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T Michael Numnum, MD International Gynecologic Cancer Society Santa Monica, CA October 15, 2006

Infectivity Enhanced Adenovirus as a Strategy for Improving the Efficiency of RNA Interference in an Ovarian Cancer Model. T Michael Numnum, MD International Gynecologic Cancer Society Santa Monica, CA October 15, 2006. Background –Ovarian Cancer.

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T Michael Numnum, MD International Gynecologic Cancer Society Santa Monica, CA October 15, 2006

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  1. Infectivity Enhanced Adenovirus as a Strategy for Improving the Efficiency of RNA Interference in an Ovarian Cancer Model T Michael Numnum, MD International Gynecologic Cancer Society Santa Monica, CA October 15, 2006

  2. Background –Ovarian Cancer • 70% complete response with radical surgery + platinum based combination chemotherapy. • Majority will recur and die of disease. • Gene knockdown strategies may a therapeutic model

  3. Gene Knockdown Strategies • Antisense Oligonucleotides • Ribozymes • Drugs • RNA interference

  4. RNA Interference (RNAi) • Short interfering RNA (siRNA) • Post transcriptional gene silencing • Inhibits gene expression via degradation of corresponding mRNA. • Cancer, infectious disease research

  5. Why RNAi? • Sequence specific • Parallels specificity of antigen-antibody • Stable • Delivery to the target still the rate limiting step. • Lipid based infection • Electroporation • Adenoviral delivery

  6. The Infectivity Enhanced Adenovirus • Genetically engineered to overcome poor infectivity of Adenovirus in cancer cells • May enhance delivery of siRNA to tumor cells CAR Normal cell Tumor cell

  7. Hypothesis • The infectivity enhanced adenovirus as a delivery mechanism for RNAi is an attractive vector for gene knockdown strategies in an in vitro ovarian cancer model.

  8. Hec1 (Highly Expressed in Cancer) • Essential in chromosome segregation • Modulates G2/M phase of the cell cycle • Disruption of Hec1 by genetic deletion leads to cell death • Potential target in actively replicating cells Martin et al., 2002

  9. Ad-siRNA Hec1 inhibits tumor growth • High Infectivity (LacZ) • 50-68% translational knockdown at 48, 72 hours • 40% in vivo knockdown Gurzov et al., 2005

  10. Expression of Hec1 in vitro Unpublished data

  11. Materials • siRNA oligo sequences designed • Hec1: Gen Bank Accession # NM_006101 • GAPDH: Gen Bank Accession # NM_002046 • Negative control • Sequences cloned into • Adenovirus 5 • Wild type • Adenovirus F5/3 • Chimeric virus designed to enhance infectivity (Kanerva et al., 2003)

  12. Reagents Ad-siRNA-Hec1 ΔE1 ΔE3 CMV >>siRNA>>polyA Ad-siRNA-Hec1 F5/3ΔE1 ΔE3 F5/3 CMV >>siRNA>>polyA Ad-siRNA-GAPDH F5/3ΔE1 ΔE3 F5/3 CMV >>siRNA>>polyA

  13. Infectivity enhancement Cell lines infected Ads DNA purified after 3 hours Quantitative PCR for Adenoviral E4 gene RNA knockdown Cell lines infected with Ads (500 vp/cell) RNA purified after 48 hours Quantitative PCR for Hec1 Translational Inhibition Cell lines infected with Ads (500 vp/cell) Protein isolated after 72 hours Western Blot for Hec1/ β-actin METHODS

  14. Apoptosis Assay Cell lines infected with Ads Cells collected after 96 hours Annexin V/PI FITC-FACS Cell Viability Cell lines infected with Ads (500 vp/cell) MTS assay performed at days 2,4,6, and 8 Crystal Violet Staining Cell lines infected with Ads Multiplicity of infection: 1000,100,10,1,0.1,0 vp/cell Crystal violet staining after 10 days Methods

  15. RESULTSINFECTIVITY ENHANCEMENT

  16. *Log scale p<0.001

  17. RESULTSmRNA KNOCKDOWN

  18. mRNA Knockdown

  19. mRNA Knockdown

  20. mRNA Knockdown

  21. Translational Inhibition

  22. Western Blot SKOV3.ip1 HEY OV4 Hec 1 βactin Mock Hec1 Hec1F5/3 GAPDH F5/3 Mock Hec1 Hec1F5/3 GAPDH F5/3 Mock Hec1 Hec1F5/3 GAPDH F5/3 *96 hours

  23. RESULTSAPOPTOSIS ASSAY

  24. Annexin V-PI AnalysisOV4 (Day 4) PI + - Annexin V - + Ad siRNA GAPDH F5/3 (500vp/cell) No Infection Ad siRNA Hec1 (500vp/cell) Ad siRNA Hec1 F5/3 (500vp/cell)

  25. Annexin V-PI AnalysisHEY (Day 4) PI + - Annexin V - + Ad siRNA GAPDH F5/3 (500vp/cell) No infection Ad siRNA Hec1 (500vp/cell) Ad siRNA Hec1 F5/3 (500vp/cell)

  26. Annexin V-PI Analysis SKOV3.ip1 (Day 4) PI + - Annexin V - + Ad siRNA GAPDH F5/3 (500vp/cell) No Infection Ad siRNA Hec1 (500vp/cell) Ad siRNA Hec1 F5/3 (500vp/cell)

  27. RESULTSCELL VIABILITY ASSAY

  28. RESULTSCRYSTAL VIOLET STAINING

  29. Crystal Violet Staining (Day 10) Vp/cell Null Hec1F5/3 Hec1 Gapdh Null Hec1F5/3 Hec1 Gapdh Null Hec1F5/3 Hec1 Gapdh 103 102 101 100 10-1 0 OV4 Hey SKOV3.ip1

  30. Conclusions • In an in vitro ovarian cancer model, RNA interference of Hec1 results in mRNA knockdown and apoptosis leading to cell death. • The infectivity enhanced adenovirus is a reasonable strategy for delivery of RNAi in an ovarian cancer model.

  31. Acknowledgements • UAB Division of Gynecologic Oncology • Sharmila Makhija, MD • Ronald Alvarez, MD • Division of Human Gene Therapy • David Curiel, MD, PhD • Zeng Bian Zhu, MD • Baogen Lu, MD • Minghui Wang, MD • Angel Rivera

  32. Infectivity Enhanced Adenovirus as a Strategy for Improving the Efficiency of RNA Interference in an Ovarian Cancer Model T Michael Numnum, MD International Gynecologic Cancer Society Los Angeles, CA October 15, 2006

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