Development of Specific Gene Silencing Methods and Reagents

特定基因沉默方法和试剂的开发

• 批准号: 8058188
• 负责人: Chiang Jia Li
• 金额: $ 31.22万
• 依托单位: BOSTON BIOMEDICAL, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8058188
• 关键词: Algorithms; Biological Sciences; Biomedical Research; Boston; Cells; Communities; Data; Development; Elements; Ensure; Gene Silencing; Gene Targeting; Genes; Goals; Guanine + Cytosine Composition; Human; Immune response; In Vitro; Interferons; Length; Libraries; Mammalian Cell; Mediating; Messenger RNA; Methods; Nucleotides; Phase; Plague; Property; RNA; RNA Interference; RNA Sequences; RNA library; Reagent; Research; Research Personnel; Selection Criteria; Small Business Innovation Research Grant; Structure; Technology; Testing; Work; base; beta catenin; biological research; design; functional genomics; gene function; genome-wide; human disease; improved; in vivo; meetings; new technology; novel; novel therapeutics; programs; research study; response; scaffold; tool

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.

描述（由申请人提供）：RNA干扰（RNAi）是一种有效的、选择性的基因沉默机制，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，并为体内功能基因组学带来了巨大的希望。 当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 19 bp 双链体组成，每个 3' 末端有 2 nt 突出端，已广泛用于体外基因功能研究，以及开发针对各种人类疾病的新型疗法。尽管 siRNA 广泛用于体外研究，但该技术受到可变基因沉默效应、先天免疫反应激活以及非靶向 mRNA 敲除（脱靶沉默）的困扰。这些众所周知的效应使依赖于特定靶基因敲低的基因功能实验的解释变得复杂。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解决。 Boston Biomedical Inc. 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 的新型专有技术。与 siRNA 相比，aiRNA 显示出更优异的体外基因沉默特性。此外，aiRNA完全消除了有义链脱靶沉默，并消除或显着降低了干扰素反应诱导。这些发现表明，aiRNA可以满足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应用的巨大潜力。 该SBIR提案的长期目标是优化aiRNA作为基因功能研究的试剂，包括全基因组功能基因组学研究。 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 基因沉默器的结构和设计。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且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 aiRNA 的相关性。在目标 3 中，我们将进行实验来确定开发 aiRNA 特定设计算法的关键要素。 我们第二阶段的总体目标是开发一种用于功能基因组学研究的全基因组验证的 aiRNA 试剂。这些研究将需要根据针对所有已知人类基因的优化 aiRNA 结构（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技术的进一步发展应该会显着提高我们分析基因功能的能力，并且可能对涉及RNAi介导的基因沉默的其他应用产生重大影响。 公共健康相关性：目前在哺乳动物细胞中诱导 RNA 干扰 (RNAi) 的范例依赖于使用短干扰 RNA (siRNA)，该技术已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其在基因功能研究中使用的显着缺点。 Boston Biomedical Inc.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的新型专有技术，与传统的siRNA相比，该技术具有优异的体外基因沉默特性，消除了有义链介导的脱靶沉默，并显着减少或消除了非特异性干扰素样反应诱导。该提案中概述的研究旨在优化aiRNA基因沉默器的结构并开发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学研究的试剂。