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WO 2015/038960 Al 19 March 2015 (19.03.2015) P O P C T

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WO 2015/038960 Al 19 March 2015 (19.03.2015) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/038960 Al 19 March 2015 (19.03.2015) P O P C T (51) International Patent Classification: (74) Agents: CANADY, Karen S. et al; canady + lortz LLP, C12Q 1/68 (2006.01) A61K 31/7105 (2006.01) 3701 Wilshire Blvd. Suite 508, Los Angeles, California C12N 15/113 (2010.01) A61P 35/00 (2006.01) 90010 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 14/055493 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) Date: International Filing BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 12 September 2014 (12.09.2014) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (26) Publication Language: English MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (30) Priority Data: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 61/877,902 13 September 2013 (13.09.2013) US SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA [US/US]; 1111 Franklin Street, Oak (84) Designated States (unless otherwise indicated, for every land, California 94607-5200 (US). kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (72) Inventors: POTHOULAKIS, Charalabos; UCLA Med- TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, div Of Digestive Diseases, Box 957019, 15 19 Mrl, Los TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Angeles, California 90095-7019 (US). ILIOPOULOS, Di- DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, mitrios; UCLA Med-div Of Digestive Diseases, Box LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, 956949, 44-133 Chs, Los Angeles, California 90095-6949 SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (US). LAW, Ka Man; UCLA Med-div Of Digestive Dis GW, KM, ML, MR, NE, SN, TD, TG). eases, Box 957019, 1519 Mrl, Los Angeles, California 90095-7019 (US). Declarations under Rule 4.17 : — of inventorship (Rule 4.17(iv)) [Continued on nextpage] (54) Title: MIR-133a AS A MARKER AND THERAPEUTIC TARGET FOR COLITIS AND INFLAMMATORY BOWEL DIS EASE (57) Abstract: The invention provides a method for detection and monitoring of in flammatory bowel disease (IBD) in a subject that comprises assaying a specimen from the subject for miR-133a, AFTPH, and, option ally NTSR1. An elevated amount of miR- n f 133a and/or NTSRl, and/or a decreased amount of AFTPH, present in the specimen compared to control sample is indicative of inflammation. The invention further provides a method of treating inflammatory disease, such as IBD or colon cancer, in a subject by administering an inhibitor of miR-133a. Published: — with sequence listing part of description (Rule 5.2(a)) — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) MIR-133a AS A MARKER AND THERAPEUTIC TARGET FOR COLITIS AND INFLAMMATORY BOWEL DISEASE [0001] This application claims priority to United States provisional patent application number 61/877,902, filed September 13, 2013, the entire contents of which are incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government support under DK060729, awarded by the National Institutes of Health. The Government has certain rights in the invention. TECHNICAL FIELD OF THE INVENTION The present invention relates generally to detection, diagnosis, and monitoring of inflammatory bowel disease. The invention more specifically pertains to use of miR-133a as a marker and treatment target for inflammatory bowel disease. BACKGROUND OF THE INVENTION Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a chronic inflammatory disease of the gastrointestinal (Gl) tract. Monoclonal antibodies against Tumor Necrosis Factor-alpha (TNF-a) remain one of the most effective treatments against IBD. In addition aminosalicylates, corticosteroids and other immunomodulators/immunosuppresants are also used as treatment modalities. However, remissions are common. IBD is a multifactorial disease, and all of the currently employed treatment modalities are associated with several and some times debilitating side effects. There is a need to identify improved methods for the detection and treatment of inflammatory bowel disease. SUMMARY OF THE INVENTION The invention provides a method for detection of inflammatory disease, such as inflammatory bowel disease, in a subject. In a typical embodiment, the method comprises contacting a specimen obtained from the subject with reagents for assaying for miR-133a; measuring the amount of miR-133a present in the specimen as compared to a control sample; and determining the presence of inflammatory bowel disease when an