US 200901 63434A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0163434 A1 BADER et al. (43) Pub. Date: Jun. 25, 2009 (54) MIR-20 REGULATED GENES AND Related U.S. Application Data PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION (60) Provisional application No. 60/915,026, filed on Apr. 30, 2007. (76) Inventors: Andreas G. BADER, Austin, TX Publication Classification (US); Mike BYROM, Austin, TX (US); Charles D. JOHNSON, (51) Int. Cl. Austin, TX (US); David BROWN, A6IR 48/00 (2006.01) Austin, TX (US) CI2O I/68 (2006.01) A6IP 43/00 (2006.01) Correspondence Address: (52) U.S. Cl. ................................ 514/44; 435/6; 977/773 Fulbright & Jaworski L.L.P. 600 Congress Avenue, Suite 2400 (57) ABSTRACT Austin, TX 78701 (US) The present invention concerns methods and compositions for identifying genes or genetic pathways modulated by miR (21) Appl. No.: 12/112,291 20a, using miR-20a to modulate a gene or gene pathway, using this profile in assessing the condition of a patient and/or (22) Filed: Apr. 30, 2008 treating the patient with an appropriate miRNA. US 2009/0163434 A1 Jun. 25, 2009 MR-2O REGULATED GENES AND cers (reviewed in Esquela-Kerscher and Slack, 2006; Calin PATHWAYS AS TARGETS FOR and Croce, 2006). Differential expression of almost all miR THERAPEUTIC INTERVENTION NAS across numerous cancer types has been observed (Lu et al., 2005). Most such studies link miRNAs to cancer only by indirect evidence. However. He et al. (2005a) has provided 0001. This application claims priority to U.S. Provisional more direct evidence that miRNAs may contribute directly to Patent application Ser. No. 60/915,026 filed Apr. 30, 2007, causing cancer, by forcing the over-expression of six miR which is incorporated herein by reference in its entirety. NAS in mice, including miR-20a, that resulted in a significant increase in B cell lymphomas. BACKGROUND OF THE INVENTION 0009. The inventors previously demonstrated that hsa 0002 I. Field of the Invention miR-20a is involved with the regulation of numerous cell 0003. The present invention relates to the fields of molecu activities that represent intervention points for cancer therapy lar biology and medicine. More specifically, the invention and for therapy of other diseases and disorders (U.S. patent relates to methods and compositions for the treatment of application Ser. No. 1 1/141,707 filed May 31, 2005 and Ser. diseases or conditions that are affected by miR-20 microR No. 1 1/273,640 filed Nov. 14, 2005, both of which are incor NAS, microRNA expression, and genes and cellular pathways porated by reference). Over-expression of miR-20a signifi directly and indirectly modulated by such. cantly reduced viability of Jurkat cells, a human T-cell line 0004 II. Background derived from leukemic peripheral blood, while significantly 0005. In 2001, several groups used a cloning method to increasing the viability and proliferation of primary normal isolate and identify a large group of “microRNAs (miRNAs) human T-cells. Cell regulators that enhance viability of nor from C. elegans, Drosophila, and humans (Lagos-Quintana et mal cells while decreasing viability of cancerous cells repre al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Several sent useful therapeutic treatments for cancer. Hsa-miR-20a hundred miRNAs have been identified in plants and ani increased apoptosis (induced death of cells with oncogenic mals—including humans—that do not appear to have endog potential) in A549 lung cancer cells and increased the per enous siRNAs. Thus, while similar to siRNAs, miRNAs are centage of BJ cells (human foreskin primary cells) in the S distinct. phase of the cell cycle while reducing the percentage of those 0006 miRNAs thus far observed have been approximately cells in the G1 phase of the cell cycle. The inventors observed 21-22 nucleotides in length, and they arise from longer pre that expression of hsa-miR-20a is higher in white blood cells cursors transcribed from non-protein-encoding genes. See from patients with chronic lymphocytic leukemia than in the review of Carrington et al. (2003). The precursors form struc same cells from normal patients. Others have shown that tures that fold back on themselves in self-complementary hsa-miR-20a regulates the translational yield of the transcrip regions; they are then processed by the nuclease Dicer (in tion factor, E2F1 (O’Donnell et al., 2005) and appears to be animals) or DCL1 (in plants) to generate the short double over-expressed in colon, pancreas, and prostate tumors while stranded miRNA. One of the miRNA strands is incorporated being down-regulated in breast cancer tumors (Volinia et al., into a complex of proteins and miRNA called the RNA 2006). induced silencing complex (RISC). The miRNA guides the 0010 Bioinformatics analyses Suggest that any given RISC complex to a target mRNA, which is then cleaved or miRNA may bind to and alter the expression of up to several translationally silenced, depending on