Nothing Special   »   [go: up one dir, main page]

EP2102342A2 - GÈNES ET VOIES RÉGULÉS PAR miR-16 UTILES COMME CIBLES POUR INTERVENTION THÉRAPEUTIQUE - Google Patents

GÈNES ET VOIES RÉGULÉS PAR miR-16 UTILES COMME CIBLES POUR INTERVENTION THÉRAPEUTIQUE

Info

Publication number
EP2102342A2
EP2102342A2 EP07871756A EP07871756A EP2102342A2 EP 2102342 A2 EP2102342 A2 EP 2102342A2 EP 07871756 A EP07871756 A EP 07871756A EP 07871756 A EP07871756 A EP 07871756A EP 2102342 A2 EP2102342 A2 EP 2102342A2
Authority
EP
European Patent Office
Prior art keywords
mir
mirna
carcinoma
nucleic acid
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07871756A
Other languages
German (de)
English (en)
Inventor
Mike Byrom
Lubna Patrawala
Charles D. Johnson
David Brown
Andreas G. Bader
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Asuragen Inc
Original Assignee
Asuragen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Asuragen Inc filed Critical Asuragen Inc
Publication of EP2102342A2 publication Critical patent/EP2102342A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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.

Definitions

  • the present invention relates to the fields of molecular biology and medicine. More specifically, the invention relates to methods and compositions for the treatment of diseases or conditions that are affected by miR-16 microRNAs, microRNA expression, and genes and cellular pathways directly and indirectly modulated by such.
  • miRNAs small molecules
  • C. elegans, Drosophila, and humans Lagos- Quintana et al, 2001; Lau et al, 2001; Lee and Ambros, 2001.
  • miRNAs Several hundreds of miRNAs have been identified in plants and animals - including humans - which do not appear to have endogenous siRNAs. Thus, while similar to siRNAs, miRNAs are distinct.
  • miRNAs thus far observed have been approximately 21-22 nucleotides in length and they arise from longer precursors, which are transcribed from non-protein-encoding genes. See review of Carrington et al. (2003). The precursors form structures that fold back on themselves in self-complementary regions; they are then processed by the nuclease Dicer in animals or DCLl in plants. miRNA molecules interrupt translation through precise or imprecise base-pairing with their targets.
  • miRNAs are conserved among diverse organisms, and this has led to the suggestion that miRNAs are involved in essential biological processes throughout the life span of an organism (Esquela-Kerscher and Slack, 2006).
  • miRNAs have been implicated in regulating cell growth, and cell and tissue differentiation; cellular processes that are associated with the development of cancer.
  • lin-4 and miR-16 both regulate passage from one larval state to another during C. elegans development (Ambros, 2001).
  • mir-14 and bantam are Drosophila miRNAs that regulate cell death, apparently by regulating the expression of genes involved in apoptosis (Brennecke et al, 2003, Xu et al, 2003).
  • miR-16 is down-regulated in B-cells from patients with chronic lymphocytic leukemia (Calin et al, 2002). Reduced expression of these miRNAs in B cell lymphomas results in overexpression of a miR-16 target gene, BCL2, and subsequent inhibition of apoptosis by the BCL2 gene product. Reduced expression of miR-16 results in uncontrolled cellular proliferation and B cell malignancy (reviewed in Calin and Croce, 2006). Together these data suggest that miR-16-1 appears to function as a tumor suppressor in human B cells.
  • hsa-miR-16 is involved with the regulation of numerous cell activities that represent intervention points for cancer therapy and for therapy of other diseases and disorders (U.S. Patent Applications serial number 11/141,707 filed May 31, 2005 and serial number 11/273,640 filed November 14, 2005).
  • Expression of miR-16 was reduced in lung tumors from numerous lung cancer patients when compared to its expression in normal adjacent lung tissues from the same patients.
  • the inventors observed increased expression of miR-16 in breast and prostate tumors as compared to expression in adjacent normal cells from the same cancer patients.
  • hsa-miR-16 activated the hTert gene that encodes the catalytic domain of telomerase.
  • Hsa-miR-16 Over 90% of human cancer samples have active telomerase (reviewed in Dong et al, 2005). Hsa-miR-16 also induces cells to enter the S phase of the cell cycle and decreases the proliferation of lung cancer cells (A549 and HTB-57 lung carcinoma cells), prostate cancer cells (22RvI), and human basal cell carcinomas (TE354T). Anti-miR inhibitors of hsa-miR-16 increased the proliferation of non-malignant human breast epithelial cells and basal cell carcinoma cells (TE354T). In addition, the inventors previously observed that hsa-miR-16 is up-regulated in patients with prion disease and Alzheimer's disease when compared to patients without those diseases.
  • genes and pathways that are altered by expression of hsa-miR-16 represent targets for therapeutic intervention in the treatment of certain diseases like Alzheimer's Disease and prion diseases, in which hsa-miR-16 likely plays a role.
  • miRNAs are thought to interact with target genes through imprecise base pairing within the 3' untranslated regions of their gene targets. Regulation of target genes by miRNAs is thought to occur primarily by translation inhibition, but mRNA instability may also be a mechanism (Reinhart et al, 2000; Bagga et al, 2005). Bioinformatics analyses suggest that any given miRNA may bind to and alter the expression of up to several hundred different genes. In addition, a single gene may be regulated by several miRNAs. Thus, each miRNA may regulate a complex interaction among genes, gene pathways, and gene networks. Mis-regulation or alteration of these regulatory pathways and networks, involving miRNAs, are likely to contribute to the development of disorders and diseases such as cancer.
  • bioinformatics tools are helpful in predicting miRNA binding targets, all have limitations. Because of the imperfect complementarity with their target binding sites, it is difficult to accurately predict miRNA targets with bioinformatics tools alone. Furthermore, the complicated interactive regulatory networks among miRNAs and target genes make it difficult to accurately predict which genes will actually be mis- regulated in response to a given miRNA.
  • the present invention provides additional compositions and methods to address problems in the art by identifying genes in cancer cells that are direct targets for hsa-miR-16 regulation or that are downstream targets of regulation following the hsa-miR-16-mediated modification of upstream gene expression. Furthermore, the invention describes gene, disease, and/or physiologic pathways and networks that are influenced by hsa-miR-16. Many of these genes and pathways are associated with various cancers and other diseases. The altered expression of miR-16 in cells would lead to changes in the expression of these key genes and contribute to the development of disease.
  • a cell may be an epithelial, stromal, or mucosal cell.
  • the cell can be, but is not limited to brain, a glial, a neuronal, a blood, an esophageal, a lung, a cardiovascular, a liver, a breast, a bone, a thyroid, a glandular, an adrenal, a pancreatic, a stomach, an intestinal, a kidney, a bladder, a prostate, a cervical, a uterus, an ovarian, a testicular, a splenic, a skin, a smooth muscle, a cardiac muscle, or a striated muscle cell.
  • the cell, tissue, or target may not be defective in miRNA expression yet may still respond therapeutically to expression or over expression of a miRNA.
  • compositions of the invention are administered to a subject having, suspected of having, or at risk of developing a metabolic, an immunologic, an infectious, a cardiovascular, a digestive, an endocrine, an ocular, a genitourinary, a blood, a musculoskeletal, a nervous system, a congenital, a respiratory, a skin, or a cancerous disease or condition.
  • a subject or patient may be selected for treatment based on expression and/or aberrant expression of one or more miRNA or mRNA.
  • a subject or patient may be selected for treatment based on aberrations in one or more biologic or physiologic pathway(s), including aberrant expression of one or more gene associated with a pathway, or the aberrant expression of one or more protein encoded by one or more gene associated with a pathway.
  • a subject or patient may be selected based on aberrations in miRNA expression, or biologic and/or physiologic pathway(s).
  • a subject may be assessed for sensitivity, resistance, and/or efficacy of a therapy or treatment regime based on the evaluation and/or analysis of miRNA or mRNA expression or lack thereof.
  • a subject may be evaluated for amenability to certain therapy prior to, during, or after administration of one or therapy to a subject or patient.
  • evaluation or assessment may be done by analysis of miRNA and/or mRNA, as well as combination of other assessment methods that include but are not limited to histology, immunohistochemistry, blood work, etc.
  • an infectious disease or condition includes a bacterial, viral, parasite, or fungal infection. Many of these genes and pathways are associated with various cancers and other diseases. Cancerous conditions include, but are not limited to astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, laryngeal squamous cell carcinoma, lung carcinoma, melanoma, medulloblastoma, mantle cell lymphoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheoch
  • the cancerous condition is prostate carcinoma, which can be positive or negative for PSA, and/or androgen dependent or androgen independent.
  • Cells of the prostate require male hormones, known as androgens, to work properly.
  • Androgens include testosterone, which is made in the testes; dehydroepiandrosterone, made in the adrenal glands; and dihydrotestosterone, which is converted from testosterone within the prostate itself.
  • Some prostate carcinomas retain androgen dependence while others are independent of androgen.
  • Prostate cancer screening is an attempt to find unsuspected cancers. Screening tests may lead to more specific follow-up tests such as a biopsy, where small pieces of the prostate are removed for closer study. Typical prostate cancer screening options include the digital rectal exam and the prostate specific antigen (PSA) blood test.
  • Prostate cancer is usually a slow-growing cancer, very common among older men.
  • a cell, tissue, or subject may be a cancer cell, a cancerous tissue, harbor cancerous tissue, or be a subject or patient diagnosed or at risk of developing a disease or condition.
  • a cancer cell is a neuronal, glial, lung, liver, brain, breast, bladder, blood, leukemic, colon, endometrial, stomach, skin, ovarian, fat, bone, cervical, esophageal, pancreatic, prostate, kidney, testicular or thyroid cell.
  • cancer includes, but is not limited to astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, laryngeal squamous cell carcinoma, lung carcinoma, melanoma, medulloblastoma, mantle cell lymphoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheochromocytoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, testicular tumor or thyroid carcinoma.
  • the gene or genes modulated comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200 or more genes or any combination of genes identified in Table 1, 2, 4 and 5.
  • the expression of a gene is down-regulated or up-regulated.
  • the gene modulated comprises or is selected from (and may even exclude) 1, 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 or all of genes identified in Table 1, 2, 4 and 5, in various combinations and permutations.
  • the invention may exclude or choose not to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200 or more genes or any combination of genes identified in Table 1, 2, 4 and 5, e.g., BCL2, RARS (arginyl-tRNA synthetase), BTG2, WTl, PPMlD, PAK7, and/or RAB9B.
  • the gene modulated or selected to modulate includes one or more genes of Table 1, 2, 4 and/or 5 provided that RARS (arginyl-tRNA synthetase), BTG2, WTl, PPMlD, PAK7, and/or RAB9B is not included.
  • Embodiments of the invention include methods of modulating gene expression, or biologic or physiologic pathways in a cell, a tissue, or a subject comprising administering to the cell, tissue, or subject an amount of an isolated nucleic acid or mimetic thereof comprising a miR-16 nucleic acid, mimetic, or inhibitor sequence in an amount sufficient to modulate the expression of a gene positively or negatively modulated by a miR-16 miRNA.
  • a “miR-16 nucleic acid sequence” or “miR-16 inhibitor” includes the full length precursor of miR-16, or complement thereof or processed (i.e., mature) sequence of miR-16 and related sequences set forth herein, as well as 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 or more nucleotides of a precursor miRNA or its processed sequence, or complement thereof, including all ranges and integers there between.
  • the miR-16 nucleic acid sequence or miR-16 inhibitor contains the full-length processed miRNA sequence or complement thereof and is referred to as the "miR-16 full- length processed nucleic acid sequence" or "miR-16 full-length processed inhibitor sequence.”
  • the miR-16 nucleic acid comprises at least one 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 nucleotide (including all ranges and integers there between) segment or complementary segment of a miR-16 that is at least 75, 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NOs provided herein.
  • the general term miR-16 includes all members of the miR-16 family that share at least part of a mature miR-16 sequence.
  • the miR-16 nucleic acid comprises at least one 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 232, 24, 25, 50 nucleotide (including all ranges and integers there between) segment of miR-16 that is at least 75, 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NOs:l-3.
  • SEQ ID NO:1 uagcagcacguaaauauuggcg (accession - MIMAT0000069), SEQ ID NO:2 (hsa-mir-16-1, accession - MI0000070) gucagcagugccuuagcagcacguaaauauuggcguuaagauucuaaaauuau cuccaguauuaacugugcugcugaaguaagguugac; SEQ ID NO:3 (hsa-mir-16-2, accession MIOOOOl 15) guuccacucuagcagcacguaaauauuggcguagugaaauauauauuaaacaccaauauuacug ugcugcuuuagugugac).
  • the gene modulated or selected to modulate is from Table 1. In further embodiments the gene modulated or selected to modulate is from Table 2. In still further embodiments the gene modulated or selected to modulate is from Table 4. In yet further embodiments the gene modulated or selected to modulate is from Table 5.
  • Embodiments of the invention may also include obtaining or assessing a gene expression profile or miRNA profile of a target cell prior to selecting the mode of treatment, e.g., administration of a miR-16 nucleic acid.
  • a miR-16 nucleic acid, or a segment or a mimetic thereof will comprise 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 or more nucleotides of the precursor miRNA or its processed sequence, including all ranges and integers there between.
  • the miR-16 nucleic acid sequence contains the full-length processed miRNA sequence and is referred to as the "miR- 16 full-length processed nucleic acid sequence.”
  • a miR-16 comprises at least one 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 nucleotide (including all ranges and integers there between) segment of miR-16 that is at least 75, 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NOs provided herein.
  • a miR-16 or miR-16 inhibitor containing nucleic acid is a hsa-miR-16 or hsa-miR-16 inhibitor, or a variation thereof.
  • a miR-16 nucleic acid or miR-16 inhibitor can be administered with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNAs or miRNA inhibitors. miRNAs or their complements can be administered concurrently, sequentially, or in an ordered progression.
  • a miR-16 or miR- 16 inhibitor can be administered in combination with one or more of let-7, miR-15, miR-126, miR-20, miR-21, miR-26a, miR-34a, miR-143, miR-147, miR-188, miR-200, miR-215, miR- 216, miR-292-3p, and/or miR-331. All or combinations of miRNAs or inhibitors thereof may be administered in a single formulation. Administration may be before, during or after a second therapy.
  • miR-16 nucleic acids or complement thereof may also include various heterologous nucleic acid sequences, i.e., those sequences not typically found operatively coupled with miR-16 in nature, such as promoters, enhancers, and the like.
  • the miR-16 nucleic acid is a recombinant nucleic acid, and can be a ribonucleic acid or a deoxyribonucleic acid.
  • the recombinant nucleic acid may comprise a miR-16 or miR-16 inhibitor expression cassette, i.e., a nucleic acid segment that expresses a nucleic acid when introduce into an environment containing components for nucleic acid synthesis.
  • the expression cassette is comprised in a viral vector, or plasmid DNA vector or other therapeutic nucleic acid vector or delivery vehicle, including liposomes and the like.
  • the miR-16 nucleic acid is a synthetic nucleic acid.
  • nucleic acids of the invention may be fully or partially synthetic.
  • viral vectors can be administered at IxIO 2 , IxIO 3 , IxIO 4 IxIO 5 , IxIO 6 , IxIO 7 , IxIO 8 , IxIO 9 , IxIO 10 , lxlO 11 , IxIO 12 , IxIO 13 , IxIO 14 pfu or viral particle (vp).
  • the miR-16 nucleic acid or miR-16 inhibitor is a synthetic nucleic acid.
  • nucleic acids of the invention may be fully or partially synthetic.
  • a nucleic acid of the invention or a DNA encoding such a nucleic acid of the invention can be administered at 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, 50, 100, 200, 400, 600, 800, 1000, 2000, to 4000 ⁇ g or mg, including all values and ranges there between.
  • nucleic acids of the invention can be administered at 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, 50, 100, to 200 ⁇ g or mg per kilogram (kg) of body weight.
  • Each of the amounts described herein may be administered over a period of time, including 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, minutes, hours, days, weeks, months or years, including all values and ranges there between.
  • administration of the composition(s) can be enteral or parenteral.
  • enteral administration is oral.
  • parenteral administration is intralesional, intravascular, intracranial, intrapleural, intratumoral, intraperitoneal, intramuscular, intralymphatic, intraglandular, subcutaneous, topical, intrabronchial, intratracheal, intranasal, inhaled, or instilled.
  • Compositions of the invention may be administered regionally or locally and not necessarily directly into a lesion.
  • a cell, tissue, or subject may be or suffer from an abnormal or pathologic condition, or in the case of a cell or tissue, the component of a pathological condition.
  • a cell, tissue, or subject is a cancer cell, a cancerous tissue or harbor cancerous tissue, or a cancer patient.
  • the cancer is neuronal, glial, lung, liver, brain, breast, bladder, blood, leukemic, cervical, testicular, colon, endometrial, stomach, skin, ovarian, esophageal, pancreatic, prostate, kidney, or thyroid cancer.
  • the database content related to all nucleic acids and genes designated by an accession number or a database submission are incorporated herein by reference as of the filing date of this application.
  • a further embodiment of the invention is directed to methods of modulating a cellular pathway comprising administering to the cell an amount of an isolated nucleic acid comprising a miR-16 nucleic acid sequence in an amount sufficient to modulate the expression, function, status, or state of a cellular pathway, in particular those pathways described in Table 2 or the pathways known to include one or more genes from Table 1, 3, 4, and/or 5.
  • Modulation of a cellular pathway includes, but is not limited to modulating the expression of one or more gene. Modulation of a gene can include inhibiting the function of an endogenous miRNA or providing a functional miRNA to a cell, tissue, or subject.
