The discovery of RNA interference (RNAi) promises to revolutionize medicine due to its unlimited potential to treat genetic, epigenetic and infectious diseases. RNAi mediated by synthetic short interfering RNA (siRNA) can theoretically be employed to target any disease gene. However, siRNA-mediated silencing is highly variable with many genes in mammalian cells being inefficiently silenced. In addition, siRNA have been reported to activate host interferon-like inflammatory responses, and mediate off-target gene silencing, both of which significantly challenge the application of RNAi in vivo. We have discovered aiRNA (asymmetric interfering RNA), a novel proprietary RNAi technology that is fundamentally different from conventional siRNA and its variations. In our preliminary studies, we have found that aiRNA has superior gene silencing properties (efficacy, potency, duration, onset of action) compared to siRNA in vitro. Moreover, aiRNAi mechanistically abolished sense strand mediated off-target silencing, and significantly reduced interferon responses. In a pilot study, we have also demonstrated that aiRNA directed against beta-catenin can target HT29 colon cancer xenografts in vivo and mediate significant gene silencing. The smaller size and higher potency of aiRNA should significantly reduce the synthesis cost, and may provide an in vivo delivery advantage compared to siRNA. The long-term goal of this SBIR proposal is to optimize aiRNA technology for gene silencing research and for developing RNAi therapeutics. In Phase I we will perform key experiments necessary for the optimization of aiRNA technology. Specifically, we will perform in vitro studies to optimize the design of aiRNA structures (Aim 1). We will examine the specificity and off-target effects of aiRNA-mediated gene silencing and compare them to (Aim 2). We will also determine the gene silencing properties of aiRNA in vivo (Aim 3). Results from these studies are needed to aid the design and development of our subsequent Phase II studies. Our overall goal in Phase II will be to develop and test a clinically compatible targeted therapy for colon cancer using the optimized aiRNA developed in Phase I. For these studies, optimized aiRNA directed against selected colon cancer target genes will be synthesized. The ability of each aiRNA to mediate therapeutic RNAi in vivo will then be assessed using xenograft and syngeneic colon cancer models. Further development of aiRNA may help to unleash the enormous potential of RNAi-based interventions against a wide spectrum of diseases. . PUBLIC HEALTH RELEVANCE: The recent discovery of RNA interference (RNAi), a natural process that allows specific genes to be "switched off" in cells, promises to revolutionize medicine;however, the development of therapies based on synthetic siRNA (short interfering RNA) has met with significant problems including the induction of non-specific effects, synthesis cost, limited efficacy, delivery difficulty in vivo. Recently, we have discovered that aiRNA (asymmetric interfering RNA), a novel proprietary RNAi technology developed at Boston Biomedical Inc., has superior gene silencing properties (efficacy, potency, specificity, duration, onset of action ) compared to siRNA. The research outlined in this proposal is designed to further investigate aiRNA with the long-term goal of optimizing this novel technology for gene silencing research and for developing RNAi therapeutics. .