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

摘要

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 achieved up to 40 percent siRNA inhibition, as well as increased apoptosis of cells transfected after cloning of the therapeutic exon component. In addition, by using a DNA-vector driven siRNA expression system, we can reduce the cost of producing siRNA and increase the duration of siRNA efficacy to achieve long term effects of gene silencing. Thus, by utilizing the combination of these 2 different targeting mechanisms from the RNA splicing process, we expected that the strategy will bring us a new affordable and promising way for efficient cancer cell targeting and treatment. Combined with the consistent and satisfactory lipid-based delivery system previously described by our lab (Li & Huang, 1997; Li et al., 2008; Li & Huang, 2006), we anticipate a greatly improved cancer targeting therapy.