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1. WO2012073253 - THERAPEUTIC USES OF MIRNAS/COMPOUNDS THAT ACTIVATE TUMOR SUPPRESSOR GENES/MIRNAS

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

2. CLAIMS

I claim:

1. A stable cell line that expresses both c-myc and tumor suppressor TAp63/p73/p53/INK4a/b/c/d/CDKN1a/b/c/ARF/RKIP/CDHl/PTEN/Ago-1to4/FBW7/RBs/CycG2/DEC2/DOK2/AML1/2/BRCA1/p38a/TSC1/MEK4/PPP2R2A/TSP1/B RMS1/E2F1/2/TIMP3/CTGF/SMAD2/RRM2B/MXI1/DMTF/CHD5/miR-15/16/let-7/miR-34/miR-145/miR-26/miR-29/miR-30/miR-23/miR-22/miR-203/miR-200/miR-134/miR-miR-192/miR-215 promoters will be generated. Any combination of c-myc promoter plus tumor suppressor promoter will be chosen (for e.g., c-myc+p53, c-myc+INK4a, c-myc+miR-145, c-myc+miR-15/16 and so on) to generate stable cell lines.

2. c-Myc promoter will be linked to renilla luciferase; and TAp63/p73/p53/INK4a/b/c/d/CDKN1a/b/c/ARF/RKIP/CDH1/PTEN/Ago1to4/FBW7/RBs/Cyc G2/DEC2/DOK2/AML1/2/BRCA1/p38a/TSC1/MEK4/PPP2R2A/TSP1/BRMS1/E2F1/2/TIMP3 /CTGF/SMAD2/RRM2B/MXI1/DMTF/CHD5/miR-15/16/let-7/miR-34/miR-145/miR-26/miR-29/miR-30/miR-23/miR-22/miR-203/miR-200/miR-134/miR-192/miR-215 promoter will be linked to firefly luciferase. These two gene fragments will be cloned into a single mammalian expression vector containing resistance markers (eg., G418, purinomycin etc.).

3. The expression vector, as stated in 2, will be used to generate stable cell lines/clones (any mammalian cell lines).

4. The stable cell lines, as stated in 3, will be used to screen for compounds that simultaneously suppress c-myc promoter and induce tumor suppressor TAp63/p73/p53/INK4a/b/c/d/CDKN1a/b/c/ARF/RKIP/CDH1/PTEN/Ago1- 4/FBW7/RBs/CycG2/DEC2/DOK2/AML1/2/BRCA1/p38a/TSC1/MEK4/PPP2R2A/TSP1/BRM S1/E2F1/2/TIMP3/CTGF/SMAD2/RRM2B/MXIl/DMTF/CHD5)/miR(microRNA)-15/16/let-7/miR-34/miR-145/miR-26/miR-29/miR-30/miR-23/miR-22/miR-203/miR-200/miR-134/miR-miR-192/miR-215 promoter will be selected for further evaluation. Compounds that induce tumor suppressor genes/miRNAs alone will also be selected for further evaluation.

5. RNPC1 promoter will be linked to renilla luciferase; and p63/p53/miR-15/16/let-7/miR-34/miR-145/miR-26/miR-29/miR-30/miR-23/miR-22/miR-203/miR-200/miR-134/miR-192/miR-215 promoter will be linked to firefly luciferase. These two gene fragments will be cloned into a single mammalian expression vector containing resistance markers (eg., G418, purinomycin etc.).

6. The stable cell line, as stated in 5, will be used to screen for compounds that simultaneously suppress RNPC1 and induce tumor suppressor p63/p53/miR-15/16/let-7/miR-34/miR-145/miR-26/miR-29/miR-30/miR-23/miR-22/miR-203/miR-200/miR-134/miR-192/miR-215 promoter activities will be selected for further evaluation.

7. A stable cell line╌any mammalian cell line╌that expresses Dicer1/let-7/Pax5/CDC6/ARK- 1/2/MYCN promoter linked to renilla/firefly luciferase gene will be generated.

8. The stable cell line, as stated in 7, will be used to screen for compounds that induce or suppress Dicer1/Pax5/let-7/CDC6/ARK-1/2/MYCN promoter activity will be selected for further evaluation.