  • Modulation refers to the expression levels or activities of a gene or its related gene product or protein, e.g., the mRNA levels may be modulated or the translation of an mRNA may be modulated, etc. Modulation may increase or up regulate a gene or gene product or it may decrease or down regulate a gene or gene product.
  • Still a further embodiment includes methods of treating a patient with a pathological condition comprising one or more of step (a) administering to the patient an amount of an isolated nucleic acid comprising a miR-16 nucleic acid sequence in an amount sufficient to modulate the expression of a cellular pathway; and (b) administering a second therapy, wherein the modulation of the cellular pathway sensitizes the patient to the second therapy.
  • a cellular pathway may include, but is not limited to one or more pathway described in Table 2 below or a pathway that is known to include one or more gene of Table 1, 3, 4, and/or 5.
  • a second therapy can include a second miRNA or other nucleic acid therapy or one or more standard therapies, such as chemotherapy, drug therapy, radiation therapy, immunotherapy, thermal therapy, and the like.
  • Embodiments of the invention include methods of treating a subject with a pathological condition comprising one or more of the steps of (a) determining an expression profile of one or more genes selected from Table 1, 3, 4, and/or 5; (b) assessing the sensitivity of the subject to therapy based on the expression profile; (c) selecting a therapy based on the assessed sensitivity; and (d) treating the subject using selected therapy.
  • the pathological condition will have as a component, indicator, or result the mis-regulation of one or more gene of Table 1, 3, 4, and/or 5.
  • Further embodiments include the identification and assessment of an expression profile indicative of miR-16 status in a cell or tissue comprising expression assessment of one or more gene from Table 1, 3, 4, and/or 5, or any combination thereof.
  • RNA refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al, 2003, which is hereby incorporated by reference. The term can be used to refer to the single-stranded RNA molecule processed from a precursor or in certain instances the precursor itself. [0030] In some embodiments, it may be useful to know whether a cell expresses a particular miRNA endogenously or whether such expression is affected under particular conditions or when it is in a particular disease state.
  • methods include assaying a cell or a sample containing a cell for the presence of one or more marker gene or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently, in some embodiments, methods include a step of generating an RNA profile for a sample.
  • RNA profile or “gene expression profile” refers to a set of data regarding the expression pattern for one or more gene or genetic marker in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers from Table 1, 3, 4, and/or 5); it is contemplated that the nucleic acid profile can be obtained using a set of RNAs, using for example nucleic acid amplification or hybridization techniques well known to one of ordinary skill in the art.
  • the difference in the expression profile in the sample from the patient and a reference expression profile, such as an expression profile from a normal or non-pathologic sample is indicative of a pathologic, disease, or cancerous condition.
  • a nucleic acid or probe set comprising or identifying a segment of a corresponding mRNA can include all or part of 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 100, 200, 500, or more, including any integer or range derivable there between, of a gene or genetic marker, or a nucleic acid, mRNA or a probe representative thereof that is listed in Table 1, 3, 4, and/or 5, or identified by the methods described herein.
  • Certain embodiments of the invention are directed to compositions and methods for assessing, prognosing, or treating a pathological condition in a patient comprising measuring or determining an expression profile of one or more marker(s) in a sample from the patient, wherein a difference in the expression profile in the sample from the patient and an expression profile of a normal sample or reference expression profile is indicative of pathological condition and particularly cancer.
  • the cellular pathway, gene, or genetic marker is or is representative of one or more pathway or marker described in Table 1, 3, 4, and/or 5, including any combination thereof and excluding 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes.
  • aspects of the invention include treating, diagnosing, or prognosing a pathologic condition or preventing a pathologic condition from manifesting.
  • the methods can be used to screen for a pathological condition; assess prognosis of a pathological condition; stage a pathological condition; assess response of a pathological condition to therapy; or to modulate the expression of a gene, genes, or related pathway as a first therapy or to render a subject sensitive or more responsive to a second therapy.
  • assessing the pathological condition of the patient can be assessing prognosis of the patient.
  • Prognosis may include, but is not limited to an estimation of the time or expected time of survival, assessment of response to a therapy, and the like.
  • the altered expression of one or more gene or marker is prognostic for a patient having a pathologic condition, wherein the marker is one or more of Table 1, 3, 4, and/or 5, including any combination thereof.
  • Certain embodiments of the invention include determining expression of one or more marker, gene, or nucleic acid representative thereof, by using an amplification assay, a hybridization assay, or protein assay, a variety of which are well known to one of ordinary skill in the art.
  • an amplification assay can be a quantitative amplification assay, such as quantitative RT-PCR or the like.
  • a hybridization assay can include array hybridization assays or solution hybridization assays. The nucleic acids from a sample may be labeled from the sample and/or hybridizing the labeled nucleic acid to one or more nucleic acid probes.
  • Nucleic acids, mRNA, and/or nucleic acid probes may be coupled to a support.
  • Such supports are well known to those of ordinary skill in the art and include, but are not limited to glass, plastic, metal, or latex.
  • the support can be planar or in the form of a bead or other geometric shapes or configurations known in the art. Proteins are typically assayed by immunoblotting, chromatography, mass spectrometry or other methods known to those of ordinary skill in the art.
  • a further embodiment of the invention is directed to methods of modulating a cellular pathway comprising administering to the cell an amount of an isolated nucleic acid comprising a miR-16 nucleic acid sequence or a miR-16 inhibitor.
  • a cell, tissue, or subject may be a cancer cell, a cancerous tissue or harbor cancerous tissue, or a cancer patient.
  • the database content related to all nucleic acids and genes designated by an accession number or a database submission are incorporated herein by reference as of the filing date of this application.
  • a further embodiment of the invention is directed to methods of modulating a cellular pathway comprising administering to the cell an amount of an isolated nucleic acid comprising a miR-16 nucleic acid sequence in an amount sufficient to modulate the expression, function, status, or state of a cellular pathway, in particular those pathways described or the pathways known to include one or more genes described herein.
  • Modulation of a cellular pathway includes, but is not limited to modulating the expression of one or more gene(s). Modulation of a gene can include inhibiting the function of an endogenous miRNA or providing a functional miRNA to a cell, tissue, or subject.
  • Modulation refers to the expression levels or activities of a gene or its related gene product (e.g., mRNA) or protein, e.g., the mRNA levels may be modulated or the translation of an mRNA may be modulated. Modulation may increase or up regulate a gene or gene product or it may decrease or down regulate a gene or gene product (e.g., protein levels or activity).
  • a gene or its related gene product e.g., mRNA
  • protein e.g., protein levels or activity
  • Still a further embodiment includes methods of administering an miRNA or mimic thereof, and/or treating a subject or patient having, suspected of having, or at risk of developing a pathological condition comprising one or more of step (a) administering to a patient or subject an amount of an isolated nucleic acid comprising a miR-16 nucleic acid sequence or a miR-16 inhibitor in an amount sufficient to modulate expression of a cellular pathway; and (b) administering a second therapy, wherein the modulation of the cellular pathway sensitizes the patient or subject, or increases the efficacy of a second therapy.
  • An increase in efficacy can include a reduction in toxicity, a reduced dosage or duration of the second therapy, or an additive or synergistic effect.
  • a cellular pathway may include, but is not limited to one or more pathway described herein or a pathway that is know to include one or more genes in the tables herein.
  • the second therapy may be administered before, during, and/or after the isolated nucleic acid or miRNA or inhibitor is administered
  • a second therapy can include administration of a second miRNA or therapeutic nucleic acid such as a siRNA or antisense oligonucleotide, or may include various standard therapies, such as pharmaceuticals, chemotherapy, radiation therapy, drug therapy, immunotherapy, and the like.
  • a second therapy is chemotherapy.
  • a chemotherapy can include, but is not limited to paclitaxel, cisplatin, carboplatin, doxorubicin, oxaliplatin, larotaxel, taxol, lapatinib, docetaxel, methotrexate, capecitabine, vinorelbine, cyclophosphamide, gemcitabine, amrubicin, cytarabine, etoposide, camptothecin, dexamethasone, dasatinib, tipifarnib, bevacizumab, sirolimus, temsirolimus, everolimus, lonafarnib, cetuximab, erlotinib, gefitinib, imatinib mesylate, rituximab, trastuzumab, nocodazole, sorafenib, sunitinib, bortezomib, alemtuzumab, gemtuzumab, to
  • Embodiments of the invention include methods of treating a subject with a disease or condition comprising one or more of the steps of (a) determining an expression profile of one or more genes selected from the tables; (b) assessing the sensitivity of the subject to therapy based on the expression profile; (c) selecting a therapy based on the assessed sensitivity; and (d) treating the subject using a selected therapy.
  • the disease or condition will have as a component, indicator, or resulting mis-regulation of one or more gene described herein.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10, or more miRNA may be used in sequence or in combination.
  • any combination of miR-16 or a miR-16 inhibitor with another miRNA may be used in sequence or in combination.
  • Further embodiments include the identification and assessment of an expression profile indicative of miR-16 status in a cell or tissue comprising expression assessment of one or more gene from the tables, or any combination thereof.
  • RNA refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al, 2003, which is hereby incorporated by reference. The term can be used to refer to the single-stranded RNA molecule processed from a precursor or in certain instances the precursor itself.
  • methods include assaying a cell or a sample containing a cell for the presence of one or more marker gene or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently, in some embodiments, methods include a step of generating an RNA profile for a sample.
  • RNA profile or “gene expression profile” refers to a set of data regarding the expression pattern for one or more gene or genetic marker or miRNA in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers from the tables; it is contemplated that the nucleic acid profile can be obtained using a set of RNAs, using for example nucleic acid amplification or hybridization techniques well know to one of ordinary skill in the art.
  • the difference in the expression profile in the sample from the patient and a reference expression profile, such as an expression profile of one or more genes or miRNAs, are indicative of which miRNAs to be administered.
  • miR-16 or miR-16 inhibitor and let-7 or let-7 inhibitor are administered to patients with astrocytoma, breast carcinoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, melanoma, medulloblastoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.
  • Further aspects include administering miR-16 or miR-16 inhibitor and miR-10 or miR-10 inhibitor to patients with astrocytoma, breast carcinoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, melanoma, mantle cell lymphoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma
  • miR-16 or miR-16 inhibitor and miR-15 or miR-15 inhibitor can be administered to patients with astrocytoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, laryngeal squamous cell carcinoma, melanoma, medulloblastoma, mantle cell lymphoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheochromocytoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.
  • miR-16 or miR-16 inhibitor and miR-20 or miR-20 inhibitor are administered to patients with astrocytoma, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.
  • miR-16 or miR-16 inhibitor and miR-21 or miR-21 inhibitor are administered to patients with astrocytoma, breast carcinoma, bladder carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, melanoma, mantle cell lymphoma, myeloid leukemia, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheochromocytoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck.
  • aspects of the invention include methods where miR-16 or miR-16 inhibitor and miR-26 or miR-26 inhibitor are administered to patients with anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, lung carcinoma, melanoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, testicular tumor.
  • miR-16 or miR-16 inhibitor and miR-34 or miR-34 inhibitor are administered to patients with astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, laryngeal squamous cell carcinoma, melanoma, medulloblastoma, mantle cell lymphoma, myeloid leukemia, multiple myeloma, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheochromocytoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor
  • miR-16 or miR-16 inhibitor and miR-124 or miR-124 inhibitor are administered to patients with astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, laryngeal squamous cell carcinoma, melanoma, medulloblastoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor.
  • miR-16 or miR-16 inhibitor and miR-126 or miR-126 inhibitor are administered to patients with astrocytoma, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, melanoma, mantle cell lymphoma, myeloid leukemia, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheochromocytoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.
  • miR-16 or miR-16 inhibitor and miR-143 or miR-143 inhibitor are administered to patients with astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, melanoma, medulloblastoma, mantle cell lymphoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor.
  • astrocytoma anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, gli
  • miR-16 or miR-16 inhibitor and miR-147 or miR-147 inhibitor are administered to patients with astrocytoma, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma.
  • miR-16 or miR-16 inhibitor and miR-188 or miR-188 inhibitor are administered to patients with astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, lung carcinoma, melanoma, multiple myeloma, non- small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor.
  • miR-16 or miR-16 inhibitor and miR-200 or miR-200 inhibitor are administered to patients with anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, lung carcinoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor.
  • miR-16 or miR-16 inhibitor and miR-215 or miR-215 inhibitor are administered to patients with astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheochromocytoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor.
  • miR-16 or miR-16 inhibitor and miR-216 or miR-216 inhibitor are administered to patients with astrocytoma, breast carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, lung carcinoma, myeloid leukemia, neurofibroma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, prostate carcinoma, pheochromocytoma, squamous cell carcinoma of the head and neck, testicular tumor.
  • miR-16 or miR-16 inhibitor and miR-292-3p or miR-292-3p inhibitor are administered to patients with astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, lung carcinoma, laryngeal squamous cell carcinoma, melanoma, myxofibrosarcoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor.
  • miR-16 or miR-16 inhibitor and miR-331 or miR-331 inhibitor are administered to patients with astrocytoma, anaplastic large cell lymphoma, breast carcinoma, B-cell lymphoma, bladder carcinoma, cervical carcinoma, chronic lymphoblastic leukemia, colorectal carcinoma, endometrial carcinoma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, lung carcinoma, laryngeal squamous cell carcinoma, melanoma, myxofibrosarcoma, myeloid leukemia, multiple myeloma, neurofibroma, ovarian carcinoma, oesophageal carcinoma, pancreatic carcinoma, prostate carcinoma, pheochromocytoma, renal cell carcinoma, rhabdomyosarcoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, testicular tumor.
  • miR-16 or a miR-16 inhibitor when given in combination with one or more other miRNA molecules, the two different miRNAs or inhibitors may be given at the same time or sequentially.
  • therapy proceeds with one miRNA or inhibitor and that therapy is followed up with therapy with the other miRNA or inhibitor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or any such combination later.
  • RNA is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al, 2003, which is hereby incorporated by reference. The term can be used to refer to the single-stranded RNA molecule processed from a precursor or in certain instances the precursor itself or a mimetic thereof.
  • methods include assaying a cell or a sample containing a cell for the presence of one or more miRNA marker gene or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently, in some embodiments, methods include a step of generating an RNA profile for a sample.
  • RNA profile or "gene expression profile” refers to a set of data regarding the expression pattern for one or more gene or genetic marker in the sample ⁇ e.g., a plurality of nucleic acid probes that identify one or more markers or genes from the tables); it is contemplated that the nucleic acid profile can be obtained using a set of RNAs, using for example nucleic acid amplification or hybridization techniques well know to one of ordinary skill in the art.
  • the difference in the expression profile in the sample from a patient and a reference expression profile, such as an expression profile from a normal or non-pathologic sample, or a digitized reference, is indicative of a pathologic, disease, or cancerous condition.
  • the expression profile is an indicator of a propensity to or probability of ⁇ i.e., risk factor for a disease or condition) developing such a condition(s).
  • a risk or propensity may indicate a treatment, increased monitoring, prophylactic measures, and the like.
  • a nucleic acid or probe set may comprise or identify a segment of a corresponding mRNA and may include all or part of 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 100, 200, 500, or more segments, including any integer or range derivable there between, of a gene or genetic marker, or a nucleic acid, mRNA or a probe representative thereof that is listed in tables or identified by the methods described herein.
  • compositions and methods for assessing, prognosing, or treating a pathological condition in a patient comprising measuring or determining an expression profile of one or more miRNA or marker(s) in a sample from the patient, wherein a difference in the expression profile in the sample from the patient and an expression profile of a normal sample or reference expression profile is indicative of pathological condition and particularly cancer (e.g.,
