Development of Specific Gene Silencing Methods and Reagents

描述(申请人提供)：RNA干扰(RNAi)，一种有效的和选择性的基因沉默机制，已经彻底改变了生物科学领域。RNAi能够特异性地下调任何基因的表达，已经对基因功能的研究产生了深远的影响，并为体内功能基因组学带来了巨大的希望。目前在哺乳动物细胞中诱导RNAi的范例依赖于21bp siRNA结构支架的使用。这种siRNA支架由19个碱基组成，每个3‘端有2个核苷酸突起，已被广泛用于体外基因功能研究，以及各种人类疾病的新疗法的开发。尽管siRNA被广泛用于体外研究，但这项技术受到各种基因沉默效应、先天免疫反应的激活和非靶向mRNA的敲除(非靶标沉默)的困扰。这些公认的效应使依赖于特定靶基因敲除的基因功能实验的解释复杂化。此外，在测试体内基因功能的大规模功能基因组学实验中，这些问题可能变得不可能处理。波士顿生物医学公司发现了不对称干扰RNA(AiRNA)，这是一种在哺乳动物细胞中诱导高效RNAi的新专利技术。与siRNA相比，aiRNA在体外表现出更好的基因沉默特性。此外，aiRNA完全消除了正义链脱靶沉默，消除或显著降低了干扰素反应诱导。这些发现表明，aiRNA可以满足对选择性敲除靶基因的技术的迫切需求，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。这项SBIR建议的长期目标是优化aiRNA作为基因功能研究的试剂，包括全基因组功能基因组研究。在第一阶段，我们将进行研究，以优化可供生物医学研究人员使用的aiRNA基因消音器的结构和设计。在目标1中，我们将检查各种aiRNA结构，以确定在各种实验条件下诱导有效和高度特异性沉默的最佳设计。在目标2中，我们将检查当前siRNA设计算法与设计有效的aiRNA的相关性。在目标3中，我们将进行实验，以确定开发特定于aiRNA的设计算法的关键要素。我们在第二阶段的总体目标将是开发一种用于功能基因组研究的全基因组验证的aiRNA试剂。这些研究将需要建立一个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)的文库。然后将评估aiRNA文库在体外介导有效和特异的基因沉默的能力。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，并可能对其他涉及RNAi介导的基因沉默的应用具有重要意义。 公共卫生相关性：目前在哺乳动物细胞中诱导RNA干扰(RNAi)的范例依赖于短干扰RNA(SiRNA)的使用，这种RNA已被广泛用于体外基因沉默；然而，与使用siRNA相关的各种非特异性效应已被认为是它们在基因功能研究中使用的一个重大缺陷。波士顿生物医学公司发现了一种在哺乳动物细胞中诱导高效RNAi的新专利技术--不对称干扰RNA，与传统的siRNA相比，它具有更优越的体外基因沉默特性，消除了正义链介导的非靶标沉默，并显著减少或消除了非特异性干扰素样反应的诱导。这项提案中概述的研究旨在优化aiRNA基因消音器的结构，并开发一种可供生物医学研究界使用的设计算法，长期目标是将这项技术开发为全基因组功能基因组研究的试剂。

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.