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WO2013106766A2 - Indications thérapeutiques du miarn-1291 - Google Patents

Indications thérapeutiques du miarn-1291 Download PDF

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Publication number
WO2013106766A2
WO2013106766A2 PCT/US2013/021307 US2013021307W WO2013106766A2 WO 2013106766 A2 WO2013106766 A2 WO 2013106766A2 US 2013021307 W US2013021307 W US 2013021307W WO 2013106766 A2 WO2013106766 A2 WO 2013106766A2
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WO
WIPO (PCT)
Prior art keywords
mir
cancer
seq
nucleic acid
nucleotides
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PCT/US2013/021307
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English (en)
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WO2013106766A3 (fr
Inventor
Aiming Yu
Yuzhuo PAN
Jingxin Qiu
Original Assignee
The Research Foundation Of State University Of New York
Health Research, Inc.
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Publication of WO2013106766A2 publication Critical patent/WO2013106766A2/fr
Publication of WO2013106766A3 publication Critical patent/WO2013106766A3/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention relates to compositions and methods for the prevention and treatment human disease.
  • cancer Prevalent as a top ten killer worldwide, cancer respectively remains the most pervasive cause of death the United States, following heart disease. In fact, it has been estimated that 1.6 million new cases of cancer will be confirmed in the U.S. for 2012, which accounts for approximately 600,000 deaths. While routine clinical management typically entails histopathological classification of tumor grade and type, carcinoma size, and lymph node stage, such clinical parameters fail to fully impart the underlying genetic events responsible for tumorigenesis, progression, and invasion. Likewise, notwithstanding the myriad of biochemical processes involved in cellular growth, regulation, differentiation, proliferation, and metastasis, refractory disease is borne out of, inter alia, tumor
  • genotypic and phenotypic tumor variability is ascribed to the multitude of cellular pathways capable of precipitating cancerous lesions, if disrupted.
  • New prophylactic and therapeutic cancer indications are therefore required for preventing and/or treating various types of cancers, especially those associated with poor prognoses.
  • pancreatic cancer to this end maintains the highest mortality rate of all major malignancies, which is in part due to the lack of approved therapeutic indications. Indeed, patients afflicted with metastatic pancreatic adenocarcinoma have a median survival period of less than 6 months, while the one-year survival rate constitutes a similar outcome with less than 20% of patients reaching the anniversary of their diagnosis. Understanding of the etiological factors linked to cancer progression and recurrence will consequently facilitate development of new approaches for effectively diagnosing, preventing, and treating pancreatic cancer and other malignancies.
  • miRNAs Various regulatory microRNAs (miRNAs) possess oncogenic or tumor suppressive functions, which therefore imparts the possibility of new therapeutic indications aimed at regulating miRNA expression and their cognate downstream targets.
  • miRNAs are expressed as RNA pol II transcripts in eukaryotic organisms and have been indicated in the regulation of gene expression, mRNA splicing, and histone formation.
  • Certain miRNAs moreover, possess developmental-specific and/or tissue tropic expression patterns. As such, studying these miRNA molecules is coterminous with the elucidation of biological processes, disease states, and the development of efficacious cancer therapies.
  • the present invention is based on the discovery that a nexus exists between miR- 1291 expression and malignant phenotypes.
  • One aspect of the present invention provides a pharmaceutical composition including a nucleic acid molecule or a complement or precursor thereof, and one or both of a pharmaceutically acceptable carrier and a stabilizing agent, where the nucleic acid molecule is composed of one or more of (i) a sequence encoding miR-1291, (ii) a sequence selected from SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, (iii) a sequence with at least 90% similarity to one or more of SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, and (iv) a sequence with at least 7 contiguous nucleotides from SEQ. ID.
  • the nucleic acid molecule further includes one or more chemical modifications selected from internucleotide linkages, internucleoside linkages, dideoxyribonucleotides, 2 '-sugar modification, 2 '-amino groups, 2'-fiuoro groups, 2'- methoxy groups, 2'-alkoxy groups, 2'- alkyl groups, 2'-deoxyribonucleotides, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, universal base nucleotides, acyclic nucleotides, 5-C-methyl nucleotides, biotin groups, terminal glyceryl incorporation, inverted deoxy abasic residue incorporation,
  • the nucleic acid molecule in some embodiments, is conjugated to a peptide, cancer cell-specific targeting ligand, or antibody derived from transferrin, vasoactive intestinal peptide, somatostatin, gastrin releasing peptide, bombesin, substance P ligand, urokinase, urokinase A chain, epidermal growth factor (EGF), transforming growth factor-alpha
  • TGFa insulin-like growth factor
  • IL-4 interleukin-4
  • IL-6 interleukin-6
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • annexin V nucleophosmin
  • HSC70, BIP Grp75
  • PDI platelet-derived growth factor
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • annexin V nucleophosmin
  • HSC70, BIP Grp75
  • TCP- 1 ⁇ , ERp29, HSP27 vimentin
  • a-internexin cytokeratin 8
  • ⁇ -actin ⁇ -actin
  • ⁇ - tubulin nm23-Hl
  • valosin containing protein tumor protein D52-like 2, ASF-2, hnRNPK, hnRNPC, 24.1
  • the nucleic acid molecule is present in, or derived from, a biological vector selected from a eukaryotic vector, a mammalian vector, a prokaryotic vector, a plasmid, a phage, a synthetic construct, and a viral vector.
  • the present invention provides methods of treating or preventing a disease or condition mediated by miR-1291 in a subject in need thereof, by administering to the subject a composition including a nucleic acid molecule or a complement or precursor thereof, under conditions effective to treat or prevent the disease or condition mediated by miR-1291 in the subject, where the nucleic acid molecule is composed of one or more of (i) a sequence encoding miR-1291, (ii) a sequence selected from SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, (iii) a sequence with at least 90% similarity to one or more of SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID.
  • the disease or condition is mediated by a decrease in miR-1291 expression compared to miR- 1291 expression in the absence of the disease or condition.
  • the decrease in miR-1291 expression alters the levels of one or more of: AKT2; Cyclin Bl; MeCP2; FOXA2; AMPKal; Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASFl; Ornithine aminotransferase (OAT); Keratin, type II cytoskeletal 8 (KRT8); Phosphoenolpyruvate carboxykinase 2 (PEPCK2); Enoyl-coenzyme A (CoA) hydratase (ECHS1); Phosphoserine aminotransferase isoform 1 (PSAT1); Dihydrolipoamide acetyltransferase (DLAT); Peroxiredoxin 3, isoform CRA a (PRDX3); Cysteine-rich protein 2 (CRIP2); Chain C, human PCNA; Fa
  • the nucleic acid molecule is provided as short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double-stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or a miR-1291 mimic, in some embodiments, where the nucleic acid molecule therapeutically regulates levels of one or more of miR-1291; AKT2; Cyclin Bl; MeCP2; FOXA2; AMPKal; Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASFl; Ornithine aminotransferase (OAT); Keratin, type II cytoskeletal 8 (KRT8); Phosphoenolpyruvate carboxykin
  • the disease or condition is selected from cancer, refractory cancer, metastatic cancer, a solid tumor, a circulating tumor, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastro intestinal stromal tumor (GIST), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, renal cell carcinoma, renal cancer, sarcoma, parotid cancer, gastric cancer, melanoma, NET (sinonasal) cancer, colon cancer, liver cancer, hepatocellular carcinoma, inflammatory bowel disease (IBD), and metabolic disease.
  • cancer refractory cancer
  • metastatic cancer a solid tumor
  • pancreatic cancer lung cancer
  • breast cancer breast cancer
  • prostate cancer ovarian cancer
  • colorectal cancer esophageal cancer
  • GIST gastro intestinal stromal tumor
  • AML acute myelogenous leukemia
  • the methods further include administering to the subject one or more second therapies selected from chemotherapy, radiotherapy, ablation therapy, hormone therapy, gene therapy, RNA therapy, epigenetic drug therapy, resection therapy, immunotherapy, anti-angiogenic therapy, stromal inhibitor therapy, extracellular matrix protein inhibitor therapy, doxorubicin, mitoxantrone, tamoxifen, imatinib mesylate, BAY43- 9006, gemcitabine, prostaglandins, retinoic acids, brostallicin, lenalidomide, thalidomide, docetaxel, erlotinib, vatalinib, VEGF-trap, fenretidine, bortezomib, bevacizumab, pertuzumab, rituximab, gefitinib, a general monoclonal antibody, where the second agent is administered separately, simultaneously, or sequentially, with the composition.
  • second therapies selected from chemotherapy, radiotherapy, ablation therapy, hormone therapy, gene
  • the methods futher entail determining the prognosis of the subject by (a) obtaining a sample from the subject prior to the administering and/or subsequent to the administering, (b) measuring the level of one or more of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 in the sample, (c) comparing the measured level in the sample to a reference level selected from (i) one or more of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 levels from a control population; (ii) one or more of native miR-1291 and native SNORNA34 levels in the subject prior to the administering, and (iii) one or more of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 levels in the subject subsequent to the administering, and (d) determining the subject's prognosis based on the
  • the measuring includes, in some embodiments, reacting the sample from the subject with a biological probe comprising at least 7 contiguous nucleotides of SEQ. ID. NO: 1 or the complement thereof, or the nucleotide sequence of SEQ. ID. NO: 2 or a complement thereof, where the biological probe is capable of hybridizing with one or more of, native or exogenous: miR-1291, miR-1291 precursors, miR12-91 RNA, miR-1291 niRNA, miR-1291 cDNA, SNORNA34, SNORNA34 precursors, SNORNA34 RNA, SNORNA34 niRNA, SNORNA34 cDNA, or complements thereof.
  • a decrease in one or more of the measured levels of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 in the sample compared to the reference level indicates an unfavorable prognosis, and wherein an increase in one or more of the measured levels of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 in the sample compared to the reference level (ii) in the subject prior to the administration, indicates that the composition possesses efficacy for the disease or condition.
  • the present invention provides methods of altering the expression level or activity of miR-1291 or a precursor or target thereof, by providing an agent that includes a nucleic acid molecule or a complement or precursor thereof, under conditions effective to increase the expression level or activity of the miR-1291 or a precursor or a target thereof, compared to a reference level or activity, wherein the nucleic acid molecule comprises one or more of (i) a sequence encoding miR-1291, (ii) a sequence selected from SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, (iii) a sequence with at least 90% similarity to one or more of SEQ. ID. NO: 1, SEQ. ID.
  • the altering modulates one or more of drug resistance, invasiveness, adherence, and metastasis in a subject and/or target cells from the subject selected from one or more cancerous cells, hyperproliferative cells, neoplastic cells, hypoplastic cells, hyperplastic cells, dysplasia cells, metaplasia cells, prosoplasia cells, desmoplasia cells, angiogenic cells, inflammatory cells, immunological cells, metabolic cells, pulmonary cells, and cardiovascular cells.
  • the providing is an administering to a subject in need thereof, and wherein the subject is a patient afflicted with cancer, refractory cancer, metastatic cancer, a solid tumor, a circulating tumor, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastro intestinal stromal tumor (GIST), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, renal cell carcinoma, renal cancer, sarcoma, parotid cancer, gastric cancer, melanoma, NET (sinonasal) cancer, colon cancer, liver cancer, hepatocellular carcinoma, inflammatory bowel disease (IBD), and metabolic disease; and/or where the subject is a patient afflicted with a disease or condition associated with aberrant expression of one or more of: miR-1291; AKT2; Cyclin Bl; Me
  • acetyltransferase DLAT
  • Peroxiredoxin 3, isoform CRA a PRDX3
  • Cysteine-rich protein 2 CRIP2
  • Chain C human PCNA
  • Fascin homolog 1, actin-bundling protein isoform CRA a (FSCN1)
  • Serpin HI precursor Protein disulfide-isomerase precursor
  • Chain A disulfide isomerase related chaperone ERP29
  • Isocitrate dehydrogenase [NAD] subunit beta IDH3B
  • AFP a- fetoprotein
  • AFP-L3% des-gamma-carboxyprothrombin
  • DCP des-gamma-carboxyprothrombin
  • CDH1 E-cadherin
  • trimethylated lysine 27 of H3 histone H3K27me3
  • the agent is a synthetic agent provided as a miR-1291 mimic, short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double- stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or any combination thereof, where the synthetic agent is a synthetic agent provided as a miR-1291 mimic, short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double- stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or any combination thereof, where the synthetic agent is a synthetic agent provided as a miR-1291 mimic, short
  • Triosephosphate isomerase isoform 2 (TPI1); Peroxiredoxin-4 (PRDX4); and Isocitrate dehydrogenase [NAD] subunit beta (IDH3B); a-fetoprotein (AFP); AFP-L3%, des-gamma- carboxyprothrombin (DCP); CDH1 (E-cadherin); trimethylated lysine 27 of H3 histone (H3K27me3); histone deacetylase -1; histone deacetylase -2; SIRT1; CD44; aldehyde dehydrogenase; KRAS2; and RREBl .
  • the synthetic agent includes one or more chemical modifications in some embodiments, where the modification is selected from internucleotide linkages,
  • internucleoside linkages dideoxyribonucleotides, 2 '-sugar modification, 2 '-amino groups, 2'- fluoro groups, 2'-methoxy groups, 2'-alkoxy groups, 2'- alkyl groups, 2'- deoxyribonucleotides, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, universal base nucleotides, acyclic nucleotides, 5-C-methyl nucleotides, biotin groups, terminal glyceryl incorporation, inverted deoxy abasic residue incorporation, sterically hindered molecules, 3 '-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3
  • the synthetic agent includes one or more of a nucleic acid molecule encoding a complement of miR-1291, a nucleic acid sequence possessing at least 90% similarity to SEQ. ID. NO: 5, and a nucleic acid sequence with at least 7 contiguous nucleotides from SEQ. ID. NO: 5.
  • the synthetic agent decreases ABC transporter activity in a target cell compared to a control cell, and where the ABC transporter is selected from the group consisting of ABCCl, ABCG2, ABCBl, ABCC2, ABCC3, and ABCC4.
  • the mR-1291 precursor includes SNORA34, while in suitable embodiments, the nucleic acid molecule is conjugated to a cell-specific targeting ligand.
  • the nucleic acid molecule is present in a biological vector, where the biological vector is a plasmid vector or viral vector in illustrative embodiments.
  • the composition also includes a delivery vehicle, where the delivery vehicle is selected from a polymeric carrier, a micelle, a liposome, a lipoplex, a polyplex, a peptide, a polymer, a dendrimer, lipids, or a nanoparticle in various embodiments.
  • the delivery vehicle is conjugated to a cell-specific targeting ligand or antibody.
  • the cell- specific targeting ligand or antibody comprises a cancer cell-specific targeting ligand or antibody.
  • the cancer cell-specific targeting ligand is selected from a peptide or ligand, such as, e.g., transferrin, vasoactive intestinal peptide, somatostatin, gastrin releasing peptide, bombesin, substance P ligand, urokinase, urokinase A chain, epidermal growth factor (EGF), transforming growth factor-alpha (TGFa), insulin-like growth factor, interleukin-4 (IL-4), interleukin-6 (IL-6), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), laminin, vascular endothelial growth factor (VEGF), annexin V, nucleophosmin, HSC70, BIP, Grp75, PDI, PDI ER60 precursor, HSP60, TCP- 1 ⁇ , ERp29, HSP27, vimentin, a-internexin, cytokeratin 8, ⁇ -act
  • the present methods provide for the modulation of an ABC transporter activity in a target cell by administering an agent that modifies the expression or activity of miR-1291 or a precursor thereof to the target cell under conditions effective to modulate ABC transporter activity in the target cell.
  • the target cell is in vitro, while in others the target cell is in vivo.
  • the ABC transporter is selected from ABCC1, ABCG2, ABCB1, ABCC2, ABCC3, and ABCC4.
  • the ABC transporter activity in the target cell is decreased, while in others it is increased.
  • the agent mimics the expression of miR-1291 or a precursor thereof.
  • the agent is a synthetic miR-1291 nucleic acid molecule or precursor thereof.
  • the agent constitutes a synthetic human miR-1291 nucleic acid molecule or precursor thereof in some embodiments, where the synthetic human miR- 1291 nucleic acid molecule or precursor thereof is a nucleotide sequence of SEQ ID NO : 1 , SEQ ID NO:3, and/or SEQ ID NO:4.
  • the synthetic miR-1291 nucleic acid molecule or precursor thereof possesses one or more chemical modifications in some embodiments, where the one or more chemical modifications is selected from a cholesterol group, 2'-0-methyl group, phosphorothioate group, 2'-fluoro group, 2'-0-methyoxyethyl group,
  • boranophosphate group 4'-thioribose group, bile acid, lipid, or a bridge connecting the 2'- oxygen and 4'-carbon.
  • the agent inhibits expression of miR-1291 or a precursor thereof.
  • the agent moreover, is a miR-1291 inhibitor in suitable embodiments.
  • the miR-1291 inhibitor is a nucleotide sequence of SEQ ID NO:5 or a chemically modified variant thereof, that is optionally contained in or conjugated to a delivery vehicle.
  • the present invention provides a method of modulating cellular drug resistance in a subject by administering an agent that modifies the expression level or activity of miR-1291 or a precursor thereof to one or more target cells of a subject under conditions effective to modulate cellular drug resistance in the subject.
  • the cellular drug resistance in the subject cell is decreased, while in others it is increased.
  • the agent is a synthetic human miR-1291 nucleic acid molecule or precursor thereof, where the synthetic human miR-1291 nucleic acid molecule or precursor thereof is a nucleotide sequence of SEQ ID NO: l, SEQ ID NO:3, and/or SEQ ID NO:4.
  • the miR-1291 inhibitor is a nucleotide sequence of SEQ ID NO: 5 or a chemically modified variant thereof, that is optionally contained in or conjugated to a delivery vehicle.
  • the one or more target cells of the subject in some embodiments, is a cancerous cell.
  • the methods further include selecting a subject having altered cellular drug resistance prior to the administering.
  • the subject has increased cellular drug resistance to cancer drugs.
  • the present invention provides a method of treating or preventing cancer in a subject by administering to the subject, a composition including a nucleic acid molecule encoding a miR-1291 or a precursor thereof under conditions effective to treat or prevent cancer in the subject.
  • the nucleic acid molecule encodes a human miR-1291 or precursor thereof.
  • the miR-1291 precursor is SNORA34, while in others it is a nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: l, SEQ ID NO:3, and/or SEQ ID NO:4.
  • the nucleic acid molecule is present in a biological vector that is a plasmid vector or viral vector.
  • the nucleic acid molecule is present in a delivery vehicle, where the delivery vehicle is selected from a polymeric carrier, a micelle, a liposome, a lipoplex, a polyplex, a peptide, a polymer, a dendrimer, lipids, or a nanoparticle.
  • the delivery vehicle is conjugated to a cancer cell- specific targeting ligand or antibody.
  • the methods of the present invention futher include, in some embodiments, selecting a subject having cancer or a subject at risk of having recurring cancer.
  • the methods further encompass administering one or more additional cancer therapies to the subject.
  • the one or more additional cancer therapies is selected from chemotherapy, radiotherapy, ablation therapy, resection therapy, immunotherapy, an anti-angiogenic therapy, a stromal inhibitor therapy, and an extracellular matrix protein inhibitor therapy.
  • the methods provide for determining the prognosis of a subject having cancer by obtaining a sample from the subject, measuring the expression level of miR-1291 and/or SNORA34 in the sample, comparing the measured miR-1291 and/or SNORA34 expression level in the sample to a reference miR-1291 and/or SNORA34 expression level, and determining the subject's prognosis based on said comparing.
  • the subject has pancreatic cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, esophageal cancer, or inflammatory bowel disease.
  • the measuring is performed by reacting the sample from the subject with a biological probe capable of detecting the amount of miR-1291 in the sample.
  • the biological probe is a nucleic acid molecule comprising 7-22 contiguous nucleotides of SEQ ID NO: 1 or the complement thereof.
  • the biological probe is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2 or a complement thereof.
  • a decrease in the measured expression level of miR-1291 and/or SNORA34 in the sample compared to a reference expression level indicates an unfavorable prognosis.
  • an unfavorable prognosis indicates a likelihood of cancer recurrence in the subject.
  • an unfavorable prognosis indicates a likelihood of cancer drug resistance.
  • the method further includes treating the subject having cancer based on the determined prognosis.
  • methods further include assessing one or more additional prognostic criteria and determining the subject's prognosis based on the combination of the comparing and assessing.
  • the additional prognostic criteria is selected from cancer lesion size, number of cancerous lesions, and vascular invasion.
  • FIG. 1 is a graph showing miR-1291 levels in human pancreatic carcinomas compared to adjacent normal pancreatic tissue. The data indicate that mature miR-1291 expression level is about 8-fold lower in pancreatic tumors.
  • FIG. 2 is a graph illustrating gain of miR-1291 expression and/or function with respect to cancer cell growth.
  • the graph details the variability of human cancer cells after transfection with SNORA/miR-1291 and control plasmids. The data indicate that gain of miR-1291 expression suppresses human cancer cell growth.
  • FIGs. 3A-D are immunoblots showing sequence expression and diagrammatic representations of the sequences and assays used in the present invention .
  • FIG. 3A represents immunoblots showing the modulation of miR-1291 expression with a miR-1291 expression plasmid and antagomir.
  • FIG. 3B shows a diagrammatic representation of miR-1291 in PANC-1 cells, as well as the up- and down-regulation of miR-1291 expression by miR-1291 expression plasmid and antagomir, respectively.
  • FIG. 3B shows a diagrammatic representation of miR-1291 in PANC-1 cells, as well as the up- and down-regulation of miR-1291 expression by miR-1291 expression plasmid and antagomir, respectively.
