SBIR Phase I: New generation of RNA interference (RNAi) research tools to study activity of genes inside living cells

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to provide researchers with more powerful and affordable tools to study activity of genes inside the living cells. RNA interference (RNAi) is a Noble Prize-winning technology that allows specific silencing of gene expression inside living cells. This technology quickly became an invaluable tool in study of gene function, functional identification of new genes, and therapeutic development. Currently, however, available RNAi techniques are limited by non-specific effects and high cost. The goal of this proposal is to develop and make available to a scientific community a novel generation of RNAi reagents, based on the use of chemically modified long double-stranded (ds) RNA molecules. The higher potency and specificity of this technology, along with lower costs, will allow researchers to significantly increase the efficacy of their functional genomics studies. The proposed RNAi technology will further the field of functional genomics and move closer toward creating a functional map of the human genome. It is anticipated that this technology has the potential to replace a significant share of currently available RNAi tools and capture a sizable share of the current RNAi-based research tools market, estimated at $120-200M. This SBIR project aims to develop a new generation of more potent and specific RNAi research tools, based on the introduction of long dsRNAs into mammalian cells. While long dsRNA is used successfully for RNAi screening in C.elegans and flies, it can't be used directly in mammalian cells due to the induction of a strong native immune response. Recent studies have demonstrated that long dsRNA has a potent and specific RNAi effect in mammalian cells, which is usually masked by dsRNA-induced toxicity. Introduction of a variety of nucleotide modifications into mRNA (i.e., long structured RNA molecules) prevents development of this innate immune response. The goal is to validate a number of RNA modifications known to block RNA interactions with Toll-receptors in the context of long dsRNA to circumvent the immune response. This will be accomplished by incorporating a number of modified nucleotides into dsRNA, and then testing them for gene silencing potency and specificity, and toxicity (ability to induce the innate immune response) in mammalian cell culture. RNAi reagents based on long dsRNAs are expected to have higher potency and specificity compared to standard siRNA-based reagents, and provide a simpler and more cost effective way of handling RNAi libraries for functional genomic screening.