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The use of DNA microarrays to determine the effect of histone deacetylyase inhibitors on host response to adenovirus gene therapy vectors Mike Waters Davidson College/Virginia Commonwealth University Mentors: A. Malcolm Campbell, Paul Fawcett Introduction A new discovery in the field of gene therapeutics resonates throughout the world of clinical medicine. Gene therapy regimens are currently under investigation as possible treatments of cancer9, 8, HIV/AIDS8, 10, cardiovascular diseases4, infectious diseases2, and neurodegenerative disorders1. Clinical progress however has been slowed due to problems regarding the vehicles of gene transduction into the patient’s genome. Today, three major transgene vectors are used in gene therapy studies: retroviral vectors, adenoviral vectors, and non-viral vectors (fig 1)8. Of the three, adenoviral vectors pose the most promise from a pharmacological standpoint because of their efficiency of gene transduction, ability to transduce genes regardless of mitotic cell status, tissue specificity, and potential duration of transgene expression6, 7, 8, 9. However, adenoviral vectors are problematic pharmacological agents due to the degree in which they elicit an innate immune response in their host3, 5, 7, 8. A robust innate immune response poses 2 major problems for gene therapy: 1) Strong cytokine activation can result in patient tissue damage through the stimulation of NK cells and in extreme cases lead to a “cytokine storm3, 7.” 2) A robust innate immune response leads to instability in Fig 1. Depicts the vectors used in gene therapy transgene expression 3, 7. clinical trials in 2003. Recent literature suggests that a transient suppression of the innate Thomas CE. 2003. Nature Reviews Genetics immune signaling cascade is possible 4:346-358. through the deacetylation inhibition of the positive regulatory domain of interferon regulatory factor 3 (IRF3)7. In mammalian cells the role of deacetylation of immune response signaling proteins is carried out by a family of histone deacetylase (HDAC) enzymes. Trichostatin A is an HDAC inhibitor and the agent we will use to attempt to transiently suppress host innate immune response in order to obtain stable transgene expression while the host is “infected” with an adenovirus gene therapy vector. 1 hr 6 hr 72 hr 4 weeks A D V A D V A D V A D V Igtp Igtp Igtp Stat1 Ifrg15 Parp9 I MyD88 Ifi203 Ifi47 II Tlr2 Csf1 Cxcl10 H3f3b Litaf Lcn2 Cxcl1 Mt1 III Mt1 Mt2 Mt2 Vcam1 Mt4 Gstp1 C1qb C1qa C1qb Cfp IV V Methods The effect of Trichostatin A on the repression of proteins involved in the innate immune response will be quantified using DNA microarray data (fig 2). The mRNA for the microarrays will be collected ex vivo, from mouse macrophage cells. The mouse macrophage cells produce a strong innate immune response that will make data quantification more apparent. Before the “infection” with replication deficient adenovirus, a dose-response experiment will be conducted where adenovirus concentration vs. transgene expression will be measured. The reporter transgene expressed in the dose-response experiment will be GFP in order to easily detect of the percentage of cells “infected” with adenovirus. Possible Results Before I can begin extracting mRNA from mouse macrophage cells I first have to familiarize myself with the subtleties of culturing mammalian cells. Once I have developed the skills necessary to work with mammalian cells and obtain mRNA without contamination I will be able to begin Fig 2. Microarray depicting the gene running microarray experiments. The expression profile of adenovirus differences in the innate immune response gene infected mouse liver cells. The top expression profile of adenovirus infected mouse two enlarged boxes show an up macrophages treated with trichostatin A and regulation of proteins involved in the that of the control mouse macrophages could innate immune response at 1 hour and vary from complete repression of innate 6 hours. immune response to little effect on the innate immune response signaling cascade, with many McCaffery & Fawcett. 2008. possible permutations in between. I am Molecular Therapy 16(5):931-941 confident that by the end of the summer I will have produced clean microarray data for analysis, and have a substantial amount of mRNA frozen down for analysis at Davidson College for the academic year. Hba1-a1 Hba1-a1 Fdx1 Hbb-b1 Hba1-a1 Hba1-a1 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. Works Consulted Nusinzon I., Horvath CM. 2005. Unexpected roles for deacetylation in interferon- and cytokine – induced transcription. Journal of Interferon and Cytokine Research 25:745-748. Akira Shizuo. 2003. Toll-like Receptor Signaling. The Journal of Biological Chemistry 278(40):38015-38108.