  • FIG. 3B shows a diagrammatic representation of SNORA34 and miR-1291 in PANC-1 cells, as well as the up- and down-regulation of miR-1291 expression by miR-1291 expression plasmid and antagomir, respectively.
  • FIG. 3B shows a diagrammatic representation of SNORA34 and miR-1291 in PANC-1 cells, as well as the up- and down-regulation of miR-1291 expression by miR-1291 expression plasmid and antagomir, respectively.
  • FIG. 3B shows a diagrammatic representation of SNORA34 and
  • FIG. 3C shows the localization and biogenesis of SNORA34/miR1291.
  • FIG. 3D is a schematic of a splinted ligation assay.
  • FIGs. 4A-C are schematic representations and graphs respectively showing the process for miR-1291 transfection, miR-1291 expression levels, and SNORA34 expression levels in miR-1291 stably transfected PANC-1 cells.
  • FIG. 4A is a schematic representation showing the process for miR-1291 transfection.
  • FIG. 4B shows the expression of miR-1291 in miR-1291 stably transfected PANC-1 cells (miR-1291) and empty vector stably transfected control PANC-1 cells is shown.
  • FIG. 4C shows the expression of SNORA34 in miR-1291 stably transfected PANC-1 cells (miR-1291) and empty vector stably transfected control PANC-1 cells is shown.
  • the data indicate that mature miR-1291 expression level in miR-1291 stably transfected PANC-1 cells is over 7-fold higher than that in control cells.
  • FIGs. 5A-B show the results from flow cytometric assays.
  • FIG. 5A illustrates that miR-1291 induces a cell cycle arrest in PANC-1 cells.
  • FIG. 5B shows a graph representing the same.
  • FIGs. 6A-B show miR-1291 's effect on apoptosis of PANC-1 cells.
  • FIG. 6A details flow cytometry results from apoptotic PANC-1 cell assays.
  • FIG. 6B shows graphs representing the same.
  • FIGs. 7A-B are results from miR-1291 migration and invasion assays using PANC- 1 cells.
  • FIG. 7 A shows that miR-1291 reduces the migration and invasion of PANC-1 cells.
  • FIG. 7B shows graphs representing the same.
  • FIGs. 8A-C show Western blot analyses and a schematic sequence representation for MeCP2 mRNA.
  • FIG. 8 A is a Western blot showing that miR-1291 suppresses the protein expression of AKT2, AKT1, Cyclin Bl, FOXA2 and AGR2 in PANC-1 cells. Protein expression was determined by Western blots, where GAPDH was employed as an internal control.
  • FIG. 8B is a Western blot showing that miR-1291 suppresses the protein expression of AKT2, Cyclin Bl, MeCP2, FOXA2 and AGR2 in PANC-1 cells. Protein expression was determined by Western blots, where GAPDH was employed as an internal control.
  • FIG. 8C shows a schematic sequence representation for MeCP2 mRNA with respect to miR-1291 MRE.
  • FIG. 9 shows AGR2 expression in human pancreatic ductal adenocarcinomas (left panel) compared to adjacent normal pancreatic tissue (right panel), as determined by immunohistochemistry.
  • the data indicate that AGR2 level in human pancreatic carcinomas is much higher than that in the adjacent normal pancreatic tissue.
  • FIGs. 10A-B are schematic representations of miR-1291 function on the 3'UTR of transcription factor FOXA2 and graphical depictions thereof, respectively.
  • FIG. 10A shows that FOXA2 is a direct target for miR-1291.
  • the 3 'UTR of FOXA2 was retrieved from NCBI ENTREZ) and was searched for miR recognized element (MRE) to miR-1291 by Targetscan and RNAhybrid.
  • FIG. 10B shows a graph representing the same.
  • FIGs. 11A-C demonstrate the impact of miR-1291 with respect to clinical symptoms that were assessed using a xenograft tumor mouse model.
  • FIG. 11 A shows pictures and graphs demonstrating suppression of tumor growth by miR-1291.
  • FIG. 1 IB concerns the pathological analysis of xenograft tumor results from a histological study at lOOx magnification, which confirms tumor progression from control PANC-1 cells.
  • FIG. 11C is an immunohistochemistry (IHC) study demonstrating that miR-1291 reduces tumor cell proliferation (Ki-67 labeling) and enhances apoptosis (Caspase-3). IHC analyses indicate that miR-1291 reduces cell proliferation and enhances apoptosis.
  • PANC-1 cells were inoculated into male athymic CD-I nude mice. Tumor growth was monitored once a week.
  • FIGs. 12A-F show diagrammatic representations and related data for miR-1291 's effect on various ABC protein expression profiles and related drug sensitivity.
  • FIG. 12A shows that miR-1291 may target the 3'UTR of ABCC1.
  • FIG. 12B shows additional representation of the same.
  • FIG. 12C shows that miR-1291 regulates ABCC1 protein expression.
  • FIG. 12D shows that miR-1291 affects ABC transporter mRNA expression.
  • FIG. 12E shows that miR-1291 modulates intracellular drug accumulation.
  • FIG. 12F shows that miR-1291 influences chemosensitivity, as labeled. DETAILED DESCRIPTION
  • compositions and methods for the treatment and prevention of disease relate to, inter alia, compositions and methods for the treatment and prevention of disease.
  • pharmaceutical formulations which impart prophylactic and therapeutic indications for subjects in need of gene regulation, e.g., patients afflicted with one or more diseases or conditions, such as, for example, cancer and related disorders, as further detailed below.
  • miRNAs may be up-regulated or down-regulated in tumor tissues, relative to the corresponding normal tissues.
  • Aberrantly expressed miRNAs are linked to various malignancy-associated pathways, indicating a role in tumorigenesis that includes promoting cell cycle progression, reducing cell death, and favoring angiogenesis and invasion.
  • the present inventors have discovered that various miRNA profiles and/or signatures impart a tool for not only differentiating cancerous from non-cancerous cells or tissues, but also as therapeutic indications when reconstituted in patients harboring certain disease conditions. Accordingly, taking advantage of such etiological indications provides for a new therapeutic regime aimed at curtailing the underlying pathology associated with miRNA expression and regulation for diseases and conditions.
  • amplification or “amplifying” refers to the production of additional copies of a nucleic acid sequence. Amplification is typically performed by using, for example, polymerase chain reaction (PCR), reverse transcription RT-PCR, qPCR, qRT- PCR, etc., technologies and/or real time PCR and/or other technologies known in the art.
  • PCR polymerase chain reaction
  • the term "amplification reaction mixture” or “PCR mixture” refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid, e.g., RNA, DNA, cDNA, etc., and the like. These may include enzymes, e.g., a thermostable polymerase, aqueous buffers, salts, amplification primers, target nucleic acid, and nucleotide
  • Amplification may be exponential or linear.
  • a nucleic acid to be amplified may be, for example, either RNA, DNA, cDNA, and the like or equivalents or complements thereof.
  • sequences amplified in this manner form an "amplicon.” While the exemplary methods described hereinafter relate to amplification using PCR, qPCR, or qRT-PCR, numerous other methods are known in the art for amplification of nucleic acids, e.g., isothermal methods, rolling circle methods, etc. The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods. See, e.g., Saiki, "Amplification of Genomic DNA” in PCR Protocols, Innis et al, Eds., Academic Press, San Diego, CA 1990, pp.
  • assessing and “evaluating” are used interchangeably to refer to any form of measurement, and include determining if a characteristic, trait, or feature is present or not.
  • the terms "determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. Assessing may be relative or absolute. "Assessing the presence of includes determining the amount of something present, as well as determining whether it is present or absent.
  • cDNA refers to complementary DNA.
  • Complementary DNA is synthesized via reverse transcription of a mR A sequence thereby forming its complementary DNA sequence.
  • clinical factors refers to any data that a medical practitioner may consider in determining a diagnosis or prognosis of disease. Such factors include, but are not limited to, the patient's medical history, a physical examination of the patient, complete blood count, analysis of the activity of enzymes ⁇ e.g., liver enzymes), examination of blood cells or bone marrow cells, cytogenetics, and immunophenotyping of blood cells.
  • the term "comparable” or “corresponding” in the context of comparing two or more samples means that the same type of sample ⁇ e.g., tissue) is used in the comparison.
  • a level of one or more miRNAs in a sample of plasma can be compared to a level of one or more miRNAs in another plasma sample.
  • comparable samples may be obtained from the same individual at different times.
  • comparable samples may be obtained from different individuals ⁇ e.g. , a patient and a healthy individual). In general, comparable samples are normalized by a common factor.
  • nucleic acids ⁇ i.e., a sequence of nucleotides such as an
  • oligonucleotide or a target nucleic acid refer to sequences that are related by base-pairing rules developed by Watson and Crick.
  • a complementary sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
  • substantially complementary means that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences comprise a contiguous sequence of bases that do not hybridize to a target or marker sequence, positioned 3 ' or 5' to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target or marker sequence.
  • the term "consensus sequence” refers to the nucleotide bases most often found at any given position when comparing a large number of similar nucleotide sequences.
  • the term “conserved region” and “conserved region of a gene in a multigene family” refers to a segment of a gene or amino acid sequence that is significantly similar between members of gene families. The degree of similarity can vary and in some embodiments, the conserved regions will be identical between family members. In other embodiments, the nucleotide sequence may vary significantly, but will still encode for amino acid segments that are conserved between family members.
  • cycle refers to the process which results in the production of a copy of a target nucleic acid.
  • a cycle includes a denaturing step, an annealing step and an extending step, i.e., during PCR.
  • diagnosis means detecting a disease or disorder or determining the stage or degree of a disease or disorder.
  • a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition.
  • Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease; i.e. there may be differential diagnoses that can be inferred from a diagnostic factor or symptom.
  • diagnosis also encompasses determining the therapeutic effect of a drug therapy, or predicting the pattern of response to a drug therapy.
  • the diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical art for a particular disease or disorder, e.g., pancreatic cancer.
  • the phrase "difference of the level” refers to differences in the quantity of a particular marker or biomarker, such as a nucleic acid or a protein, in a sample as compared to a control or reference level, or one or more RNAs or nucleic acids, in a sample as compared to a control or reference level.
  • a particular marker or biomarker such as a nucleic acid or a protein
  • the quantity of a particular RNA may be present at an elevated amount or at a decreased amount in samples of patients with a disease compared to a reference level.
  • a "difference of a level” may be a difference between the quantity of a particular biomarker present in a sample as compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%>, at least about 25%, at least about 30%), at least about 35%, at least about 40%>, at least about 50%>, at least about 60%>, at least about 75%, at least about 80% or more.
  • a "difference of a level” may be a statistically significant difference between the quantity of a biomarker present in a sample as compared to a control. For example, a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.
  • a "disease” is a pathological condition; for example, one that can be identified by symptoms or other identifying factors as diverging from a healthy or a normal state.
  • the term “disease” includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases. Diseases also include neural, immune system, muscular, reproductive,
  • a disease is one or more cancers.
  • pancreatic cancer is a disease mediated by the expression level of one or more miRNA molecules, such as, for example, miR-1291.
  • drug As used herein, the terms "drug,” “prodrug”, “compound,” “active agent,” “agent,” “actives,” “pharmaceutical composition,” “pharmaceutical formulation,” and
  • pharmaceutically active agent are used interchangeably and refer to any chemical or biological compound, complex, or composition, that is suitable for administration and that has a beneficial biological effect, suitably a therapeutic effect in the treatment of a disease or abnormal physiological condition, although the effect may also be prophylactic in nature.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs, and the like.
  • active agent active pharmaceutical ingredient
  • pharmaceutically active agent active pharmaceutical ingredient
  • API active pharmaceutical ingredient
  • duplex region refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary.
  • an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the "duplex region” consists of 19 base pairs.
  • the remaining base pairs may, for example, exist as 5' and/or 3' overhangs.
  • the terms "effective amount” or “pharmaceutically effective amount” or “therapeutically effective amount” of a composition is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated.
  • the amount of a composition of the invention administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions of the present invention can also be administered in combination with one or more additional therapeutic compounds.
  • control levels i.e., normal levels
  • control levels refer to a range of miRNA levels that would be normally be expected to be observed in a mammal that does not have a disease or condition, such as, e.g., cancer.
  • a control level may be used as a reference level for comparative purposes.
  • Elevated levels refer to miR A levels that are above the range of control levels. The ranges accepted as “elevated levels” or “control levels” are dependent on a number of factors.
  • one laboratory may routinely determine the level of miRNA in a sample that is different than the miRNA obtained for the same sample by another laboratory.
  • different assay methods may achieve different value ranges. Value ranges may also differ in various sample types, for example, different body fluids or by different treatments of the sample.
  • One of ordinary skill in the art is capable of considering the relevant factors and establishing appropriate reference ranges for "control values” and "elevated values” of the present invention. For example, a series of samples from control subjects and subjects diagnosed with pancreatic cancer, for example, can be used to establish ranges that are "normal” or "control” levels and ranges that are “elevated” or “higher” than the control range.
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA, such as, e.g., total RNA, mRNA, rRNA, tRNA, snRNA, snoRNAs, miRNA, pri-RNA, through transcription of the gene, i.e., via the enzymatic action of an RNA polymerase, and for protein encoding genes, into protein through translation of mRNA.
  • Gene expression can be regulated at many stages in the process. Up-regulation or activation refers to regulation that increases the production of gene expression products, i.e., RNA or protein, while down-regulation, repression or knockdown refers to regulation that decrease production.
  • Molecules e.g., transcription factors that are involved in up-regulation or down-regulation are often called activators and repressors, respectively.
  • hybridization or “annealing” are used in reference to the pairing of complementary nucleic acids.
  • Hybridization and the strength of hybridization is influenced by the degree of complementarity between the nucleic acids, stringency of the hybridization conditions involved, the melting temperature of the formed hybrid, the G/C ratio within the nucleic acids, and other annealing factors.
  • inhibitor means that the level of RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA) is reduced below that observed in the absence of the inhibitor.
  • RNA isoforms are mature RNAs that are of nearly identical sequences, usually differing by about 1 , 2, 3, 4, or 5 nucleotides. For example, these families are designated with a letter (e.g. let-7b and let-7c).
  • the second type of isoforms are miRNA genes that produce the identical mature miRNA from a different precursor gene (e.g. let-7a-l and let-7a-2). These are designated with a number implying that both genes, let-7a-l and let-7a-2, produce the identical mature miRNA (let-7a).
  • Each isoform is usually located in different regions of the genome
  • nucleic acid e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer
  • a nucleic acid that is apart from a substantial portion of the genome in which it naturally occurs and/or is substantially separated from other cellular components which naturally accompany such nucleic acid.
  • any nucleic acid that has been produced synthetically e.g., by serial base condensation
  • nucleic acids that are recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or otherwise excised from a genome are also considered to be isolated.
  • label refers to any physical molecule directly or indirectly associated with a specific binding agent or antigen which provides a means for detection for that antibody or antigen.
  • a "detectable label” as used herein refers any moiety used to achieve signal to measure the amount of complex formation between a target and a binding agent. These labels are detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, electrochemilummescence or any other appropriate means. Suitable detectable labels include fluorescent dye molecules or f uorophores.
  • microarray refers to an arrangement of a collection of nucleic acids, e.g., nucleotide sequences in a centralized location.
  • Arrays can be on a solid substrate, such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane.
  • the nucleotide sequences can be DNA, RNA, or any combination or permutations thereof.
  • the nucleotide sequences can also be partial sequences or fragments from a gene, primers, whole gene sequences, non- coding sequences, coding sequences, published sequences, known sequences, or novel sequences.
  • Tissue microarrays are well known in the art and can be performed as described. See e.g., Camp, R. L., et al, J Clin Oncol, 26, 5630-5637 (2008).
  • a "microRNA,” “miR,” or “miRNA” refer to the unprocessed or processed RNA transcript from a miRNA gene.
  • the unprocessed miRNA gene transcript is also called a “miRNA precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length.
  • the miRNA precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the "processed" miRNA gene transcript or "mature" miRNA.
  • miR- 1291 is a nucleic acid molecule including ribonucleotides, deoxyribonucleotides, and/or modified nucleotides exemplified by SEQ. ID. NO: 3, i.e., GGTAGAATTCCAGTGGCCCTGACTGAAGACCAGCAGTTGTACTGTGGCTGTTGGT TTCAAGCAGAGGCCTAAAGGACTGTCTTCCTG, or substantially identical sequences related thereto.
  • SEQ. ID. NO: 3 i.e., GGTAGAATTCCAGTGGCCCTGACTGAAGACCAGCAGTTGTACTGTGGCTGTTGGT TTCAAGCAGAGGCCTAAAGGACTGTCTTCCTG, or substantially identical sequences related thereto.
  • SEQ. ID. NO: 3 i.e., GGTAGAATTCCAGTGGCCCTGACTGAAGACCAGCAGTTGTACTGTGGCTGTTGGT TTCAAGCAGAGGCCTAAAGGACTGTCTTCCTG, or substantially identical sequences related
  • a miR- 1291 nucleic acid is similarly provided by the compositions and methods of the present invention when it is expressed or delineated as, or contains, fully or in part, RNA sequences, DNA sequences, a mixture of RNA and DNA, and/or incorporate one or more modified bases or base substitutes.
  • nucleotide refers to a chemical moiety having a sugar (modified, unmodified, or an analog thereof), a nucleotide base (modified, unmodified, or an analog thereof), and a phosphate group (modified, unmodified, or an analog thereof).
  • Nucleotides include deoxyribonucleotides, ribonucleotides, and modified nucleotide analogs including, for example, locked nucleic acids (“LNAs”), peptide nucleic acids (“PNAs”), L- nucleotides, ethylene -bridged nucleic acids (“EN As”), arabinoside, and nucleotide analogs (including abasic nucleotides).
  • LNAs locked nucleic acids
  • PNAs peptide nucleic acids
  • EN As ethylene -bridged nucleic acids
  • nucleotide analogs including abasic nucleotides.
  • nucleic acid refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof and to naturally occurring or synthetic molecules.
  • RNA may be used in the methods described herein and/or may be converted to cDNA by reverse- transcription and/or RNA for use in the methods described herein.
  • sequence and “oligonucleotide” are used interchangeably and refer to a polymers composed of deoxyribonucleotides, ribonucleotides or any combination thereof. Oligonucleotides are generally between about 10 and about 100 nucleotides in length.
  • Oligonucleotides are typically 15 to 70 nucleotides long, with 20 to 26 nucleotides being the most common, while sequences may be longer as delineated.
  • An oligonucleotide may be used as a primer or as a probe.
  • An oligonucleotide is "specific" for a nucleic acid if the oligonucleotide has at least 50% sequence identity with a portion of the nucleic acid when the oligonucleotide and the nucleic acid are aligned.
  • An oligonucleotide that is specific for a nucleic acid is one that, under the appropriate hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%>, at least 85%, at least 90%, or at least 95% sequence identity.
  • polypeptide “protein,” and “peptide” are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds.
  • the amino acid chains can be of any length of greater than two amino acids.
  • the terms “polypeptide,” “protein,” and “peptide” also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinated forms, etc.
  • Modifications also include intra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc.
  • modifications may also include cyclization, branching and cross-linking.
  • amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.
  • a "probe” for ascertaining the presence, absence, level, expression, abundance, and the like is an oligonucleotide that specifically anneals to a target or marker nucleotide sequence. Probes of the present invention have one or more labels as futher described herein, in some embodiments.
  • a probe can also be a "primer” for amplification as described herein, where the primer is one or more oligonucleotides that specifically anneal to a target or marker nucleotide sequence and forms a substrate for a nucleic acid polymerase.
  • primer includes "primer pairs" required to amplify a nucleic acid sequence. The 3' nucleotide of the primer should be identical to the target or marker sequence at a
  • a "forward primer” is a primer that anneals to the anti-sense strand of double stranded DNA (dsDNA).
  • a “reverse primer” anneals to the sense-strand of dsDNA or an appropriate cDNA or RNA or other nucleic acids with similar polarity.
  • prognosis refers to a prediction of the probable course and outcome of a clinical condition or disease.
  • a prognosis is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease.
  • determining the prognosis refers to the process by which the skilled artisan can predict the course or outcome of a condition in a patient.
  • prognosis does not refer to the ability to predict the course or outcome of a condition with 100% accuracy.
  • prognosis refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.
  • prognosis and positive prognosis or “unfavorable prognosis” and “negative prognosis” as used herein are relative terms for the prediction of the probable course and/or likely outcome of a condition or a disease. A favorable or positive prognosis predicts a better outcome for a condition than an unfavorable or negative prognosis.
  • a "favorable prognosis" is an outcome that is relatively better than many other possible prognoses that could be associated with a particular condition, whereas an unfavorable prognosis predicts an outcome that is relatively worse than many other possible prognoses that could be associated with a particular condition.
  • Typical examples of a favorable or positive prognosis include a better than average cure rate, a lower propensity for metastasis, a longer than expected life expectancy, differentiation of a benign process from a cancerous process, and the like.
  • a positive prognosis is one where a patient has a 50% probability of being cured of a particular disease, e.g., cancer, after treatment, while the average patient with the same cancer has only a 25% probability of being cured.
  • the terms "reference level,” “reference standard,” “control” or “control sample” are used interchangeably and refer to a sample having a level or amount of a substance which may be of interest for comparative purposes.
  • a reference level is the average of the amount of, and/or expressions of, certain nucleic acids, e.g., miR As, from one or more biological samples taken from a control population of one or more healthy (disease-free) subjects.