  • the miRNAs, cellular pathway, gene, or genetic marker is or is representative of one or more pathway or marker described in the tables, including any combination thereof.
  • aspects of the invention include diagnosing, assessing, or treating a pathologic condition or preventing a pathologic condition from manifesting.
  • the methods can be used to screen for a pathological condition; assess prognosis of a pathological condition; stage a pathological condition; assess response of a pathological condition to therapy; or to modulate the expression of a gene, genes, or related pathway as a first therapy or to render a subject sensitive or more responsive to a second therapy.
  • assessing the pathological condition of the patient can be assessing prognosis of the patient. Prognosis may include, but is not limited to an estimation of the time or expected time of survival, assessment of response to a therapy, and the like.
  • the altered expression of one or more gene or marker is prognostic for a patient having a pathologic condition, wherein the marker is one or more of the tables, including any combination thereof.
  • kits containing compositions of the invention or compositions to implement methods of the invention.
  • kits can be used to evaluate one or more marker molecules, and/or express one or more miRNA or miRNA inhibitor.
  • a kit contains, contains at least or contains at most 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 100, 150, 200 or more probes, recombinant nucleic acid, or synthetic nucleic acid molecules related to the markers to be assessed or an miRNA or miRNA inhibitor to be expressed or modulated, and may include any range or combination derivable therein.
  • Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as Ix, 2x, 5x, 10x, or 2Ox or more. Kits for using probes, synthetic nucleic acids, recombinant nucleic acids, or non-synthetic nucleic acids of the invention for therapeutic, prognostic, or diagnostic applications are included as part of the invention.
  • negative and/or positive controls are included in some kit embodiments.
  • the control molecules can be used to verify transfection efficiency and/or control for transfection- induced changes in cells. .
  • kits for assessment of a pathological condition or the risk of developing a pathological condition in a patient by nucleic acid profiling of a sample comprising, in suitable container means, two or more nucleic acid hybridization or amplification reagents.
  • the kit can comprise reagents for labeling nucleic acids in a sample and/or nucleic acid hybridization reagents.
  • the hybridization reagents typically comprise hybridization probes.
  • Amplification reagents include, but are not limited to amplification primers, reagents, and enzymes.
  • an expression profile is generated by steps that include: (a) labeling nucleic acid in the sample; (b) hybridizing the nucleic acid to a number of probes, or amplifying a number of nucleic acids, and (c) determining and/or quantitating nucleic acid hybridization to the probes or detecting and quantitating amplification products, wherein an expression profile is generated.
  • Methods of the invention involve diagnosing and/or assessing the prognosis of a patient based on a miRNA and/or a marker nucleic acid expression profile.
  • the elevation or reduction in the level of expression of a particular gene or genetic pathway or set of nucleic acids in a cell is correlated with a disease state or pathological condition compared to the expression level of the same in a normal or non- pathologic cell or tissue sample. This correlation allows for diagnostic and/or prognostic methods to be carried out when the expression level of one or more nucleic acid is measured in a biological sample being assessed and then compared to the expression level of a normal or non-pathologic cell or tissue sample.
  • expression profiles for patients can be generated by evaluating any of or sets of the miRNAs and/or nucleic acids discussed in this application.
  • the expression profile that is generated from the patient will be one that provides information regarding the particular disease or condition.
  • the profile is generated using nucleic acid hybridization or amplification, (e.g., array hybridization or RT-PCR).
  • an expression profile can be used in conjunction with other diagnostic and/or prognostic tests, such as histology, protein profiles in the serum and/or cytogenetic assessment.
  • the methods can further comprise one or more of the steps including: (a) obtaining a sample from the patient, (b) isolating nucleic acids from the sample, (c) labeling the nucleic acids isolated from the sample, and (d) hybridizing the labeled nucleic acids to one or more probes.
  • Nucleic acids of the invention include one or more nucleic acid comprising at least one segment having a sequence or complementary sequence of to a nucleic acid representative of one or more of genes or markers in the tables.
  • any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. It is specifically contemplated that any methods and compositions discussed herein with respect to miRNA molecules, miRNA, genes and nucleic acids representative of genes may be implemented with respect to synthetic nucleic acids. In some embodiments the synthetic nucleic acid is exposed to the proper conditions to allow it to become a processed or mature nucleic acid, such as a miRNA under physiological circumstances.
  • the claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.
  • any embodiment of the invention involving specific genes (including representative fragments thereof), mRNA, or miRNAs by name is contemplated also to cover embodiments involving miRNAs whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the sequence or mature sequence of the specified miRNA, mRNA, gene, or representative nucleic acid.
  • miRNA members of a gene family are identified by a number following the initial designation.
  • miR-16-1 and miR-16-2 are members of the miR-16 gene family and "mir-7" refers to miR-7-1, miR-7-2 and miR-7-3.
  • a shorthand notation refers to related miRNAs (distinguished by a letter).
  • let-7 refers to let-7a, let-7b, let-7c, etc. Exceptions to this shorthand notation will be otherwise identified.
  • FIG. 1 Percent (%) proliferation of hsa-miR-16 treated cells relative to cells treated with negative control miRNA (100%).
  • Cell lines used include the prostate cancer cell lines PPC-I, Dul45 and RWPE2.
  • the present invention is directed to compositions and methods relating to the identification and characterization of genes and biological pathways related to these genes as represented by the expression of the identified genes, as well as use of miRNAs related to such, for therapeutic, prognostic, and diagnostic applications.
  • the present invention is directed to those methods and compositions related to assessing and/or identifying pathological conditions directly or indirectly related to miR-16 expression or the aberrant expression thereof.
  • the mature sequence of miR-16 is typically comprised of uagcagcacguaaauauuggcg SEQ ID NO:1 (MIMAT0000069).
  • the invention is directed to methods for the assessment, analysis, and/or therapy of a cell or subject where certain genes have a reduced expression (relative to normal) as a result of an increased or decreased expression of miR-16 and/or genes with an increased expression (relative to normal) as a result of an increased or decreased expression of miR-16.
  • the expression profile and/or response to miR-16 expression or lack of expression are indicative of an individual with a pathological condition, e.g., cancer.
  • Prognostic assays featuring any one or combination of the miRNAs listed or the markers listed (including nucleic acids representative thereof) could be used to assess a patient to determine what if any treatment regimen is justified.
  • the absolute values that define low expression will depend on the platform used to measure the miRNA(s). The same methods described for the diagnostic assays could be used for a prognostic assays.
  • Embodiments of the invention concern nucleic acids that perform the activities of or inhibit endogenous miRNAs when introduced into cells.
  • nucleic acids are synthetic or non-synthetic miRNA.
  • Sequence-specific miRNA inhibitors can be used to inhibit sequentially or in combination the activities of one or more endogenous miRNAs in cells, as well those genes and associated pathways modulated by the endogenous miRNA.
  • the present invention concerns, in some embodiments, short nucleic acid molecules that function as miRNAs or as inhibitors of miRNA in a cell.
  • short refers to a length of a single polynucleotide that is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, or 150 nucleotides or fewer, including all integers or ranges range derivable there between.
  • the nucleic acid molecules are typically synthetic.
  • synthetic refers to a nucleic acid molecule that is isolated and not produced naturally in a cell.
  • sequence deviates from a naturally- occurring nucleic acid molecule, such as an endogenous precursor miRNA or miRNA molecule or complement thereof.
  • nucleic acids of the invention do not have an entire sequence that is identical or complementary to a sequence of a naturally-occurring nucleic acid, such molecules may encompass all or part of a naturally- occurring sequence or a complement thereof. It is contemplated, however, that a synthetic nucleic acid administered to a cell may subsequently be modified or altered in the cell such that its structure or sequence is the same as non-synthetic or naturally occurring nucleic acid, such as a mature miRNA sequence.
  • a synthetic nucleic acid may have a sequence that differs from the sequence of a precursor miRNA, but that sequence may be altered once in a cell to be the same as an endogenous, processed miRNA or an inhibitor thereof.
  • isolated means that the nucleic acid molecules of the invention are initially separated from different (in terms of sequence or structure) and unwanted nucleic acid molecules such that a population of isolated nucleic acids is at least about 90% homogenous, and may be at least about 95, 96, 97, 98, 99, or 100% homogenous with respect to other polynucleotide molecules.
  • a nucleic acid is isolated by virtue of it having been synthesized in vitro separate from endogenous nucleic acids in a cell. It will be understood, however, that isolated nucleic acids may be subsequently mixed or pooled together.
  • synthetic miRNA of the invention are RNA or RNA analogs. miRNA inhibitors may be DNA or RNA, or analogs thereof. miRNA and miRNA inhibitors of the invention are collectively referred to as "synthetic nucleic acids.”
  • a miRNA or a synthetic miRNA having a length of between 17 and 130 residues.
  • the present invention concerns miRNA or synthetic miRNA molecules that are, are at least, or are at most 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105
  • synthetic miRNA have (a) a "miRNA region” whose sequence or binding region from 5' to 3' is identical or complementary to all or a segment of a mature miRNA sequence, and (b) a "complementary region” whose sequence from 5' to 3' is between 60% and 100% complementary to the miRNA sequence in (a).
  • these synthetic miRNA are also isolated, as defined above.
  • the term "miRNA region” refers to a region on the synthetic miRNA that is at least 75, 80, 85, 90, 95, or 100% identical, including all integers there between, to the entire sequence of a mature, naturally occurring miRNA sequence or a complement thereof.
  • the miRNA region is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to the sequence of a naturally-occurring miRNA or complement thereof.
  • complementary region refers to a region of a nucleic acid or mimetic that is or is at least 60% complementary to the mature, naturally occurring miRNA sequence.
  • the complementary region is or is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein.
  • the complementary region is on a different nucleic acid molecule than the miRNA region, in which case the complementary region is on the complementary strand and the miRNA region is on the active strand.
  • a miRNA inhibitor is between about 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature miRNA.
  • a miRNA inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein.
  • an miRNA inhibitor may have a sequence (from 5' to 3') that is or is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5' to 3' sequence of a mature miRNA, particularly a mature, naturally occurring miRNA.
  • One of skill in the art could use a portion of the miRNA sequence that is complementary to the sequence of a mature miRNA as the sequence for a miRNA inhibitor.
  • that portion of the nucleic acid sequence can be altered so that it is still comprises the appropriate percentage of complementarity to the sequence of a mature miRNA.
  • a synthetic miRNA or inhibitor contains one or more design element(s).
  • design elements include, but are not limited to: (i) a replacement group for the phosphate or hydroxyl of the nucleotide at the 5' terminus of the complementary region; (ii) one or more sugar modifications in the first or last 1 to 6 residues of the complementary region; or, (iii) noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3' end of the complementary region and the corresponding nucleotides of the miRNA region.
  • design modifications include, but are not limited to: (i) a replacement group for the phosphate or hydroxyl of the nucleotide at the 5' terminus of the complementary region; (ii) one or more sugar modifications in the first or last 1 to 6 residues of the complementary region; or, (iii) noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3' end of the complementary region and the corresponding nucleo
  • a synthetic miRNA has a nucleotide at its 5' end of the complementary region in which the phosphate and/or hydroxyl group has been replaced with another chemical group (referred to as the "replacement design").
  • the replacement design referred to as the "replacement design”.
  • the phosphate group is replaced, while in others, the hydroxyl group has been replaced.
  • the replacement group is biotin, an amine group, a lower alkylamine group, an acetyl group, 2'0-Me (2'oxygen-methyl), DMTO (4,4'-dimethoxytrityl with oxygen), fluoroscein, a thiol, or acridine, though other replacement groups are well known to those of skill in the art and can be used as well.
  • This design element can also be used with a miRNA inhibitor.
  • Additional embodiments concern a synthetic miRNA having one or more sugar modifications in the first or last 1 to 6 residues of the complementary region (referred to as the "sugar replacement design").
  • sugar modifications in the first 1, 2, 3, 4, 5, 6 or more residues of the complementary region, or any range derivable therein there are one or more sugar modifications in the last 1, 2, 3, 4, 5, 6 or more residues of the complementary region, or any range derivable therein, have a sugar modification.
  • first and “last” are with respect to the order of residues from the 5' end to the 3' end of the region.
  • the sugar modification is a 2'0-Me modification, a 2 'F modification, a 2'H modification, a 2 'amino modification, a 4'thioribose modification or a phosphorothioate modification on the carboxy group linked to the carbon at position 6'.
  • This design element can also be used with a miRNA inhibitor.
  • a miRNA inhibitor can have this design clement and/or a replacement group on the nucleotide at the 5' terminus, as discussed above.
  • noncomplementarity design there is a synthetic miRNA or inhibitor in which one or more nucleotides in the last 1 to 5 residues at the 3 ' end of the complementary region are not complementary to the corresponding nucleotides of the miRNA region.
  • the noncomplementarity may be in the last 1, 2, 3, 4, and/or 5 residues of the complementary miRNA.
  • synthetic miRNA of the invention have one or more of the replacement, sugar modification, or noncomplementarity designs.
  • synthetic RNA molecules have two of them, while in others these molecules have all three designs in place.
  • the miRNA region and the complementary region may be on the same or separate polynucleotides. In cases in which they are contained on or in the same polynucleotide, the miRNA molecule will be considered a single polynucleotide. In embodiments in which the different regions are on separate polynucleotides, the synthetic miRNA will be considered to be comprised of two polynucleotides.
  • the RNA molecule is a single polynucleotide
  • the single polynucleotide is capable of forming a hairpin loop structure as a result of bonding between the miRNA region and the complementary region.
  • the linker constitutes the hairpin loop. It is contemplated that in some embodiments, the linker region is, is at least, or is 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, or 40 residues in length, or any range derivable therein. In certain embodiments, the linker is between 3 and 30 residues (inclusive) in length.
  • flanking sequences as well at either the 5' or 3' end of the region.
  • Methods of the invention include reducing or eliminating activity of one or more miRNAs in a cell comprising introducing into a cell a miRNA inhibitor (which may be described generally herein as an miRNA, so that a description of miRNA, where appropriate, also will refer to a miRNA inhibitor); or supplying or enhancing the activity of one or more miRNAs in a cell.
  • a miRNA inhibitor which may be described generally herein as an miRNA, so that a description of miRNA, where appropriate, also will refer to a miRNA inhibitor
  • the present invention also concerns inducing certain cellular characteristics by providing to a cell a particular nucleic acid, such as a specific synthetic miRNA molecule or a synthetic miRNA inhibitor molecule.
  • the miRNA molecule or miRNA inhibitor need not be synthetic. They may have a sequence that is identical to a naturally occurring miRNA or they may not have any design modifications.
  • the miRNA molecule and/or the miRNA inhibitor are synthetic, as discussed above.
  • the particular nucleic acid molecule provided to the cell is understood to correspond to a particular miRNA in the cell, and thus, the miRNA in the cell is referred to as the "corresponding miRNA.”
  • the corresponding miRNA will be understood to be the induced or inhibited miRNA or induced or inhibited miRNA function. It is contemplated, however, that the miRNA molecule introduced into a cell is not a mature miRNA but is capable of becoming or functioning as a mature miRNA under the appropriate physiological conditions.
  • the particular miRNA will be referred to as the "targeted miRNA.” It is contemplated that multiple corresponding miRNAs may be involved. In particular embodiments, more than one miRNA molecule is introduced into a cell. Moreover, in other embodiments, more than one miRNA inhibitor is introduced into a cell. Furthermore, a combination of miRNA molecule(s) and miRNA inhibitor(s) may be introduced into a cell. The inventors contemplate that a combination of miRNA may act at one or more points in cellular pathways of cells with aberrant phenotypes and that such combination may have increased efficacy on the target cell while not adversely effecting normal cells.
  • a combination of miRNA may have a minimal adverse effect on a subject or patient while supplying a sufficient therapeutic effect, such as amelioration of a condition, growth inhibition of a cell, death of a targeted cell, alteration of cell phenotype or physiology, slowing of cellular growth, sensitization to a second therapy, sensitization to a particular therapy, and the like.
  • Methods include identifying a cell or patient in need of inducing those cellular characteristics. Also, it will be understood that an amount of a synthetic nucleic acid that is provided to a cell or organism is an "effective amount,” which refers to an amount needed (or a sufficient amount) to achieve a desired goal, such as inducing a particular cellular characteristic(s). [00101] In certain embodiments of the methods include providing or introducing to a cell a nucleic acid molecule corresponding to a mature miRNA in the cell in an amount effective to achieve a desired physiological result.