elevated amount of miR-133a is present in the specimen compared to the control sample. The invention additionally provides a method of detecting or monitoring inflammatory disease by assaying for levels of miR-133a, aftiphilin (AFTPH) and/or neurotensin receptor 1 (NTSR1). The method can be used to distinguish between ulcerative colitis and Crohn's disease. In one embodiment, the specimen is intestinal biopsy tissue, such as, for example, colon tissue, or intestinal fluid. Representative examples of inflammatory bowel disease include, but are not limited to, ulcerative colitis, Crohn's disease or Clostridium difficile colitis. In a typical embodiment, the measuring comprises polymerase chain reaction (PCR) assay, such as rea l time PCR. In another embodiment, the measuring comprises an immunoassay (e.g., for AFTPH or NTSR1). In one embodiment, the immunoassay detects cytokines deregulated during inflammatory bowel disease, including, but not limited to, ulcerative colitis, Crohn's disease or Clostridium difficile colitis. The method for monitoring the efficacy of treatment of inflammatory disease, such as inflammatory bowel disease, in a subject typically comprises contacting a specimen obtained from the subject at a first time point with reagents for assaying for miR-133a, AFTPH and/or NTSR1 ; contacting a specimen obtained from the subject at a second time point with reagents for assaying for miR-1 33a, AFTPH and/or NTSR1 , wherein the subject has been treated for inflammatory bowel disease prior to the second time point. The method further comprises measuring the amount of miR-1 33a, AFTPH and/or NTSR1 present in the specimens obtained at the first and second time points; and determining whether an increased or decreased amount of miR-1 33a, AFTPH and/or NTSR1 is present in the specimen obtained at the second time point compared to the specimen obtained at the first time point, which decreased amount of miR- 133a or NTSR1 , or increased amount of AFTPH, is indicative of effective amelioration of the inflammatory bowel disease. In one embodiment, the above method is modified to monitor the progression of inflammatory bowel disease in a subject, optionally performed in the absence of treatment, by comparing measurements obtained at the two time points. An increase in the amount of miR-1 33a and/or NTSR1 (or decrease in the amount of AFTPH) at the second time point compared to the first time point is indicative of disease progression. The specimen can be blood or other bodily fluid, such as peritoneal fluid, or a tissue specimen. Typically, the specimen is intestinal fluid or tissue. Examples of specimens include intestinal biopsy tissue, such as colon biopsy. Examples of inflammatory bowel disease include, but are not limited to, ulcerative colitis, Crohn's disease or Clostridium difficile colitis. The measuring typically comprises PCR or immunoassay. The invention additionally provides a method of treating inflammatory disease, such as inflammatory bowel disease, or cancer, in a subject. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of an inhibitor of miR-1 33a. In one embodiment, the administering is intracolonic or intravenous. Examples of inhibitors of miR- 133a include an antisense miR-1 33a oligonucleotide. The antisense oligonucleotide can be provided in a more stabilized form, such as, in one example, a locked-nucleic acid-based antisense miR-1 33a oligonucleotide. The method can comprise administering the antisense miR-1 33a oligonucleotide either directly, or via a lentiviral vector. The inflammatory bowel disease can be, for example, ulcerative colitis, Crohn's disease or Clostridium difficile colitis. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1C. MiR-1 33a down-regulation inhibits receptor recycling. (1A) NTSR1 localization in NCM460-NTSR1 cells transfected with control or antisense-miR-133a. Cells were incubated with vehicle or NTS (100 nM) for 1 h , washed and recovered in NTS-free medium for 3 h . (arrows, intracellular NTSR1; arrowheads, membrane-associated NTSR1). Scale, 10 µηη . (1B) Mean Fluorescence Intensity (MFI) was measured in the intracellular and cell surface- associated NTSR1 labeling and the surface and intracellular values were expressed as a ratio. (1C) Membrane-associated NTSR1 in biotinylation assay after vehicle, NTS (100 nM) treatment and recovery for 3 h were analyzed in an NTSR1 -specific ELISA. * P<0.05 when compared to antisense miR-control group. Figures 2A-2F. AFTPH is the binding target of miR-1 33a. (2A) Diagram showing complementary binding site of miR-1 33a in AFTPH 3' UTR in different species.
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