  • the reference level is the amount of, and/or expression of, certain nucleic acids, e.g., miRNAs, from the same subject at a different time, e.g., prior to the subject developing the disease and/or prior to and/or after and/or during therapy, and/or before and after a sample preparation procedure, and/or before and after sample storage.
  • samples are normalized by a common factor.
  • sample biological sample
  • test sample test sample
  • clinical sample laboratory sample
  • laboratory sample and/or “biospecimen” are used interchangeably and in the broadest sense.
  • a sample may include a bodily tissue or a bodily fluid including but not limited to tissue samples, blood (or a fraction of blood such as plasma or serum), lymph, mucus, tears, urine, stool and saliva.
  • a sample may include an extract from an animal or plant cell, a chromosome, organelle, or a virus.
  • a sample may be a "cell-free” sample, meaning that the volume of cells in the sample are less than about 2% of the total sample volume (preferably less than about 1% of the total sample volume).
  • a sample may comprise RNA, e.g., miRNA or cDNA.
  • a sample may be obtained from any subject or any patient.
  • siRNA refers to any nucleic acid molecule capable of down regulating ⁇ i.e., inhibiting) gene expression in a mammalian cells (preferably a human cell).
  • siRNA includes without limitation nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA).
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • the sense strand of a siRNA molecule may also include additional nucleotides not complementary to the antisense region of the siRNA molecule.
  • the term "antisense region” refers to a nucleotide sequence of a siRNA molecule complementary (partially or fully) to a target nucleic acid sequence.
  • the antisense strand of a siRNA molecule may include additional nucleotides not complementary to the sense region of the siRNA molecule.
  • the term "subject” refers to a mammal, such as a human, but can also be another animal such as a domestic animal (e.g., a dog, cat, or the like), a farm animal (e.g., a cow, a sheep, a pig, a horse, or the like) or a laboratory animal (e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like).
  • a domestic animal e.g., a dog, cat, or the like
  • a farm animal e.g., a cow, a sheep, a pig, a horse, or the like
  • a laboratory animal e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like.
  • patient refers to a subject who is, or is suspected to be, afflicted with a disease.
  • sequences that are "substantially similar” or “substantially identical” to each other have similar or identical nucleotides, respectively, at least at about from 50% of aligned nucleotide positions, preferably at least at about from 75%, 85%, or 90% of aligned nucleotide positions, and more preferably at least at about 95% of aligned nucleotide positions.
  • target nucleic acid refers to a nucleotide sequence of interest which is the subject of amplification or detection under the parameters described and/or to be detected or quantified in a method or system as disclosed herein.
  • Target nucleic acids contain the target nucleic acid sequences that are actually assayed during an assay procedure. The target can be directly or indirectly assayed.
  • the target nucleic acid if present in the sample, is used as a template for amplification according to the methods disclosed herein.
  • Target nucleic acid may include a miRNA molecule, including without limitation, a miRNA, a siRNA, a shRNA, or other ncRNA.
  • Threshold cycle or “CT” is used in reference to quantitative or real-time analysis methods and indicates the fractional cycle number at which the amount of amplification products, reaches a fixed threshold or limit. Thresholds can be set manually by the user or determined by the software of a real-time instrument.
  • Tm refers to the melting temperature at which half of the designated primers are annealed to a target nucleic acid or region.
  • the terms “treating” or “treatment” or “alleviation” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • a subject is successfully "treated” for a disorder if, after receiving a therapeutic agent according to the methods of the present invention, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of a particular disease or condition.
  • MicroRNAs are short non-coding RNAs that govern post-transcriptional regulation of target genes.
  • miRNA precursors pre -miRNA that is approximately 70 nucleotides in length
  • pri-miRNAs double-stranded primary miRNAs
  • mRNA precursor splicing pathways some miRNAs may originated from small nucleolar RNAs (snoRNAs).
  • snoRNAs small nucleolar RNAs
  • the resultant pre-miRNA is subsequently transported to the cytoplasm, cleaved by Dicer and then processed into mature miRNA.
  • the mature, single stranded miRNA is consequently loaded onto the micro -ribonucleoprotein complex (miRNP), where it hybridizes to its cognate mRNA targets via complementary Watson-Crick base pairings, which therefore imparts translational inhibition.
  • miRNP micro -ribonucleoprotein complex
  • miRNAs control various critical biological processes including cancer initiation, progression, and metastasis. See Voorhoeve et al, 2006; Mayr et al, 2007; Kota et al, 2009; Korpal et al, 2011; Liu et al, 2011; Garofalo et al, 2012; and Png et al, 2012.
  • Oncogenic miRNAs may be used as therapeutic targets, while, tumor suppressive miRNAs serve as therapeutic agents for the prevention or treatment of diseases, such as, e.g., cancers, according to various aspects of the present invention.
  • diseases such as, e.g., cancers
  • biopsies are considered the gold standard for assessment of various solid tumors, such procedure are invasive and carry the risk of complications. Moreover, biopsies may yield misleading results when a representative sample is not obtained. Alternative tests that are simple, reliable, and non-invasive are therefore needed with respect to diagnosing, staging, preventing and treating various cancers.
  • miRNA-mediated regulation of tumorigenesis is a new paradigm in the field of cancer biology. Specific subsets of miRNAs that are deregulated or aberrantly expressed in certain cells and/or tissues are the etiological precursors to tumor development in some embodiments. miRNA profiles to this end may reflect tumor lineage and differentiation state in accord with the ability to
  • RNAs signature genotypes or phenotypes differing between poorly differentiated tumors and genetically disparate cancers.
  • RNAs such as, miRNAs
  • becuase miRNA precursor genes are usually nested within other protein coding genes, often within intron sequences, misregulation, by, for example, genetic, epigenetic, and phenotypic mechanisms, of these protein-coding genes may also cause aberrant regulation of miRNA target genes.
  • miRNAs miR-221/222 down-regulate p27Kipl/CDKNlB and the c-KIT receptor mRNA levels, thereby controlling the progression of neoplasia, leading to enhanced proliferation and reduced differentiation in such cancer cells (Felicetti et al, 2008).
  • miR-137 moreover, down-regulates the expression of MITF, a master regulator of cell growth, maturation, and pigmentation in certain cancers (Bemis et al, 2008). It has recently been shown that several miRNA genes are differentially regulated in certain cells types, and therefore, lead to cancer.
  • miRNA is consistently reduced in certain cancers and is capable of targeting KCNMA1 mRNA, encoding a potassium transporter (Mazar et al., 2010), which is associated with increased invasiveness and high proliferation rates in such cells.
  • oncogenic miRNAs such as, e.g., miR-21 and miR-372
  • miR-1291 is oncogenic with respect to various head and neck cancers. See Chen et al, "Novel Dysregulated MicroRNAs in Primary Laryngeal Squamous Cell Cancer.” Int. J. of Head and Neck Surg. Vol. 3(2) 76-81 (2012). Therefore, in some embodiments of the present invention, restoration of one or more tumor suppressive miRNAs depleted in tumors, alone or in concert with inhibition of one or more oncogenic miRNAs elevated in tumors are employed to treat various cancers.
  • miR-1291 is reconstituted as therapeutic or preventative treatment for a subject afflicted with cancer, including, but not limited to, e.g., pancreatic cancers, such as, e.g., human pancreatic ductal adenocarcinoma (PDAC). Accordingly, the present inventors have expanded druggable targets to virtually the whole human genome for cancer treatment.
  • pancreatic cancers such as, e.g., human pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • SNORNA34, SNORA34, and/or ACA34 which are related to specific nucleic acid sequences, such as, e.g., UGGCC CUG ACUG AAG AC C AGC AGU (SEQ. ID. NO: 1) and/or SEQ. ID. NOs: 2-5, as provided below in Table 1, for the treatment and/or prevention of diseases and conditions, such as, e.g., cancer, and/or reducing the risk of developing or recurrence of a cancer.
  • Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, which underscores the need for more effective therapies to treat fatal pancreatic cancers.
  • the present inventors have identified a tumor suppressor miRNA— miR- 1291— that is expressed at reduced levels in certain diseases, such as, e.g., cancer, including, but not limited to, e.g., pancreatic cancers, such as, e.g., human pancreatic ductal
  • adenocarcinoma PDAC
  • the reconstitution of miR-1291 "mimics" to PDAC cells induces cancer cell cycle arrest and the suppression of xenografts pancreatic tumor growth in certain aspects of the present invention, as futher provided below.
  • miR-1291 possesses tumor suppressive functionality
  • its loss-of- function in PDAC imparts one aspect of the present invention, i.e., a new "miR-1291 replacement therapy" technology for the treatment of cancer, including, but not limited to, e.g., pancreatic cancers, such as, e.g., human pancreatic ductal
  • PDAC adenocarcinoma
  • miRNA replacement therapy constitutes tumor growth management, treatment, and prevention, which is contrary to the vast majority of current miRNA therapies that focus on miRNA antagonism.
  • the miRNA mimics of the present invention are employed as miRNA replacement therapy inasmuch as such mimics possess a similar sequence as a naturally occurring miRNA, e.g., miR-1291, and are therefore unlikely to possess non-specific, and unwanted, effects.
  • the mimics possess an identical sequence to a naturally occurring miRNA, e.g., miR-1291.
  • the mimics possess a sequence related to or similar to any of the sequences from Table 1, or as known in the art concerning miR-1291.
  • miRNAs are normal constituents of healthy cells, the reintroduction of therapeutic miRNAs into healthy cells possess little to no toxicity concerns.
  • miR-34a mimics, using a lipid-based delivery vehicle, possess a good safety profile is demonstrated for this miR-34aformulation, as manifested by the tolerance in mouse models and the lack of change in blood chemistries, immune response, liver the kidney functions (Bader, 2012).
  • miR- 1291 reactivates miRNA regulatory pathways in human cancer cells, and consequently inhibit uncontrolled cell proliferation, induce apoptotic cascade, and suppress tumor growth.
  • a miR-1291 is expressed at lower levels in human pancreatic ductal adenocarcinoma (PDAC) compared to normal pancreatic tissues.
  • the expression is decreased from about 0.5, 1, 5, 10, 15, 20, 30, 50, 75, or 100-fold lower to about from 5, 10, 15, 20, 30, 50, 75, 100, 200, 300, 500, or 1000-fold lower level in human pancreatic ductal adenocarcinoma (PDAC) than the paired normal pancreatic tissues.
  • the expression is decreased from about 0.5, 1, or 5 -fold lower to about from 1, 5, or 10-fold lower level in human pancreatic ductal adenocarcinoma (PDAC) than the paired normal pancreatic tissues.
  • PDAC pancreatic ductal adenocarcinoma
  • the expression is decreased about 8-fold lower level in human pancreatic ductal adenocarcinoma (PDAC) than the paired normal pancreatic tissues.
  • PDAC pancreatic ductal adenocarcinoma
  • pancreatic cancer remains a highly lethal cancer disease in the United States and treatment options are limited to chemotherapy, radiation, and/or surgery, which possess limited efficacy in relieving symptoms or extending survival for the pancreatic cancer patients (Bayraktar and Rocha-Lima, 2010).
  • ACS American Cancer Society
  • more than 44,000 people in the U.S. were diagnosed with pancreatic cancer in 2011. Only 15-20% of patients are candidates for potentially curative surgery but even with such surgery, metastases often occur.
  • the current standard of care is Eli Lilly's Gemzar® (gemcitabine) despite only having a median survival of less than 6 months and a one-year survival rate of around 18% (Berlin et al., 2002).
  • miRNAs have been implicated, for example, in cellular post- transcriptional regulation, regulation of developmental timing and pattern formation, restriction of differentiation potential, the differential expression at various developmental stages, cell types, and tissues in mammalian organs and embryonic stem cells, regulation of insulin secretion, resistance to viral infection, such as, e.g., Epstein Barr virus infections, metabolic disease, obesity, genomic rearrangements associated with genetic disorders, such as, e.g., fragile X syndrome, and tumorigenesis. See, e.g., Wiemer, Eur. J Cancer 43: 1529- 1544 (2007).
  • miRNAs are associated with cancer and are potential biomarkers and/or therapeutic targets for prognosis and prevention or treatment, respectively, little is known about how they are altered during systemic therapy and the effect on the response of cancer to treatment.
  • Hormone therapy and chemotherapeutic agents such as doxorubicin and paclitaxe, alters expression profiles of various miRNAs, which consequently can affect chemosensitivity, while radiation may possess similar effects.
  • identification and characterization of druggable miRNA targets remains an important consideration in the prevention and treatment of various diseases. Examples of such characterization are indicated below.
  • compositions and methods of the present invention may be used to respectively treat subjects and/or measure differential expression of miRNAs that are known to be associated with certain diseases.
  • a target miRNA may be selected from human miRNAs including but not limited to miR- 10b, miR-21, miR-29b, miR-17-5p, miR-125b, miR-145, miR-146, and miR-155.
  • a target miRNA may be selected from human miRNAs including but not limited to miR-155, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92.
  • a target miRNA may be selected from human miRNAs including but not limited to the let-7 family, miR-lOa, miR-20a, miR-24, miR-29b, miR-31, miR-96, miR-133b, miR-135b, miR-143, miR-145, miR-183, miR-17, miR-18a, miR-19a, miR-19b and miR-92.
  • the target miRNA may be selected from human miRNAs including but not limited to miR-18, miR-125a, miR-195, miR-199a, miR-200a, and miR-224.
  • the target miRNA may be selected from human miRNAs including but not limited to miR-21, miR-24, miR-34a, miR-100, miR-101, miR-103, miR-107, miR-125b, miR-143, miR-145, miR-148b, miR-200, and miR-155.
  • the target miRNA may be selected from human miRNAs including but not limited to let-7d, miR-128a, miR-195, and miR-203.
  • the target miRNA may be selected from human miRNAs including but not limited to the let-7 family, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92, miR-21, miR-126*, miR-155, miR-200b, miR-205, and miR-210.
  • let-7 human miRNAs
  • miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92, miR-21, miR-126*, miR-155, miR-200b, miR-205, and miR-210 See Wiemer, Eur. J Cancer 43: 1529-1544 (2007); Zhao et al., "MiR-148b functions as a tumor suppressor in pancreatic cancer by targeting AMPKal" DOL lO. l 158/1535-7163.MCT-12-0534-T, American Assoc. or Cancer Res.
  • miRNAs miR-221/222 down-regulate p27Kipl/CDKNlB and the c-KIT receptor mRNA levels, thereby controlling the progression of neoplasia, leading to enhanced proliferation and reduced differentiation in such cancers cells (Felicetti et al., 2008).
  • miR-137 moreover, down-regulates the expression of MITF, a master regulator of cell growth, maturation, and pigmentation in melanoma (Bemis et al., 2008). It has recently been shown that several miRNA genes are differentially regulated in melanoma cells, and therefore, lead to cancer.
  • miRNA-211 is consistently reduced in melanoma ⁇ see Mazar et al, 2010), which is associated with increased invasiveness and high proliferation rates in susceptible cells.
  • a group of epigenetically regulated miRNA genes has been associated with melanomas, e.g., miR-34b, -489, -375, -132, -142-3p, -200a, -145, -452, -21, -34c, -496, -let7e, -654, and - 519b.
  • Breast tumors moreover, comprise heterogeneous miRNA profiles and miRNA signature of, e.g., let-7 family, mir-lOb, mir -18a, mir-106a, mirl25-a, mirl25-b, mir-126, mir-130a, mir-145, mir-155, mir-141, mir-214, mir-205, mir-206, mir-210, mir-126, mir-335, mir-213, mir-203, 17-5p, miR-30, mir-34, and mir-342, have been proposed to affect breast cancer outcomes. See, e.g., Wiemer, Eur. J Cancer 43: 1529-1544 (2007).
  • AFP hepatocellular carcinoma
  • CLD non-HCC chronic liver disease
  • AFP is currently the most commonly used serum tumor marker for screening HCC
  • the American Association for the Study of Liver Diseases has recommended against its use as a sole marker for HCC screening unless ultrasonography is not available.
  • the conventional HCC markers usually provide good detection of HCC patients with large tumors, however, early detection of HCC is a key factor for HCC therapeutic response.
  • miR-16, miR-199a, and/or miR-195 have been employed as biomarkers for various liver cancers.
  • the present invention provides a target nucleic acid, such as, e.g., a miRNA, such as, for example, but not limited to, sequences which miR-1291 hybridizes.
  • the present invention provides compositions and methods relating to the prevention, treatment, detection and alteration of the expression levels or activity of miR- 1291 or a precursor or target thereof, by providing an agent comprising a nucleic acid molecule or a complement or precursor thereof, under conditions effective to increase the expression level or activity of the miR- 1291 or a precursor or a target thereof, compared to a reference level or activity, where the nucleic acid molecule that interacts (binds, hybridizes, recognizes, etc.) with a target sequence is one or more of (i) a sequence encoding miR-1291, (ii) a sequence selected from SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID.
  • the target is a nucleotide sequence within a cell, where the cell is a drug resistant cell, cancer cell, invasive cell, and/or metastatic cell in a subject
  • the cell is a cancerous cell, hyperproliferative cell, neoplastic cell, hypoplastic cell, hyperplastic cell, dysplasia cell, metaplasia cell, prosoplasia cell, desmoplasia cell, angiogenic cell, inflammatory cell, immunological cell, metabolic cell, pulmonary cell, and/or a cardiovascular cell in a subject or sample.
  • the target sequence in suitable embodiments, is associated with a disease or condition linked to the aberrant expression of one or more of: miR-1291; AKT2; Cyclin Bl; MeCP2; FOXA2; AMPKal; Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASF1; Ornithine
  • OAT aminotransferase
  • Keratin, type II cytoskeletal 8 KRT8
  • Phosphoenolpyruvate carboxykinase 2 PEPCK2
  • Enoyl-coenzyme A Co A) hydratase
  • PSAT1 Phosphoserine aminotransferase isoform 1
  • DLAT Dihydrolipoamide acetyltransferase
  • Peroxiredoxin 3 isoform CRA a (PRDX3); Cysteine -rich protein 2 (CRIP2); Chain C, human PCNA; Fascin homolog 1, actin-bundling protein, isoform CRA a (FSCN1); Serpin HI precursor; Protein disulfide -isomerase precursor; Chain A, disulfide isomerase related chaperone ERP29; Triosephosphate isomerase isoform 2 (TPIl); Peroxiredoxin-4 (PRDX4); and Isocitrate dehydrogenase [NAD] subunit beta (IDH3B); a-fetoprotein (AFP); AFP-L3%, des-gamma-carboxyprothrombin (DCP); CDH1 (E-cadherin); trimethylated lysine 27 of H3 histone (H3K27me3); histone deacetylase -1; histone deacetylase -2; SIRT1; CD44; aldehyde dehydrogen
  • a subject harbors the sequence to be targeted, when the subject is a patient afflicted with cancer, refractory cancer, metastatic cancer, a solid tumor, a circulating tumor, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastro intestinal stromal tumor (GIST), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, renal cell carcinoma, renal cancer, sarcoma, parotid cancer, gastric cancer, melanoma, NET (sinonasal) cancer, colon cancer, liver cancer, hepatocellular carcinoma, inflammatory bowel disease (IBD), and/or metabolic disease.
  • GIST gastro intestinal stromal tumor
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • NET parotid cancer
  • gastric cancer melanoma
  • NET in
  • Synthetic agents provided as miR-1291 mimics, short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double-stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or any combination thereof, as further detailed herein, are suitable for identifying, interacting (binding, hybridizing, recognizing, etc.), treating, preventing and/or regulating a target sequence.
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • shRNA short hairpin RNA
  • pri-miRNAs primary miRNAs
  • snoRNAs small nucleolar RNAs
  • the synthetic agent is one or more of a nucleic acid molecule encoding a complement of miR-1291, a nucleic acid sequence possessing at least 90% similarity to SEQ. ID. NO: 5, and a nucleic acid sequence with at least 7 contiguous nucleotides from SEQ. ID. NO: 5 as the agent that targets ABC transporter activity in a cell.
  • the ABC transporter is selected from ABCC1, ABCG2, ABCB1, ABCC2, ABCC3, and ABCC4, in some embodiments.
  • a miR-1291 nucleic acid molecule and/or miR-1291 precursor such as, e.g., SNORA34, is conjugated to a cell-specific targeting ligand or peptide in suitable
  • the nucleic acid molecule is present in a biological vector, where the biological vector is a plasmid vector or viral vector in illustrative embodiments.
  • a delivery vehicle such as, but not limited to, a polymeric carrier, a micelle, a liposome, a lipoplex, a polyplex, a peptide, a polymer, a dendrimer, lipids, or a nanoparticle, in some embodiments, is conjugated to a cell-specific targeting ligand or antibody.
  • the cell-specific targeting peptide, ligand or antibody includes a cancer cell-specific targeting ligand or antibody.
  • the cancer cell-specific targeting ligand, peptide, or antibody is selected from one or more peptides or ligands, such as, but not limited to, e.g., transferrin, vasoactive intestinal peptide, somatostatin, gastrin releasing peptide, bombesin, substance P ligand, urokinase, urokinase A chain, epidermal growth factor (EGF), transforming growth factor-alpha (TGFa), insulin-like growth factor, interleukin-4 (IL-4), interleukin-6 (IL-6), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), laminin, vascular endothelial growth factor (VEGF), annexin V, nucleophosmin, HSC70, BIP, Grp75, PDI, PDI ER60 precursor, HSP60, TCP- 1 ⁇ , ERp29, HSP27, vimentin, a-inter
  • RT-PCR reverse transcriptase PCR
  • the methods and compositions provided herein are used to detect miRNA in a biological sample, in illustrative embodiments.
  • the biological sample may be from any organism that has endogenous miRNA.
