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 cellTumor 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 Expression of Hec1 in vitro Unpublished data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 F5/3 ΔE1 ΔE3 F5/3 Ad-siRNA-GAPDH F5/3 ΔE1 ΔE3 F5/3 CMV >>siRNA>>polyA

13. METHODS 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

14. Methods 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

15. RESULTS INFECTIVITY ENHANCEMENT

16. p<0.001 *Log scale

17. RESULTS mRNA KNOCKDOWN

18. mRNA Knockdown

21. Translational Inhibition

22. Western Blot SKOV3.ip1 HEYOV4 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. RESULTS APOPTOSIS ASSAY

24. Annexin V-PI Analysis OV4 (Day 4) No Infection Ad siRNA Hec1 (500vp/cell)Ad siRNA Hec1 F5/3 (500vp/cell) Ad siRNA GAPDH F5/3 (500vp/cell) PI + - Annexin V - +

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

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

27. RESULTS CELL VIABILITY ASSAY

31. RESULTS CRYSTAL VIOLET STAINING

32. Null Hec1F5/3 Hec1 Gapdh OV4SKOV3.ip1Hey Crystal Violet Staining (Day 10) Null Hec1F5/3 Hec1 Gapdh 10 3 10 2 10 1 10 0 10 -1 0 Vp/cell

33. 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.

34. 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

35. 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