The Impact of Nucleotide Modification Patterns on Therapeutic Small Interfering RNA Activity in the Central Nervous System

项目总结 亨廷顿病(HD)是由亨廷顿蛋白(HTT)基因第一外显子CAG三核苷酸重复扩增引起的 产生突变的HTT mRNA和蛋白质的基因。突变型HTT的表达导致进展性 通过鲜为人知的机制导致的神经退化。因为HD是一种基因定义的疾病，它 是通过小干扰RNA(SiRNAs)进行研究和治疗干预的理想候选者 整合到RNA诱导沉默复合体(RISC)中，以靶向和降解致病基因。使用 二价(Di)-siRNA化学结构的发展，siRNA可以传递到整个中心 啮齿动物和非人灵长类的神经系统。为了确保CNS的稳定性，di-siRNAs需要 对每个核苷酸进行化学修饰。然而，化学修饰会影响siRNA的活性和 细胞定位--限制了双siRNAs在中枢神经系统中的实用性和灵活性。 SiRNA中最常见的核苷酸修饰是用2‘-氟(2’F)或2‘-O-取代核糖的2’-羟基。 甲基(2‘OMe)。最近的工作表明，在某些核苷酸位置掺入2‘OME和2’F可能 阻止siRNA进入RISC或靶向RISC的结合/切割，并可能改变核质 SiRNA的本地化。然而，由于这项工作的范围有限，很难确定总体设计 有效的隔室特异性siRNA的参数。由Anastasia Khvorova博士(SiRNA)指导 化学)，Neil Aronin(HD)，Phillip Zamore(RNA生物化学)和Athma Pai(RNA测序)，这 提案将系统地评估修饰模式对siRNA有效性和细胞定位的影响 在中枢神经系统中，优化siRNAs作为HD治疗和研究工具，以解剖HD病理。 目标1将表征siRNA化学修饰如何影响体内RISC负载和靶向结合 体外卵裂。为了衡量修饰如何改变RISC负载，差异修饰的siRNA池将 注射到小鼠的中枢神经系统，RISC将被拉下并对加载的siRNA进行测序。要确定 SiRNA化学修饰对RISC-靶相互作用、靶结合和切割动力学的影响将 用单分子全内反射荧光法测量一组修饰的siRNA 显微镜。对修饰如何影响CNS中siRNA有效性的机械性洞察将提供 用于设计优化的siRNAs来治疗HD和其他中枢神经系统疾病的框架。Aim 2将使用相同的 来自Aim 1的修饰siRNA池以确定增强核定位的最佳修饰模式 中枢神经系统中的siRNA。这些数据将被用来设计有效的针对核的化学修饰的双siRNAs。 或仅含细胞质的HTT RNA。然后将这些双干扰RNA注射到YAC128 HD小鼠体内，并观察其对 将评估运动缺陷、神经变性和纹状体mRNA的表达。这些结果将提供 对HD的生物学有价值的见解并确定以核RNA为靶点的siRNA作为 具有潜在RNA毒性的重复扩张性疾病的治疗范例。

PROJECT SUMMARY Huntington’s Disease (HD) is caused by a CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT) gene that produces mutant HTT mRNA and protein. Expression of mutant HTT leads to progressive neurodegeneration via mechanisms that are poorly understood. Because HD is a genetically-defined disease, it is an ideal candidate for study and therapeutic intervention by small interfering RNAs (siRNAs) – which incorporate into the RNA-induced silencing complex (RISC) to target and degrade disease-causing genes. With the development of a divalent (di)-siRNA chemical architecture, siRNAs can be delivered throughout the central nervous system (CNS) of rodents and non-human primates. To ensure stability in the CNS, di-siRNAs require chemical modifications on every nucleotide. However, chemical modifications can affect siRNA activity and cellular localization – limiting the utility and flexibility of di-siRNAs in the CNS. The most common nucleotide modifications in siRNA replace the 2′OH of the ribose with 2′-Fluoro (2′F) or 2′-O- Methyl (2′OMe). Recent work suggests that incorporation of 2′OMe and 2′F at certain nucleotide positions may hinder siRNA loading into RISC or target binding/cleavage by RISC, and may alter the nuclear-to-cytoplasmic localization of siRNAs. Yet, the limited scope of this work has made it difficult to identify general design parameters for efficacious, compartment-specific siRNA. With guidance from Drs. Anastasia Khvorova (siRNA chemistry), Neil Aronin (HD), Phillip Zamore (RNA biochemistry) and Athma Pai (RNA sequencing), this proposal will systematically assess the impact of modification patterns on siRNA efficacy and cellular localization in the CNS to optimize siRNAs as an HD therapy and research tool for dissecting HD pathology. Aim 1 will characterize how siRNA chemical modifications impact RISC loading in vivo and target binding and cleavage in vitro. To measure how modifications alter RISC loading, a pool of differentially-modified siRNAs will be injected into the CNS of mice, RISC will be pulled down and loaded siRNAs will be sequenced. To determine the effect of siRNA chemical modifications on RISC-target interactions, target binding and cleavage kinetics will be measured for a panel of modified siRNAs using single-molecule total internal reflection fluorescence microscopy. Mechanistic insight into how modifications impact siRNA efficacy in the CNS will provide a framework with which to design optimized siRNAs to treat HD and other CNS diseases. Aim 2 will use the same modified siRNA pool from Aim 1 to identify optimal modification patterns for enhanced nuclear localization of siRNA in the CNS. These data will be used to design efficacious chemically-modified di-siRNAs targeting nuclear or cytoplasmic-only HTT RNA. These di-siRNA will then be injected into YAC128 HD mice and the effect on motor deficits, neurodegeneration, and striatal mRNA expression will be assessed. These results will provide valuable insight into the biology of HD and determine the potential of nuclear RNA-targeting siRNAs as a therapeutic paradigm for repeat expansion disorders with underlying RNA toxicity.