  • Organisms include, but are not limited to, arthopods (Drosophila melanogaster); nematodes (Caenorhabditis elegans and Caenorhabditis briggsae); vertebrates (Homo sapiens, Mus musculus, Rattus norvegicus); plants (Arabidopsis thaliana and Oryza sativa), all of which have miRNAs that has been sequenced.
  • miRNA may be recombinant, such that it is obtained from a cell-free system or reaction mixture or from a recombinant host cell, which may or may not have endogenous miRNA.
  • miRNA may be evaluated in samples that were previously fixed.
  • the sample may be fixed in formaldehyde or paraformaldehyde prior to taking steps to evaluate its miRNA.
  • samples that can be used according to the invention include those in which RNA in the sample has been degraded. Such samples include those in which about or at least about 50%, 60%>, 70%>, 80%>, 90%>, 95%> or more, or any range derivable therein, of the miRNA and/or rRNA in the sample is degraded. In particular embodiments, samples in which there has been substantial
  • degradation that is, at least about 80% degradation of miRNA and/or rRNA in the sample- are analyzed according to methods and compositions of the invention.
  • miRNA used in an assay or reaction may be obtained by a variety of methods and from a variety of sources.
  • the miRNA may be obtained from a biological sample, such as a cell, tissue, or organ. It may be isolated from a biological sample that contains other RNA molecules as well, such as mRNA, tRNA, and/or rRNA. In certain instances, total RNA is first isolated from the sample and then the miRNA is separated from the other RNA, thereby enriching for miRNA.
  • the miRNA has been isolated away from other RNA to enrich for the miRNA, such that the miRNA is substantially pure, meaning it is at least about 80%>, 85%, 90%>, 95% pure or more, but less than 100% pure, with respect to other RNA molecules.
  • RNA molecules Various kits for the extraction of RNA from biological samples are available commercially.
  • a cell from which a nucleic acid is obtained for use in the methods can be a normal cell or a cell displaying one or more symptom of a particular disease or condition.
  • a biological sample used in a method of the present invention can be obtained from a cancer cell, neoplastic cell, necrotic cell or cell experiencing a disease or condition set forth below.
  • Exemplary methods that can be used to isolate a particular cell from other cells in a population include, but are not limited to, Fluorescent Activated Cell Sorting (FACS) as described, for example, in Shapiro, Practical Flow Cytometry, 3rd edition Wiley-Liss; (1995), density gradient centrifugation, or manual separation using micromanipulation methods with microscope assistance.
  • FACS Fluorescent Activated Cell Sorting
  • Exemplary cell separation devices that are useful in the invention include, without limitation, a Beckman JE-6 centrifugal elutriation system, Beckman Coulter EPICS ALTRA computer-controlled Flow Cytometer-cell sorter, Modular Flow Cytometer from Cytomation, Inc., Coulter counter and channelyzer system, density gradient apparatus, cytocentrifuge, Beckman J-6 centrifuge, EPICS V dual laser cell sorter, or EPICS PROFILE flow cytometer.
  • a tissue or population of cells can also be removed by surgical techniques. For example, a tumor or cells from a tumor can be removed from a tissue by surgical methods, or conversely non-cancerous cells can be removed from the vicinity of a tumor.
  • a biological sample can be prepared for use in the methods of the present invention by lysing a cell that contains one or more desired nucleic acids.
  • a cell is lysed under conditions that substantially preserve the integrity of the desired nucleic acid.
  • cells can be lysed or subfractions obtained under conditions that stabilize R A integrity.
  • Such conditions include, for example, cell lysis in strong denaturants, including chaotropic salts such as guanidine thiocyanate, ionic detergents such as sodium dodecyl sulfate, organic solvents such as phenol, high lithium chloride concentrations or other conditions known in the art to be effective in limiting the activity of endogenous R ases during RNA purification.
  • RNA can be obtained from a cell lysed by an enzyme that degrades the cell wall.
  • Cells lacking a cell wall either naturally or due to enzymatic removal can also be lysed by exposure to osmotic stress.
  • Other conditions that can be used to lyse a cell include exposure to detergents, mechanical disruption, sonication, heat, pressure differential such as in a French press device, or Dounce homogenization.
  • the nucleic acid to be detected may be from a biological sample such as a tissue sample and the like.
  • a biological sample such as a tissue sample and the like.
  • Various methods of extraction are suitable for isolating the DNA or RNA. Suitable methods include phenol and chloroform extraction. See Maniatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, pp. 16- 54 (1989). Numerous commercial kits also yield suitable DNA and RNA including, but not limited to, QIAampTM mini blood kit, Agencourt GenfmdTM, Roche Cobas® Roche MagNA Pure® or phenol: chloroform extraction using Eppendorf Phase Lock Gels®, and the
  • RNA is isolated from patient serum on the NucliSens easyMAG system (Biomeriux SA, France) according to the manufacturer's protocol.
  • Presence of the target nucleic acid in the sample is determined by correlating the presence of the labeled sequence or oligonucleotide in the amplification product.
  • Suitable detection and visualization methods are used to detect the target nucleic acid.
  • biotin-labeled oligonucleotide(s) can be detected using non-isotopic detection methods which employ avidin conjugates such as streptavidin-alkaline phosphatase conjugates.
  • Flurophore- labeled oligonucleotide(s) can be detected using a fiuorescence-imager.
  • the presence of the target nucleic acid can then be determined by detecting the label by fluorescence or other visualization method. Fluorescence polarization, for example, can be used to detect the incorporation of the reporter into the amplification product.
  • a reporter is a sequence complementary to a probe or primer sequences used to detect a target sequence (the interrogation sequence), where the reporter has a quencher.
  • the interrogation sequence includes a fluorophore.
  • Incorporation of the reporter brings the quencher into proximity with the fluorophore. This, in turn, reduces the signal output of the fluorophore, and this reduction in signal can be detected and correlated with the presence of the target nucleic acid.
  • Suitable fluorophore -quencher pairs are discussed below.
  • a dye-dye pair can be used for fluorescence induction.
  • PCR creates a duplexed product that places the two dyes in close proximity, and the fluorescent output of the label changes. The change is detectable by bench-top fluorescent plate readers or using a real-time PCR detection system.
  • a duplexed product is created that places the first and second labels (e.g. fiuorophore/quencher pair) into close proximity. And when the two labels are in close proximity, the fluorescent output of the reporter molecule label changes. The change is detectable by most bench-top fluorescent plate readers.
  • the label pair comprises a quencher-label pair in close proximity. In this embodiment, the fluorescent output of the reporter molecule label changes, and this change is detectable.
  • Other suitable detection methods are contemplated for used in the disclosed methods and kits.
  • the sequences are detected after further processing.
  • the reporter oligonucleotide or sequence fragments can be separated from the reaction using any of the many techniques known in the art useful for separating oligonucleotides.
  • the reporter oligonucleotide fragments can be separated from the reaction mixture by solid phase extraction.
  • the reporter oligonucleotide fragments can be separated by electrophoresis or by methods other than electrophoresis.
  • biotin- labeled oligonucleotides can be separated from nucleic acid present in the reaction mixture using paramagnetic or magnetic beads, or particles which are coated with avidin (or streptavidin).
  • biotinylated oligonucleotide/avidin-magnetic bead complex can be physically separated from the other components in the mixture by exposing the complexes to a magnetic field.
  • reporter oligonucleotide fragments are analyzed by mass spectrometry.
  • the intended PCR product from non-specific PCR products can be differentiated using a melting-curve analysis.
  • Increasing the temperature of the reaction which contains the non-specific reaction products to above the T m of the duplexed DNAs and intended product may melt the DNA duplex of the non-specific products and disrupt the interaction of the labels giving a measurable change in fluorescence.
  • By measuring the change in fluorescence while gradually increasing the temperature of the reaction subsequent to amplification and signal generation it may be possible to determine the T m of the intended product as well as that of the nonspecific product.
  • a non-specific product may also be detected during amplification by reading the signal during the extension step (e.g., at 72°C). If the T m of the non-specific product is less than the temperature, then the signal from one or more labels will be detected.
  • labels which are appropriate for use in the methods and kits, as well as methods for their inclusion in the probe, are disclosed herein and are known in the art. These include, but are not limited to, enzyme substrates, fluorescent dyes, chromophores, chemiluminescent labels, electrochemiluminescent labels, such as ORI-TAGTM (Igen), ligands having specific binding partners, or any other labels that can interact with each other to enhance, alter, or diminish a signal. It is understood that, should the PCR be practiced using a thermocycler instrument, a label should be selected to survive the temperature cycling required in this automated process.
  • the primers and probes used in the methods are labeled in some embodiments.
  • the oligonucleotides may include a label that emits a detectable signal.
  • the label system may be used to produce a detectable signal based on a change in fluorescence, fluorescence resonance energy transfer (FRET), fluorescence quenching, phosphorescence, bioluminescence resonance energy transfer (BRET), or chemiluminescence resonance energy transfer (CRET).
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • CRET chemiluminescence resonance energy transfer
  • two interactive labels may be used on a single oligonucleotide with due consideration given for maintaining an appropriate spacing of the labels. In other embodiments, two interactive labels on different
  • oligonucleotides may be used, such as, for example, the reporter and the second region of the second primer or probe.
  • the reporter and the second region are designed to hybridize to each other. Again, consideration is given to maintaining an appropriate spacing of the labels between the oligonucleotides when hybridized.
  • sequences, oligonucleotides, and nucleotides of the disclosed methods may be labeled with a "fluorescent dye” or a "fluorophore.”
  • a "fluorescent dye” or a “fluorophore” is a chemical group that can be excited by light to emit fluorescence. Some suitable fluorophores may be excited by light to emit phosphorescence. Dyes may include acceptor dyes that are capable of quenching a fluorescent signal from a fluorescent donor dye.
  • Dyes that may be used in the disclosed methods include, but are not limited to, the following dyes and/or dyes sold under the following tradenames: 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxytetramethylrhodamine (5-TAMRA) ; 5-ROX (carboxy-X-rhodamine); 6- Carboxyrhodamine 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7- AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acrif
  • AMCA Aminomethylcoumarin
  • AMCA-X Aminoactinomycin D
  • Aminocoumarin Aminocoumarin
  • AMCA Aminomethylcoumarin
  • Anilin Blue Anthrocyl stearate
  • APC Allophycocyanin
  • APC-Cy7 APC-Cy7
  • APTS Astrazon Brilliant Red 4G
  • Astrazon Orange R Astrazon Red 6B;
  • Astrazon Yellow 7 GLL Atabrine; ATTO-TAGTM CBQCA; ATTO-TAGTM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); Blancophor FFG; Blancophor SV;
  • GeneBlazer CCF2; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid ;
  • Granular Blue Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1;
  • Mitotracker Red Mitramycin ; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; NEDTM; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green; Oregon Green 488-X; Oregon GreenTM; Oregon GreenTM 488; Oregon GreenTM 500; Oregon GreenTM 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;
  • Rhodamine Rhodamine
  • Rhodamine 110 Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G;
  • Rhodamine B Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red;
  • Rhodamine WT Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); RsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFPTM; sgBFPTM (super glow BFP); sgGFPTM; sgGFPTM (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF 1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange;
  • Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO 11 ; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; TETTM; Tetracycline; Tetramethylrhod
  • TetramethylRodaminelsoThioCyanate True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; VIC®; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3; and salts thereof.
  • Fluorescent dyes or fluorophores may also include derivatives that have been modified to facilitate conjugation to another reactive molecule.
  • fluorescent dyes or fluorophores may include amine-reactive derivatives such as isothiocyanate derivatives and/or succinimidyl ester derivatives of the fluorophore.
  • the sequences, oligonucleotides and nucleotides of the disclosed compositions and methods may be labeled with a quencher.
  • Quenching may include dynamic quenching (e.g. , by FRET), static quenching, or both.
  • Suitable quenchers may include Dabcyl.
  • Suitable quenchers may also include black hole quenchers sold under the tradename "BHQ” (e.g., BHQ-0, BHQ-1, BHQ-2, and BHQ-3, Biosearch Technologies, Novato, CA). Dark quenchers also may include quenchers sold under the tradename "QXLTM" (Anaspec, San Jose, CA).
  • Dark quenchers also may include DNP-type non-fluorophores that include a 2,4- dinitrophenyl group.
  • the sequences, oligonucleotides and nucleotides of the disclosed methods may also be labeled with a donor fluorophore and an acceptor fluorophore (or quencher dye) that are present in the oligonucleotides at positions that are suitable to permit FRET (or quenching).
  • Labeled oligonucleotides that are suitable for the present methods may include but are not limited to oligonucleotides designed to function as LightCycler primers or probes, Taqman® Probes, Molecular Beacon Probes, Amplifluor® Primers, Scorpion® Primers, and LuxTM Primers.
  • the labels can be attached to the sequences, nucleotides, or oligonucleotides directly or indirectly by a variety of techniques. Depending upon the precise type of label used, the label can be located at the 5 Or 3' end of the oligonucleotide, located internally in the oligonucleotide sequence, or attached to spacer arms extending from the oligonucleotide and having various sizes and compositions to facilitate signal interactions.
  • oligonucleotides containing functional groups e.g., thiols or primary amines
  • functional groups e.g., thiols or primary amines
  • oligonucleotides containing functional groups e.g., thiols or primary amines
  • a phosphoramidite dye to the 5' hydroxyl of the 5' base by the formation of a phosphate bond, or internally, via an appropriately protected phosphoramidite
  • the label can be indirectly attached to a nucleotide or oligonucleotide using a suitable spacer or chemical linker.
  • oligonucleotide functionalizing reagents having one or more sulfhydryl, amino or hydroxyl moieties into the oligonucleotide reporter sequence, typically at the 5' terminus, are described in U.S. Pat. No. 4,914,210, incorporated herein by reference.
  • 5' phosphate group can be incorporated as a radioisotope by using
  • polynucleotide kinase and [ ⁇ P]ATP to provide a reporter group.
  • Biotin can be added to the 5' end by reacting an aminothymidine residue, introduced during synthesis, with an N- hydroxysuccinimide ester of biotin.
  • Labels at the 3' terminus can employ polynucleotide terminal transferase to add the desired moiety, such as for example, cordycepin, 35 S-dATP, and biotinylated dUTP.
  • Oligonucleotide derivatives are also available as labels.
  • etheno-dA and etheno-A are known fluorescent adenine nucleotides which can be incorporated into a an oligonucleotide.
  • etheno-dC is another analog that can be used in reporter synthesis.
  • the oligonucleotides containing such nucleotide derivatives can be hydrolyzed to release much more strongly fluorescent mononucleotides by the polymerase's 5' to 3' nuclease activity as nucleic acid polymerase extends a primer during PCR.
  • the system for detection comprises a pair of interactive signal-generating labels effectively positioned on the oligonucleotide and on a second component of the assay (such as the labeled nucleotide triphosphate), so as to quench the generation of detectable signal when the interactive signal-generating labels are in
  • Such labels include dye/quencher pairs or two dye pairs (where the emission of one dye stimulates emission by the second dye).
  • the interactive signal generating pair comprises a fluorophore and a quencher that can quench the fluorescent emission of the fluorophore, as described herein.
  • a quencher may include dimethylaminoazobenzen
  • fluorophore aminoexal-3-acryinido (Dabcyl, also known as Methyl Red) and the fluorophore may be FAM or HEX.
  • FAM aminoexal-3-acryinido
  • HEX HEX
  • fluorophore-quencher pairs have been described in Morrison, Detection of Energy Transfer and Fluorescence Quenching in Nonisotopic Probing, Blotting and Sequencing, Academic Press, 1995.
  • FRET fluorescence resonance energy transfer
  • FRET fluorescence polarization
  • EDANSTM/Dabcyl Fluorescein/Fluorescein (Molecular Probes, Eugene, OR), BODIPYTM FL/BODIPYTM FL (Molecular Probes, Eugene, OR), and Fluorescein/QSY7TM.
  • determining the melting temperature of the detected nucleic acid may include observing a signal from a second fluorescent dye that is different from the first fluorescent dye.
  • the second fluorescent dye for determining the melting temperature of the detected nucleic acid is an intercalating agent.
  • Suitable intercalating agents may include, but are not limited to SYBRTM Green 1 dye, SYBR dyes, Pico Green, SYTO dyes, SYTOX dyes, ethidium bromide, ethidium homodimer-1, ethidium homodimer-2, ethidium derivatives, acridine, acridine orange, acridine derivatives, ethidium-acridine heterodimer, ethidium monoazide, propidium iodide, cyanine monomers, 7-aminoactinomycin D, YOYO-1, TOTO- 1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1, cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5, PO-PRO-1, BO-PRO- 1, PO-PRO-3, BO-PRO-3, LO
  • an intercalating agent used in the method will exhibit a change in fluorescence when intercalated in double-stranded nucleic acid.
  • a change in fluorescence may include an increase in fluorescence intensity or a decrease in fluorescence intensity.
  • the intercalating agent may exhibit an increase in fluorescence when intercalated in double-stranded nucleic acid, and a decrease in fluorescence when the double-stranded nucleic acid is melted.
  • a change in fluorescence may include a shift in fluorescence spectra (i.e., a shift to the left or a shift to the right in maximum absorbance wavelength or maximum emission wavelength).
  • the intercalating agent may emit a fluorescent signal of a first wavelength (e.g., green) when intercalated in double-stranded nucleic and emit a fluorescent signal of a second wavelength (e.g., red) when not intercalated in double-stranded nucleic acid.
  • a change in fluorescence of an intercalating agent may be monitored at a gradient of temperatures to determine the melting temperature of the nucleic acid (where the intercalating agent exhibits a change in fluorescence when the nucleic acid melts).
  • the miRNA e.g., miRNA-1291, is detected using a nucleic acid amplification process.
  • Nucleic acid extracted from a sample can be amplified using nucleic acid amplification techniques well known in the art.
  • these techniques can include the polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • nested PCR ligase chain reaction.
  • NASBA isothermal nucleic acid sequence based amplification
  • RNA to cDNA Some methods employ reverse transcription of RNA to cDNA.
  • the method of reverse transcription and amplification may be performed by previously published or recommended procedures.
  • Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptase from Thermus thermophilus .
  • MMLV RT RNase H mutants of MMLV RT
  • AMV RT thermostable reverse transcriptase from Thermus thermophilus
  • one method which may be used to convert RNA to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), as described by Rashtchian, A., PCR Methods Applic, 4:S83-S91, (1994).
  • PCR methods include, for example, denaturation, or melting of double- stranded nucleic acids; annealing of primers; and extension of the primers using a
  • RT-PCR method further includes an initial reverse transcription step to produce cDNA from RNA.
  • a first primer ⁇ i.e., a cDNA primer
  • a primer extension reaction using a nucleic acid polymerase i.e., a reverse transcriptase
  • the first step of extending is performed at a temperature from about 15°C to about 50°C. The actual temperature of extension may be calculated by one of skill in the art based on the T m of the target binding region of the cDNA primer.
  • the cDNA is amplified using PCR.
  • the partially-double stranded product is denatured by heating and then the second primer is allowed to anneal to the cDNA.
  • the first annealing step is performed at a temperature from about 35°C to about 45°C or from about 35°C to about 55°C.
  • the actual temperature of annealing may be calculated by one of skill in the art based on the T m of the target binding region of the second primer.
  • Subsequent annealing steps may be performed at a higher temperature, e.g., at least about 55°C, because the tail regions of the first and second primers increase the T m of the primers relative to the amplicon.
  • the amplification methods described herein may include “real-time monitoring” or “continuous monitoring.” These terms refer to monitoring multiple times during a cycle of PCR, such as during temperature transitions and/or temperature hold steps.
  • real-time monitoring or “continuous monitoring.” These terms refer to monitoring multiple times during a cycle of PCR, such as during temperature transitions and/or temperature hold steps.
  • homogeneous detection assay is used to describe an assay that includes coupled
  • end-point monitoring refers to the detection of amplification at the termination of a reaction.
  • end-point monitoring may include melting curve analysis and gel electrophoresis and visualization with dyes or autoradiography.
  • the level of a miRNA gene product in a sample can be measured using any technique that is suitable for detecting RNA expression levels in a biological sample. Suitable techniques for determining RNA expression levels in a biological sample are well known to those of skill in the art. These include, for example, Northern blot analysis, RT-PCR, and in situ hybridization.
  • the level of at least one miRNA gene product is detected using Northern blot analysis.
  • total RNA can be purified from a sample in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters. The RNA is then immobilized on the filters by heating. Detection and quantification of specific R A is accomplished using appropriately labeled DNA or RNA probes complementary to the miRNA in question. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al, eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7.
  • Suitable probes for Northern blot hybridization of a given miRNA gene product can be produced from the known nucleic acid sequences and include, but are not limited to, probes having at least about 70%, 75%>, 80%>, 85%>, 90%>, 95%>, 98% or 99% complementarity to a miRNA gene product of interest, as well as probes that have complete complementarity to a miRNA gene product of interest. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11.
  • the nucleic acid probe can be labeled with, e.g., a radionuclide, such as
  • Probes can be labeled to high specific activity by either the nick translation method or by the random priming method. Autoradiographic detection of hybridization can then be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of miRNA levels. Using another approach, miRNA gene transcript levels can be quantified by computerized imaging systems.
  • the primer is a probe generated from any one of SEQ ID NOs: 1-5. In some embodiments, the probe is SEQ ID NO: 5.
  • a sample is suspected to contain multiple isoforms of a miRNA.
  • mature miRNA isoforms of the same family may differ from one another by 1, 2, 3, 4, or 5 nucleotides.
  • the detection methods described herein are capable of discriminating between related miRNA isoforms.
  • a first specific primer for a first miRNA isoform and a second specific primer for a second miRNA isoform are added to the sample, along with a universal primer.
  • One or more of the primers may further comprise a non-natural nucleotide base having a label.