the degree of sequence hundred different genes. In addition, a single gene may be complementarity of the miRNA to its target mRNA. Cur regulated by several miRNAs. Thus, each miRNA may regu rently, it is believed that perfect or nearly perfect complemen late a complex interaction among genes, gene pathways, and tarity leads to mRNA degradation, as is most commonly gene networks. Mis-regulation or alteration of these regula observed in plants. In contrast, imperfect base pairing, as is tory pathways and networks, involving miRNAS, are likely to primarily found in animals, leads to translational silencing. contribute to the development of disorders and diseases such However, recent data Suggest additional complexity (Bagga as cancer. Although bioinformatics tools are helpful in pre et al., 2005; Lim et al., 2005), and mechanisms of gene dicting miRNA binding targets, all have limitations. Because silencing by miRNAS remain under intense study. of the imperfect complementarity with their target binding 0007. Many miRNAs are conserved among diverse organ sites, it is difficult to accurately predict the mRNA targets of isms, and this has led to the Suggestion that miRNAS are miRNAs with bioinformatics tools alone. Furthermore, the involved in essential biological processes throughout the life complicated interactive regulatory networks among miRNAS span of an organism (Esquela-Kerscher and Slack, 2006). In and target genes make it difficult to accurately predict which particular, miRNAS have been implicated in regulating cell genes will actually be mis-regulated in response to a given growth and cell and tissue differentiation—cellular processes miRNA. that are associated with the development of cancer. For 0011 Correcting gene expression errors or modulating instance, lin-4 and let-7 both regulate passage from one larval gene expression by manipulating miRNA expression or by state to another during C. elegans development (Ambros, repairing miRNA mis-regulation represent promising meth 2001). mir-14 and bantam are Drosophila miRNAs that regu ods to repair genetic disorders and cure diseases like cancer. late cell death, apparently by regulating the expression of A current, disabling limitation of this approach is that, as genes involved in apoptosis (Brennecke et al., 2003, Xu et al., mentioned above, the details of the regulatory pathways and 2003). networks that are affected by any given miRNA remain gen 0008 Research on microRNAs is increasing as scientists erally unidentified. Besides E2F1, the genes, gene pathways, are beginning to appreciate the broad role that these mol and gene networks that are regulated by miR-20 in cancerous ecules play in the regulation of eukaryotic gene expression. In cells remain largely unknown. Currently, this represents a particular, several recent studies have shown that expression significant limitation for treatment of cancers in which miR levels of numerous miRNAs are associated with various can 20 may play a role. A need exists to identify the genes, genetic US 2009/0163434 A1 Jun. 25, 2009 pathways, and genetic networks that are regulated by or that tissues or subjects would resultina therapeutic response. The may regulate hsa-miR-20 expression. identities of key genes that are regulated directly or indirectly by miR-20 and the disease with which they are associated are SUMMARY OF THE INVENTION provided herein. In certain aspects a cell may be an epithelial, 0012. The present invention provides additional composi stromal, or mucosal cell. The cell can be, but is not limited to tions and methods by identifying genes that are direct targets brain, a neuronal, a blood, an esophageal, a lung, a cardiovas for miR-20 regulation or that are indirect or downstream cular, a liver, a breast, a bone, a thyroid, a glandular, an targets of regulation following the miR-20-mediated modifi adrenal, a pancreatic, a stomach, a intestinal, a kidney, a cation of another gene(s) expression. Furthermore, the inven bladder, a prostate, a uterus, an ovarian, a testicular, a splenic, tion describes gene, disease, and/or physiologic pathways askin, a smooth muscle, a cardiac muscle, or a striated muscle and networks that are influenced by miR-20 and its family cell. In certain aspects, the cell, tissue, or target may not be members. In certain aspects, compositions of the invention defective in miRNA expression yet may still respond thera are administered to a Subject having, Suspected of having, or peutically to expression or over expression of an miRNA. at risk of developing a metabolic, an immunologic, an infec miR-20 could be used as a therapeutic target for any of these tious, a cardiovascular, a digestive, an endocrine, an ocular, a diseases. genitourinary, a blood, a musculoskeletal, a nervous system, 0016. In certain aspects, the cell, tissue, or target may not a congenital, a respiratory, a skin, or a cancerous disease or be defective in miRNA expression yet may still respond condition.