9. The components of the biological pathways mentioned below will be used for diagnosis, prognosis, and treatment of a number of disease conditions, including cancer.

a. p53/TA-p73/p63-miR-145-c-myc-lin-28/miR-17-92-let-7 (e.g., lung cancer)

b. p53/TA-p73/p63-miR-145/let7/miR-34-c-myc- miR-17-92

c. TA-p73/p63-miR-145/let7/miR-34-c-myc- miR-17-92/

CDKN1c/CDKN1a/CycG2/DEC2/AMLl/DOK2/p105/CD82/Dicer/DMTF/CHD5/BCMS1 d. p53/TA-p73/p63-TRIM32- c-myc-let-7

e. p53/TA-p73/p63/miR-145/miR-34/let-7/FBXW7- c-myc

f. p53/TA-p73/p63-miR-145/miR-34/let-7/FBXW7-c-myc-let-7/miR-15/16/miR-34/miR-26/miR-23/miR-29/miR-30/miR-146/miR-22/miR-150

g. p53/TA-p73/p63-miRs-192, 215, 145, 200-ZEB2/SIP1-E-cadherin

h. p53/TA-p73/p63-miR-145/34/let-7-c-myc-miR-9-3-E-cadherin

i. p53/TA-p73/p63-miR-145/34/let47-c-myc- miR-221/222-TIMP3/PTEN/CDKN1b, c j. p63-AML1/Runx-1-ARF

k. E2F-1-p73/miR-15/16-JunB-INK4a/ARF

l. p73/p63-JunB/DMTF-ARF

m. E2F-1-TA-p73/p63/p53-Snail-RKIP-c-myc-lin-28-let-7a/g-HMGA2-ras(Ha/N/K)

n. p53/p73/p63-JunB/AP-2/KAI1-KiSS

o. TA-p73/p53/p63-JunB-miR-203-Snail1/Slug/E-cadherin/PTEN

p. TA-p73/p53/p63-JunB-miR-203-BMI- INK4a/ARF

q. p53/p73/p63-let-7-c-myc/Skp-2/CKS1B/CDK1-p130/RhoA-CDKN1-A/-B/p57Kip2/INK4-a,-b-,c,-d

r. p53/TA-p73/p63-miR-200-ZEB1-TA-p73/E-cadherin/INK4B/CDKNlA

s. p53/p73/p63-miR-34/130-SRC-1-ETS-2-c-myc-Twist1—E-cadherin

t.p53/p73/p63-c-myc-miR-26-EZH2-INK4a/ARF/p130/CDKN1B/C-DNMT3b/Klf-4/HOXA9/ HMGA2/Jagged-1/HIF-2α/AIB

u. p53/p73-c-myc-miR-30-WWP1-p63/let-7/CDKN1B/C/p130/E-cadherin

v. p53/TA-p73/p63-let-7/miR-145-c-myc-miR-23-HIF-2α/ZEB1/CDH1/INK4b/Skp2

w. p53/TA-p73/p63-let-7/miR-145-c-myc- miR-146 -β-catenin

x.E2F-1/2-TA-p73/p63-p57kip2/LZTS1/TSC1/PTEN1/RBs/14-3-3o/AML2/INK4-miR

145/143/let-7/101/29/34(eg. lung cancer; and bladder cancer)

y. p53/p73/p63-c-myc-miR-26- HDM2, HMGA2, and Skp2-p53, INK4a, ARF, p130, and CDKN1B/C.

10. BMI1/WWP1 promoter will be linked to renilla luciferase; and miR-15/16/30/CDKNlA/INK4A/PTEN/TA-p63 promoter will be linked to firefly lucifease. These gene fragments will be cloned into a single mammalian expression vector containing resistance markers (e.g., G418, purinomycin etc.). This expression vector will be used to generate stable cell lines/clones (any mammalian cell lines). These stable cell lines will be used to screen for compounds that simultaneously suppress BMI-1/WWP1 and induce miR-15/16/CDKN1A/INK4A/PTEN/TA-p63 promoter activities will be selected for evaluation (for e.g., WWP1+p63; BMI1+INK4a; BMI1+PTEN promoters and so on).

3. DATE AND SIGNATURE (to be given at the end of last page of specification)