  • the sequence of each specific primer may differ from another at one terminus or near a terminus ⁇ e.g.
  • each specific primer may differ from another (e.g., by a single nucleotide, by two nucleotides, or by three nucleotides).
  • Each specific primer may include an identical non-natural nucleotide base and a label (e.g., a fluorescent label).
  • a label e.g., a fluorescent label.
  • Each of the at least two labels may be different from the other.
  • one label may be fluorescein (FAM) and the other label may be hexachlorof uorescein (HEX).
  • the specific primers or probes have a 5' tail sequence in the 5' region of the primer or probe.
  • the tail region comprises at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length, or any range derivable therein.
  • the sequence of the tail region is typically not complementary to any nucleic acid in the sample.
  • the tails may be designed to improve the specificity of the primers by reducing mispriming during PCR, i.e., the tail sequences can be designed to add about 10°C to the T m of the specific primers.
  • the annealing temperature used in the first 1 to 5 cycles of PCR with tailed primers may be about 5°C to 15°C lower than the annealing temperature in subsequent PCR cycles.
  • the length of the primer or probe can affect the temperature at which the primer will hybridize to the target nucleic acid. Generally, a longer primer or probe will form a sufficiently stable hybrid to the target nucleic acid sequence at a higher temperature than will a shorter primer or probe. Further, the presence of high proportion of G or C or of particular non-natural bases in the primer can enhance the stability of a hybrid formed between the primer or probe and the target nucleic acid. This increased stability can be due to, for example, the presence of three hydrogen bonds in a G-C interaction or other non- natural base pair interaction compared to two hydrogen bonds in an A-T interaction.
  • Stability of a nucleic acid duplex can be estimated or represented by the melting temperature, or "T m .”
  • T m of a particular nucleic acid duplex under specified conditions is the temperature at which 50% of the population of the nucleic acid duplexes dissociate into single-stranded nucleic acid molecules.
  • the T m of a particular nucleic acid duplex can be predicted by any suitable method. Suitable methods for determining the T m of a particular nucleic acid duplex include the OligoTM software program. Primers suitable for use in the methods and kits disclosed herein can be predetermined based on the predicted T m of an oligonucleotide duplex that comprises the primer.
  • detection of a miRNA is performed using the TaqMan® assay, which is also known as the 5' nuclease assay (U.S. Pat. Nos. 5,210,015 and 5,538,848).
  • the TaqMan® assay detects the accumulation of a specific amplified product during PCR.
  • the TaqMan® assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye.
  • the reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET). When attached to the probe, the excited reporter dye does not emit a signal.
  • FRET fluorescence resonance energy transfer
  • the proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter.
  • the reporter dye and quencher dye may be at the 5' most and the 3' most ends, respectively or vice versa.
  • the reporter dye may be at the 5 ' or 3' most end while the quencher dye is attached to an internal nucleotide, or vice versa.
  • both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced.
  • DNA polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye.
  • the DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target-containing template which is amplified during PCR.
  • TaqMan® primer and probe sequences can readily be determined using the nucleic acid sequence information of the miRNA of interest.
  • a number of computer programs such as Primer Express (Applied Biosystems, Foster City, Calif), can be used to rapidly obtain optimal primer/probe sets. It will be apparent to one of skill in the art that such primers and probes for detecting the target nucleic acids are useful in diagnostic assays for neoplastic disorders, such as HCC, and can be readily incorporated into a kit format.
  • the present invention also includes modifications of the TaqMan® assay well known in the art such as the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and 5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and 6,117,635).
  • real time PCR is performed using TaqMan® Assays in combination with a suitable amplification/analyzer such as the ABI Prism® 7900HT Sequence Detection System.
  • the ABI PRISM® 7900HT Sequence Detection System is a high-throughput real-time PCR system that detects and quantitates nucleic acid sequences.
  • Real-time detection on the ABI Prism 7900HT or 7900HT Sequence Detector monitors fluorescence and calculates Rn during each PCR cycle.
  • the threshold cycle, or Ct value is the cycle at which fluorescence intersects the threshold value.
  • the threshold value is determined by the sequence detection system software or manually.
  • the Ct can be correlated to the initial amount of nucleic acids or number of starting cells using a standard curve.
  • TaqMan® MiR Assays are used to detect the miRNA.
  • TaqMan® MiR Assays are predesigned assays that are available for the majority of content found on the miRBase miRNA sequence repository.
  • the mirVanaTM qRT-PCR miRNA Detection Kit (Ambion) is a used to detect and quantify the miRNA.
  • This is a quantitative reverse transcription-PCR (qRT-PCR) kit enabling sensitive, rapid quantification of miRNA (miRNA) expression from total RNA samples.
  • an internal amplification control may be included in one or more samples to be extracted and amplified.
  • the skilled artisan will understand that any detectable sequence that is not typically present in the sample can be used as the control sequence.
  • a control sequence can be produced synthetically. If PCR amplification is successful, the internal amplification control amplicons can then be detected. Additionally, if included in the sample prior to purification of nucleic acids, the control sequences can also act as a positive purification control.
  • Suitable PCR-amplification competent nucleic acid polymerases include, for example, polymerases capable of extending an oligonucleotide by incorporating nucleic acids complementary to a template oligonucleotide.
  • the polymerase can be a DNA polymerase. Enzymes having polymerase activity catalyze the formation of a bond between the 3' hydroxyl group at the growing end of a nucleic acid primer and the 5' phosphate group of a nucleotide triphosphate.
  • nucleotide triphosphates are usually selected from deoxyadenosine triphosphate (A), deoxythymidine triphosphate (T), deoxycytosine triphosphate (C) and deoxyguanosine triphosphate (G).
  • A deoxyadenosine triphosphate
  • T deoxythymidine triphosphate
  • C deoxycytosine triphosphate
  • G deoxyguanosine triphosphate
  • polymerases useful for the methods disclosed herein also may incorporate non-natural bases using nucleotide triphosphates of those non-natural bases.
  • nucleic acid polymerase enzymes useful for performing the methods disclosed herein preferably retain sufficient polymerase activity to complete the reaction when subjected to the temperature extremes of methods such as PCR.
  • the nucleic acid polymerase enzymes useful for the methods disclosed herein are thermostable nucleic acid polymerases.
  • Suitable thermostable nucleic acid polymerases include, but are not limited to, enzymes derived from thermophilic organisms. Examples of thermophilic organisms from which suitable thermostable nucleic acid polymerase can be derived include, but are not limited to, Thermus aquaticus, Thermus thermophilus, Thermus flavus,
  • Thermostable polymerases may also be derived from thermostable bacteriophage isolated from thermophilic organisms. Nucleic acid polymerases can be purified directly from these thermophilic organisms and thermostable phages and/or may be recombinantly expressed and isolated from a suitable system such as Escherichia coli cells. Suitable thermostable nucleic acid polymerases, such as those described above, are commercially available.
  • Polymerases can "misincorporate" bases during PCR.
  • the polymerase can incorporate a nucleotide (for example adenine) at the 3' position on the synthesized strand that does not form canonical hydrogen base pairing with the paired nucleotide (for example, cytosine) on the template nucleic acid strand.
  • the PCR conditions can be altered to decrease the occurrence of misincorporation of bases.
  • reaction conditions such as temperature, salt concentration, pH, detergent concentration, type of metal, concentration of metal, and the like can be altered to decrease the likelihood that polymerase will incorporate a base that is not complementary to the template strand.
  • any of the methods described herein can be performed using multiple enzymes.
  • a reverse transcriptase can be used to transcribe the RNA to cDNA (e.g., AMV or MMLv). The reverse transcription may occur prior to PCR amplification.
  • the present invention provides a pharmaceutical composition including a nucleic acid molecule or a complement or precursor thereof, and one or both of a pharmaceutically acceptable carrier and a stabilizing agent, wherein the nucleic acid molecule is composed of one or more of (i) a sequence encoding miR-1291, (ii) a sequence selected from SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, (iii) a sequence with at least 90% similarity to one or more of SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, and (iv) a sequence with at least 7 contiguous nucleotides from SEQ. ID.
  • compositions of the present invention may contain other therapeutic agents as described below, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration, for example, excipients, binders, preservatives, stabilizers, flavors, etc., according to techniques such as those well known in the art of pharmaceutical formulation. See, e.g., Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005).
  • the nucleic acid molecule in some embodiments, is conjugated to a peptide, cancer cell-specific targeting ligand, or antibody derived from transferrin, vasoactive intestinal peptide, somatostatin, gastrin releasing peptide, bombesin, substance P ligand, urokinase, urokinase A chain, epidermal growth factor (EGF), transforming growth factor-alpha (TGFa), insulin-like growth factor, interleukin-4 (IL-4), interleukin-6 (IL-6), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), laminin, vascular endothelial growth factor (VEGF), annexin V, nucleophosmin, HSC70, BIP, Grp75, PDI, PDI ER60 precursor, HSP60, TCP- 1 ⁇ , ERp29, HSP27, vimentin, a-internexin, cytokeratin 8, ⁇ -
  • the nucleic acid molecule is present in, or derived from, a biological vector selected from the group consisting of a eukaryotic vector, a mammalian vector, a prokaryotic vector, a plasmid, a phage, a synthetic construct, and a viral vector.
  • a biological vector selected from the group consisting of a eukaryotic vector, a mammalian vector, a prokaryotic vector, a plasmid, a phage, a synthetic construct, and a viral vector.
  • compositions are typically formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral (e.g. , intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course.
  • compositions of the present disclosure are administered by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders;
  • parenterally such as by subcutaneous, intravenous, intramuscular, intra(trans)dermal, or intracisternal injection or infusion techniques, e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions, nasally such as by inhalation spray or insufflation, topically, such as in the form of a cream or ointment ocularly in the form of a solution or suspension, vaginally in the form of pessaries, tampons or creams, or rectally such as in the form of suppositories, in unit dosage formulations containing nontoxic,
  • compositions may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the present compositions, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
  • compositions for the administration of the active agents are presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy, in some embodiments. These methods typically include bringing the active agent into association with the carrier which constitutes one or more accessory ingredients.
  • the active agents e.g., the nucleic acid molecule, sequence, or sequences.
  • compositions are prepared by uniformly and intimately bringing the active agent into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the active agent is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • compositions containing the active agent in some embodiments, in some
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents such as sweetening agents, flavoring agents, coloring agents and preserving agents, e.g., to provide pharmaceutically stable and palatable preparations.
  • Tablets contain the compositions of the present invention in admixture forms with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • the tablets are further coated to form osmotic therapeutic tablets for control release.
  • Formulations for oral use may also be presented as hard gelatin capsules where the compositions are mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active agent is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • the active agent is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymetfiylcellulose, methylcellulose, hydroxy- propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as
  • polyoxyethylene sorbitol monooleate or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active agent in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active agent in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
  • the pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally- occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions are sterile injectable solutions of aqueous or oleagenous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation is a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol, in some embodiments.
  • vehicles and solvents that are employed include, but are not limited to, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectable formulations.
  • the active agent may be administered by any of the methods and formulations employed in the art for administration to the respiratory tract.
  • the active agent may be administered in the form of a solution, suspension, or as a dry powder, in some embodiments.
  • agents according to this aspect of the present invention may also be any agent.
  • compositions of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • the propellant-driven inhalation aerosols which may be used according to the invention may also contain other ingredients such as co-solvents, stabilizers, surfactants, antioxidants, lubricants and pH adjusters.
  • the propellant-driven inhalation aerosols according to the invention which may be used according to the invention may be administered using inhalers known in the art, e.g., metered dose inhalers.
  • the compositions and agents of the present invention may be administered to the airways in the form of a lung surfactant formulation.
  • the lung surfactant formulation can include exogenous lung surfactant formulations (e.g., Infasurf ® (Forest Laboratories), Survanta ® (Ross Products), and Curosurf ® (DEY, California, USA) or synthetic lung surfactant formulations (e.g., Exosurf ® (Glaxo Wellcome Inc.) and ALEC). These surfactant formulations are typically administered via airway instillation (i.e., after intubation) or intratracheally.
  • exogenous lung surfactant formulations e.g., Infasurf ® (Forest Laboratories), Survanta ® (Ross Products), and Curosurf ® (DEY, California, USA
  • synthetic lung surfactant formulations e.g., Exosurf ® (Glaxo Wellcome Inc.) and ALEC.
  • compositions agents of the present invention may be administered to the airways in the form of an inhalable powder.
  • the powder formulation may include physiologically acceptable excipients such as monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g. dextrane), polyalcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these excipients with one another.
  • monosaccharides e.g. glucose or arabinose
  • disaccharides e.g. lactose, saccharose, maltose
  • oligo- and polysaccharides e.g. dextrane
  • polyalcohols e.g. sorbitol, mannitol, xylitol
  • salts e
  • disaccharides are used, while the use of lactose or glucose is preferred, particularly, but not exclusively, in the form of their hydrates.
  • the excipients have a maximum average particle size of up to 250 ⁇ , preferably between 10 and 150 ⁇ , most preferably between 15 and 80 ⁇ . It may sometimes seem appropriate to add finer excipient fractions with an average particle size of 1 to 9 ⁇ to the excipients mentioned above. These finer excipients are also selected from the group of possible excipients listed hereinbefore. Finally, in order to prepare the inhalable powders according to the invention, micronised formulations, preferably with an average particle size of 0.5 to 10 ⁇ is added to the excipient mixture. Processes for producing the inhalable powders according to the invention by grinding and micronizing and by finally mixing the ingredients together are known from the prior art.
  • Inhalable powders according to the invention which contain a physiologically acceptable excipient in addition to the active formulation may be administered, for example, by means of inhalers which deliver a single dose from a supply using a measuring chamber as described in U.S. Pat. No. 4,570,630, or by other means as described in DE 36 25 685 A, each of which is hereby incorporated by reference in its entirety.
  • the agents of the present invention may be administered to the airways in the form of a propellant-free inhalable solution and suspension.
  • the solvent used may be an aqueous or alcoholic, preferably an ethanolic solution.
  • the solvent may be water on its own or a mixture of water and ethanol.
  • the relative proportion of ethanol compared with water is not limited but the maximum is up to 70 percent by volume, more particularly up to 60 percent by volume and most preferably up to 30 percent by volume. The remainder of the volume is made up of water.
  • the solutions or suspensions containing the active formulation are adjusted to a pH of 2 to 7, preferably 2 to 5, using suitable acids.
  • the pH may be adjusted using acids selected from inorganic or organic acids. Examples of particularly suitable inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid and/or phosphoric acid.
  • organic acids examples include ascorbic acid, citric acid, malic acid, tartaric acid, maleic acid, succinic acid, fumaric acid, acetic acid, formic acid and/or propionic acid etc.
  • Preferred inorganic acids are hydrochloric and sulphuric acids. It is also possible to use the acids which have already formed an acid addition salt with one of the active substances.
  • ascorbic acid, fumaric acid and citric acid are preferred.
  • mixtures of the above acids may be used, particularly in the case of acids which have other properties in addition to their acidifying qualities, e.g., as flavorings, antioxidants or complexing agents, such as citric acid or ascorbic acid, for example.
  • hydrochloric acid it is particularly preferred to use hydrochloric acid to adjust the pH.
  • Co-solvents and/or other excipients may be added to the propellant-free inhalable solutions which may be used according to the invention.
  • Preferred co-solvents are those which contain hydroxyl groups or other polar groups, e.g. alcohols-particularly isopropyl alcohol, glycols-particularly propyleneglycol, polyethyleneglycol, polypropyleneglycol, glycolether, glycerol, polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • excipients and additives in this context denote any pharmacologically acceptable substance which is not an active substance but which can be formulated with the active substance or substances in the pharmacologically suitable solvent in order to improve the qualitative properties of the active substance formulation.
  • these substances have no pharmacological effect or, in connection with the desired therapy, no appreciable or at least no undesirable pharmacological effect.
  • the excipients and additives include, for example, surfactants such as soya lecithin, oleic acid, sorbitan esters, such as polysorbates, polyvinylpyrrolidone, other stabilisers, complexing agents, antioxidants and/or preservatives which guarantee or prolong the shelf life of the finished pharmaceutical formulation, flavorings, vitamins and/or other additives known in the art.
  • the additives also include pharmacologically acceptable salts such as sodium chloride as isotonic agents.
  • the preferred excipients include antioxidants such as ascorbic acid, for example, provided that it has not already been used to adjust the pH, vitamin A, vitamin E, tocopherols and similar vitamins and provitamins occurring in the human body.
  • Preservatives may be used to protect the formulation from contamination with pathogens. Suitable preservatives are those which are known in the art, particularly cetyl pyridinium chloride, benzalkonium chloride or benzoic acid or benzoates such as sodium benzoate in the concentration known from the prior art.
  • the preservatives mentioned above are preferably present in
  • concentrations of up to 50 mg/100 ml, more preferably between 5 and 20 mg/100 ml.
  • Solutions and suspensions will generally be aqueous, for example prepared from water alone, e.g., sterile or pyrogen-free water, or water and a physiologically acceptable co- solvent, e.g., ethanol, propylene glycol or polyethylene glycols such as PEG 400.
  • a physiologically acceptable co- solvent e.g., ethanol, propylene glycol or polyethylene glycols such as PEG 400.
  • Such solutions or suspensions may additionally contain other excipients, e.g., preservatives such as benzalkonium chloride, solubilising agents/surfactants such as polysorbates, e.g., Tween 80, Span 80, and benzalkonium chloride, buffering agents, isotonicity-adjusting agents, e.g., sodium chloride, absorption enhancers and viscosity enhancers.
  • Suspensions may be e.g., benzalkonium chloride, solubilising agents/surfactants such
  • suspending agents e.g., microcrystalline cellulose and carboxymethyl cellulose sodium.
  • Solutions or suspensions are applied directly to the nasal cavity by conventional means, e.g., with a dropper, pipette or spray, in some embodiments.
  • the formulations are provided in single or multidose form in some embodiments.
  • administration is achieved by appropriate, predetermined volume of the solution or suspension.
  • spray administration is achieved, e.g., via metering atomising spray pump.
  • Inhalation administration to the respiratory tract is achieved using an aerosol formulation in which the composition is provided in a pressurized pack with a suitable propellant, such as a chlorofluorocarbon (CFC), e.g., dichlorodifluoromethane,
  • a suitable propellant such as a chlorofluorocarbon (CFC), e.g., dichlorodifluoromethane,
  • the aerosol may also contain a surfactant such as, e.g., lecithin.
  • the active composition dose is controlled by a valve.
  • the active composition is provided in the form of a dry powder, e.g., a powder mix of the composition in a suitable powder base such as, e.g., lactose, starch, starch derivatives such as, e.g., hydroxypropylmethyl cellulose and/or polyvinylpyrrolidine (PVP).
  • the powder carrier forms a gel in the nasal cavity in some embodiments.
  • the powder composition is
  • unit dose form e.g., in capsules or cartridges of, e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler, in some embodiments.
  • the active agent is typically configured to have a small particle size, e.g., approximately 5 microns or less, via micronisation techniques and the like. Sustained release formulations of the active agents are employed in some embodiments.
  • the active agents in other embodiments, is administered by oral inhalation as a free-flow powder using an aerosol inhaler.
  • compositions of the present disclosure are administered in the form of suppositories for rectal administration in some embodiments.
  • Such compositions are prepared by mixing the formulation with a suitable non-irritating excipient, which is solid at room temperature, but liquid at body temperature, and will therefore be released subsequent to supposition.
  • Compositions suitable for vaginal administration are configured as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, suitable carriers known in the art.
  • creams, ointments, jellies, solutions or suspensions, etc. containing the compositions of the present disclosure, are employed.
  • the active agents may be in the form of a solution or suspension in a suitable sterile aqueous or non-aqueous vehicle.
  • Additives for instance buffers, preservatives including bactericidal and fungicidal agents, such as phenyl mercuric acetate or nitrate, benzalkonium chloride, or chlorohexidine and thickening agents such as hypromellose may also be included.
  • compositions of the present disclosure are administered in liposome form in some embodiments.
  • liposomes are generally derived from
  • Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a composition of the present invention, stabilizers, preservatives, excipients and the like.
  • Suitable lipids in some embodiments, are phospholipids and phosphatidyl cholines, both natural and synthetic.
  • the active agents of the compositions of the present invention are one or more nucleic acid molecules in some embodiments.
  • Such molecules are provided as short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double-stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or one or more miR-1291 mimics, in some embodiments.
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • shRNA short hairpin RNA
  • pri-miRNAs primary miRNAs
  • small nucleolar RNAs a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or one or more miR-1291 mimics, in some embodiments.
  • These molecules possess efficacy against cancer, refractory cancer, metastatic cancer, a solid tumor, a circulating tumor, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastro intestinal stromal tumor (GIST), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, renal cell carcinoma, renal cancer, sarcoma, parotid cancer, gastric cancer, melanoma, NET (sinonasal) cancer, colon cancer, liver cancer, hepatocellular carcinoma, inflammatory bowel disease (IBD), and metabolic disease.
  • GIST gastro intestinal stromal tumor
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • NET parotid cancer
  • gastric cancer melanoma
  • NET inonasal cancer
  • colon cancer colon cancer
  • liver cancer hepat
  • compositions and methods of the present disclosure further include additional therapeutically active agents or therapies (second therapies), as noted herein and/or known in the art, which are typically employed for treating one or more pathological conditions in concert with the compositions comprising the active agents of the present disclosure.
