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1. DNA microarrays to determine the effect of histone deacetylase inhibition on host response to adenovirus gene therapy vectors http://www.kimicontrol.com/microorg/adenovirus.jpg Mike Waters Davidson College Biology Department VCU BBSI Laboratory of Dr. A. Malcolm Campbell Laboratory of Dr. Paul Fawcett

2. Gene Therapy- What Is It? • Delivery of a transgene into a host genome • Requires a vector • Retroviral, adenoviral, others Thomas CE. 2003. Nature Reviews Genetics 4:346-358. http://www.ngxinc.com/image/AdHTS_01.jpg

3. Neurodegenerative Disorders Baekelandt V., DeStrooper B., Nuttin B., Debyser Z. 2000.  Gene therapeutic strategies for neurodegenerative diseases. Current Opinion in Molecular Therapeutics 2:540-554 Cancer Tseng JF & Mulligan RC. 2002. Gene therapy for pancreatic cancer. Surgical Oncology Clinics of North America 11(3):537-569. HIV/AIDS Wolkowicz R., Nolan GP. 2005.  Gene therapy progress and prospects: Novel gene therapy approaches for AIDS. Gene Therapy 12: 467-476. Cardiovascular Disease Isner, J.M. Myocardial gene therapy. 2002. Nature 415, 234-239. Infectious Diseases Bunnell B & Morgan RA. 1998.  Gene therapy for infectious deceases. Clinical Microbiology Reviews 11:42-56. Gene Therapy- Possibilities

4. Viral vectors stimulate an innate immune response Attenuates transgene expression Cytokine storm Can be fatal http://www.abc.net.au/science/news/stories/s234410.htm Gene Therapy- A Problem http://www.jyi.org/articleimages/294/originals/img0.jpg

5. Responsible for activation of adaptive immmunity Toll-like receptors recognize Pathogen Associated Molecular Patterns (PAMPs) LPS, petidoglycan,dsRNA, CpG motifs MyD88 dependent pathway signals type-I interferon response Biology of Innate Immunity With Respect to Adenoviral Infection

6. MyD88 Pathway

7. STAT= signal transduction and activator of transcription Recognizes interferons to produce antiviral state Upregulate genes involved in immune response STAT Pathway http://www.nature.com/nri/journal/v5/n9/pdf/nri1684.pdf

8. TLR recognizes presence of PAMPs Stimulates MyD88 dependent pathway MyD88 dependent pathway produces an interferon and inflammatory response Interferons activate STAT pathway STAT pathway up regulates ISRE controlled genes Take Home Biology

9. Acetylation = important modulator of cellular response/ signal transduction HAT/HDAC HDAC inhibition has recently been shown to modulate interferon response Histone Deacetylase Inhibitors http://www.phy.mtu.edu/images/menu/HistoneAcetylation.jpg

10. Can treatment with an HDAC inhibitor transiently suppress the innate immune system to obtain stable gene expression? Compare the host response to adenoviral infection in the presence and absence of an HDAC inhibitor Big Question

11. GOAL - A quantifiable image of relative cDNA amounts between two conditions + TSA and –TSA TSA=HDAC inhibitor DNA Microarrays

12. Prep Hepatocytes Treat selected conditions (add TSA) Infect with Adenovirus Harvest RNA using lysis buffer according to experimental time course Prep and purify RNA Amplify RNA Clean aRNA Dye Couple Hybridize RNA to post-processed microarray slide Clean hybridized array Scan Grid RAM horn database Technique- DNA Microarrays

13. DNA Microarrays Adenovirus transduced hepatocytes Adenovirus transduced hepatocytes + TSA

14. MOI Cell Type Hepatocyte RAW (macrophage like) BMDM (bone marrow derived macrophages) Initial Conditions

15. Results • I was successful in obtaining a “gridable” microarray • Next step= make sense of data

16. Work in progress The host responose to adenovirus, helper-dependent adenovirus and adeno-associated virus in mouse liver McCaffrey AP., Fawcett., Nakai H., McCaffrey L., Ehrhardt A., Pham T., Pandy K., Xu H., Feuss S., Storm T., Kay MA. 2008. The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Molecular Therapy 16(5):931-941 Results analysis- Preface

17. Immune Response Genes Signal Transduction Transcription Cell Cycle Apoptosis Chromatin Stucture Cell Structure Cell metabolism Results

18. Immune Response Genes • Upregulation upon infection (Fawcett)

19. Signal Transduction • Upregulated upon adenoviral infection

20. Future Directions • 1064 genes whose expression level was changed by log base 2 or greater • Analyze data

21. Other hosts “Blue Sky” applications (Pathway related) Other HDAC inhibitors Different effects for different methods of deacetylation Drug Development Future Directions

22. NSF Fawcett Lab Jen Drake, Jen Feitweiss, Joe Bertsche, Nusara Satproedprai Barbour Lab Valerie Lab Beth Rosenburg Heilemann Lab Pat Bohdan Jeff, Sheryl, Jenn, and Billy Acknowledgments

23. References 1. Baekelandt V., DeStrooper B., Nuttin B., Debyser Z. 2000. Gene therapeutic strategies for neurodegenerative diseases. Current Opinion in Molecular Therapeutics 2:540-554. 2. Bunnell B & Morgan RA. 1998. Gene therapy for infectious deceases. Clinical Microbiology Reviews 11:42-56. 3. Decker T., Muller Mathias., Stockinger Silvia. 2005. The yin and yang of type I interferon activity in bacterial infection. Nature Reviews Immunology 5:675-687. 4. Isner, J.M. Myocardial gene therapy. 2002. Nature 415, 234-239. 5. Marshall E. 1999. Clinical Trials- Gene therapy death prompts review of adenovirus vector. Science 286:2244-2245. 6. McCaffrey AP., Fawcett., Nakai H., McCaffrey L., Ehrhardt A., Pham T., Pandy K., Xu H., Feuss S., Storm T., Kay MA. 2008. The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Molecular Therapy 16(5):931-941. 7. Nusionzon I & Horvath CM. 2006. Positive and negative regulation of the innate antiviral response and beta interferon gene expression by deacetylation. Molecular and Cellular Biology 26(8):3106-3113. 8. Thomas CE., Ehrhardt A., Kay MA. 2003. Progress and problems with the use of viral vectors for gene therapy. Nature Reviews Genetics 4:346-358. 9. Tseng JF & Mulligan RC. 2002. Gene therapy for pancreatic cancer. Surgical Oncology Clinics of North America 11(3):537-569. 10. Wolkowicz R., Nolan GP. 2005. Gene therapy progress and prospects: Novel gene therapy approaches for AIDS. Gene Therapy 12: 467-476. 11. Nusinzon I., Horvath CM. 2005. Unexpected roles for deacetylation in interferon- and cytokine – induced transcription. Journal of Interferon and Cytokine Research 25:745-748. 12. Akira Shizuo. 2003. Toll-like Receptor Signaling. The Journal of Biological Chemistry 278(40):38015-38108. Presentation References

24. Questions?