437. Alternative Strategies To Deliver Multiple shRNAs in Order to Simultaneously Inhibit Multiple Targets

Abstract

RNA interference is a new promising technology for therapeutic drug development. It can be initiated either by chemically synthesized double-stranded RNA delivered to a cell, or by transcription of a short hairpin RNA (shRNA) in the cell nucleus from a DNA template. We are interested in developing RNAi therapies for HCV, which is difficult infection to treat due to the generation of viral escape mutants. RNAi is uniquely positioned to address this issue because it has the ability to simultaneously inhibit multiple viral targets. This can be accomplished by delivering several shRNAs that target different sequences of the viral genome. One approach to accomplish this is to include several promoter-shRNA cassettes in a single DNA vector. The disadvantages of this strategy are the possible interference of the promoters, potential differences in promoter activity, and vector size limitations. Alternatively, multiple shRNAs could be expressed from a single promoter as a long shRNA precursor. Processing of this pre-shRNA by Drosha/Dicer complex may result in the generation of multiple siRNAs; however it may be difficult to optimize the pre-shRNA sequences for correct processing, and there is the potential for toxic effects attributed to the generation of a long double-stranded RNA. In this work we present a third alternative to express multiple shRNAs from a single promoter which includes the tandem positioning of short shRNAs (19-20 nucl) separated by short spacers. We identified several individual shRNA sequences that efficiently inhibit replication of HCV in the replicon system. We then combined these validated shRNAs in a triple construct under the expression of a single promoter and demonstrated functional activity of each of the individual shRNAs against their cognate targets. Results comparing these different strategies and their implication for shRNA-based drug development will be discussed.