  • methods can involve providing synthetic or nonsynthetic miRNA molecules. It is contemplated that in these embodiments, that the methods may or may not be limited to providing only one or more synthetic miRNA molecules or only one or more nonsynthetic miRNA molecules. Thus, in certain embodiments, methods may involve providing both synthetic and nonsynthetic miRNA molecules. In this situation, a cell or cells are most likely provided a synthetic miRNA molecule corresponding to a particular miRNA and a nonsynthetic miRNA molecule corresponding to a different miRNA. Furthermore, any method articulated using a list of miRNAs using Markush group language may be articulated without the Markush group language and a disjunctive article (i.e., or) instead, and vice versa.
  • a method for reducing or inhibiting cell proliferation comprising introducing into or providing to the cell an effective amount of (i) a miRNA inhibitor molecule or (ii) a synthetic or nonsynthetic miRNA molecule that corresponds to a miRNA sequence.
  • the methods involves introducing into the cell an effective amount of (i) an miRNA inhibitor molecule having a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of one or more mature miRNA.
  • Certain embodiments of the invention include methods of treating a pathologic condition, in particular cancer, e.g., lung or liver cancer.
  • the method comprises contacting a target cell with one or more nucleic acid, synthetic miRNA, or miRNA comprising at least one nucleic acid segment having all or a portion of a miRNA sequence.
  • the segment may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides or nucleotide analog, including all integers there between.
  • An aspect of the invention includes the modulation of gene expression, miRNA expression or function or mRNA expression or function within a target cell, such as a cancer cell.
  • an endogenous gene, miRNA or mRNA is modulated in the cell.
  • the nucleic acid sequence comprises at least one segment that is at least 70, 75, 80, 85, 90, 95, or 100% identical in nucleic acid sequence to one or more miRNA or gene sequence.
  • Modulation of the expression or processing of an endogenous gene, miRNA, or mRNA can be through modulation of the processing of a mRNA, such processing including transcription, transportation and/or translation with in a cell. Modulation may also be effected by the inhibition or enhancement of miRNA activity with a cell, tissue, or organ. Such processing may affect the expression of an encoded product or the stability of the mRNA.
  • a nucleic acid sequence can comprise a modified nucleic acid sequence.
  • one or more miRNA sequence may include or comprise a modified nucleobase or nucleic acid sequence.
  • a cell or other biological matter such as an organism (including patients) can be provided a miRNA or miRNA molecule corresponding to a particular miRNA by administering to the cell or organism a nucleic acid molecule that functions as the corresponding miRNA once inside the cell.
  • the form of the molecule provided to the cell may not be the form that acts a miRNA once inside the cell.
  • a synthetic miRNA or a nonsynthetic miRNA is provided a synthetic miRNA or a nonsynthetic miRNA, such as one that becomes processed into a mature and active miRNA once it has access to the cell's miRNA processing machinery.
  • the miRNA molecule provided to the biological matter is not a mature miRNA molecule but a nucleic acid molecule that can be processed into the mature miRNA once it is accessible to miRNA processing machinery.
  • nonsynthetic in the context of miRNA means that the miRNA is not “synthetic,” as defined herein.
  • the use of corresponding nonsynthetic miRNAs is also considered an aspect of the invention, and vice versa. It will be understand that the term “providing” an agent is used to include “administering" the agent to a patient.
  • methods also include targeting a miRNA to modulate in a cell or organism.
  • targeting a miRNA to modulate means a nucleic acid of the invention will be employed so as to modulate the selected miRNA.
  • the modulation is achieved with a synthetic or non-synthetic miRNA that corresponds to the targeted miRNA, which effectively provides the targeted miRNA to the cell or organism (positive modulation).
  • the modulation is achieved with a miRNA inhibitor, which effectively inhibits the targeted miRNA in the cell or organism (negative modulation).
  • the miRNA targeted to be modulated is a miRNA that affects a disease, condition, or pathway.
  • the miRNA is targeted because a treatment can be provided by negative modulation of the targeted miRNA.
  • the miRNA is targeted because a treatment can be provided by positive modulation of the targeted miRNA or its targets.
  • a further step of administering the selected miRNA modulator to a cell, tissue, organ, or organism in need of treatment related to modulation of the targeted miRNA or in need of the physiological or biological results discussed herein (such as with respect to a particular cellular pathway or result like decrease in cell viability). Consequently, in some methods of the invention there is a step of identifying a patient in need of treatment that can be provided by the miRNA modulator(s). It is contemplated that an effective amount of a miRNA modulator can be administered in some embodiments.
  • a therapeutic benefit refers to an improvement in the one or more conditions or symptoms associated with a disease or condition or an improvement in the prognosis, duration, or status with respect to the disease. It is contemplated that a therapeutic benefit includes, but is not limited to, a decrease in pain, a decrease in morbidity, a decrease in a symptom.
  • a therapeutic benefit can be inhibition of tumor growth, prevention of metastasis, reduction in number of metastases, inhibition of cancer cell proliferation, induction of cell death in cancer cells, inhibition of angiogenesis near cancer cells, induction of apoptosis of cancer cells, reduction in pain, reduction in risk of recurrence, induction of chemo- or radiosensitivity in cancer cells, prolongation of life, and/or delay of death directly or indirectly related to cancer.
  • the miRNA compositions may be provided as part of a therapy to a patient, in conjunction with traditional therapies or preventative agents.
  • any method discussed in the context of therapy may be applied as preventatively, particularly in a patient identified to be potentially in need of the therapy or at risk of the condition or disease for which a therapy is needed.
  • methods of the invention concern employing one or more nucleic acids corresponding to a miRNA and a therapeutic drug.
  • the nucleic acid can enhance the effect or efficacy of the drug, reduce any side effects or toxicity, modify its bioavailability, and/or decrease the dosage or frequency needed.
  • the therapeutic drug is a cancer therapeutic. Consequently, in some embodiments, there is a method of treating cancer in a patient comprising administering to the patient the cancer therapeutic and an effective amount of at least one miRNA molecule that improves the efficacy of the cancer therapeutic or protects non-cancer cells.
  • Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments.
  • Combination chemotherapies include but are not limited to, for example, 5-fluorouracil, alemtuzumab, amrubicin, bevacizumab, bleomycin, bortezomib, busulfan, camptothecin, capecitabine, cisplatin (CDDP), carboplatin, cetuximab, chlorambucil, cisplatin (CDDP), EGFR inhibitors (gefitinib and cetuximab), procarbazine, mechlorethamine, cyclophosphamide, camptothecin, COX-2 inhibitors (e.g., celecoxib), cyclophosphamide, cytarabine, ) ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, dasatinib, daunorubicin, dexamethasone, docetaxel, doxorubicin (
  • inhibitors of miRNAs can be given to decrease the activity of an endogenous miRNA.
  • inhibitors of miRNA molecules that increase cell proliferation can be provided to cells to increase proliferation or inhibitors of such molecules can be provided to cells to decrease cell proliferation.
  • the present invention contemplates these embodiments in the context of the different physiological effects observed with the different miRNA molecules and miRNA inhibitors disclosed herein.
  • Methods of the invention include providing or introducing one or more different nucleic acid molecules corresponding to one or more different miRNA molecules.
  • nucleic acid or miRNA molecules may be provided or introduced: 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range derivable therein. This also applies to the number of different miRNA molecules. This also applies to the number of different miRNA molecules. This also applies to the number
  • Methods of the present invention include the delivery of an effective amount of a miRNA or an expression construct encoding the same.
  • An "effective amount" of the pharmaceutical composition generally, is defined as that amount sufficient to detectably and repeatedly to achieve the stated desired result, for example, to ameliorate, reduce, minimize or limit the extent of the disease or its symptoms. Other more rigorous definitions may apply, including elimination, eradication or cure of disease.
  • the routes of administration will vary, naturally, with the location and nature of the lesion or site to be targeted, and include, e.g., intradermal, subcutaneous, regional, parenteral, intravenous, intramuscular, intranasal, systemic, and oral administration and formulation. Direct injection, intratumoral injection, or injection into tumor vasculature is specifically contemplated for discrete, solid, accessible tumors, or other accessible target areas. Local, regional, or systemic administration also may be appropriate. For tumors of >4 cm, the volume to be administered will be about 4-10 ml (preferably 10 ml), while for tumors of ⁇ 4 cm, a volume of about 1-3 ml will be used (preferably 3 ml).
  • compositions of the invention may be administered in multiple injections to a tumor or a targeted site. In certain aspects, injections may be spaced at approximately 1 cm intervals.
  • the present invention may be used preoperatively, to render an inoperable tumor subject to resection.
  • the present invention may be used at the time of surgery, and/or thereafter, to treat residual or metastatic disease.
  • a resected tumor bed may be injected or perfused with a formulation comprising a miRNA or combinations thereof.
  • Administration may be continued post- resection, for example, by leaving a catheter implanted at the site of the surgery. Periodic post-surgical treatment also is envisioned. Continuous perfusion of an expression construct or a viral construct also is contemplated.
  • Continuous administration also may be applied where appropriate, for example, where a tumor or other undesired affected area is excised and the tumor bed or targeted site is treated to eliminate residual, microscopic disease. Delivery via syringe or catherization is contemplated. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.
  • Treatment regimens may vary as well and often depend on tumor type, tumor location, immune condition, target site, disease progression, and health and age of the patient. Certain tumor types will require more aggressive treatment. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations.
  • the tumor or affected area being treated may not, at least initially, be resectable.
  • Treatments with compositions of the invention may increase the resectability of the tumor due to shrinkage at the margins or by elimination of certain particularly invasive portions. Following treatments, resection may be possible. Additional treatments subsequent to resection may serve to eliminate microscopic residual disease at the tumor or targeted site.
  • Treatments may include various "unit doses.”
  • a unit dose is defined as containing a predetermined quantity of a therapeutic composition(s). The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. With respect to a viral component of the present invention, a unit dose may conveniently be described in terms of ⁇ g or mg of miRNA or miRNA mimetic. Alternatively, the amount specified may be the amount administered as the average daily, average weekly, or average monthly dose.
  • miRNA can be administered to the patient in a dose or doses of about or of at least about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 800, 810, 820, 820, 810, 810
  • the amount specified may be the amount administered as the average daily, average weekly, or average monthly dose, or it may be expressed in terms of mg/kg, where kg refers to the weight of the patient and the mg is specified above. In other embodiments, the amount specified is any number discussed above but expressed as mg/m 2 (with respect to tumor size or patient surface area).
  • the method for the delivery of a miRNA or an expression construct encoding such or combinations thereof is via systemic administration.
  • the pharmaceutical compositions disclosed herein may also be administered parenterally, subcutaneously, directly, intratracheally, intravenously, intradermally, intramuscularly, or even intraperitoneally as described in U.S. Patents 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • nucleic acids may be delivered by syringe or any other method used for injection of a solution, as long as the nucleic acid and any associated components can pass through the particular gauge of needle required for injection.
  • a syringe system has also been described for use in gene therapy that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Patent 5,846,225).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and in oils.
  • compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • sterile aqueous solutions or dispersions include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • a water-based formulation is employed while in others, it may be lipid-based.
  • a composition comprising a tumor suppressor protein or a nucleic acid encoding the same is in a water-based formulation.
  • the formulation is lipid based.
  • aqueous solutions for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, intralesional, and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologies standards.
  • a “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the nucleic acid(s) are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the aggressiveness of the disease or cancer, the size of any tumor(s) or lesions, the previous or other courses of treatment. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and subsequent administration are also variable, but are typified by an initial administration followed by other administrations.
  • Such administration may be systemic, as a single dose, continuous over a period of time spanning 10, 20, 30, 40, 50, 60 minutes, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and/or 1, 2, 3, 4, 5, 6, 7, days or more.
  • administration may be through a time release or sustained release mechanism, implemented by formulation and/or mode of administration.
  • compositions and methods of the present invention involve a miRNA, or expression construct encoding such.
  • miRNA compositions can be used in combination with a second therapy to enhance the effect of the miRNA therapy, or increase the therapeutic effect of another therapy being employed.
  • These compositions would be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with the miRNA or second therapy at the same or different time.
  • compositions or pharmacological formulation that includes or more of the agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition provides (1) miRNA; and/or (2) a second therapy.
  • a second composition or method may be administered that includes a chemotherapy, radiotherapy, surgical therapy, immunotherapy, or gene therapy.
  • a course of treatment will last 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more.
  • one agent may be given on day 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any combination thereof, and another agent is given on day 1, 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,
  • the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no treatment is administered. This time period may last 1 , 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as their prognosis, strength, health, etc.
  • miRNA therapy is “A” and a second therapy is “B”:
  • Administration of any compound or therapy of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the vector or any protein or other agent. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
  • a second therapy such as chemotherapy, radiotherapy, immunotherapy, surgical therapy or other gene therapy, is employed in combination with the miRNA therapy, as described herein.
  • chemotherapeutic agents may be used in accordance with the present invention.
  • the term "chemotherapy” refers to the use of drugs to treat cancer.
  • a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
  • Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific.
  • Alkylating agents can be implemented to treat chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and particular cancers of the breast, lung, and ovary. They include: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan. Troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents.
  • Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. They have been used to combat chronic leukemias in addition to tumors of breast, ovary and the gastrointestinal tract. Antimetabolites include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.
  • 5-fluorouracil 5-FU
  • cytarabine Ara-C
  • fludarabine gemcitabine
  • gemcitabine methotrexate
  • 5-Fluorouracil has the chemical name of 5-fluoro-2,4(lH,3H)- pyrimidinedione. Its mechanism of action is thought to be by blocking the methylation reaction of deoxyuridylic acid to thymidylic acid. Thus, 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and proliferation, it is thought that the effect of 5-FU is to create a thymidine deficiency leading to cell death. Thus, the effect of 5-FU is found in cells that rapidly divide, a characteristic of metastatic cancers.
  • Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers. Examples of antitumor antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), and idarubicin, some of which are discussed in more detail below.
  • these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m at 21 day intervals for adriamycin, to 35- 100 mg/m for etoposide intravenously or orally.
  • Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors comprise docetaxel, etoposide (VP 16), paclitaxel, taxol, taxotere, vinblastine, vincristine, and vinorelbine.
  • Nitrosureas like alkylating agents, inhibit DNA repair proteins. They are used to treat non-Hodgkin's lymphomas, multiple myeloma, malignant melanoma, in addition to brain tumors. Examples include carmustine and lomustine.
  • Radiotherapy also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly. Radiotherapy may be used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, or cervix. It can also be used to treat leukemia and lymphoma (cancers of the blood-forming cells and lymphatic system, respectively).
  • Radiation therapy used according to the present invention may include, but is not limited to, the use of ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmuno therapy). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.
  • Stereotactic radio-surgery for brain and other tumors does not use a knife, but very precisely targeted beams of gamma radiotherapy from hundreds of different angles. Only one session of radiotherapy, taking about four to five hours, is needed. For this treatment a specially made metal frame is attached to the head. Then, several scans and x- rays are carried out to find the precise area where the treatment is needed.