  • second therapies additional therapeutically active agents or therapies
  • Such second agents include, but are not limited to, chemotherapy, radiotherapy, ablation therapy, hormone therapy, gene therapy, RNA therapy, epigenetic drug therapy, resection therapy, immunotherapy, anti-angiogenic therapy, stromal inhibitor therapy, extracellular matrix protein inhibitor therapy, doxorubicin, mitoxantrone, tamoxifen, imatinib mesylate, BAY43-9006, gemcitabine, prostaglandins, retinoic acids, brostallicin, lenalidomide, thalidomide, docetaxel, erlotinib, vatalinib, VEGF-trap, fenretidine,
  • bortezomib bevacizumab, pertuzumab, rituximab, gefitinib, a general monoclonal antibody, where the second agent is administered separately, simultaneously, or sequentially, with the composition.
  • the second therapy is an agent other than the nucleic acid molecules of the present invention, e.g., SEQ ID NOs: 1-5, which nevertheless therapeutically regulates levels of one or more of miR-1291; A T2; Cyclin Bl; MeCP2; FOXA2; AMPKal; Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASF1; Ornithine aminotransferase (OAT); Keratin, type II cytoskeletal 8 (KRT8); Phosphoenolpyruvate carboxykinase 2 (PEPCK2); Enoyl-co enzyme A (CoA) hydratase ( ECHS1); Phosphoserine aminotransferase isoform 1 (PSAT1); Dihydrolipoamide acetyltransferase (DLAT); Peroxiredoxin 3, isoform CRA
  • Triosephosphate isomerase isoform 2 (TPI1); Peroxiredoxin-4 (PRDX4); and Isocitrate dehydrogenase [NAD] subunit beta (IDH3B); a-fetoprotein (AFP); AFP-L3%, des-gamma- carboxyprothrombin (DCP); CDH1 (E-cadherin); trimethylated lysine 27 of H3 histone (H3K27me3); histone deacetylase -1; histone deacetylase -2; SIRT1; CD44; aldehyde dehydrogenase; KRAS2; RREBl; PTEN, and 3,4-difluoro-benzo-curcumin (CDF), in suitable embodiments.
  • TPI1 Triosephosphate isomerase isoform 2
  • PRDX4 Peroxiredoxin-4
  • AFP a-fetoprotein
  • combination therapies include, but are not limited to, compositions having one or more formulations of miRNA or the complimentary sequence thereof, selected from, but not limited to miR-lOb, miR-21, miR-29b, miR-17-5p, miR-125b, miR-145, miR-146, miR-155, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92, let-7 family, miR-lOa, miR-20a, miR-24, miR-31, miR-96, miR-133b, miR-135b, miR-143, miR-145, miR-183, miR-19b, miR-18, miR-125a, miR-195, miR-199a, miR-200a, miR-224, miR-34a, miR-100, miR-101, miR-103, miR-107, miR-125b, miR-148b
  • Still other combination therapies include, but are not limited to, prostanoids, endothelin antagonists, cytoplasmic kinase inhibitors, receptor kinase inhibitors, endothelin receptor antagonists, e.g., ambrisentan, bosentan, and sitaxsentan, PDE5 (PDE-V) inhibitors, e.g., sildenafil, tadalafil, and vardenafil, calcium channel blockers, e.g., amlodipine, felodipine, varepamil, diltiazem, and menthol, prostacyclin, treprostinil, iloprost, beraprost, nitric oxide, oxygen, heparin, warfarin, diuretics, digoxin, cyclosporins, e.g., cyclosporin A, CTLA4-Ig, antibodies such as ICAM-3,
  • camptothecin camptothecin, cytarabine, gemcitabine, fluorodeoxyuridine, melphalan and
  • cyclophosphamide antimetabolites such as methotrexate, topoisomerase inhibitors such as camptothecin, DNA alkylators such as cisplatin, kinase inhibitors such as sorafenib, microtubule poisons such as paclitaxel, TNF- a inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor, hydroxy urea and rapamycin (sirolimus or Rapamune) or derivatives thereof.
  • compositions of the invention may also be prepared as salts which are pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure at least to the extent that such salts are useful as intermediates in the preparation of pharmaceutically acceptable salts.
  • salts include, but are not limited to, sulfates, phosphates, mesylates, bismesylates, tosylates, lactates, tartrates, malates, bis-acetates, citrates, bishydrochloride salts, salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic,
  • Salts of amine groups may also comprise quaternary ammonium salts in which the amino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety.
  • the salts may be formed by conventional means, such as by reacting the free base form with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying or by exchanging the anions of an existing salt for another anion on a suitable ion exchange resin.
  • the salt is a sulfate, phosphate, mesylate, bismesylate, tosylate, lactate, tartrate, malate, bis- acetate, citrate, or bishydrochloride salt.
  • compositions of the present disclosure are formulated as prodrugs.
  • composition having free amino, amido, hydroxy or carboxylic acid groups can be converted into prodrugs.
  • Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy and carboxylic acid groups of compositions of the invention.
  • the amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4- hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta- alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone.
  • Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of
  • compositions of the present invention through the carbonyl carbon prodrug sidechain.
  • Prodrugs also include phosphate derivatives of compounds (such as acids, salts of acids, or esters) joined through a phosphorus-oxygen bond to a free hydroxyl of present compositions.
  • Prodrugs may also include N-oxides, and S-oxides of the appropriate nitrogen and sulfur atoms in the compositions of the present invention.
  • the miR A molecules, agents, drugs, prodrugs, and the like, of the present invention contain one or more chemical modifications in some embodiment. There is no requirement that chemical modifications are of the same type or in the same location on each of the miPvNAs.
  • each of the sense and antisense strands of an miRNA may contain a mixture of modified and unmodified nucleotides. Modifications may be made for any suitable purpose including, for example, to increase RNAi activity, increase the in vivo stability of the molecules ⁇ e.g., when present in the blood), and/or to increase bioavailability.
  • Suitable modifications include, for example, internucleotide linkages, internucleoside linkages, dideoxyribonucleotides, 2 '-sugar modification, 2 '-amino groups, 2'- fluoro groups, 2'-methoxy groups, 2'-alkoxy groups, 2'- alkyl groups, 2'- deoxyribonucleotides, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, universal base nucleotides, acyclic nucleotides, 5-C-methyl nucleotides, biotin groups, terminal glyceryl incorporation, inverted deoxy abasic residue incorporation, sterically hindered molecules, 3 '-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2'
  • Chemical modifications also include terminal modifications on the 5' and/or 3' part of the oligonucleotides and are also known as capping moieties. Such terminal modifications are selected from a nucleotide, a modified nucleotide, a lipid, a peptide, and a sugar.
  • L-nucleotides may further include at least one sugar or base modification and/or a backbone modification as described herein.
  • Nucleic acid molecules disclosed herein may be administered with a carrier or diluent or with a delivery vehicle which facilitate entry to the cell.
  • Suitable delivery vehicles include, for example, viral vectors, viral particles, liposome formulations, and lipofectin.
  • the nucleic acid molecule is present in a biological vector that is a plasmid vector or viral vector.
  • the nucleic acid molecule is present in a delivery vehicle, where the delivery vehicle is selected from a polymeric carrier, a micelle, a liposome, a lipoplex, a polyplex, a peptide, a polymer, a dendrimer, lipids, or a nanoparticle.
  • the delivery vehicle is selected from a polymeric carrier, a micelle, a liposome, a lipoplex, a polyplex, a peptide, a polymer, a dendrimer, lipids, or a nanoparticle.
  • Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins ⁇ see e.g., Gonzalez et al, Bioconjugate Chem., 10: 1068-1074 (1999); WO 03/47518; and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres ⁇ see for example U.S. 6,447,796 and U.S.
  • PLGA poly(lactic-co-glycolic)acid
  • PLCA microspheres see for example U.S. 6,447,796 and U.S.
  • nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Corny et al, Clin. Cancer Res., 5: 2330-2337 (1999) and WO 99/31262.
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • Nucleic acid molecules may be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
  • the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. Delivery systems include surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).
  • Formulations of the nucleic acid molecules may be complexed with
  • polyethylenimine ⁇ e.g., linear or branched PEI
  • polyethylenimine derivatives including for example polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI- PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG- triGAL) derivatives, grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof ⁇ see, for example Ogris et al, 2001, AAPA PharmSci, 3, 1-11; Furgeson et al, 2003, Bioconjugate Chem., 14, 840-847; Kunath et al, 2002, Pharmaceutical Research, 19, 810-817; Choi et al., 2001, Bull.
  • PEI- PEG-GAL polyethyleneimine-polyethylenegly
  • Delivery systems may include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers ⁇ e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers ⁇ e.g., polycarbophil and polyvinylpyrolidone).
  • the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.
  • liposomes which can be used in this invention include the following: (1) CellFectin, 1 : 1.5 (M/M) liposome formulation of the cationic lipid ⁇ , ⁇ , ⁇ , ⁇ - tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2: 1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[l-(2,3-dioleoyloxy)-N,N,N-tri-methyl- ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3 : 1 (M/M) liposome formulation of the polycationic lipid DOSPA, the neutral lipid DOPE (GIBCO BRL) and Di- Alkylated Amino Acid (DiLA2).
  • nucleic acid molecules may be expressed from transcription units inserted into DNA or RNA vectors.
  • Recombinant vectors can be DNA plasmids or viral vectors.
  • Nucleic acid molecule expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors are capable of expressing the nucleic acid molecules either permanently or transiently in target cells. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous, subcutaneous, or intramuscular administration.
  • Expression vectors may include a nucleic acid sequence encoding at least one nucleic acid molecule disclosed herein, in a manner which allows expression of the nucleic acid molecule.
  • the vector may contain sequence(s) encoding both strands of a nucleic acid molecule that include a duplex.
  • the vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a nucleic acid molecule.
  • An expression vector may encode one or both strands of a nucleic acid duplex, or a single self-complementary strand that self hybridizes into a nucleic acid duplex.
  • the nucleic acid sequences encoding nucleic acid molecules can be operably linked to a transcriptional regulatory element that results expression of the nucleic acid molecule in the target cell.
  • Transcriptional regulatory elements may include one or more transcription initiation regions (e.g., eukaryotic pol I, II or III initiation region) and/or transcription termination regions (e.g., eukaryotic pol I, II or III termination region).
  • the vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5 ' side or the 3 '-side of the sequence encoding the nucleic acid molecule; and/or an intron
  • the nucleic acid molecules or the vector construct can be introduced into the cell using suitable formulations.
  • suitable formulations are with a lipid formulation such as in LipofectamineTM 2000 (Invitrogen, CA, USA), vitamin A coupled liposomes (Sato et al. Nat Biotechnol 2008; 26:431-442, PCT Patent Publication No. WO 2006/068232).
  • Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art.
  • the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable.
  • compositions for administering oligonucleotides are known and can be used.
  • the direct injection of dsRNA duplexes may also be done.
  • Suitable methods of introducing dsRNA are provided, for example, in U.S. 2004/0203145 and U.S. 20070265220.
  • Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate.
  • the polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and
  • Nucleic acid moles may be formulated as a microemulsion.
  • a microemulsion is a system of water, oil and amphiphile which is a single optically isotropic and
  • microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a 4th component, generally an intermediate chain-length alcohol to form a transparent system.
  • surfactants that may be used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers,
  • polyglycerol fatty acid esters tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),
  • decaglycerol monocaprate MCA750
  • decaglycerol monooleate MO750
  • decaglycerol sequioleate SO750
  • decaglycerol decaoleate DA0750
  • cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • the present invention provides methods of treating or preventing a disease or condition mediated by miR-1291 in a subject in need thereof by administering to the subject a composition including a nucleic acid molecule or a complement or precursor thereof, under conditions effective to treat or prevent the disease or condition mediated by miR-1291 in the subject, where the nucleic acid molecule is composed of one or more of (i) a sequence encoding miR-1291, (ii) a sequence selected from the group consisting of SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, (iii) a sequence with at least 90% similarity to one or more of SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ.
  • the disease or condition is mediated by a decrease in miR-1291 expression compared to miR-1291 expression in the absence of the disease or condition.
  • the decrease is at least a 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease.
  • the decrease is at least a 40% decrease.
  • the decrease in miR-1291 expression alters the levels of one or more of: AKT2; Cyclin Bl; MeCP2; FOXA2; AMPKal; Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASFl; Ornithine aminotransferase (OAT); Keratin, type II cytoskeletal 8 (KRT8); Phosphoenolpyruvate carboxykinase 2 (PEPCK2); Enoyl-coenzyme A (CoA) hydratase (ECHS1); Phosphoserine aminotransferase isoform 1 (PSAT1); Dihydrolipoamide acetyltransferase (DLAT); Peroxiredoxin 3, isoform CRA a (PRDX3); Cysteine-rich protein 2 (CRIP2); Chain C, human PCNA; Fa
  • the disease or condition mediated by, or associated with, one or more miRNA nucleic acids of the present invention includes, but is not limited to, cancer, refractory cancer, metastatic cancer, a solid tumor, a circulating tumor, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastro intestinal stromal tumor (GIST), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, renal cell carcinoma, renal cancer, sarcoma, parotid cancer, gastric cancer, melanoma, NET (sinonasal) cancer, colon cancer, liver cancer, hepatocellular carcinoma, inflammatory bowel disease (IBD), and metabolic disease.
  • cancer refractory cancer
  • metastatic cancer e.g., metastatic cancer
  • a solid tumor e.g., circulating tumor
  • pancreatic cancer lung cancer
  • breast cancer breast cancer
  • the disease or condition mediated by, or associated with, one or more miRNA nucleic acids of the present invention includes, but is not limited to, systemic sclerosis, mixed connective tissue disease, cancer, refractory cancer, metastatic cancer, neoplasia, hypoplasia, hyperplasia, dysplasia, metaplasia, prosoplasia, desmoplasia, angiogenic disease, pulmonary function disorders, cardiovascular function disorders, HIV infection, hepatitis, portal hypertension, pulmonary hypertension, congenital heart disease, hypoxia, chronic hemolytic anemia, newborn persistent pulmonary hypertension, pulmonary veno-occlusive disease (PVOD), pulmonary capillary
  • PCH hemangiomatosis
  • left heart disease pulmonary hypertension pulmonary hypertension
  • systolic dysfunction diastolic dysfunction
  • valvular disease lung disease
  • interstitial lung disease pulmonary fibrosis
  • schistosomiasis chronic obstructive pulmonary disease (COPD)
  • COPD chronic obstructive pulmonary disease
  • CTEPH chronic thromboembolic pulmonary hypertension
  • pulmonary hypertension with unclear multifactorial mechanisms hematologic disorders, myeloproliferative disorders, splenectomy, systemic disorders, sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleimoyomatosis, neurofibromatosis, vasculitis, metabolic disorders, glycogen storage disease, Gaucher disease, thyroid disorders, tumoral obstruction, fibrosing mediastinitis, and chronic renal failure on dialysis; and diseases such as pulmonary hypertension, congenital heart disease, hypoxia, chronic hemolytic anemia, newborn persistent pulmonary hypertension, pulmonary veno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH), left heart disease pulmonary hypertension, systolic dysfunction, diastolic dysfunction, valvular disease, lung disease, interstitial lung disease, pulmonary fibrosis, schistosomiasis
  • the present invention further contemplate the prevention or treatment of subjects afflicted with idiopathic or primary pulmonary hypertension, familial hypertension, pulmonary hypertension secondary to, but not limited to, connective tissue disease, congenital heart defects (shunts), pulmonary fibrosis, portal hypertension, HIV infection, sickle cell disease, drugs and toxins, e.g., anorexigens, cocaine, chronic hypoxia, chronic pulmonary obstructive disease, sleep apnea, and schistosomiasis, pulmonary hypertension associated with significant venous or capillary involvement (pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis), secondary pulmonary hypertension that is out of proportion to the degree of left ventricular dysfunction, and/or persistent pulmonary hypertension in newborn babies, especially in subjects that previously failed prior PAH therapy.
  • shunts congenital heart defects
  • pulmonary fibrosis portal hypertension
  • HIV infection sickle cell disease
  • drugs and toxins e.g.,
  • the present disclosure provides a composition for treating one or more diseases associated with hyperproliferation, neoplasia, hypoplasia, hyperplasia, dysplasia, metaplasia, prosoplasia, desmoplasia, angiogenesis, inflammation, pulmonary function, and cardiovascular function.
  • Hyperproliferative diseases include, but are not limited to, cancer and diseases involving neo- and myeloproliferative disease states such as cellular-proliferative disease states, including, but not limited to, cardiac sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma, pulmonary bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hanlartoma, mesothelioma, gastrointestinal (GI) diseases effecting the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lympho
  • fibrosarcoma malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfrorna (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors, nervous system diseases effecting the skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges
  • meningioma meningiosarcoma, gliomatosis
  • brain astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors
  • spinal cord neurofibroma meningioma, glioma, sarcoma
  • gynecological conditions effecting the uterus endometrial carcinoma
  • cervix cervical carcinoma, pre-tumor cervical dysplasia
  • ovaries ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma),
  • rhabdomyosarcoma fallopian tubes (carcinoma), hematologic diseases such as those associated with the blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, myelodysplasia syndrome), Hodgkin's disease, non- Hodgkin's lymphoma [malignant lymphomaj, CJI, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; Adrenal glands: neuroblastoma, and myleoproliferative diseases such as polycythemia rubra vera, primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), agnoneic myeloid metap
  • immunological and inflammatory diseases include, but are not limited to, an immunological, inflammatory or autoimmune disease, including, but not limited to rheumatoid arthritis, polyarthritis, rheumatoid spondylitis, osteoarthritis, gout, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, cystic fibrosis,
  • an immunological, inflammatory or autoimmune disease including, but not limited to rheumatoid arthritis, polyarthritis, rheumatoid spondylitis, osteoarthritis, gout, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, cystic fibrosis,
  • inflammatory bowel disease irritable bowel syndrome, mucous colitis, ulcerative colitis, diabrotic colitis, Crohn's disease, autoimmune thyroid disorders, gastritis, esophagitis, hepatitis, pancreatitis, nephritis, psoriasis, eczema, acne vulgaris, dermatitis, hives, multiple sclerosis, Alzheimer's disease, Lou Gehrig's disease, Paget's disease, sepsis, conjunctivitis, neranl catarrh, chronic arthrorheumatism, systemic inflammatory response syndrome (SIRS), polymyositis, dermatomyositis (DM), Polaritis nodoa (PN), mixed connective tissue disorder (MCTD), Sjoegren's syndrome, Crouzon syndrome, achondroplasia, systemic lupus
  • erythematosus erythematosus, scleroderma, vasculitis, thanatophoric dysplasia, insulin resistance, Type I diabetes and complications from diabetes and metabolic syndrome.
  • compositions and methods of the present disclosure are employed for preventing or treating metabolic diseases, such as, e.g., atherosclerosis, metabolic syndrome, obesity, hepatic steatosis, high cholesterol, high plasma lipids, insulin resistance, type 2 diabetes, hypoglycemia, diabetic ketoacidosis, nonketotic hyperosmolar syndrome, acid- base imbalance, metabolic acidosis, metabolic alkalosis, amyloidosis, calcium metabolism disorders, iron metabolism disorders, malabsorption disorders, phosphorus metabolism disorders, porphyries, metabolic skin diseases, wasting syndrome, water-electrolyte imbalance, lipid metabolism disorders, metabolic syndrome X,
  • metabolic diseases such as, e.g., atherosclerosis, metabolic syndrome, obesity, hepatic steatosis, high cholesterol, high plasma lipids, insulin resistance, type 2 diabetes, hypoglycemia, diabetic ketoacidosis, nonketotic hyperosmolar syndrome, acid- base imbalance, metabolic acidosis, metabolic alkalosis,
  • compositions, and methods of the present disclosure are employed in some embodiments for preventing or treating vascular diseases, including, but not limited to, cardiovascular diseases, hypertension, hypertrophy, hypercholesterolemia, hyperlipidemia, thrombotic disorders, stroke, Raynaud's phenomenon, POEMS syndrome, angina, ischemia, migraine, peripheral arterial disease, heart failure, restenosis, atherosclerosis, left ventricular hypertrophy, myocardial infarction, ischemic diseases of heart, kidney, liver and brain, and PAH.
  • cardiovascular diseases including, but not limited to, cardiovascular diseases, hypertension, hypertrophy, hypercholesterolemia, hyperlipidemia, thrombotic disorders, stroke, Raynaud's phenomenon, POEMS syndrome, angina, ischemia, migraine, peripheral arterial disease, heart failure, restenosis, atherosclerosis, left ventricular hypertrophy, myocardial infarction, ischemic diseases of heart, kidney, liver and brain, and PAH.
  • the methods of the present disclosure further include additional therapeutically active agents or therapies (second therapies), as noted herein and/or known in the art, which are typically employed for treating one or more pathological conditions in concert with the compositions comprising the active agents of the present disclosure.
  • second agents include, but are not limited to chemotherapy, radiotherapy, ablation therapy, hormone therapy, gene therapy, RNA therapy, epigenetic drug therapy, resection therapy,
  • immunotherapy anti-angiogenic therapy, stromal inhibitor therapy, extracellular matrix protein inhibitor therapy, doxorubicin, mitoxantrone, tamoxifen, imatinib mesylate, BAY43- 9006, gemcitabine, prostaglandins, retinoic acids, brostallicin, lenalidomide, thalidomide, docetaxel, erlotinib, vatalinib, VEGF-trap, fenretidine, bortezomib, bevacizumab,
  • pertuzumab pertuzumab, rituximab, gefitinib, a general monoclonal antibody, where the second agent is administered separately, simultaneously, or sequentially, with the composition.
