1023. New Generation of shRNA for Targeted Cancer Gene Therapy

Small interfering RNA (siRNA) molecules are powerful mediators for post-transcrptional gene silencing known as RNA interference. Short hairpin (shRNA) encoded within an expression vector can effectively harnessing the RNA interference process in mammalian cells. RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Argonaute2 (Ago2) has been found to be the catalytic endonuclease of human RISC that mediates cleavage of targeted mRNA. Interestingly, the other Argonaute family of proteins (Ago1, Ago3, Ago4) lack of endonuclease activity, but are nonetheless capable of loaded with single-stranded guide RNAs. RISC assembled with Ago1, Ago3, or Ago4 may be cleavage-independent pathway for which mediates translational repressions. Translational repression via sequestration of mRNA in the P-body is proposed to be the major gene silencing pathway for micro RNAs (miRNAs). We are actively developing targeted cancer gene therapy strategy through the application of shRNAs. In the past, siRNA and shRNA design strategies have been focused on the RNA degradation pathway (Ago2 cleavage-dependent). In this study, we seek to expand the shRNA design strategy to simultaneously mediate targeted RNA degradation and translational repression. Simultaneously mediate RNA degradation and translational repression is predicted to produce much quicker and sustained knock-down effect on targeted mRNA. We will present data to support our novel shRNA design strategy, examine the efficacy of novel shRNA design process and compare knock-down efficiency with conventional shRNA molecules.