  • the patient lies with their head in a large helmet, which has hundreds of holes in it to allow the radiotherapy beams through.
  • Related approaches permit positioning for the treatment of tumors in other areas of the body.
  • immunotherapeutics In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Trastuzumab (HerceptinTM) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.
  • the tumor or disease cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), g ⁇ 68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL- 12, GM-CSF, gamma-IFN, and chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand.
  • cytokines such as IL-2, IL-4, IL- 12, GM-CSF, gamma-IFN, and chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand.
  • MDA-7 tumor suppressor
  • antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.
  • immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998), cytokine therapy e.g., interferons ⁇ , ⁇ and ⁇ ; IL-I, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998) gene therapy e.g., TNF, IL-I, IL-2, p53 (Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S.
  • immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds
  • cytokine therapy e.g.
  • Herceptin is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It possesses anti-tumor activity and has been approved for use in the treatment of malignant tumors (Dillman, 1999).
  • a non-limiting list of several known anti-cancer immunotherapeutic agents and their targets includes, but is not limted to (Generic Name (Target)) Cetuximab (EGFR), Panitumumab (EGFR), Trastuzumab (erbB2 receptor), Bevacizumab (VEGF), Alemtuzumab (CD52), Gemtuzumab ozogamicin (CD33), Rituximab (CD20), Tositumomab (CD20), Matuzumab (EGFR), Ibritumomab tiuxetan (CD20), Tositumomab (CD20), HuP AM4 (MUCl), MORAb-009 (Mesothelin), G250 (carbonic anhydrase IX), mAb 8H9 (8H9 antigen), Ml 95 (CD33), Ipilimumab (CTLA4), HuLuc63 (CSl), Alemtuzumab (CD53
  • a combination treatment involves gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as one or more therapeutic miRNA. Delivery of a therapeutic polypeptide or encoding nucleic acid in conjunction with a miRNA may have a combined therapeutic effect on target tissues.
  • a variety of proteins are encompassed within the invention, some of which are described below.
  • Various genes that may be targeted for gene therapy of some form in combination with the present invention include, but are not limited to inducers of cellular proliferation, inhibitors of cellular proliferation, regulators of programmed cell death, cytokines and other therapeutic nucleic acids or nucleic acid that encode therapeutic proteins.
  • the tumor suppressor oncogenes function to inhibit excessive cellular proliferation.
  • the inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation.
  • the tumor suppressors e.g., therapeutic polypeptides
  • p53, FHIT, pi 6 and C-CAM can be employed.
  • CDK cyclin-dependent kinases
  • CDK4 cyclin-dependent kinase 4
  • the activity of this enzyme may be to phosphorylate Rb at late Gl.
  • the activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pl6INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et ah, 1993; Serrano et al, 1995). Since the pl6INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6.
  • pl6INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes p!6B, pi 9, ⁇ 2 IWAFl, and p27KIPl .
  • the pl6INK4 gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pl6INK4 gene are frequent in human tumor cell lines. This evidence suggests that the pl6INK4 gene is a tumor suppressor gene.
  • genes that may be employed according to the present invention include Rb, APC, DCC, NF-I, NF-2, WT-I, MEN-I, MEN-II, zacl, p73, VHL, MMACl / PTEN, DBCCR-I, FCC, rsk-3, p27, p27/pl6 fusions, p21/p27 fusions, antithrombotic genes (e.g., COX-I, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fins, trk, ret, gsp, hst, abl, ElA, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-I, GDAIF, or their receptors) and MCC.
  • angiogenesis e.g., VEGF, FGF, thrombospondin
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy.
  • Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • These treatments may be of varying dosages as well.
  • agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment.
  • additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents.
  • Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-I, MIP-lbeta, MCP-I, RANTES, and other chemokines.
  • cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
  • Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. TRAIL activates rapid apoptosis in many types of cancer cells, yet is not toxic to normal cells. TRAIL mRNA occurs in a wide variety of tissues. Most normal cells appear to be resistant to TRAIL'S cytotoxic action, suggesting the existence of mechanisms that can protect against apoptosis induction by TRAIL. The first receptor described for TRAIL, called death receptor 4 (DR4), contains a cytoplasmic "death domain"; DR4 transmits the apoptosis signal carried by TRAIL. Additional receptors have been identified that bind to TRAIL.
  • DR4 death receptor 4
  • DR5 One receptor, called DR5, contains a cytoplasmic death domain and signals apoptosis much like DR4.
  • the DR4 and DR5 mRNAs are expressed in many normal tissues and tumor cell lines.
  • decoy receptors such as DcRl and DcR2 have been identified that prevent TRAIL from inducing apoptosis through DR4 and DR5.
  • These decoy receptors thus represent a novel mechanism for regulating sensitivity to a pro- apoptotic cytokine directly at the cell's surface.
  • the preferential expression of these inhibitory receptors in normal tissues suggests that TRAIL may be useful as an anticancer agent that induces apoptosis in cancer cells while sparing normal cells. (Marsters et al, 1999).
  • hyperthermia is a procedure in which a patient's tissue is exposed to high temperatures (up to 106 0 F).
  • External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia.
  • Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.
  • a patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets.
  • some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated.
  • Whole-body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.
  • Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described.
  • the use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
  • miRNAs are generally 21 to 22 nucleotides in length, though lengths of 19 and up to 23 nucleotides have been reported.
  • the miRNAs are each processed from a longer precursor RNA molecule ("precursor miRNA").
  • Precursor miRNAs are transcribed from non-protein-encoding genes.
  • the precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back-like structure, which is cleaved in animals by a ribonuclease Ill-like nuclease enzyme called Dicer.
  • the processed miRNA is typically a portion of the stem.
  • the processed miRNA (also referred to as "mature miRNA”) becomes part of a large complex to down-regulate a particular target gene or its gene product.
  • animal miRNAs include those that imperfectly basepair with the target, which halts translation (Olsen et ah, 1999; Seggerson et ah, 2002).
  • siRNA molecules also are processed by Dicer, but from a long, double-stranded RNA molecule. siRNAs are not naturally found in animal cells, but they can direct the sequence-specific cleavage of an mRNA target through a RNA-induced silencing complex (RISC) (Denli et ah, 2003).
  • RISC RNA-induced silencing complex
  • Certain embodiments of the present invention concerns the preparation and use of mRNA or nucleic acid arrays, miRNA or nucleic acid arrays, and/or miRNA or nucleic acid probe arrays, which are macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary (over the length of the prove) or identical (over the length of the prove) to a plurality of nucleic acid, mRNA or miRNA molecules, precursor miRNA molecules, or nucleic acids derived from the various genes and gene pathways modulated by miR-16 miRNAs and that are positioned on a support or support material in a spatially separated organization.
  • Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
  • Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
  • Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of marker RNA and/or miRNA-complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.
  • a variety of different array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art.
  • Useful substrates for arrays include nylon, glass, metal, plastic, latex, and silicon.
  • Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.
  • the labeling and screening methods of the present invention and the arrays are not limited in its utility with respect to any parameter except that the probes detect miRNA, or genes or nucleic acid representative of genes; consequently, methods and compositions may be used with a variety of different types of nucleic acid arrays.
  • the arrays can be high density arrays, such that they contain 2, 20, 25, 50, 80, 100 or more different probes. It is contemplated that they may contain 1000, 16,000, 65,000, 250,000 or 1,000,000 or more different probes.
  • the probes can be directed to mRNA and/or miRNA targets in one or more different organisms or cell types.
  • the oligonucleotide probes range from 5 to 50, 5 to 45, 10 to 40, 9 to 34, or 15 to 40 nucleotides in length in some embodiments. In certain embodiments, the oligonucleotide probes are 5, 10, 15, 20 to 20, 25, 30, 35, 40 nucleotides in length including all integers and ranges there between.
  • each different probe sequence in the array are generally known. Moreover, the large number of different probes can occupy a relatively small area providing a high density array having a probe density of generally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes per cm 2 .
  • the surface area of the array can be about or less than about 1, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm 2 .
  • RNA and/or miRNA of a wide variety of samples can be analyzed using the arrays, index of probes, or array technology of the invention. While endogenous miRNA is contemplated for use with compositions and methods of the invention, recombinant miRNA - including nucleic acids that are complementary or identical to endogenous miRNA or precursor miRNA - can also be handled and analyzed as described herein. Samples may be biological samples, in which case, they can be from biopsy, fine needle aspirates, exfoliates, blood, tissue, organs, semen, saliva, tears, other bodily fluid, hair follicles, skin, or any sample containing or constituting biological cells, particularly cancer or hyperproliferative cells.
  • samples may be, but are not limited to, biopsy, or cells purified or enriched to some extent from a biopsy or other bodily fluids or tissues.
  • the sample may not be a biological sample, but be a chemical mixture, such as a cell-free reaction mixture (which may contain one or more biological enzymes).
  • the population of target nucleic acids is contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed.
  • Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001) and WO 95/21944. Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.
  • a single array or set of probes may be contacted with multiple samples.
  • the samples may be labeled with different labels to distinguish the samples.
  • a single array can be contacted with a tumor tissue sample labeled with Cy3, and normal tissue sample labeled with Cy5. Differences between the samples for particular miRNAs corresponding to probes on the array can be readily ascertained and quantified.
  • the small surface area of the array permits uniform hybridization conditions, such as temperature regulation and salt content. Moreover, because of the small area occupied by the high density arrays, hybridization may be carried out in extremely small fluid volumes ⁇ e.g., about 250 ⁇ l or less, including volumes of about or less than about 5, 10, 25, 50, 60, 70, 80, 90, 100 ⁇ l, or any range derivable therein). In small volumes, hybridization may proceed very rapidly.
  • Arrays of the invention can be used to detect differences between two samples. Specifically contemplated applications include identifying and/or quantifying differences between miRNA or gene expression from a sample that is normal and from a sample that is not normal, between a disease or condition and a cell not exhibiting such a disease or condition, or between two differently treated samples. Also, miRNA or gene expression may be compared between a sample believed to be susceptible to a particular disease or condition and one believed to be not susceptible or resistant to that disease or condition. A sample that is not normal is one exhibiting phenotypic or genotypic trait(s) of a disease or condition, or one believed to be not normal with respect to that disease or condition. It may be compared to a cell that is normal with respect to that disease or condition. Phenotypic traits include symptoms of, or susceptibility to, a disease or condition of which a component is or may or may not be genetic, or caused by a hyperproliferative or neoplastic cell or cells.
  • An array comprises a solid support with nucleic acid probes attached to the support.
  • Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations.
  • These arrays also described as “microarrays” or colloquially “chips” have been generally described in the art, for example, U.S. Patents 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al, (1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S.
  • Patent 5,384,261 incorporated herein by reference in its entirety for all purposes.
  • the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces.
  • Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Patents 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated in their entirety for all purposes.
  • Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all inclusive device, see for example, U.S.
  • Patents 5,856,174 and 5,922,591 incorporated in their entirety by reference for all purposes. See also U.S. patent application Ser. No. 09/545,207, filed April, 7, 2000 for additional information concerning arrays, their manufacture, and their characteristics, which is incorporated by reference in its entirety for all purposes.
  • arrays can be used to evaluate samples with respect to pathological condition such as cancer and related conditions. It is specifically contemplated that the invention can be used to evaluate differences between stages or sub-classifications of disease, such as between benign, cancerous, and metastatic tissues or tumors.
  • Phenotypic traits to be assessed include characteristics such as longevity, morbidity, expected survival, susceptibility or receptivity to particular drugs or therapeutic treatments (drug efficacy), and risk of drug toxicity. Samples that differ in these phenotypic traits may also be evaluated using the compositions and methods described.
  • miRNA and/or expression profiles may be generated to evaluate and correlate those profiles with pharmacokinetics or therapies. For example, these profiles may be created and evaluated for patient tumor and blood samples prior to the patient's being treated or during treatment to determine if there are miRNA or genes whose expression correlates with the outcome of the patient's treatment.
  • Identification of differential miRNAs or genes can lead to a diagnostic assay for evaluation of tumor and/or blood samples to determine what drug regimen the patient should be provided. In addition, it can be used to identify or select patients suitable for a particular clinical trial. If an expression profile is determined to be correlated with drug efficacy or drug toxicity, that profile is relevant to whether that patient is an appropriate patient for receiving a drug, for receiving a combination of drugs, or for receiving a particular dosage of the drug.
  • samples from patients with a variety of diseases can be evaluated to determine if different diseases can be identified based on miRNA and/or related gene expression levels.
  • a diagnostic assay can be created based on the profiles that doctors can use to identify individuals with a disease or who are at risk to develop a disease.
  • treatments can be designed based on miRNA profiling. Examples of such methods and compositions are described in the U.S. Provisional Patent Application entitled "Methods and Compositions Involving miRNA and miRNA Inhibitor Molecules" filed on May 23, 2005 in the names of David Brown, Lance Ford, Angie Cheng and Rich Jarvis, which is hereby incorporated by reference in its entirety.
  • assays include, but are not limited to, nucleic acid amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Bridge Litigation Assay (Genaco).
  • HPA hybridization protection assay
  • bDNA branched DNA
  • RCA rolling circle amplification
  • US Genomics Invader assay
  • GTWave Technologies Three ave Technologies
  • Bridge Litigation Assay Geneaco
  • the present invention concerns nucleic acids, modified or mimetic nucleic acids, miRNAs, mRNAs, genes, and representative fragments thereof that can be labeled, used in array analysis, or employed in diagnostic, therapeutic, or prognostic applications, particularly those related to pathological conditions such as cancer.
  • the molecules may have been endogenously produced by a cell, or been synthesized or produced chemically or recombinantly. They may be isolated and/or purified.
  • Each of the miRNAs described herein and includes the corresponding SEQ ID NO and accession numbers for these miRNA sequences.
  • the name of a miRNA is often abbreviated and referred to without a "hsa-" prefix and will be understood as such, depending on the context.
  • miRNAs referred to in the application are human sequences identified as miR-X or let-X, where X is a number and/or letter.
  • a miRNA probe designated by a suffix "5P” or “3P” can be used.
  • “5P” indicates that the mature miRNA derives from the 5' end of the precursor and a corresponding "3P” indicates that it derives from the 3' end of the precursor, as described on the world wide web at sanger.ac.uk.
  • a miRNA probe is used that does not correspond to a known human miRNA. It is contemplated that these non- human miRNA probes may be used in embodiments of the invention or that there may exist a human miRNA that is homologous to the non-human miRNA. In other embodiments, any mammalian cell, biological sample, or preparation thereof may be employed.
  • methods and compositions involving miRNA may concern miRNA, markers (e.g., mRNAs), and/or other nucleic acids.
  • Nucleic acids may be, be at least, or be at most 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98
  • miRNA are 19-24 nucleotides in length, while miRNA probes are 19-35 nucleotides in length, depending on the length of the processed miRNA and any flanking regions added. miRNA precursors are generally between 62 and 110 nucleotides in humans.
  • Nucleic acids of the invention may have regions of identity or complementarity to another nucleic acid. It is contemplated that the region of complementarity or identity can be at least 5 contiguous residues, though it is specifically contemplated that the region is, is at least, or is at most 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
  • complementarity within a precursor miRNA or other nucleic acid or between a miRNA probe and a miRNA or a miRNA gene are such lengths.
  • the complementarity may be expressed as a percentage, meaning that the complementarity between a probe and its target is 90% or greater over the length of the probe. In some embodiments, complementarity is or is at least 90%, 95% or 100%.
  • such lengths may be applied to any nucleic acid comprising a nucleic acid sequence identified in any of SEQ ID NOs described herein, accession number, or any other sequence disclosed herein.
  • miRNA probe refers to a nucleic acid probe that can identify a particular miRNA or structurally related miRNAs.
  • nucleic acids are derived from genomic sequences or a gene.
  • the term "gene” is used for simplicity to refer to the genomic sequence encoding the precursor nucleic acid or miRNA for a given miRNA or gene.
