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

项目摘要 亨廷顿病（HD）是由亨廷顿蛋白（HTT）外显子1中CAG三核苷酸重复扩增引起的 产生突变HTT mRNA和蛋白质的基因。突变体HTT的表达导致进行性 神经退行性变的机制尚不清楚。因为HD是一种遗传定义的疾病， 是通过小干扰RNA（siRNA）进行研究和治疗干预的理想候选者， 整合到RNA诱导的沉默复合物（RISC）中，以靶向和降解致病基因。与 随着二价（双）-siRNA化学结构的发展，siRNA可以被递送到整个中央区域， 啮齿类动物和非人灵长类动物的神经系统（CNS）。为了确保在CNS中的稳定性，di-siRNA需要 每一个核苷酸上都有化学修饰然而，化学修饰可以影响siRNA活性， 细胞定位-限制了di-siRNA在CNS中的效用和灵活性。 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′OH， 甲基（2′OMe）。最近的工作表明，在某些核苷酸位置上掺入2′OMe和2′F可能会 阻碍siRNA加载到RISC中或RISC的靶结合/切割，并可能改变细胞核到细胞质 siRNA的定位。然而，这项工作的范围有限，难以确定一般设计 有效的、隔室特异性siRNA的参数。在Anastasia Khvorova博士的指导下（siRNA 化学）、尼尔·阿罗宁（HD）、菲利普·扎莫尔（RNA生物化学）和阿特玛·派（RNA测序），这 该提案将系统地评估修饰模式对siRNA功效和细胞定位的影响 在CNS中优化siRNA作为HD治疗和研究工具，用于解剖HD病理。 目的1将表征siRNA化学修饰如何影响RISC体内负载和靶结合， 体外卵裂。为了测量修饰如何改变RISC加载，将差异修饰的siRNA库与其他siRNA库进行比较。 将其注射到小鼠的CNS中，RISC将被拉下并对加载的siRNA进行测序。以确定 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 使用单分子全内反射荧光测量一组修饰的siRNA 显微镜对修饰如何影响siRNA在CNS中的功效的机理洞察将提供一个新的视角。 设计优化的siRNA来治疗HD和其他CNS疾病的框架。目标2将使用相同的 来自Aim 1的经修饰的siRNA库，以鉴定用于增强细胞核定位的最佳修饰模式。 CNS中的siRNA。这些数据将用于设计有效的化学修饰的双siRNA靶向细胞核， 或仅存在于细胞质中的HTT RNA。然后将这些di-siRNA注射到YAC 128 HD小鼠中，并观察其对YAC 128 HD小鼠的影响 评估运动缺陷、神经变性和纹状体mRNA表达。这些结果将提供 对HD生物学的有价值的见解，并确定核RNA靶向siRNA作为治疗HD的潜力。 具有潜在RNA毒性的重复扩增病症的治疗范例。

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.