  • NLRP3 NLR family pyrin domain containing 3
  • ARR3 arrestin 3 retinal (X-arrestin)
  • GRIP2 glutamate receptor interacting protein 2 Docket No. 27527-0076WO1
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • IFITM5 interferon induced transmembrane protein 5
  • PRAMEF7 PRAME family member 7
  • PRAMEF7 PRAME family member 7
  • PRAMEF7 PRAME family member 7
  • MCCD1 mitochondrial coiled-coil domain 1
  • ZNF548 zinc finger protein 548
  • KIR2DS2 killer cell immunoglobulin-like receptor two domains, short
  • FXYD3 FXYD domain containing ion transport regulator 3
  • NEK6 NIMA severe in mitosis gene a
  • MCCD1 mitochondrial coiled-coil domain 1
  • ABCA10 ATP-binding cassette sub-family A (ABC1 ), member 10
  • MAPK8IP3 mitogen-activated protein kinase 8 interacting protein 3
  • MDK midkine (neurite growth-promoting factor 2)
  • NAGLU N-acetylglucosaminidase, alpha-
  • ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)
  • MIXL1 Mix1 homeobox-like 1 (Xenopus laevis)
  • SLC28A3 solute carrier family 28 sodium-coupled nucleoside transporter
  • PROCR protein C receptor endothelial (EPCR)
  • GPR141 G protein-coupled receptor 141
  • ATHL1 ATH1 ATHL1 ATH1 , acid trehalase-like 1 (yeast)
  • GIPC3 GIPC PDZ domain containing family, member 3
  • KCNG1 potassium voltage-gated channel, subfamily G, member 1
  • GJD4 gap junction protein delta 4, 40.1 kDa
  • GOLGA8D golgi autoantigen, golgin subfamily a, 8D
  • GOLGA8D golgi autoantigen, golgin subfamily a, 8D

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Abstract

Cette invention concerne des inhibiteurs de micro-ARN et des méthodes pour leur utilisation. Plus particulièrement, l'invention concerne des compositions et des méthodes de traitement de carcinomes hépatocellulaires à l'aide d'inhibiteurs de molécules de micro-ARN surexprimées dans le locus chromosomique 19q13.41 (C19MC).
PCT/US2011/042436 2010-06-29 2011-06-29 Compositions et procédés pour l'inhibition de micro-arn oncogènes et le traitement du cancer Ceased WO2012006181A2 (fr)

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WO2017210735A1 (fr) * 2016-06-07 2017-12-14 Garvan Institute Of Medical Research Méthodes de traitement du neuroblastome et réactifs à cet effet
WO2017218689A1 (fr) * 2016-06-14 2017-12-21 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Expression de protéines de ligand d'activation de nkg2d pour sensibiliser les cellules cancéreuses à une attaque par des cellules immunitaires cytotoxiques
WO2018047148A1 (fr) * 2016-09-12 2018-03-15 Novartis Ag Composés pour inhibition du miarn
WO2018161031A1 (fr) * 2017-03-02 2018-09-07 Youhealth Biotech, Limited Marqueurs de méthylation pour diagnostiquer un carcinome hépatocellulaire et un cancer du poumon
US10280422B2 (en) 2015-01-20 2019-05-07 MiRagen Therapeutics, Inc. MiR-92 inhibitors and uses thereof
US10576115B2 (en) 2013-10-28 2020-03-03 University of Pittsburgh—of the Commonwealth System of Higher Education Oncolytic HSV vector
US11078280B2 (en) 2016-06-30 2021-08-03 Oncorus, Inc. Oncolytic viral delivery of therapeutic polypeptides
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CN114480399A (zh) * 2022-03-17 2022-05-13 江苏医药职业学院 降低CPB1基因表达的siRNA、重组载体及其应用
US11419926B2 (en) 2010-04-16 2022-08-23 University of Pittsburgh—of the Commonwealth System of Higher Education Identification of mutations in herpes simplex virus envelope glycoproteins that enable or enhance vector retargeting to novel non-HSV receptors
US11452750B2 (en) 2016-01-27 2022-09-27 Oncorus, Inc. Oncolytic viral vectors and uses thereof
US11612625B2 (en) 2017-07-26 2023-03-28 Oncorus, Inc. Oncolytic viral vectors and uses thereof
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JP2011513238A (ja) * 2008-02-21 2011-04-28 ザ ボード オブ リージェンツ オブ ザ ユニバーシティ オブ テキサス システム 平滑筋の増殖および分化を調節するマイクロｒｎａならびにこれらの使用
WO2009111375A2 (fr) * 2008-03-01 2009-09-11 Abraxis Bioscience, Llc Traitement, diagnostic et procédé pour découvrir un antagoniste à l'aide d'arnmi spécifique de sparc

