Methods for controlling small RNA stability

 描述(由申请人提供)：小RNA构成了人类生物学中转录后调控的主要类别。人工合成的siRNAs已被广泛用于哺乳动物细胞的反向基因实验，是治疗包括肝癌、卵巢癌和肺癌在内的多种人类疾病的一种有前途的治疗途径。同样，抑制特定的microRNAs(MiRNAs)--人类最丰富的内源性小RNA的形式--可以对细胞生理学产生深远的影响，越来越多的用于治疗各种形式癌症的“抗miR”化合物已进入临床和临床前试验。尽管围绕着小RNA有明显的治疗机会 在生物学领域，基础知识的空白阻碍了该领域发挥其全部潜力。一个重要的差距是对如何控制RNA诱导沉默复合体(RISC)的稳定性的洞察。RISC是小RNA发挥作用的RNA/蛋白质复合体。这项应用的目的是将对介导小RNA代谢的酶的了解应用于开发控制人类细胞中小RNA稳定性的方法。这一目标将通过追求两个具体目标来实现：1)开发优化siRNA效力和持久性的方法；以及2)开发破坏内源性miRNAs稳定的方法。在目标1下，我们将应用我们在小RNA生物化学方面的专业知识来生成不同的siRNA文库，从中可以选择哺乳动物细胞中最稳定的序列。这个目标很重要，因为目前还没有方法预测，更不用说控制siRNA的稳定性了。我们的理性是，识别具有延长细胞半衰期的siRNA的能力将大大有助于开发有效的治疗性siRNA。在目标2下，我们将使用我们开发的用于监测miRNAs与其伴侣蛋白ArgAerte2(Ago2)的相关性的方法来确定抗miRs如何影响miRNA的稳定性，ArgAerte2(Ago2)构成RISC的核心蛋白亚单位。这是一个重要的目标，因为尽管已经开发了化学修饰来增加核酸酶的抗性和改善anit-miRs的PK/PD谱，但这些修饰如何影响RISC的稳定性仍不清楚。我们的理性是，通过专注于破坏Ago2-miRNA复合体稳定和促进miRNA降解的修饰，有可能开发出能够执行多轮miRNA破坏的抗miRs。原则上，这种催化抗MIR在远低于常规抗MIR的剂量下有效。由于向靶组织传递的挑战是开发治疗性抗miRs的主要限制因素，因此对提高效力的见解将是一个重大的进步。

 DESCRIPTION (provided by applicant): Small RNAs constitute a major class of posttranscriptional regulators in human biology. Synthetic siRNAs have been used extensively for reverse-genetic experiments in mammalian cells and are a promising therapeutic avenue for the treatment of diverse human diseases, including liver cancer, ovarian cancer, and lung cancer. Similarly, inhibition of specific microRNAs (miRNAs), the most abundant form of endogenous small RNAs in humans, can have profound effects on cellular physiology and a growing list of "anti-miR" compounds for the treatment of diverse forms of cancer have entered clinical and pre-clinical trials. Despite the clear therapeutic opportunities surrounding small RNA biology, gaps in basic knowledge have prevented the field from reaching its full potential. One significant gap is insight into how to control the stability of the RNA-Induced Silencing Complex (RISC), the RNA/protein complex in which small RNAs function. The objective of this application is to apply understanding of the enzymes mediating small RNA metabolism towards development of methods for controlling small RNA stability in human cells. This objective will be achieved by pursuing two specific aims: 1) Develop approaches for optimizing siRNA potency and persistence; and, 2) Develop approaches for destabilizing endogenous miRNAs. Under Aim 1 we will apply our expertise in small RNA biochemistry to generate diverse libraries of siRNAs, from which the most stable sequences can be selected in mammalian cells. This goal is significant because there is currently no way of predicting, much less controlling, siRNA stability Our rational is that the ability to identify siRNAs with extended cellular half-lives would significantly benefit efforts to develop effective therapeutic siRNAs. Under Aim 2 we will determine how anti-miRs influence miRNA stability using assays we developed for monitoring the association of miRNAs with their partner protein, Argonaute2 (Ago2), which forms the core protein subunit of RISC. This is an important goal because, although chemical modifications have been developed to increase the nuclease resistance and improve the PK/PD profile of anit-miRs, exactly how these modifications impact the stability of RISC remains unclear. Our rational is that by focusing on modifications that destabilize the Ago2-miRNA complex and promote miRNA degradation it may be possible to develop anti-miRs that can perform multiple rounds of miRNA destruction. In principle, such catalytic anti-miRs would be effective at far lower doses than conventional anti-miRs. Because the challenge of delivering into target tissues is the major limiting factor in development of therapeutic anti- miRs, insights for improving potency would be a significant advance.