Combined gene therapy of siRNA and apoptosis inducing factor generated from a single transcriptional event

shaping the way I approach scientific research and acting as a mentor to provide me lots of suggestion and advice about life. I feel very lucky to have worked under his direction and to have enjoyed the intelligent freedom to study a novel approach of siRNA delivery system from a plasmid vector that utilizes the RNA splicing system. I am also thankful to my thesis committee members, Drs. X i a o Xiao and Moo Cho for facilitating the progress of this research project with their insightful suggestions and expertise. Also, thanks goes to the entire Huang lab: Dr. Feng Liu's excellent work in maintaining the lab in a good condition and many perspicacious scientific discussions; Lisa Shollenberger's intelligent suggestions and help in teaching me how to use many pieces of lab equipment; also all other members in the lab for support and help. As to the administration part of the Division, I would like to thank Angela Lyght, Kathryn Fiscelli, and Lee Daub, especially for Angela's always ready help in taking care of miscellaneous matters. I would also like to thank Taiwan government for awarding me 3 years scholarship and giving me the opportunity to come to the US to study. Special thanks to Dr. Anthony iv Hickey for always listening, advising and continuous encouragement. Finally, I would like to appreciate my parents, Shan-Nan Hung and Li-Fang Yie for their always warm support, also Dr. Biao Dong, Becky, Grace, Thomas and all the friends for their invaluable friendship and ever-present love. v ABSTRACT Hsin-I (Cindy) Hung The goal of this project is to test the hypothesis that a single transcriptional event driven by RNA polymerase II promoter driven siRNA expression plasmid that utilized RNA splicing mechanism. During this event, a specific siRNA to a target oncogene, forms from the spliced intron; meanwhile a therapeutic protein is established after rejoining of the exon fragments. In this case, the well-known, strong RNA polymerase II (CMV) promoter was used to produce an siRNA against several well known oncogenes and a therapeutic protein, Apoptosis Inducing Factor (AIF), as a dual therapeutic to induce tumor cells apoptosis simultaneously from a single plasmid construct. By using this splicing approach with 2 different targeting mechanisms (i.e., oncogene inhibition by siRNA and therapeutic protein expression), we expect to see a synergistic effect from our novel RNA polymerase II promoter-driven plasmid. It was demonstrated that the plasmid construct …