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主要的一类转录后调节因子。合成的siRNA已被广泛用于哺乳动物细胞中的反向遗传实验，并且是用于治疗多种人类疾病（包括肝癌、卵巢癌和肺癌）的有前景的治疗途径。类似地，特异性微小RNA（miRNA）（人类中内源性小RNA的最丰富形式）的抑制可对细胞生理学具有深远影响，并且用于治疗多种形式的癌症的“抗miR”化合物的列表越来越多，已经进入临床和临床前试验。尽管围绕小RNA有明确的治疗机会， 在生物学方面，基础知识的差距阻碍了该领域充分发挥其潜力。一个重要的差距是如何控制RNA诱导沉默复合物（RISC）的稳定性，RISC是小RNA发挥作用的RNA/蛋白质复合物。本申请的目的是将对介导小RNA代谢的酶的理解应用于开发用于控制人细胞中小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）开发用于优化siRNA效力和持久性的方法;以及2）开发用于使内源性miRNA去稳定的方法。在目标1下，我们将运用我们在小RNA生物化学方面的专业知识来产生不同的siRNA文库，从中可以在哺乳动物细胞中选择最稳定的序列。这一目标是重要的，因为目前没有预测，更不用说控制siRNA稳定性的方法。我们的理由是，鉴定具有延长的细胞半衰期的siRNA的能力将显著有益于开发有效的治疗性siRNA的努力。在目标2下，我们将使用我们开发的用于监测miRNA与其伴侣蛋白Argonaute 2（Ago 2）（其形成RISC的核心蛋白亚基）的缔合的测定来确定抗miR如何影响miRNA稳定性。这是一个重要的目标，因为尽管已经开发了化学修饰以增加核酸酶抗性并改善抗miR的PK/PD谱，但这些修饰究竟如何影响RISC的稳定性仍不清楚。我们的理由是，通过专注于使Ago 2-miRNA复合物不稳定并促进miRNA降解的修饰，有可能开发出能够进行多轮miRNA破坏的抗miR。原则上，这样的催化性抗miR将在比常规抗miR低得多的剂量下有效。由于递送到靶组织中的挑战是治疗性抗miR开发的主要限制因素，因此提高效力的见解将是一个重大进步。

 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.