  • embodiments of the invention may involve genomic sequences of a miRNA that are involved in its expression, such as a promoter or other regulatory sequences.
  • the term "recombinant” may be used and this generally refers to a molecule that has been manipulated in vitro or that is a replicated or expressed product of such a molecule.
  • nucleic acid is well known in the art.
  • a “nucleic acid” as used herein will generally refer to a molecule (one or more strands) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • a nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA ⁇ e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA ⁇ e.g., an A, a G, an uracil "U” or a C).
  • the term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.”
  • miRNA generally refers to a single-stranded molecule, but in specific embodiments, molecules implemented in the invention will also encompass a region or an additional strand that is partially (between 10 and 50% complementary across length of strand), substantially (greater than 50% but less than 100% complementary across length of strand) or fully complementary to another region of the same single-stranded molecule or to another nucleic acid.
  • miRNA nucleic acids may encompass a molecule that comprises one or more complementary or self-complementary strand(s) or "complement(s)" of a particular sequence.
  • precursor miRNA may have a self-complementary region, which is up to 100% complementary.
  • miRNA probes or nucleic acids of the invention can include, can be or can be at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% complementary to their target.
  • a "synthetic nucleic acid” of the invention means that the nucleic acid does not have all or part of a chemical structure or sequence of a naturally occurring nucleic acid. Consequently, it will be understood that the term “synthetic miRNA” refers to a “synthetic nucleic acid” that functions in a cell or under physiological conditions as a naturally occurring miRNA.
  • nucleic acid molecule(s) need not be "synthetic.”
  • a non-synthetic nucleic acid or miRNA employed in methods and compositions of the invention may have the entire sequence and structure of a naturally occurring mRNA or miRNA precursor or the mature mRNA or miRNA.
  • non-synthetic miRNAs used in methods and compositions of the invention may not have one or more modified nucleotides or nucleotide analogs.
  • the non-synthetic miRNA may or may not be recombinantly produced.
  • the nucleic acid in methods and/or compositions of the invention is specifically a synthetic miRNA and not a non-synthetic miRNA (that is, not a miRNA that qualifies as "synthetic"); though in other embodiments, the invention specifically involves a non- synthetic miRNA and not a synthetic miRNA. Any embodiments discussed with respect to the use of synthetic miRNAs can be applied with respect to non-synthetic miRNAs, and vice versa.
  • a synthetic miRNA molecule does not have the sequence of a naturally occurring miRNA molecule.
  • a synthetic miRNA molecule may have the sequence of a naturally occurring miRNA molecule, but the chemical structure of the molecule, particularly in the part unrelated specifically to the precise sequence (non-sequence chemical structure) differs from chemical structure of the naturally occurring miRNA molecule with that sequence.
  • the synthetic miRNA has both a sequence and non-sequence chemical structure that are not found in a naturally-occurring miRNA.
  • the sequence of the synthetic molecules will identify which miRNA is effectively being provided or inhibited; the endogenous miRNA will be referred to as the "corresponding miRNA.”
  • Corresponding miRNA sequences that can be used in the context of the invention include, but are not limited to, all or a portion of those sequences in the SEQ IDs provided herein, as well as any other miRNA sequence, miRNA precursor sequence, or any sequence complementary thereof.
  • the sequence is or is derived from or contains all or part of a sequence identified herein to target a particular miRNA (or set of miRNAs) that can be used with that sequence.
  • Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or any number or range of sequences there between may be selected to the exclusion of all non-selected sequences.
  • hybridization As used herein, “hybridization”, “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature.
  • anneal as used herein is synonymous with “hybridize.”
  • hybridization “hybridize(s)” or “capable of hybridizing” encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”
  • stringent condition(s) or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
  • Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.5 M NaCl at temperatures of about 42°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.
  • low stringency or “low stringency conditions”
  • non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 2O 0 C to about 50 0 C.
  • hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 2O 0 C to about 50 0 C.
  • nucleobase refers to a heterocyclic base, such as for example a naturally occurring nucleobase (i.e., an A, T, G, C or U) found in at least one naturally occurring nucleic acid (i.e., DNA and RNA), and naturally or non-naturally occurring derivative(s) and analogs of such a nucleobase.
  • a nucleobase generally can form one or more hydrogen bonds (“anneal” or “hybridize”) with at least one naturally occurring nucleobase in a manner that may substitute for naturally occurring nucleobase pairing (e.g., the hydrogen bonding between A and T, G and C, and A and U).
  • Preferredine and/or "pyrimidine” nucleobase(s) encompass naturally occurring purine and/or pyrimidine nucleobases and also derivative(s) and analog(s) thereof, including but not limited to, those a purine or pyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol or alkylthiol moiety.
  • Preferred alkyl (e.g., alkyl, carboxyalkyl, etc.) moieties comprise of from about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms.
  • a purine or pyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil, a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, a bromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a 8-thioguanine, an azaguanine, a 2-aminopurine, a 5- ethylcytosine, a 5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a 5- chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, a methylthio adenine, a N,N- diemethyladenine, an azaguanine
  • nucleoside refers to an individual chemical unit comprising a nucleobase covalently attached to a nucleobase linker moiety.
  • nucleobase linker moiety is a sugar comprising 5-carbon atoms (i.e., a "5-carbon sugar"), including but not limited to a deoxyribose, a ribose, an arabinose, or a derivative or an analog of a 5-carbon sugar.
  • Non-limiting examples of a derivative or an analog of a 5-carbon sugar include a 2'-fluoro-2'-deoxyribose or a carbocyclic sugar where a carbon is substituted for an oxygen atom in the sugar ring.
  • a nucleobase linker moiety Different types of covalent attachment(s) of a nucleobase to a nucleobase linker moiety are known in the art (Kornberg and Baker, 1992).
  • nucleotide refers to a nucleoside further comprising a "backbone moiety".
  • a backbone moiety generally covalently attaches a nucleotide to another molecule comprising a nucleotide, or to another nucleotide to form a nucleic acid.
  • the "backbone moiety” in naturally occurring nucleotides typically comprises a phosphorus moiety, which is covalently attached to a 5-carbon sugar. The attachment of the backbone moiety typically occurs at either the 3'- or 5'-position of the 5-carbon sugar.
  • other types of attachments are known in the art, particularly when a nucleotide comprises derivatives or analogs of a naturally occurring 5-carbon sugar or phosphorus moiety.
  • a nucleic acid may comprise, or be composed entirely of, a derivative or analog of a nucleobase, a nucleobase linker moiety and/or backbone moiety that may be present in a naturally occurring nucleic acid.
  • RNA with nucleic acid analogs may also be labeled according to methods of the invention.
  • a "derivative” refers to a chemically modified or altered form of a naturally occurring molecule, while the terms “mimic” or “analog” refer to a molecule that may or may not structurally resemble a naturally occurring molecule or moiety, but possesses similar functions.
  • a "moiety” generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure.
  • Nucleobase, nucleoside and nucleotide analogs or derivatives are well known in the art, and have been described (see for example, Scheit, 1980, incorporated herein by reference).
  • nucleosides, nucleotides or nucleic acids include those in: U.S. Patents 5,681,947, 5,652,099 and 5,763,167, 5,614,617, 5,670,663, 5,872,232, 5,859,221, 5,446,137, 5,886,165, 5,714,606, 5,672,697, 5,466,786, 5,792,847, 5,223,618, 5,470,967, 5,378,825, 5,777,092, 5,623,070, 5,610,289, 5,602,240, 5,858,988, 5,214,136, 5,700,922, 5,708,154, 5,728,525, 5,637,683, 6,251,666, 5,480,980, and 5,728,525, each of which is incorporated herein by reference in its entirety.
  • Labeling methods and kits of the invention specifically contemplate the use of nucleotides that are both modified for attachment of a label and can be incorporated into a miRNA molecule.
  • Such nucleotides include those that can be labeled with a dye, including a fluorescent dye, or with a molecule such as biotin. Labeled nucleotides are readily available; they can be acquired commercially or they can be synthesized by reactions known to those of skill in the art.
  • Modified nucleotides for use in the invention are not naturally occurring nucleotides, but instead, refer to prepared nucleotides that have a reactive moiety on them.
  • Specific reactive functionalities of interest include: amino, sulfhydryl, sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate, isocyanate, anhydride, monochlorotriazine, dichlorotriazine, mono-or dihalogen substituted pyridine, mono- or disubstituted diazine, maleimide, epoxide, aziridine, sulfonyl halide, acid halide, alkyl halide, aryl halide, alkylsulfonate, N-hydroxysuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-propionamide, g
  • the reactive functionality may be bonded directly to a nucleotide, or it may be bonded to the nucleotide through a linking group.
  • the functional moiety and any linker cannot substantially impair the ability of the nucleotide to be added to the miRNA or to be labeled.
  • Representative linking groups include carbon containing linking groups, typically ranging from about 2 to 18, usually from about 2 to 8 carbon atoms, where the carbon containing linking groups may or may not include one or more heteroatoms, e.g. S, O, N etc., and may or may not include one or more sites of unsaturation.
  • alkyl linking groups typically lower alkyl linking groups of 1 to 16, usually 1 to 4 carbon atoms, where the linking groups may include one or more sites of unsaturation.
  • the functionalized nucleotides (or primers) used in the above methods of functionalized target generation may be fabricated using known protocols or purchased from commercial vendors, e.g., Sigma, Roche, Ambion, Biosearch Technologies and NEN.
  • Functional groups may be prepared according to ways known to those of skill in the art, including the representative information found in U.S. Patents 4,404,289; 4,405,711; 4,337,063 and 5,268,486, and U.K.. Patent 1,529,202, which are all incorporated by reference.
  • Amine-modified nucleotides are used in several embodiments of the invention.
  • the amine-modified nucleotide is a nucleotide that has a reactive amine group for attachment of the label. It is contemplated that any ribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T, or C) can be modified for labeling.
  • Examples include, but are not limited to, the following modified ribo- and deoxyribo-nucleo tides: 5-(3-aminoallyl)- UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4- amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP; N6-(6- Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5- propargylamino-UTP; 5-(3-aminoallyl)-dUTP; 8-[(4-amino)butyl]-amino-dATP and 8-[(6- amino)butyl]-amino-dATP
  • nucleotides can be prepared according to methods known to those of skill in the art. Moreover, a person of ordinary skill in the art could prepare other nucleotide entities with the same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP, dCTP, dGTP, dTTP, or dUTP in place of a 5-(3- aminoallyl)-UTP.
  • a nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production, or biological production. It is specifically contemplated that miRNA probes of the invention are chemically synthesized.
  • miRNAs are recovered or isolated from a biological sample.
  • the miRNA may be recombinant or it may be natural or endogenous to the cell (produced from the cell's genome). It is contemplated that a biological sample may be treated in a way so as to enhance the recovery of small RNA molecules such as miRNA.
  • U.S. Patent Application Serial No. 10/667,126 describes such methods and it is specifically incorporated by reference herein. Generally, methods involve lysing cells with a solution having guanidinium and a detergent.
  • nucleic acid synthesis is performed according to standard methods. See, for example, Itakura and Riggs (1980) and U.S. Patents 4,704,362, 5,221,619, and 5,583,013, each of which is incorporated herein by reference.
  • Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemically synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al, 1986 and U.S. Patent 5,705,629, each incorporated herein by reference.
  • Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
  • a non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Patents 4,683,202 and 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference. See also Sambrook et al, 2001 , incorporated herein by reference).
  • Oligonucleotide synthesis is well known to those of skill in the art. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
  • Recombinant methods for producing nucleic acids in a cell are well known to those of skill in the art. These include the use of vectors (viral and non-viral), plasmids, cosmids, and other vehicles for delivering a nucleic acid to a cell, which may be the target cell ⁇ e.g. , a cancer cell) or simply a host cell (to produce large quantities of the desired RNA molecule). Alternatively, such vehicles can be used in the context of a cell free system so long as the reagents for generating the RNA molecule are present. Such methods include those described in Sambrook, 2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporated by reference.
  • Nucleic acids may be isolated using techniques well known to those of skill in the art, though in particular embodiments, methods for isolating small nucleic acid molecules, and/or isolating RNA molecules can be employed. Chromatography is a process often used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography. If miRNA from cells is to be used or evaluated, methods generally involve lysing the cells with a chaotropic (e.g. , guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA.
  • a chaotropic e.g. , guanidinium isothiocyanate
  • detergent e.g., N-lauroyl sarcosine
  • a gel matrix is prepared using polyacrylamide, though agarose can also be used.
  • the gels may be graded by concentration or they may be uniform. Plates or tubing can be used to hold the gel matrix for electrophoresis. Usually one-dimensional electrophoresis is employed for the separation of nucleic acids. Plates are used to prepare a slab gel, while the tubing (glass or rubber, typically) can be used to prepare a tube gel.
  • the phrase "tube electrophoresis” refers to the use of a tube or tubing, instead of plates, to form the gel. Materials for implementing tube electrophoresis can be readily prepared by a person of skill in the art or purchased, such as from CB. S. Scientific Co., Inc. or Scie-Plas.
  • Methods may involve the use of organic solvents and/or alcohol to isolate nucleic acids, particularly miRNA used in methods and compositions of the invention.
  • Some embodiments are described in U.S. Patent Application Serial No. 10/667,126, which is hereby incorporated by reference.
  • this disclosure provides methods for efficiently isolating small RNA molecules from cells comprising: adding an alcohol solution to a cell lysate and applying the alcohol/lysate mixture to a solid support before eluting the RNA molecules from the solid support.
  • the amount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well.
  • a solid support may be any structure, and it includes beads, filters, and columns, which may include a mineral or polymer support with electronegative groups. A glass fiber filter or column has worked particularly well for such isolation procedures.
  • miRNA isolation processes include: a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about 1 M guanidinium is produced; b) extracting miRNA molecules from the lysate with an extraction solution comprising phenol; c) adding to the lysate an alcohol solution for forming a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%; d) applying the lysate/alcohol mixture to a solid support; e) eluting the miRNA molecules from the solid support with an ionic solution; and, f) capturing the miRNA molecules.
  • the sample is dried and resuspended in a liquid and volume appropriate for subsequent manipulation.
  • the present invention concerns miRNA that are labeled. It is contemplated that miRNA may first be isolated and/or purified prior to labeling. This may achieve a reaction that more efficiently labels the miRNA, as opposed to other RNA in a sample in which the miRNA is not isolated or purified prior to labeling.
  • the label is non-radioactive.
  • nucleic acids may be labeled by adding labeled nucleotides (one-step process) or adding nucleotides and labeling the added nucleotides (two-step process).
  • nucleic acids are labeled by catalytically adding to the nucleic acid an already labeled nucleotide or nucleotides.
  • One or more labeled nucleotides can be added to miRNA molecules. See U.S. Patent 6,723,509, which is hereby incorporated by reference.
  • an unlabeled nucleotide or nucleotides is catalytically added to a miRNA, and the unlabeled nucleotide is modified with a chemical moiety that enables it to be subsequently labeled.
  • the chemical moiety is a reactive amine such that the nucleotide is an amine-modified nucleotide. Examples of amine-modified nucleotides are well known to those of skill in the art, many being commercially available such as from Ambion, Sigma, Jena Bioscience, and TriLink.
  • the issue for labeling miRNA is how to label the already existing molecule.
  • the present invention concerns the use of an enzyme capable of using a di- or tri-phosphate ribonucleotide or deoxyribonucleotide as a substrate for its addition to a miRNA. Moreover, in specific embodiments, it involves using a modified di- or tri-phosphate ribonucleotide, which is added to the 3' end of a miRNA.
  • Enzymes capable of adding such nucleotides include, but are not limited to, poly(A) polymerase, terminal transferase, and polynucleotide phosphorylase.
  • a ligase is contemplated as not being the enzyme used to add the label, and instead, a non-ligase enzyme is employed.
  • Terminal transferase catalyzes the addition of nucleotides to the 3' terminus of a nucleic acid.
  • Polynucleotide phosphorylase can polymerize nucleotide diphosphates without the need for a primer.
  • Labels on miRNA or miRNA probes may be colorimetric (includes visible and UV spectrum, including fluorescent), luminescent, enzymatic, or positron emitting (including radioactive).
  • the label may be detected directly or indirectly.
  • Radioactive labels include 125 1, 32 P, 33 P, and 35 S.
  • Examples of enzymatic labels include alkaline phosphatase, luciferase, horseradish peroxidase, and ⁇ -galactosidase. Labels can also be proteins with luminescent properties, e.g., green fluorescent protein and phycoerythrin.
