Development of Specific Gene Silencing Methods and Reagents

描述（申请人提供）：RNA干扰（RNAi）是一种有效的选择性基因沉默机制，已经彻底改变了生物科学领域。RNAi特异性下调任何基因表达的能力对基因功能的研究产生了深远的影响，并在体内功能基因组学中具有很大的前景。目前在哺乳动物细胞中诱导RNAi的范例依赖于使用21 bp siRNA结构支架。这种siRNA支架由一个19 bp的双链组成，每个3'端有2-nt悬垂，已广泛用于体外基因功能研究，以及开发各种人类疾病的新疗法。尽管siRNA在体外研究中广泛使用，但该技术受到可变基因沉默效应、先天免疫反应的激活和非靶向mRNA的敲低（脱靶沉默）的困扰。这些众所周知的效应使依赖特定靶基因敲除的基因功能实验的解释复杂化。此外，在体内测试基因功能的大规模功能基因组学实验中，这些问题可能变得无法管理。波士顿生物医学公司发现了aiRNA（不对称干扰RNA），这是一种在哺乳动物细胞中诱导高效RNAi的新颖专利技术。与siRNA相比，aiRNA已显示出更优越的体外基因沉默特性。此外，aiRNA完全消除了义链脱靶沉默，消除或显著降低了干扰素应答诱导。这些发现表明，aiRNA可以满足对选择性敲低靶基因技术的迫切需求，并表明aiRNA在包括功能基因组学在内的生物学研究中具有广泛的应用潜力。本SBIR提案的长期目标是优化aiRNA作为基因功能研究试剂，包括全基因组功能基因组学研究。在I期，我们将进行研究以优化aiRNA基因沉默子的结构和设计，以供生物医学研究人员使用。在Aim 1中，我们将研究各种aiRNA结构，以确定在各种实验条件下诱导强效和高特异性沉默的最佳设计。在目标2中，我们将研究设计有效aiRNA的当前siRNA设计算法的相关性。在目标3中，我们将进行实验，以确定开发airna特定设计算法的关键要素。我们在II期的总体目标是开发一种全基因组验证的aiRNA试剂，用于功能基因组学研究。这些研究将需要基于针对所有已知人类基因的优化的aiRNA结构（在第一阶段开发）构建一个文库。然后将评估aiRNA文库在体外介导有效和特异性基因沉默的能力。aiRNA技术的进一步发展将显著提高我们分析基因功能的能力，并可能对涉及rnai介导的基因沉默的其他应用产生重大影响。

DESCRIPTION (provided by applicant): RNA interference (RNAi), a potent and selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any gene has had a profound impact on the study of gene function, and holds great promise for in vivo functional genomics. The current paradigm for inducing RNAi in mammalian cells relies on the use of a 21-bp siRNA structural scaffold. This siRNA scaffold, which consists of a 19-bp duplex with 2-nt overhangs at each 3' end, has been widely used for in vitro gene function studies, as well as for the development of novel therapeutics for various human diseases. Despite the extensive use of siRNA for in vitro studies, this technology is plagued by variable gene silencing effects, the activation of innate immune responses, and the knockdown of non-targeted mRNA (off-target silencing). These well-recognized effects complicate the interpretation of gene function experiments that rely on specific target gene knockdown. Moreover, these problems can become impossible to manage in large scale functional genomics experiments that test gene function in vivo. Boston Biomedical Inc. has discovered aiRNA (asymmetric interfering RNA), a novel proprietary technology for inducing highly efficient RNAi in mammalian cells. aiRNA has shown superior in vitro gene silencing properties compared to siRNA. Moreover, aiRNA completely abolished sense-strand off-target silencing, and eliminated or significantly reduced interferon response induction. These findings indicate that aiRNA can meet the urgent need for a technology to selectively knockdown target genes, and suggest that aiRNA holds significant potential for broad applications in biological research, including functional genomics. The long-term goal of this SBIR proposal is to optimize aiRNA as a reagent for gene function research, including genome-wide functional genomics studies. In Phase I we will perform studies to optimize the structure and design of aiRNA gene silencers that can be used by biomedical researchers. In Aim 1, we will examine various aiRNA structures to determine the best design for inducing potent and highly specific silencing under various experimental conditions. In Aim 2, we will examine relevance of current siRNA design algorithms for devising efficacious aiRNA. In Aim 3, we will perform experiments to identify key elements for the development of an aiRNA-specific design algorithm. Our overall goal in Phase II will be to develop a genome-wide validated aiRNA reagent for functional genomics research. These studies will entail the construction of a library based on the optimized aiRNA structures (developed in Phase I) against all known human genes. The ability of the aiRNA library to mediate efficacious and specific gene silencing in vitro will then be evaluated. The further development of aiRNA technology should significantly improve our ability to analyze gene function, and may have significant implications for other applications involving RNAi-mediated gene silencing. PUBLIC HEALTH RELEVANCE: The current paradigm for inducing RNA interference (RNAi) in mammalian cells relies on the use of short interfering RNA (siRNA) that have been used extensively for in vitro gene silencing; however, various non- specific effects relating to the use of siRNA have been recognized as a significant drawback to their use in gene function research. Boston Biomedical Inc. has discovered aiRNA (asymmetric interfering RNA), a novel proprietary technology for inducing highly efficient RNAi in mammalian cells that has superior in vitro gene silencing properties, eliminated sense-strand mediated off-target silencing, and markedly reduced or eliminated non-specific interferon-like response induction compared to conventional siRNA. The research outlined in this proposal is designed to optimize the structure of, and develop a design algorithm for, aiRNA gene silencers that can be used by the biomedical research community, with the long-term goal of developing this technology as a reagent for genome-wide functional genomics studies.