  • the second therapy is an agent other than the nucleic acid molecules of the present invention, e.g., SEQ ID NOs: 1-5, which nevertheless
  • Triosephosphate isomerase isoform 2 (TPI1); Peroxiredoxin-4 (PRDX4); and Isocitrate dehydrogenase [NAD] subunit beta (IDH3B); a-fetoprotein (AFP); AFP-L3%, des-gamma- carboxyprothrombin (DCP); CDH1 (E-cadherin); trimethylated lysine 27 of H3 histone (H3K27me3); histone deacetylase -1; histone deacetylase -2; SIRT1; CD44; aldehyde dehydrogenase; KRAS2; RREBl; PTEN, and 3,4-difluoro-benzo-curcumin (CDF), in suitable embodiments.
  • TPI1 Triosephosphate isomerase isoform 2
  • PRDX4 Peroxiredoxin-4
  • AFP a-fetoprotein
  • combination therapies include, but are not limited to, compositions having one or more formulations of miRNA or the complimentary sequence thereof, selected from, but not limited to miR-lOb, miR-21, miR-29b, miR-17-5p, miR-125b, miR-145, miR-146, miR-155, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92, let-7 family, miR-lOa, miR-20a, miR-24, miR-31, miR-96, miR-133b, miR-135b, miR-143, miR-145, miR-183, miR-19b, miR-18, miR-125a, miR-195, miR-199a, miR-200a, miR-224, miR-34a, miR-100, miR-101, miR-103, miR-107, miR-125b, miR-148b
  • Still other combination therapies include, but are not limited to, of prostanoids, endothelin antagonists, cytoplasmic kinase inhibitors, receptor kinase inhibitors, endothelin receptor antagonists, e.g., ambrisentan, bosentan, and sitaxsentan, PDE5 (PDE-V) inhibitors, e.g., sildenafil, tadalafil, and vardenafil, calcium channel blockers, e.g., amlodipine, felodipine, varepamil, diltiazem, and menthol, prostacyclin, treprostinil, iloprost, beraprost, nitric oxide, oxygen, heparin, warfarin, diuretics, digoxin, cyclosporins, e.g., cyclosporin A, CTLA4-Ig, antibodies such as ICAM-3
  • camptothecin camptothecin, cytarabine, gemcitabine, fluorodeoxyuridine, melphalan and
  • cyclophosphamide antimetabolites such as methotrexate, topoisomerase inhibitors such as camptothecin, DNA alkylators such as cisplatin, kinase inhibitors such as sorafenib, microtubule poisons such as paclitaxel, TNF- a inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor, hydroxy urea and rapamycin (sirolimus or Rapamune) or derivatives thereof.
  • the present invention provides methods of altering the expression level or activity of miR-1291 or a precursor or target thereof, by providing an agent comprising a nucleic acid molecule or a complement or precursor thereof, under conditions effective to increase the expression level or activity of the miR-1291 or a precursor or a target thereof, compared to a reference level or activity, wherein the nucleic acid molecule comprises one or more of (i) a sequence encoding miR-1291, (ii) a sequence selected from SEQ. ID. NO: 1, SEQ. ID. NO: 3, and SEQ. ID. NO: 4, (iii) a sequence with at least 90% similarity to one or more of SEQ. ID. NO: 1, SEQ. ID.
  • the altering modulates one or more of drug resistance, invasiveness, adherence, and metastasis in a subject and/or target cells from the subject selected from one or more cancerous cells, hyperproliferative cells, neoplastic cells, hypoplastic cells, hyperplastic cells, dysplasia cells, metaplasia cells, prosoplasia cells, desmoplasia cells, angiogenic cells, inflammatory cells, immunological cells, metabolic cells, pulmonary cells, and cardiovascular cells.
  • the providing is an administering to a subject in need thereof, where the subject is a patient afflicted with cancer, refractory cancer, metastatic cancer, a solid tumor, a circulating tumor, pancreatic cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastro intestinal stromal tumor (GIST), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, renal cell carcinoma, renal cancer, sarcoma, parotid cancer, gastric cancer, melanoma, NET (sinonasal) cancer, colon cancer, liver cancer, hepatocellular carcinoma, inflammatory bowel disease (IBD), and metabolic disease; and/or wherein the subject is a patient afflicted with a disease or condition associated with aberrant expression of one or more of: miR-1291; AKT2; Cyclin Bl; MeCP
  • acetyltransferase DLAT
  • Peroxiredoxin 3, isoform CRA a PRDX3
  • Cysteine-rich protein 2 CRIP2
  • Chain C human PCNA
  • Fascin homolog 1, actin-bundling protein isoform CRA a (FSCN1)
  • Serpin HI precursor Protein disulfide-isomerase precursor
  • Chain A disulfide isomerase related chaperone ERP29
  • Isocitrate dehydrogenase [NAD] subunit beta IDH3B
  • AFP a- fetoprotein
  • AFP-L3% des-gamma-carboxyprothrombin
  • DCP des-gamma-carboxyprothrombin
  • CDH1 E-cadherin
  • trimethylated lysine 27 of H3 histone H3K27me3
  • the providing may be an administering to a subject in need thereof, where the subject is a patient afflicted with a disease or condition associated with, mediated by, or related to levels of one or more of miR-1291; AKT2; Cyclin Bl; MeCP2; FOXA2;
  • AMPKal Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASFl; Ornithine aminotransferase (OAT); Keratin, type II cytoskeletal 8 (KRT8); Phosphoenolpyruvate carboxykinase 2 (PEPCK2); Enoyl- coenzyme A (CoA) hydratase ( ECHS1); Phosphoserine aminotransferase isoform 1
  • PSAT1 Dihydrolipoamide acetyltransferase
  • DLAT Dihydrolipoamide acetyltransferase
  • Peroxiredoxin 3 isoform CRA a (PRDX3)
  • Cysteine-rich protein 2 CRIP2
  • Chain C human PCNA
  • Fascin homolog 1, actin-bundling protein isoform CRA a (FSCN1)
  • Serpin HI precursor Protein disulfide- isomerase precursor
  • Chain A disulfide isomerase related chaperone ERP29;
  • Triosephosphate isomerase isoform 2 (TPIl); Peroxiredoxin-4 (PRDX4); and Isocitrate dehydrogenase [NAD] subunit beta (IDH3B); a-fetoprotein (AFP); AFP-L3%, des-gamma- carboxyprothrombin (DCP); CDH1 (E-cadherin); trimethylated lysine 27 of H3 histone (H3K27me3); histone deacetylase -1; histone deacetylase -2; SIRT1; CD44; aldehyde dehydrogenase; KRAS2; RREB1; PTEN, and 3,4-difluoro-benzo-curcumin (CDF), in suitable embodiments.
  • TPIl Triosephosphate isomerase isoform 2
  • PRDX4 Peroxiredoxin-4
  • AFP a-fetoprotein
  • the providing may be an administering to a subject in need thereof, where the subject is a patient afflicted with a disease or condition associated with, mediated by, or related to levels of one or more of miR-lOb, miR-21, miR-29b, miR-17-5p, miR-125b, miR- 145, miR-146, miR-155, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92, let-7 family, miR-lOa, miR-20a, miR-24, miR-31, miR-96, miR-133b, miR-135b, miR-143, miR- 145, miR-183, miR-19b, miR-18, miR-125a, miR-195, miR-199a, miR-200a, miR-224, miR- 34a, miR-100, miR-101, miR-103, miR-107, miR-125b
  • the agent is a synthetic agent provided as a miR-1291 mimic, short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double- stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or any combination thereof, where the synthetic agent is a synthetic agent provided as a miR-1291 mimic, short interfering RNA (siRNA), a RNA interference (RNAi) molecule, double- stranded RNA (dsRNA), short hairpin RNA (shRNA), primary miRNAs (pri-miRNAs), small nucleolar RNAs (snoRNAs), a molecule capable of sequence-specific post-transcriptional gene silencing of miRNA, or any combination thereof, where the synthetic agent is a synthetic agent provided as a miR-1291 mimic, short
  • Triosephosphate isomerase isoform 2 (TPI1); Peroxiredoxin-4 (PRDX4); and Isocitrate dehydrogenase [NAD] subunit beta (IDH3B); a-fetoprotein (AFP); AFP-L3%, des-gamma- carboxyprothrombin (DCP); CDH1 (E-cadherin); trimethylated lysine 27 of H3 histone (H3K27me3); histone deacetylase -1; histone deacetylase -2; SIRT1; CD44; aldehyde dehydrogenase; KRAS2; and RREBl .
  • the synthetic agent includes one or more chemical modifications in some embodiments, where the modification is selected from internucleotide linkages,
  • internucleoside linkages dideoxyribonucleotides, 2 '-sugar modification, 2 '-amino groups, 2'- fluoro groups, 2'-methoxy groups, 2'-alkoxy groups, 2'- alkyl groups, 2'- deoxyribonucleotides, 2'-0-methyl ribonucleotides, 2 '-deoxy-2' -fluoro ribonucleotides, universal base nucleotides, acyclic nucleotides, 5-C-methyl nucleotides, biotin groups, terminal glyceryl incorporation, inverted deoxy abasic residue incorporation, sterically hindered molecules, 3 '-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2'
  • the synthetic agent comprises one or more of a nucleic acid molecule encoding a complement of miR-1291, a nucleic acid sequence possessing at least 90% similarity to SEQ. ID. NO: 5, and a nucleic acid sequence with at least 7 contiguous nucleotides from SEQ. ID. NO: 5.
  • the synthetic agent decreases ABC transporter activity in a target cell compared to a control cell, and wherein the ABC transporter is selected from the group consisting of ABCCl, ABCG2, ABCBl, ABCC2, ABCC3, and ABCC4.
  • compositions of the present invention are administered in a therapeutically effective amount.
  • Such an administration imparts a response associated with, e.g., cells, tissues, fluids, of a subject being sought by the clinician.
  • a response associated with e.g., cells, tissues, fluids, of a subject being sought by the clinician.
  • appropriate dosage level is administered. In some embodiments, from about 0.01 to
  • dosage levels are from about 0.1 to about 250 mg/kg per day in some embodiments, while in other embodiments from about 0.5 to about 100 mg/kg per day is administered to the subject. Suitable dosage levels include, for example, from about 0.01 to 250 mg/kg per day, from about 0.05 to 100 mg/kg per day, or from about 0.1 to 50 mg/kg per day. Within this range, in some embodiments, the dosage is from about 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. [0246]
  • the compositions are provided in the form of tablets containing 1.0 to lOOOmg of the active ingredient, including, but not limited to, 1, 5, 10, 15.
  • compositions of the present disclosure are administered by inhalation as described in, e.g., US 8257741, US 8263128, WO
  • compositions of the present disclosure are administered from 1 to 5 times daily.
  • Methods of treating, preventing, lessening the risk, decreasing the severity, and/or alleviating the symptoms of a disease or condition as described herein include, but are not limited to, the following exemplary therapeutic modalities and indications for the
  • compositions of the present invention such as, for example, RNAi.
  • R A interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering R As (siR As) (Zamore et al, 2000, Cell, 101, 25-33; Fire et al, 1998, Nature, 391, 806; Hamilton et al, 1999, Science, 286, 950- 951; Lin et al, 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13 : 139-141; and Strauss, 1999, Science, 286, 886).
  • SiR As short interfering R As
  • Post-transcriptional gene silencing is believed to be an evolutionarily-conserved cellular mechanism for preventing expression of foreign genes that may be introduced into the host cell (Fire et al, 1999, Trends Genet., 15, 358).
  • Post- transcriptional gene silencing may be an evolutionary response to the production of double- stranded RNAs (dsRNAs) resulting from viral infection or from the random integration of transposable elements (transposons) into a host genome.
  • dsRNAs double- stranded RNAs
  • transposons transposable elements
  • RNAi response that appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2',5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L ⁇ see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al, 1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et al, 2001, Curr. Med.
  • Dicer processes long dsRNA into double-stranded short interfering R As (siRNAs) which are typically about 21 to about 23 nucleotides in length and include about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Elbashir et al, 2001, Genes Dev., 15, 188).
  • siRNAs double-stranded short interfering R As
  • RNA-induced silencing complex Single-stranded RNA, including the sense strand of siRNA, trigger an RNAi response mediated by an endonuclease complex known as an RNA-induced silencing complex (RISC).
  • RISC mediates cleavage of this single-stranded RNA in the middle of the siRNA duplex region ⁇ i.e., the region complementary to the antisense strand of the siRNA duplex) (Elbashir et al, 2001, Genes Dev., 15, 188).
  • the siRNAs may be a substrate for the cytoplasmic Dicer enzyme ⁇ i.e., a "Dicer substrate") which is characterized as a double stranded nucleic acid capable of being processed in vivo by Dicer to produce an active nucleic acid molecules.
  • Dicer substrate a substrate for the cytoplasmic Dicer enzyme
  • the activity of Dicer and requirements for Dicer substrates are described, for example, U.S. 2005/0244858. Briefly, it has been found that dsRNA, having about 25 to about 30 nucleotides, effective result in a down-regulation of gene expression.
  • Dicer cleaves the longer double stranded nucleic acid into shorter segments and facilitates the incorporation of the single-stranded cleavage products into the RNA-induced silencing complex (RISC complex).
  • RISC complex RNA-induced silencing complex
  • the active RISC complex, containing a single-stranded nucleic acid cleaves the cytoplasmic RNA having complementary sequences.
  • Dicer substrates must conform to certain general requirements in order to be processed by Dicer.
  • the Dicer substrates must of a sufficient length (about 25 to about 30 nucleotides) to produce an active nucleic acid molecule and may further include one or more of the following properties: (i) the dsRNA is asymmetric, e.g., has a 3' overhang on the first strand (antisense strand) and (ii) the dsRNA has a modified 3' end on the antisense strand (sense strand) to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA.
  • the Dicer substrates may be symmetric or asymmetric.
  • Dicer substrates may have a sense strand includes 22-28 nucleotides and the antisense strand may include 24-30 nucleotides, resulting in duplex regions of about 25 to about 30 nucleotides, optionally having 3'-overhangs of 1-3 nucleotides.
  • Dicer substrates may have any modifications to the nucleotide base, sugar or phosphate backbone as known in the art and/or as described herein for other nucleic acid molecules (such as siR A molecules).
  • the R Ai pathway may be induced in mammalian and other cells by the introduction of synthetic siRNAs that are ⁇ 20 nucleotides in length (Elbashir et al, 2001, Nature, 411, 494 and Tuschl et al, WO 01/75164; incorporated by reference in their entirety).
  • RNAi RNAi-dependent requirements necessary to induce the down-regulation of gene expression by RNAi are described in Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Kreutzer et al, WO 00/44895; Zernicka-Goetz et al, WO 01/36646; Fire, WO 99/32619; Plaetinck et al, WO 00/01846; Mello and Fire, WO 01/29058;
  • an siRNA nucleic acid molecule can be assembled from two separate polynucleotide strands (a sense strand and an antisense strand) that are at least partially complementary and capable of forming stable duplexes.
  • the length of the duplex region may vary from about 15 to about 49 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides).
  • the antisense strand includes nucleotide sequence that is
  • an siRNA is "RISC length" and/or may be a substrate for the Dicer enzyme.
  • an siRNA nucleic acid molecule may be assembled from a single polynucleotide, where the sense and antisense regions of the nucleic acid molecules are linked such that the antisense region and sense region fold to form a duplex region (i.e., forming a hairpin structure).
  • siRNAs may be blunt-ended on both sides, have overhangs on both sides or a combination of blunt and overhang ends. Overhangs may occur on either the 5'- or 3 '- end of the sense or antisense strand. Overhangs typically consist of 1-8 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides each) and are not necessarily the same length on the 5'- and 3 '- end of the siRNA duplex. The nucleotide(s) forming the overhang need not be of the same character as those of the duplex region and may include deoxyribonucleotide(s),
  • the 5 '- and/or 3 '-end of one or both strands of the nucleic acid may include a free hydroxyl group or may contain a chemical modification to improve stability.
  • end modifications e.g., terminal caps
  • end modifications include, but are not limited to, abasic, deoxy abasic, inverted (deoxy) abasic, glyceryl, dinucleotide, acyclic nucleotide, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, CI to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02CH3; ON02; N02, N3; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;
  • polyalkylamino or substituted silyl as, among others, described in European patents EP 586,520 and EP 618,925.
  • association between a pathological state (e.g., pancreatic cancer) and the aberration of the level of one or more miRNAs can be readily determined by comparative analysis in a normal population and an abnormal or affected population.
  • a pathological state e.g., pancreatic cancer
  • association between a pathological state and the aberration of the level of one or more miRNAs can be readily determined by comparative analysis in a normal population and an abnormal or affected population.
  • a pathological state e.g., pancreatic cancer
  • association between a pathological state e.g., pancreatic cancer
  • association between a pathological state e.g., pancreatic cancer
  • the aberration of the level of one or more miRNAs can be readily determined by comparative analysis in a normal population and an abnormal or affected population.
  • the study results can be compared and analyzed by statistical means. Any detected statistically significant difference in the two populations would indicate an association. For example, if the level of miRNAs is statistically significantly lower in the affected population than
  • the methods can be applied to quantify the relative expression (i.e. up-regulation or down-regulation) of certain target miRNA sequences in biological samples.
  • Embodiments of the invention include methods for diagnosing and/or assessing a condition or potential condition in a patient comprising determining the amount of a target miRNA sequence and the amount of a reference sequence in a sample from a patient, for example.
  • the difference in the miRNA in the sample from a patient and the miRNA in a reference sample is indicative of a pathology, prognosis, disease, or condition, or risk thereof, for example.
  • the invention may also be applied in methods to quantify miR A that are indicative of infectious disease, such as a viral, fungal, or bacterial infection.
  • this disclosure provides for the comparison of quantity of miRNA in a subject sample to a reference level.
  • miRNA may be biomarkers, that is, biochemical features or facets that pertain to a subject and can be used to measure the progression or regression of a disease and/or the effect of a treatment.
  • the expression level of biomarkers described herein can be correlative to a subject's disease state.
  • biomarkers can be indicative of a diseased or healthy state of a subject.
  • similar expression of miRNA in a sample from a subject relative to the reference level indicates the absence of a disease or medical condition in the subject.
  • a difference in the expression of miRNA in a sample from a subject relative to the reference level indicates the presence or severity of the disease or medical condition in the subject.
  • miRNA expression can be compared to a reference level at different times to monitor disease progression or regression.
  • the confidence interval is expressed in the same units as the estimate or calculated value. Wider intervals indicate lower precision; narrow intervals indicate greater precision. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%.
  • the particular physiological state can be diagnosed or detected by determining whether a patient has the particular aberration, i.e. elevated or reduced miRNA levels.
  • the level of one or more miRNAs measured in a sample is normalized, such as by comparison to an internal reference nucleic acid.
  • the levels of the one or more miRNAs may then be compared to a reference value to determine if the levels of the one or more miRNAs are elevated or reduced relative to the reference value.
  • the reference value is the level measured in a comparable sample from one or more healthy individuals.
  • An increase or decrease in the level of the one or more miRNAs may be used in conjunction with clinical factors to diagnose various cancers, such as, e.g., pancreatic cancer.
  • the level of one or more miRNAs is combined with one or more additional pancreatic cancer markers to improve diagnostic sensitivity and specificity.
  • a subject's response to the administration of one or more of the compositions of the present invention may also indicate a disease state.
  • Association between a pathological state ⁇ e.g., cancer and/or chemotherapy drug-resistance) and the failure to obtain a positive response can be readily determined by comparative analysis in a normal population and an abnormal or affected population.
  • miRNA levels, activity, degradation, presence or absence thereof, the presence or existence of a disease and/or disease associated with a RTK, and/or degree of disease regression, alleviation, remission, or and other biomarkers of disease associated therewith, etc. in the subject by administering to the subject a composition of the present invention and subsequently measuring the outcome and or biomarker response or index.
  • the response in both normal populations and a population affected with a particular pathological state are ascertained.
  • the study results can be compared and analyzed by statistical means. Any detected statistically significant difference in the two populations would indicate an association.
  • the present invention provides for methods futher including determining the prognosis of the subject by (a) obtaining a sample from the subject prior to the administering and/or subsequent to the administering, (b) measuring the level of one or more of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 in the sample, (c) comparing the measured level in the sample to a reference level selected from the group consisting of: (i) one or more of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 levels from a control population; (ii) one or more of native miR-1291 and native SNORNA34 levels in the subject prior to the administering, and (iii) one or more of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 levels in the subject subsequent to the administering, and (d) determining the subject's prognosis based
  • the measuring includes, in some embodiments, reacting the sample from the subject with a biological probe including at least 7 contiguous nucleotides of SEQ. ID NO: 1 or the complement thereof, or the nucleotide sequence of SEQ. ID. NO: 2 or a complement thereof, wherein the biological probe is capable of hybridizing with one or more of, native or exogenous: miR-1291, miR-1291 precursors, miR12-91 RNA, miR-1291 niRNA, miR-1291 cDNA, SNORNA34, SNORNA34 precursors, SNORNA34 RNA, SNORNA34 niRNA, SNORNA34 cDNA, or complements thereof.
  • a decrease in one or more of the measured levels of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 in the sample compared to the reference level indicates an unfavorable prognosis, and wherein an increase in one or more of the measured levels of native miR-1291, native SNORNA34, exogenous miR-1291, and exogenous SNORNA34 in the sample compared to the reference level (ii) in the subject prior to the administration, indicates that the composition possesses efficacy for the disease or condition.
  • the level of one or more miRNAs in a biological sample of a patient is used to monitor the effectiveness of treatment or the prognosis of disease.
  • the level of one or more miRNAs in a test sample obtained from a treated patient can be compared to the level from a reference sample obtained from that patient prior to initiation of a treatment.
  • Clinical monitoring of treatment typically entails that each patient serve as his or her own baseline control.
