Lack Of Interferon (IFN) Response To T7 Transcribed pppG (n)(n = 2,3)-shRNA

Abstract

RNA interference (RNAi) mediated by siRNAs has proved to be a highly effective gene silencing mechanism with great potential for gene therapeutic applications. However, siRNA agents have been shown to exert non-target-related biological effects and toxicities, including immune stimulation. Specifically, siRNA synthesized from a T7 RNA polymerase system can trigger the potent induction of type I IFN in a variety of cells. The single-stranded RNA can also stimulate innate cytokine responses in mammals. We found that pppGn (n = 1–3), associated with the 5′ end of the shRNA produced from the T7 RNA polymerase system, did not induce detectable levels of IFN. The residual amount of G associated with the 5′-end of the transcript was proportional to the reduction of the interferon response. We describe a T7 pppGn (n = 1–3) shRNA synthesis system that alleviates the IFN response, which will facilitate the design of siRNAs while maintaining their full efficacy.