  • the colorimetric and fluorescent labels contemplated for use as conjugates include, but are not limited to, Alexa Fluor dyes, BODIPY dyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-amino-4- methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum DyeTM; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G; Texas Red; , fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.
  • Alexa Fluor dyes such as BODIPY FL
  • dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR,
  • fiuorescently labeled ribonucleotides are available from Molecular Probes, and these include, Alexa Fluor 488-5-UTP, Fluorescein- 12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14- UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP.
  • Other fluorescent ribonucleotides are available from Amersham Biosciences, such as Cy3-UTP and Cy5-UTP.
  • Examples of fluorescently labeled deoxyribonucleotides include Dinitrophenyl (DNP)-11-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546- 14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR- 14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14- dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546
  • nucleic acids may be labeled with two different labels.
  • fluorescence resonance energy transfer FRET may be employed in methods of the invention (e.g., Kleinmeier et al, 2002; Emptage, 2001 ; Didenko, 2001, each incorporated by reference).
  • the label may not be detectable per se, but indirectly detectable or allowing for the isolation or separation of the targeted nucleic acid.
  • the label could be biotin, digoxigenin, polyvalent cations, chelator groups and the other ligands, include ligands for an antibody.
  • a number of techniques for visualizing or detecting labeled nucleic acids are readily available. Such techniques include, microscopy, arrays, Fluorometry, Light cyclers or other real time PCR machines, FACS analysis, scintillation counters, Phosphoimagers, Geiger counters, MRI, CAT, antibody-based detection methods (Westerns, immunofluorescence, immunohistochemistry), histochemical techniques, HPLC (Griffey et al., 1997), spectroscopy, capillary gel electrophoresis (Cummins et al, 1996), spectroscopy; mass spectroscopy; radiological techniques; and mass balance techniques.
  • FRET fluorescent resonance energy transfer
  • compositions described herein may be comprised in a kit.
  • reagents for isolating miRNA, labeling miRNA, and/or evaluating a miRNA population using an array, nucleic acid amplification, and/or hybridization can be included in a kit, as well reagents for preparation of samples from blood samples.
  • the kit may further include reagents for creating or synthesizing miRNA probes.
  • the kits will thus comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled.
  • the kit can include amplification reagents.
  • the kit may include various supports, such as glass, nylon, polymeric beads, and the like, and/or reagents for coupling any probes and/or target nucleic acids. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, and components for isolating miRNA. Other kits of the invention may include components for making a nucleic acid array comprising miRNA, and thus, may include, for example, a solid support.
  • Kits for implementing methods of the invention described herein are specifically contemplated.
  • kits for preparing miRNA for multi-labeling and kits for preparing miRNA probes and/or miRNA arrays.
  • kit comprise, in suitable container means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of the following: (1) poly(A) polymerase; (2) unmodified nucleotides (G, A, T, C, and/or U); (3) a modified nucleotide (labeled or unlabeled); (4) poly(A) polymerase buffer; and, (5) at least one microfilter; (6) label that can be attached to a nucleotide; (7) at least one miRNA probe; (8) reaction buffer; (9) a miRNA array or components for making such an array; (10) acetic acid; (11) alcohol; (12) solutions for preparing, isolating, enriching, and purifying miRNAs or miRNA probes or arrays.
  • kits of the invention include an array containing miRNA probes, as described in the application.
  • An array may have probes corresponding to all known miRNAs of an organism or a particular tissue or organ in particular conditions, or to a subset of such probes.
  • the subset of probes on arrays of the invention may be or include those identified as relevant to a particular diagnostic, therapeutic, or prognostic application.
  • the array may contain one or more probes that is indicative or suggestive of (1) a disease or condition (acute myeloid leukemia), (2) susceptibility or resistance to a particular drug or treatment; (3) susceptibility to toxicity from a drug or substance; (4) the stage of development or severity of a disease or condition (prognosis); and (5) genetic predisposition to a disease or condition.
  • a disease or condition acute myeloid leukemia
  • susceptibility or resistance to a particular drug or treatment susceptibility to a particular drug or treatment
  • susceptibility to toxicity from a drug or substance susceptibility to toxicity from a drug or substance
  • (4) the stage of development or severity of a disease or condition prognosis
  • genetic predisposition to a disease or condition genetic predisposition to a disease or condition.
  • kits embodiment including an array
  • nucleic acid molecules that contain or can be used to amplify a sequence that is a variant of, identical to or complementary to all or part of any of SEQ IDs described herein.
  • a kit or array of the invention can contain one or more probes for the miRNAs identified by the SEQ IDs described herein. Any nucleic acid discussed above may be implemented as part of a kit.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • 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 means.
  • labeling dyes are provided as a dried power.
  • kits of the invention 10-20 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ⁇ g or at least or at most those amounts of dried dye are provided in kits of the invention.
  • the dye may then be resuspended in any suitable solvent, such as DMSO.
  • kits may also include components that facilitate isolation of the labeled miRNA. It may also include components that preserve or maintain the miRNA or that protect against its degradation. Such components may be RNAse-free or protect against RNAses. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
  • kits will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
  • Kits of the invention may also include one or more of the following: Control RNA; nuclease- free water; RNase-free containers, such as 1.5 ml tubes; RNase-free elution tubes; PEG or dextran; ethanol; acetic acid; sodium acetate; ammonium acetate; guanidinium; detergent; nucleic acid size marker; RNase-free tube tips; and RNase or DNase inhibitors.
  • kits of the invention are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of miRNA.
  • miRNAs are believed to primarily influence gene expression at the level of translation. Translational regulation leading to an up or down change in protein expression may lead to changes in activity and expression of downstream gene products and genes that are in turn regulated by those proteins. These regulatory effects would be revealed as changes in the global mRNA expression profile. Furthermore, it has recently been reported that, in some instances, miRNAs may reduce the mRNA levels of their direct targets (Bagga et al, 2005; Lim et al., 2005), and such changes can be observed upon microarray gene expression analysis. Microarray gene expression analyses were performed to identify genes that are mis-regulated by hsa-miR-16.
  • Synthetic Pre-miR-16 was reverse transfected into quadruplicate samples of A549 cells for each of three time points.
  • Cells were transfected using siPORT NeoFX (Ambion) according to the manufacturer's recommendations using the following parameters: 200,000 cells per well in a 6 well plate, 5.0 ⁇ l of NeoFX, 30 nM final concentration of miRNA in 2.5 ml.
  • Cells were harvested at 4 h, 24 h, and 72 h post transfection.
  • Total RNA was extracted using RNAqueous-4PCR (Ambion) according to the manufacturer's recommended protocol.
  • mRNA array analyses were performed by Asuragen Services (Austin,TX), according to the company's standard operating procedures. Using the MessageAmpTM 11-96 aRNA Amplification Kit (Ambion, cat #1819) 2 ⁇ g of total RNA were used for target preparation and labeling with biotin. cRNA yields were quantified using an Agilent Bioanalyzer 2100 capillary electrophoresis protocol. Labeled target was hybridized to Affymetrix mRNA arrays (Human HG-Ul 33 A 2.0 arrays) using the manufacturer's recommendations and the following parameters. Hybridizations were carried out at 45 0 C for 16 hr in an Affymetrix Model 640 hybridization oven.
  • Arrays were washed and stained on an Affymetrix FS450 Fluidics station, running the wash script Midi_euk2v3_450. The arrays were scanned on a Affymetrix GeneChip Scanner 3000. Summaries of the image signal data, group mean values, p-values with significance flags, log ratios and gene annotations for every gene on the array were generated using the Affymetrix Statistical Algorithm MAS 5.0 (GCOS vl.3). Data were reported in a file (cabinet) containing the Affymetrix data and result files and in files (.eel) containing the primary image and processed cell intensities of the arrays.
  • GCOS vl.3 Affymetrix Statistical Algorithm MAS 5.0
  • hsa-miR-16 The mis-regulation of gene expression by hsa-miR-16 affects many cellular pathways that represent potential therapeutic targets for the control of cancer and other diseases and disorders.
  • the inventors determined the identity and nature of the cellular genetic pathways affected by the regulatory cascade induced by hsa-miR-16 expression.
  • Cellular pathway analyses were performed using Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA). The most significantly affected pathways following over- expression of hsa-miR-16 in A549 cells are shown in Table 2.
  • hsa-miR-16 directly or indirectly affects the expression of numerous metabolic-, cellular proliferation-, cellular development-, and cell cycle-related genes and thus primarily affects functional pathways related to cellular growth, development, and proliferation. Those cellular processes all have integral roles in the development and progression of various cancers. Manipulation of the expression levels of genes in the cellular pathways shown in Table 2 represents a potentially useful therapy for cancer and other diseases in which increased or reduced expression of hsa-miR-16 has a role in the disease.
  • Predicted hsa-miR-16 targets that exhibited altered mRNA expression levels in human cancer cells after transfection with pre-miR hsa-miR-16.
  • the predicted gene targets of hsa-miR-16 whose mRNA expression levels are affected by hsa-miR-16 represent particularly useful candidate targets for cancer therapy and therapy of other diseases through manipulation of their expression levels.
  • Hsa-miR-16 directly or indirectly regulates the transcripts of proteins that are critical in the regulation of these pathways. Many of these targets have inherent oncogenic or tumor suppressor activity. Hsa- miR-16 targets that are associated with various cancer types are shown in Table 5.
  • cyclin Dl cyclin Dl
  • CCNG2 cyclin G2
  • TACCl transforming acidic coiled coil 1 protein
  • cyclin G2 is down-regulated in multiple cancers, such as oral cancer and papillary carcinomas (Alevizos et al, 2001; Ito et al, 2003). Since hsa-miR-16 over-expression leads to suppression of the cyclin Dl transcript and up regulation of cyclin G2, hsa-miR-16 may function as a tumor suppressor. This view is supported by the fact that hsa-miR-16 negatively regulates the TACCl message which encodes a putative oncogene located within a breast cancer amplicon on chromosome 8pl l (Cully et al, 2005). Over- expression of TACCl induces oncogenic transformation of fibroblasts in culture and cooperates with Ras to form tumors in mice with a PTEN+/- background (Cully et al, 2005).
  • FGF2 fibroblast growth factor 2
  • FGFR4 fibroblast growth factor receptor 4
  • IKKalpha, CHUK IkappaB kinase alpha
  • FGFR4 is up- regulated in numerous types of cancer (Chandler et al, 1999).
  • IKKalpha is a positive regulator of the intracellular signaling cascade and functions to activate the transcription factor nuclear factor kappa B (NFkappaB) (Karin et al 2002). NFkappaB is constitutively activated in several cancer types and promotes anti-apoptotic and survival pathways.
  • hsa-miR-16 negatively regulates these proteins and therefore is likely to function as a tumor-suppressor.
  • hsa-miR-16 may also have oncogenic activity. This view is supported by the observation that hsa-miR-16 negatively regulates the tumor-suppressor RBL-I (plO7) and induces an up-regulation of MCLl, thioredoxin (TXN) and the oncogenic E3 ubiquitin ligase Skp2 (Gstaiger et al., 2001; Huang et al, 2005; Jiang et al, 2005).
  • Skp2 is a component of the multi-subunit E3 ubiquitin ligase complex that earmarks proteins for proteasomal degradation.
  • a well characterized target is the CDK inhibitor p27 which offers an explanation for the cell cycle promoting activity of Skp2 (Carrano et al., 1999).
  • Skp2 is inherently oncogenic and shows elevated levels in various cancer types (Gstaiger et al., 2001; Kamata et al., 2005; Saigusa et al., 2005; Einama et al., 2006).
  • MCLl is a member of the BCL-2 (B cell lymphoma 2) gene family.
  • MCLl gives rise to two alternatively spliced gene products with opposing functions (Bae et al., 2000).
  • the predominant species is MCLl-L that has anti-apoptotic activity.
  • High levels of MCLl are correlated with poor prognosis of patients with ovarian carcinoma and is indicative for leukemic relapse (Kaufmann et al., 1998; Shigemasa et al., 2002).
  • RNA interference against MCLl induces a therapeutic response in gastric and hepatocellular carcinoma cells (Schulze- Bergkamen et al., 2006; Zangemeister-Wittke and Huwiler, 2006).
  • Thioredoxin is a 12-kDa thiol reductase targeting various proteins and multiple pathways. Thioredoxin modulates the activity of transcription factors, induces the expression of angiogenic Hif-1 alpha (hypoxia induced factor 1 alpha) as well as VEGF (vascular endothelial growth factor) and can act as a proliferative and anti-apoptotic agent (Marks, 2006). In accord, carcinomas of the lung, pancreas, cervix and liver show increased levels of thioredoxin. Thioredoxin expression is also correlated with aggressive tumor growth, poor prognosis and chemoresistance (Marks, 2006).
  • hsa-miR-16 regulates genes that may have either oncogenic or growth-inhibitory activity, depending on the cellular context: among these are connective tissue growth factor (CTGF) and neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin-2 (LCN2) (Croci et al, 2004; Hishikawa et al, 1999; Lin et al, 2005; Yang et al, 2005; Fernandez et al, 2005; Lee et al, 2Q06).
  • CTGF connective tissue growth factor
  • NGAL neutrophil gelatinase-associated lipocalin
  • LN2 lipocalin-2
  • hsa-miR-16 governs the activity of proteins that are critical regulators of cell proliferation and survival. These targets are frequently deregulated in human cancer. Based on this review of the genes and related pathways that are regulated by miR-16, introduction of hsa-miR-16 or an anti-hsa-miR-16 into a variety of cancer cell types would likely result in a therapeutic response.
  • AC astrocytoma
  • ALCL anaplastic large cell lymphoma
  • BC breast carcinoma
  • BCL B-cell lymphoma
  • BIdC bladder carcinoma
  • CeC cervical carcinoma
  • CLL chronic lymphoblastic leukemia
  • CRC colorectal carcinoma
  • EC endometrial carcinoma
  • G glioma
  • GB glioblastoma
  • GC gastric carcinoma
  • HB hepatoblastoma
  • HCC hepatocellular carcinoma
  • HL Hodgkin lymphoma
  • LC lung carcinoma
  • LSCC laryngeal squamous cell carcinoma
  • M melanoma
  • MB medulloblastoma
  • MCL mantle cell lymphoma
  • MFS myxofibrosarcoma
  • ML myeloid leukemia
  • MM multiple myeloma
  • NF neurofibroma
  • NSCLC non-small cell lung carcinoma
  • hsa-miR-16 is involved in the regulation of numerous cell activities that represent intervention points for cancer therapy and for therapy of other diseases and disorders (U.S. Patent Applications serial number 11/141,707 filed May 31, 2005 and serial number 11/273,640 filed November 14, 2005).
  • overexpression of hsa-miR-16 decreases the proliferation and/or viability of certain normal or cancerous cell lines.
  • hsa-miR-16 assessed the therapeutic effect of hsa-miR-16 for prostate cancer by using the prostate cancer cell lines PPC-I, Dul45, and RWPE2.
  • Synthetic hsa-miR-16 Pre- miRTM-hsa-miR-16, Ambion cat. no. AM17100
  • NC negative control miRNA
  • Pre- miRTM microRNA Precursor Molecule-Negative Control #2; Ambion cat. no. AM17111 was delivered via lipid-based reverse transfections in triplicate according to a published protocol and the following parameters: 6000-7000 cells per 96 well, 0.2 ⁇ l LipofectamineTM 2000 (cat. no.
  • siEg5 was used in lipid-based transfection following the same experimental parameters that apply to miRNA. Percent (%) proliferation values from the Alamar Blue assay were normalized to values from cells treated with negative control miRNA. Percent proliferation of hsa-miR-16 treated cells relative to cells treated with negative control miRNA (100%) is shown in Table 6 and in FIG. 1.
  • hsa-miR-16 inhibits cellular proliferation of prostate cancer cells PPC- 1, Dul45 and RWPE2 (Table 6 and FIG. 1). On average, hsa-miR-16 inhibits cellular proliferation by 25% (Table 6 and FIG. 1). Hsa-miR-16 has maximal inhibitory activity in PPC-I cells, reducing proliferation by 37%. Since hsa-miR-16 induces a therapeutic response in all prostate cancer cells tested, hsa-miR-16 may provide therapeutic benefit to patients with prostate cancer and other malignancies.
  • hsa-miR-16 To evaluate the inhibitory phenotype of hsa-miR-16 over an extended period of time, the inventors conducted growth curve experiments in the presence of hsa-miR-16 for up to 22 days in PPC-I cells. Since in vitro transfections of naked interfering RNAs, such as synthetic miRNA, are transient by nature and compromised by the dilution of the oligonucleotide during ongoing cell divisions, hsa-miR-16 was administered at multiple time points via electroporation (Bartlett et al, 2006, Bartlett et al, 2007).
  • Equal numbers of PPC- 1 cells were electroporated with 1.6 ⁇ M synthetic hsa-miR-16 (Pre-miRTM-hsa-miR-16, Ambion cat. no. AMI 7100) or negative control miRNA (Pre-miRTM microRNA Precursor Molecule-Negative Control #2; Ambion cat. no. AM17111) in 200 ⁇ l OptiMEM (Invitrogen) on days 0, 4, and 11 using the the BioRad GenePulserXcellTM instrument (BioRad Laboratories, Inc.; Hercules, CA, USA).
  • Patent 6,368,799 U.S. Patent 6,383,749 U.S. Patent 6,617,112 U.S. Patent 6,638,717 U.S. Patent 6,720,138 U.S. Patent 6,723,509 U.S. Patent Serial 09/545,207 U.S. Patent Serial 10/667,126 U.S. Patent Serial 11/141,707 U.S. Prov. Appln. 60/575,743 U.S. Prov. Appln. 60/649,584
  • EP 266,032 EP 373 203 EP 785 280 EP 799 897 PCT Appln. WO 0168255 PCT Appln. WO 03020898 PCT Appln. WO 03022421 PCT Appln. WO 03023058 PCT Appln. WO 03029485 PCT Appln. WO 03040410 PCT Appln. WO 03053586 PCT Appln. WO 03066906 PCT Appln. WO 03067217 PCT Appln. WO 03076928 PCT Appln. WO 03087297 PCT Appln. WO 03091426 PCT Appln. WO 03093810 PCT Appln.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés et des compositions pour l'identification de gènes ou de voies génétiques modulés par miR-16, utilisant miR-16 pour moduler un gène ou une voie génétique, en utilisant ce profil pour l'évaluation de l'état d'un patient et/ou le traitement du patient avec un ARNmi approprié.
EP07871756A 2006-12-08 2007-12-31 GÈNES ET VOIES RÉGULÉS PAR miR-16 UTILES COMME CIBLES POUR INTERVENTION THÉRAPEUTIQUE Withdrawn EP2102342A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US86929506P 2006-12-08 2006-12-08
US88275806P 2006-12-29 2006-12-29
PCT/US2007/087038 WO2008073923A2 (fr) 2006-12-08 2007-12-10 Gènes et voies régulés par miarn comme cibles d'intervention thérapeutique
PCT/US2007/089206 WO2008085797A2 (fr) 2006-12-08 2007-12-31 Gènes et voies régulés par mir-16 utiles comme cibles pour intervention thérapeutique