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US9873739B2 (en) 2012-08-01 2018-01-23 Ikaika Therapeutics, Llc Mitigating tissue damage and fibrosis via latent transforming growth factor beta binding protein (LTBP4)
AU2013313282B2 (en) * 2012-08-01 2018-02-01 Ikaika Therapeutics, Llc Mitigating tissue damage and fibrosis via latent transforming growth factor beta binding protein (LTBP4)
WO2014039189A1 (fr) 2012-08-01 2014-03-13 Mcnally Elizabeth Réduction des lésions tissulaires et de la fibrose via la protéine-4 de liaison au tgf bêta latent (ltbp4)
US11883448B2 (en) 2013-10-28 2024-01-30 University of Pittsburgh—of the Commonwealth System of Higher Education Oncolytic HSV vector
US10576115B2 (en) 2013-10-28 2020-03-03 University of Pittsburgh—of the Commonwealth System of Higher Education Oncolytic HSV vector
US10280422B2 (en) 2015-01-20 2019-05-07 MiRagen Therapeutics, Inc. MiR-92 inhibitors and uses thereof
US11452750B2 (en) 2016-01-27 2022-09-27 Oncorus, Inc. Oncolytic viral vectors and uses thereof
WO2017210735A1 (fr) * 2016-06-07 2017-12-14 Garvan Institute Of Medical Research Méthodes de traitement du neuroblastome et réactifs à cet effet
JP2019517815A (ja) * 2016-06-14 2019-06-27 ユニバーシティ オブ ピッツバーグ −オブ ザ コモンウェルス システム オブ ハイヤー エデュケイション 細胞傷害性免疫細胞による攻撃に対してがん細胞を増感させるためのｎｋｇ２ｄ活性化リガンドタンパク質の発現
JP7184337B2 (ja) 2016-06-14 2022-12-06 ユニバーシティ オブ ピッツバーグ －オブ ザ コモンウェルス システム オブ ハイヤー エデュケイション 細胞傷害性免疫細胞による攻撃に対してがん細胞を増感させるためのｎｋｇ２ｄ活性化リガンドタンパク質の発現
WO2017218689A1 (fr) * 2016-06-14 2017-12-21 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Expression de protéines de ligand d'activation de nkg2d pour sensibiliser les cellules cancéreuses à une attaque par des cellules immunitaires cytotoxiques
US11427625B2 (en) 2016-06-14 2022-08-30 University of Pittsburgh—of the Commonwealth System of Higher Education Expression of NKG2D activating ligand proteins for sensitizing cancer cells to attack by cytotoxic immune cells
US11078280B2 (en) 2016-06-30 2021-08-03 Oncorus, Inc. Oncolytic viral delivery of therapeutic polypeptides
WO2018047148A1 (fr) * 2016-09-12 2018-03-15 Novartis Ag Composés pour inhibition du miarn
US10513739B2 (en) 2017-03-02 2019-12-24 Youhealth Oncotech, Limited Methylation markers for diagnosing hepatocellular carcinoma and lung cancer
WO2018161031A1 (fr) * 2017-03-02 2018-09-07 Youhealth Biotech, Limited Marqueurs de méthylation pour diagnostiquer un carcinome hépatocellulaire et un cancer du poumon
US11612625B2 (en) 2017-07-26 2023-03-28 Oncorus, Inc. Oncolytic viral vectors and uses thereof
US12208126B2 (en) 2017-07-26 2025-01-28 Virogin Biotech Canada Ltd. Oncolytic viral vectors and uses thereof
US11865081B2 (en) 2017-12-29 2024-01-09 Virogin Biotech Canada Ltd. Oncolytic viral delivery of therapeutic polypeptides
CN113917146A (zh) * 2020-07-10 2022-01-11 上海吉凯基因医学科技股份有限公司 人pole2基因的用途及相关产品
CN114480399A (zh) * 2022-03-17 2022-05-13 江苏医药职业学院 降低CPB1基因表达的siRNA、重组载体及其应用

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