Improvement of RNAi efficacy by blocking RNAi inhibitors

描述（由申请人提供）：RNA干扰（RNAi）有望成为合理药物设计的有力方法。RNAi的一个重要缺点是它对基因表达的影响很少是100%有效的。当期望或需要完全停止基因表达时，广泛观察到的这种限制是有问题的。本提案的主要目标是制定有针对性的策略，以提高商业RNAi应用期间基因沉默的功效。我们的目标是开发小分子模拟物和基于rna的化合物，以确定RNAi疗效的重要核心途径为目标。将这些化合物与基于rna的药物或治疗结合使用将提高该药物或治疗的性能。我们最初的目标是发现一种果蝇突变体，它表现出增强的RNAi活性——比正常情况强三倍——没有任何可检测到的副作用。这是因为突变体缺乏一个重要的抑制基因，自然地限制了RNAi的效力。该抑制剂特异性地减弱短干扰rna （sirna）与降解靶mrna的复合物组装的一个步骤。这种基因的存在说明自然生理机制限制了RNAi的功效。此外，高度保守的同源基因存在于所有已测序的脊椎动物基因组中，包括人类。我们的假设是，这些基因也限制了这些物种的RNAi活性。因此，阻断该抑制剂的活性可以提高RNAi的疗效。我们建议测试开发sirna的可行性，以阻断该抑制剂，从而提高所给RNAi药物或治疗的性能。我们将评估该抑制剂在小鼠细胞系突变体中的RNAi活性。我们将评估用siRNA或shRNA处理的人和小鼠细胞系的RNAi活性，以敲除抑制剂。我们将评估抑制剂耗竭（通过siRNA）对促进基于rna的疗法（如杀死前列腺肿瘤细胞和阻断流感病毒复制）的影响。提高RNAi性能的化合物的开发将对公众健康产生重大影响。

DESCRIPTION (provided by applicant): RNA Interference (RNAi) promises to become a powerful approach to rational drug design. One significant drawback to RNAi is that its effect on gene expression is rarely 100% potent. This limitation, observed widely, is problematic when a complete cessation of gene expression is desired or required. A major goal of this proposal is to develop targeted strategies to enhance the efficacy of gene silencing during commercial RNAi applications. Our goal is to develop small molecule mimetics and RNA-based compounds that target core pathways important in determining RNAi efficacy. Application of such compounds in conjunction with an RNA-based drug or treatment will boost performance of that drug or treatment. Our initial target was discovered as a Drosophila mutant that exhibits enhanced RNAi activity - three times stronger than normal - without any detectable side-effects. This occurs because the mutant lacks an important inhibitory gene that naturally limits RNAi efficacy. The inhibitor specifically attenuates one step in the assembly of short interfering RNAs (siRNAs) with the complex that degrades target mRNAs. The existence of such a gene argues that natural physiological mechanisms restrict RNAi efficacy. Moreover, highly conserved homologous genes exist in all sequenced vertebrate genomes, including humans. It is our hypothesis that these genes also restrict RNAi activity in these species. Thus, blocking the inhibitor's activity should improve RNAi efficacy. We propose to test the feasibility of developing siRNAs that block the inhibitor and thereby boost performance of an administered RNAi drug or treatment. We will evaluate the RNAi activity in mouse cell lines mutant for the inhibitor. We will evaluate the RNAi activity of human and mouse cell lines treated with siRNA or shRNA to knockdown the inhibitor. We will evaluate the effect of inhibitor depletion (by siRNA) on boosting RNA-based therapies such as killing prostate tumor cells and blocking influenza virus replication. Development of compounds that boost RNAi performance in general will have significant impact on public health.