  • test samples are obtained at multiple time points following administration of the treatment. In these embodiments, measurement of level of one or more miRNAs in the test samples provides an indication of the extent and duration of in vivo effect of the treatment.
  • a prognosis may be expressed as the amount of time a patient can be expected to survive.
  • a prognosis may refer to the likelihood that the disease goes into remission or to the amount of time the disease can be expected to remain in remission.
  • Prognosis can be expressed in various ways; for example, prognosis can be expressed as a percent chance that a patient will survive after one year, five years, ten years or the like. Alternatively, prognosis may be expressed as the number of years, on average that a patient can expect to survive as a result of a condition or disease. The prognosis of a patient may be considered as an expression of relativism, with many factors affecting the ultimate outcome. For example, for patients with certain conditions, prognosis can be appropriately expressed as the likelihood that a condition may be treatable or curable, or the likelihood that a disease will go into remission, whereas for patients with more severe conditions prognosis may be more appropriately expressed as likelihood of survival for a specified period of time.
  • a change in a clinical factor from a baseline level may impact a patient's prognosis, and the degree of change in level of the clinical factor may be related to the severity of adverse events.
  • Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value.
  • miRNA levels can be made, and a temporal change in activity can be used to determine a prognosis. For example, comparative measurements are made of the miRNA of an acellular body fluid in a patient at multiple time points, and a comparison of a miRNA value at two or more time points may be indicative of a particular prognosis.
  • the levels of activity of one or more miRNAs are used as indicators of an unfavorable prognosis.
  • the determination of prognosis can be performed by comparing the measured miRNA level to levels determined in comparable samples from healthy individuals or to levels known to corresponding with favorable or unfavorable outcomes.
  • the miRNA levels obtained may depend on an number of factors, including, but not limited to, the laboratory performing the assays, the assay methods used, the type of body fluid sample used and the type of disease a patient is afflicted with.
  • values can be collected from a series of patients with a particular disorder to determine appropriate reference ranges of miRNA for that disorder.
  • One of ordinary skill in the art is capable of performing a retrospective study that compares the determined levels to the observed outcome of the patients and establishing ranges of levels that can be used to designate the prognosis of the patients with a particular disorder. For example, levels in the lowest range would be indicative of a more favorable prognosis, while miRNA levels in the highest ranges would be indicative of an unfavorable prognosis.
  • the term “elevated levels” refers to levels of that are above the range of the reference value. In some embodiments patients with "high” or “elevated” levels have levels that are higher than the median activity in a population of patients with that disease.
  • “high” or “elevated” levels for a patient with a particular disease refers to levels that are above the median values for patients with that disorder and are in the upper 40% of patients with the disorder, or to levels that are in the upper 20% of patients with the disorder, or to levels that are in the upper 10%> of patients with the disorder, or to levels that are in the upper 5% of patients with the disorder.
  • the determination of prognosis can be performed using statistical analyses to relate the determined miRNA levels to the prognosis of the patient.
  • a skilled artisan is capable of designing appropriate statistical methods.
  • the methods may employ the chi-squared test, the Kaplan-Meier method, the log-rank test, multivariate logistic regression analysis, Cox's proportional-hazard model and the like in determining the prognosis.
  • Computers and computer software programs may be used in organizing data and performing statistical analyses. The approach by Giles et. al., British Journal of Hemotology, 121 :578-585, is exemplary.
  • association between categorical variables ⁇ e.g., miRNA levels and clinical characteristics can be assessed via cross-tabulation and Fisher's exact test. Unadjusted survival probabilities can be estimated using the method of Kaplan and Meier.
  • the Cox proportional hazards regression model also can be used to assess the ability of patient characteristics (such as miRNA levels) to predict survival, with 'goodness of fit' assessed by the Grambsch-Therneau test, Schoenfeld residual plots, martingale residual plots and likelihood ratio statistics ⁇ see Grambsch et al, 1995).
  • this approach can be adapted as a simple computer program that can be used with personal computers or personal digital assistants (PDA).
  • the prediction of patients' survival time in based on their miRNA levels can be performed via the use of a visual basic for applications (VBA) computer program developed within Microsoft ® Excel.
  • VBA visual basic for applications
  • the core construction and analysis may be based on the Cox proportional hazard models.
  • the VBA application can be developed by obtaining a base hazard rate and parameter estimates. These statistical analyses can be performed using a statistical program such as the SAS®
  • proportional hazards regression PHREG, procedure. Estimates can then be used to obtain probabilities of surviving from one to 24 months given the patient's covariates.
  • the program can make use of estimated probabilities to create a graphical representation of a given patient's predicted survival curve. In certain embodiments, the program also provides 6- month, 1-year and 18 -month survival probabilities.
  • a graphical interface can be used to input patient characteristics in a user- friendly manner.
  • multiple prognostic factors are considered when determining the prognosis of a patient.
  • the prognosis of a cancer patient may be determined based on miRNA and one or more prognostic factors selected from the group consisting of cytogenetics, performance status, age, gender and previous diagnosis.
  • other prognostic factors may be combined with the miRNA level or other biomarkers in the algorithm to determine prognosis with greater accuracy.
  • prognostic and diagnostic factors include, but are not limited to levels of one or more of miR-1291; AKT2; Cyclin Bl; MeCP2; FOXA2; AMPKal; Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASF1; Ornithine aminotransferase (OAT); Keratin, type II cytoskeletal 8 (KRT8); Phosphoenolpyruvate carboxykinase 2 (PEPCK2); Enoyl-coenzyme A (CoA) hydratase ( ECHS1); Phosphoserine aminotransferase isoform 1 (PSAT1); Dihydrolipoamide
  • acetyltransferase DLAT
  • Peroxiredoxin 3, isoform CRA a PRDX3
  • Cysteine-rich protein 2 CRIP2
  • Chain C human PCNA
  • Fascin homolog 1, actin-bundling protein isoform CRA a (FSCN1)
  • Serpin HI precursor Protein disulfide-isomerase precursor
  • Chain A disulfide isomerase related chaperone ERP29
  • Isocitrate dehydrogenase [NAD] subunit beta IDH3B
  • AFP a- fetoprotein
  • AFP-L3% des-gamma-carboxyprothrombin
  • DCP des-gamma-carboxyprothrombin
  • CDH1 E-cadherin
  • trimethylated lysine 27 of H3 histone H3K27me3
  • kits include at least one sequence of SEQ. ID. NOs: 1-5 or related sequences, and/or related probes and primers.
  • the kit can also contain other suitably packaged reagents and materials needed for amplification, for example, buffers, dNTPs, or polymerizing enzymes, and for detection analysis, for example, enzymes and solid phase extractants.
  • the kits may comprise multiple amplification primer sets, wherein at least one of the primers in each of the primer sets comprises a sequence that is complementary to a portion of at least two miRNAs, such as a target miRNA.
  • kits include one or more of the following (consistent with methods, reagents, and compositions discussed above): components for sample purification, including a lysis buffer with a chaotropic agent; a glass-fiber filter or column; an elution buffer; a wash buffer; an alcohol solution; and a nuclease inhibitor.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form, for example, and will be provided in a suitable container.
  • the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
  • the container will generally include at least one vial, test tube, flask, bottle, syringe, and/or other container means, into which the solvent is placed, optionally aliquoted.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other solvent.
  • Reagents useful for the disclosed methods can be stored in solution or can be lyophilized. When lyophilized, some or all of the reagents can be readily stored in microtiter plate wells for easy use after reconstitution. It is contemplated that any method for lyophilizing reagents known in the art would be suitable for preparing dried down reagents useful for the disclosed methods.
  • DMEM Dulbecco's modified Eagle's medium
  • RPMI 1640 medium penicillin sodium and streptomycin sulfate solution were purchased from Mediatech
  • Fetal bovine serum (FBS), Lipofectamine 2000 and Trizol reagent were bought from Life Technologies (Carlsbad, CA).
  • Rhodaminel23 and methylthiazolyldiphenyl- tetrazolium bromide (MTT) were purchased from Sigma- Aldrich (St. Louis, MO).
  • Primers and Digoxin (Dig) labeled oligos were bought from Integrated DNA Technologies
  • the hsa-miR-1291 miRIDIAN miR Hairpin Inhibitors (Antagomir miR-1291) and Negative control (control antagomir), enhanced chemiluminescence (ECL) Western Blotting Substrate, BCA Protein Assay Kit and Pierce CL-Xposure Film were from Thermo Scientific (Rockford, IL).
  • RIPA buffer was purchased from Rockland Immunochemicals (GilbertsviUe, PA).
  • Complete protease inhibitor cocktail was purchased from Roche Diagnostics (Mannheim, Germany).
  • Plasmids The coding region of SNORA34 comprising hsa-miR-1291 was amplified by PCR from human genome DNA. The PCR products were digested with Mlul and Xhol, and inserted into pCMV-Globin expression vector, resulting in pCMV-globin-miR-1291. The pCMV-Globin empty vector was named as control.
  • Target prediction miR- 1291 targeted genes were predicted using online algorithms, Targetscan and RNAhybrid).
  • miR- 1291 stably transfected cell line.
  • the PANC-1 cells were transfected with pCMV-globin-miR-1291 plasmid or pCMV-globin vector using lipofectamine 2000. Medium was replaced 24 hours after transfection, and then the medium containing 500 ⁇ g/mL G418 was used for selection. About 4 weeks later, G418 -resistant clones were selected and established as stably transfected cell lines.
  • RNA isolation Total RNA was isolated from cells with Trizol and miRNA was isolated with the mirPremier miR isolation kit (Sigma- Aldrich, St. Louis, MO) according to the manufacturers' instructions.
  • RT-qPCR Reverse transcription real-time polymerase chain reaction
  • 1 ⁇ g of total RNA was reversed transcribed using Oligo dT, hsa-miR-1291 stem-loop primer, or U74 primer (internal control for miRNA).
  • SYBR Green Real-time quantitative PCR (qPCR) reactions were conducted on a MylQ real-time PCR system (Bio-Rad, Hercules, CA).
  • the cycle number (C T ) at which the amplicon crossed a defined threshold was defined for each mRNA or miRNA.
  • the relative level of each mRNA or miRNA over internal standard was calculated using the formula 2 " , where AC T was the difference in C T values between mRNA or miRNA and internal standard, 18S or U74 small nucleolar RNA.
  • Annexin-V apoptosis assay was assessed using the ApoDetectTM
  • Annexin-V FITC Apoptosis Kit (Invitrogen, Carlsbad, CA). This assay employs fluorescein- labeled Annexin-V in concert with propidium iodide (PI) to detect the cells undergoing apoptosis. Briefly, cells were trypsinized, washed with PBS and resuspended at 2-3 x 10 6 cells/mL in 1 x Annexin-V Binding Buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ). Annexin-V FITC and Propidium Iodide Buffer were added to the cells, and then the cells were incubated at room temperature for 15 min in the dark. Cells were thus analyzed by flow cytometry.
  • PI propidium iodide
  • H&E Hematoxylin and eosin
  • MALDI matrix-assisted laser desorption/ionization
  • TOF time of flight
  • MS/MS tandem MS
  • 2D-DIGE and MS proteomic analysis were conducted at Applied Biomics (Hayward, CA). Briefly, equal amounts of Cy3- and Cy5 -labeled cell lysates (50 ⁇ g each) were mixed with rehydration buffer. After adding destreak solution (and 1% pH 3-10 pharmalyte (GE Healthcare), the samples were loaded onto an isoelectric focusing (IEF) strip (pH 3-10 linear range; GE Healthcare).
  • IEF isoelectric focusing
  • IEF was done for a total of 25,000 V/h with standard conditions using Ettan IPGPhore II. After the IEF, electrophoresis was performed at 16 °C on 10% SDS-PAGE. The resulting 2D gel images were acquired using a Typhoon Trio scanner (Amersham
  • miR- 1291 was expressed in human pancreatic ductal adenocarcinoma (PDAC) at 8-fold lower levels than the paired adjacent normal pancreatic tissues (FIG. 1) and 3 -fold lower level than unpaired normal pancreatic tissues, respectively.
  • PDAC pancreatic ductal adenocarcinoma
  • RT-qPCR stem-loop reverse transcription real-time quantitative PCR
  • miR-1291 expression was generally depleted in human pancreatic carcinoma cell lines including PANC-1, AsPC-1 and BxPC-3. The miR-1291 expression in these pancreatic carcinoma cell lines, however, was readily elevated after trans fection with a miR-1291 expression plasmid, and restoration of miR-1291 expression reduced pancreatic cancer cell proliferation (see below).
  • FIG. 2 shows that cell variability was determined using a MTT assay as described above. As shown in FIG. 2, gain of miR-1291 expression suppresses human cancer cell growth.
  • FIG. 2 graph moreover, graph details the variability of human cancer cells after transfection with SNORA/miR-1291 and control plasmids. Likewise, FIG. 3 details representative
  • RNAs were detected by splinted ligation assay. Briefly, total RNA was isolated from cells with Trizol. MiRNAs was ligated with digoxin-labeled probe through a miRNA specific bridge oligonucleotide, separated by electrophoresis, transferred onto Nylon+ membrane and detected with anti-Dig antibody.
  • FIG. 4 The process for miR-1291 transfection and miR-1291 expression levels in miR- 1291 stably transfected PANC-1 cells, respectively, is shown in FIG. 4.
  • a miR-1291 stably transfected PANC-1 cell line was established, in which miR- 1291 levels were approximately 9-fold higher than the control plasmid stably transfected PANC-1 cell lines.
  • Cells were then subjected to flow cytometric analysis. Briefly, cells were harvested, trypsinized, washed in ice-cold saline and fixed in 80% ice-cold ethanol. After treated with Bovine pancreatic RNAase, cells were stained with propidium iodide (PI) and analyzed via fluorescence-activated cell sorting (FACS). As shown in FIG 5, miR-1291 induces a cell cycle arrest in PANC-1 cells, where miR-1291 specifically induced a cell cycle arrest at both Gl/S and G2/M phases. Apoptosis was assessed using the ApoDetectTM
  • FIG. 6 shows miR-1291 's positive effect on apoptosis of PANC-1 cells.
  • FIG. 8 is a western blot showing that miR-1291 suppresses the protein expression of AKT2, Cyclin Bl, FOXA2 and AGR2 in PANC-1 cells. Protein expression was determined by Western blots, as noted above, where GAPDH was employed as an internal control.
  • AGR2 expression in human pancreatic ductal adenocarcinomas was compared to adjacent normal pancreatic tissue, as shown in FIG. 9's left and right panels, respectively. The histological determinations are described above, and the data indicate that AGR2 level in human pancreatic carcinomas is much higher than that in the adjacent normal pancreatic tissue. Protein expression was studied by immunohistochemistry (IHC), noted above.
  • IHC immunohistochemistry
  • the 3 'UTR of FOXA2 was retrieved from NCBI ENTREZ and was searched for miR recognized element (MRE) to miR-1291 by Targetscan and RNAhybrid.
  • FIG. 10 shows a schematic representation of miR-1291 functions on the 3 'UTR of transcription factor FOXA2 in concert with graphical depictions thereof. FOXA2 was accordingly shown to be a direct target for miR- 1291.
  • Pancreatic cancer xenografts were generated by surgical implantation or subcutaneous injection of human pancreatic carcinoma cells ⁇ e.g., PANC-1, AsPC-1 or BxPC-3) in athymic mice, and growth of orthotopic and subcutaneous tumors was monitored by ultrasonograpy and caliper measurement, respectively.
  • Formulated miR-1291 (non-toxic dose levels 3-10 mg/kg) was administrated intravenously via tail vein injection three times per week for 1-3 weeks after tumors reach a volume of 150-200 mm . Tumors were collected in the end of the study, weighted and divided into two halves. The samples were subjected to qPCR (see above), miR-1291 accumulation and target gene expression, while
  • mice (N 12) inoculated with the miR- 1291 stably transfected PANC-1 cells showed minimal or no tumor growth, in contrast to sites injected with a control. PANC-1 cells produced common xenograft tumors (FIG. 11) that were further confirmed by histological and pathological analyses, detailed above. Additional proteomic and biochemical studies demonstrated that miR-1291 suppressed cancer cell growth and tumorigenesis through targeting a number of proto-oncogenes in pancreatic cancer cells is shown in Table 2 below.
  • FIG. 11 further shows the influence that miR-1291 possesses with respect to clinical symptoms that were assessed using a xenograft tumor mouse model.
  • FIG. 11 A shows pictures and graphs demonstrating suppression of tumor growth by miR-1291.
  • FIG. 1 IB concerns the pathological analysis of xenograft tumor results from a histological study at lOOx magnification, which confirms tumor progression from control PANC-1 cells.
  • FIG. 11C is an immunohistochemistry (IHC) study, as explained above, demonstrating that miR-1291 reduces tumor cell proliferation (Ki-67 labeling) and enhances apoptosis (Caspase-3). IHC analyses indicate that miR-1291 reduces cell proliferation and enhances apoptosis.
  • PANC-1 cells were inoculated into male athymic CD-I nude mice. Tumor growth was monitored once a week. At the end of the study, tumors were harvested, weighted and subjected to pathological and IHC studies.
  • FIG. 12 shows that multidrug resistance-associated protein (MRP1/ABCC1) is expressed ubiquitously in human tissues including the lungs, testis, kidney, pancreas, and placenta, and contributes to a variety of physiological functions including the extrusion of endobiotic and xenobiotic toxins.
  • ABCC1 may also confer multidrug resistance (MDR) in cancer cells.
  • MDR multidrug resistance
  • SNORA34 was found to be processed to miR-1291 in PANC-1 human pancreases cancer cells. Luciferase assays supported the action of miR-1291 on ABCC1 3'UTR.
  • miR-1291 suppresses pancreatic xenografts tumor growth through the inhibition of (proto)-oncogenes and induction of cell cycle arrest. These findings teach that miR-1291 is a tumor suppressor and restoration of miR-1291 function can control human pancreatic cancer disease.
  • the role of miR-1291 in suppression of pancreatic cancer cell growth and tumorigenesis was defined by using a stably transfected cell line and transient transfection with miR-1291 expression plasmid.
  • Controls include the scrambled RNA, vehicle only and saline treatments.
  • xenograft tumors are collected in the end of studies for pathological confirmation, and immunohistochemical analyses of cell proliferation (e.g., Ki-67) and apoptosis (e.g., caspase- 3), as well as biochemical evaluation of miR-1291 accumulation and target gene expression.
  • Ki-67 cell proliferation
  • apoptosis e.g., caspase- 3
  • Liver and kidney functions are evaluated in conjunction with immune responses through examination of blood chemistries and cytokine levels, respectively, in immune- compromised mice treated with multiple doses of miR-1291.
  • a single dose intravenous bolus of miR-1291 complexed with delivery indications in fully immune competent Balb/c mice at 3 dose levels addresses pharmacokinetic considerations, serum cytokine levels and basic safety parameters (i.e. blood chemistries).
  • Mice treated with intravenous bolus saline, vehicle (empty liposomes) and scrambled RNA are used as controls to distinguish specific effects caused by the sequence of miR-1291.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • BUN blood urea nitrogen
  • ALP alkaline phosphatase
  • CK creatinine and creatine kinase
  • cytokine levels e.g., IL-2, IL-4, IL-6, IFN- ⁇ and TNF-a, etc.
  • serum samples collected at various time points e.g., pre-dose, 3 min, 15 min, 30min, 90 min, 3 hr, 6 hr, 24 hr and 48 hr.
  • Blood collected at these time points are employed for RNA isolation and PCR analysis to establish the PK profile of the miR-1291 formulation in whole blood.
  • major organs including liver, kidney, lung, heart, brain, spleen
  • mice e.g., BALB/c
  • formulated miR-1291 e.g., a separate group of animals for positive induction of cytokines is given 0.5-1.0 mg/kg of LPS isolated from Escherichia coli 0111 :B4. Blood chemistries and cytokine levels in response to systemically delivered control miRNA and miR-1291 are determined.
  • Serum cytokine levels are quantified using a mouse Fluorokine Multianalyte Profiling Kit (R&D Systems). Whole blood chemistry is determined by the Comparative Pathology Laboratory (University of California - Davis), and the profiles are utilized for the assessment of hepatic and renal functions. miR-1291 concentrations are quantified by stem-loop qPCR assay, as described above. Animal organs are collected, fixed in 10% formalin, embedded in paraffin-wax and subjected to histological analyses by experienced pathologists at the Core Research Facilities at RPCI.

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Abstract

La présente invention concerne dans des compositions et des procédés destinés à la prévention et au traitement de maladies et/ou d'états humain(e)s. La présente invention concerne, entre autres, la reconstitution de niveaux de miARN-1291 dans des échantillons et chez des sujets. Des procédés destinés au diagnostic et au traitement de maladies et d'états humain(e)s par l'évaluation des niveaux d'expression du miARN -1291 sont également décrits. Des procédés de traitement de cancers humains par modification ou régulation des voies du miARN sont également décrits.
PCT/US2013/021307 2012-01-13 2013-01-11 Indications thérapeutiques du miarn-1291 WO2013106766A2 (fr)

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US10619156B2 (en) 2014-05-28 2020-04-14 The Regents Of The University Of California Hybrid tRNA/pre-miRNA molecules and methods of use
US10422003B2 (en) 2015-03-23 2019-09-24 The Regents Of The University Of California Methods for detection of RNase activity
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CN110088278B (zh) * 2016-10-31 2023-08-11 e-NA生物科技公司 双链核酸分子及其用途
CN113498437A (zh) * 2019-02-26 2021-10-12 首尔大学校产学协力团 包含末端尿苷酰基转移酶4/7表达调控因子的用于预防或治疗癌症的药学组合物
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