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

这个小企业创新研究（SBIR）项目的更广泛的影响/商业潜力是为研究人员提供更强大和负担得起的工具来研究活细胞内基因的活动。RNA干扰（RNAi）是一项获得诺贝尔奖的技术，允许活细胞内基因表达的特异性沉默。这项技术迅速成为研究基因功能、新基因功能鉴定和治疗开发的宝贵工具。 然而，目前可用的RNAi技术受到非特异性效应和高成本的限制。该提案的目标是开发并向科学界提供新一代RNAi试剂，该试剂基于使用化学修饰的长双链（ds）RNA分子。这项技术的更高效力和特异性，沿着更低的成本，将使研究人员能够显着提高他们的功能基因组学研究的功效。拟议的RNAi技术将进一步推进功能基因组学领域，并朝着创建人类基因组功能图谱的方向迈进。预计这项技术有可能取代目前可用的RNAi工具的很大一部分，并在目前基于RNAi的研究工具市场中占据相当大的份额，估计为1.2亿至2亿美元。该SBIR项目旨在开发新一代更有效和更特异的RNAi研究工具，基于将长dsRNA引入哺乳动物细胞。虽然长dsRNA成功地用于秀丽隐杆线虫和果蝇中的RNAi筛选，但由于诱导强烈的天然免疫应答，它不能直接用于哺乳动物细胞。最近的研究表明，长dsRNA在哺乳动物细胞中具有有效和特异性的RNAi效应，这种效应通常被dsRNA诱导的毒性所掩盖。将多种核苷酸修饰引入mRNA（即，长结构的RNA分子）阻止这种先天免疫应答的发展。目的是验证已知在长dsRNA的背景下阻断RNA与Toll受体相互作用以规避免疫应答的许多RNA修饰。 这将通过将许多修饰的核苷酸掺入dsRNA中，然后在哺乳动物细胞培养物中测试它们的基因沉默效力和特异性以及毒性（诱导先天免疫应答的能力）来实现。 与基于标准siRNA的试剂相比，基于长dsRNA的RNAi试剂预期具有更高的效力和特异性，并且提供了处理RNAi文库以进行功能基因组筛选的更简单且更具成本效益的方式。

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.