Publications (1)

Publication Number Publication Date
EP2102342A2 true EP2102342A2 (fr) 2009-09-23

Family

ID=39512449

Family Applications (2)

Application Number Title Priority Date Filing Date
EP07871694A Withdrawn EP2102341A2 (fr) 2006-12-08 2007-12-10 Gènes et voies régules par miarn comme cibles d'intervention thérapeutique
EP07871756A Withdrawn EP2102342A2 (fr) 2006-12-08 2007-12-31 GÈNES ET VOIES RÉGULÉS PAR miR-16 UTILES COMME CIBLES POUR INTERVENTION THÉRAPEUTIQUE

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP07871694A Withdrawn EP2102341A2 (fr) 2006-12-08 2007-12-10 Gènes et voies régules par miarn comme cibles d'intervention thérapeutique

Country Status (5)

Country Link
EP (2) EP2102341A2 (fr)
AU (2) AU2007333110A1 (fr)
CA (1) CA2671302A1 (fr)
IL (1) IL199161A0 (fr)
WO (2) WO2008073923A2 (fr)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007039256A2 (fr) 2005-09-30 2007-04-12 Abbott Gmbh & Co. Kg Domaines de liaison de proteines de la famille proteinique des molecules de guidage repulsif (rgm), fragments fonctionnels de ces domaines et leur utilisation
US8513209B2 (en) 2007-11-09 2013-08-20 The Board Of Regents, The University Of Texas System Micro-RNAS of the MIR-15 family modulate cardiomyocyte survival and cardiac repair
WO2009075799A2 (fr) 2007-12-05 2009-06-18 The Wistar Institute Of Anatomy And Biology Procédé de diagnostic des cancers du poumon à l'aide de profils d'expression génétique dans des cellules mononucléaires de sang périphérique
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
JP2010094122A (ja) 2008-06-12 2010-04-30 Keio Gijuku バイオマーカーとしてのマイクロｒｎａを用いた頭頸部腫瘍の診断・治療選択
WO2010054233A1 (fr) 2008-11-08 2010-05-14 The Wistar Institute Of Anatomy And Biology Marqueurs biologiques dans des cellules mononucléaires du sang périphérique pour diagnostiquer ou détecter des cancers du poumon
MX2012006560A (es) 2009-12-08 2012-10-05 Abbott Gmbh & Co Kg Anticuerpos monoclonales contra la proteina rgm a para utilizarse en el tratamiento de degeneracion de capa de fibra de nervio retinal.
WO2013055911A1 (fr) 2011-10-14 2013-04-18 Dana-Farber Cancer Institute, Inc. Biomarqueur znf365/zfp365 pouvant prévoir une réponse anticancéreuse
SG10201600316SA (en) 2012-01-27 2016-02-26 Abbvie Deutschland Composition and method for diagnosis and treatment of diseases associated with neurite degeneration
US9163235B2 (en) 2012-06-21 2015-10-20 MiRagen Therapeutics, Inc. Inhibitors of the miR-15 family of micro-RNAs
WO2017005773A1 (fr) * 2015-07-07 2017-01-12 Universite de Bordeaux Utilisation de micro-arn ciblant la bêta-caténine pour le traitement du cancer du foie
US11692226B2 (en) 2017-07-31 2023-07-04 Arizona Board Of Regents On Behalf Of The University Of Arizona Genotyping of SNPs to stratify cancer risk
KR102200428B1 (ko) * 2018-05-16 2021-01-08 주식회사 지놈앤컴퍼니 Lrit2 억제제를 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물
CN111972399B (zh) * 2020-08-06 2021-11-30 温州医科大学 维持肝细胞活性的保存液

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4939055B2 (ja) * 2002-11-13 2012-05-23 トマス ジェファソン ユニバーシティ 癌の診断および治療のための組成物および方法
AU2006291165B2 (en) * 2005-09-12 2013-03-14 The Ohio State University Research Foundation Compositions and methods for the diagnosis and therapy of BCL2-associated cancers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2008085797A3 *

Also Published As

Publication number Publication date
WO2008085797A8 (fr) 2009-07-16
IL199161A0 (en) 2010-03-28
WO2008085797A3 (fr) 2008-10-09
CA2671302A1 (fr) 2008-06-19
AU2007342068A1 (en) 2008-07-17
WO2008073923A2 (fr) 2008-06-19
AU2007333110A1 (en) 2008-06-19
WO2008073923A3 (fr) 2008-12-11
EP2102341A2 (fr) 2009-09-23
WO2008085797A2 (fr) 2008-07-17

Similar Documents

Publication Publication Date Title
EP2104735A2 (fr) Gènes et voies génétiques régulés par mir-21 utilisés en tant que cibles pour une intervention thérapeutique
US20090131354A1 (en) miR-126 REGULATED GENES AND PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION
EP2076599A2 (fr) Gènes et voies régulés par mir-200 servant de cibles dans le cadre d'une intervention thérapeutique
EP2102342A2 (fr) GÈNES ET VOIES RÉGULÉS PAR miR-16 UTILES COMME CIBLES POUR INTERVENTION THÉRAPEUTIQUE
WO2008073922A2 (fr) Fonctions et cibles de microarn let-7
EP2104736B1 (fr) Gènes et voies régulés par mir-126 comme cibles d'intervention thérapeutique
EP2145001A2 (fr) Gènes régulés mir-15, mir-26, mir -31,mir -145, mir-147, mir-188, mir-215, mir-216 mir-331, mmu-mir-292-3p et voies de signalisation utiles comme cibles dans une intervention thérapeutique
EP2104734A2 (fr) Gènes et voies génétiques régulés par le mir-20 en tant que cibles en vue d'une intervention thérapeutique
US20090192114A1 (en) miR-10 Regulated Genes and Pathways as Targets for Therapeutic Intervention
WO2008154333A2 (fr) Gènes et chemins régulés par mir-34 en tant que cibles pour une intervention thérapeutique
WO2009070805A2 (fr) Gènes régulés par le mir-124 et cheminements servant de cibles pour une intervention thérapeutique
EP2990487A1 (fr) Compositions et procédés relatifs à la modulation de miarn de néovascularisation ou angiogenèse
CN101622350A (zh) 作为干预治疗靶标的miR-126调控基因和通路
EP2260110A2 (fr) Micro arn (mirna) exprimés différentiellement dans des noeuds lymphoïdes prélevés chez des patients atteints d'un cancer
EP2271757A2 (fr) Compositions et procédés liés à mir-16 et à la thérapie contre le cancer de la prostate
US20090186015A1 (en) Micrornas differentially expressed in lung diseases and uses thereof
WO2009126726A1 (fr) Procédés et compositions pour diagnostiquer et moduler le papillomavirus humain (hpv)
EP2094848A2 (fr) GÈNES ET TRAJETS RÉGULÉS PAR miR-143 COMME CIBLES D'INTERVENTION THÉRAPEUTIQUE

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20090706

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20091020

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20101111