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

核苷酸修饰模式对中枢神经系统治疗性小干扰 RNA 活性的影响

基本信息

批准号：
10389434
负责人：
Samuel Hildebrand
金额：
$ 3.15万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-19 至 2024-08-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10389434
关键词：
Affect Architecture Bar Codes Behavioral Bilateral Binding Biochemical Biology Brain Brain region CAG repeat Cell Nucleus Central Nervous System Diseases Chemicals Chemistry Corpus striatum structure Cytoplasm Data Development Disease Disease Progression Ensure Exons Fluorescence Microscopy Functional disorder Gene Silencing Genes High-Throughput Nucleotide Sequencing Huntington Disease Huntington gene Huntington protein In Vitro Injections Kinetics Length Libraries Measures Mediating Messenger RNA Methods Microscopy Modification Molecular Conformation Mus Nature Nerve Degeneration Neuraxis Neurodegenerative Disorders Nuclear Nuclear RNA Nucleotides Pathogenicity Pathology Pattern Pharmaceutical Preparations Physiological Positioning Attribute Preparation Production Proteins RNA RNA Biochemistry RNA Interference RNA Splicing RNA-Induced Silencing Complex Research Ribose Rodent Role Small Interfering RNA Small RNA Testing Therapeutic Therapeutic Intervention Therapeutic Studies Time Toxic effect Trinucleotide Repeat Expansion Work design flexibility gait examination in vivo insight mRNA Expression motor deficit mutant neuron loss nonhuman primate novel therapeutics optimal treatments polyglutamine progressive neurodegeneration single molecule tool transcriptome sequencing treatment strategy uptake

项目摘要

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.

项目摘要亨廷顿氏病（HD）是由亨廷顿（Huntingtin 产生突变体HTT mRNA和蛋白质的基因。突变体HTT的表达导致渐进式神经退行性通过不理理解的机制。因为高清是一种遗传定义的疾病，所以是小型干扰RNA（siRNA）进行研究和治疗干预的理想候选者 - 纳入RNA诱导的沉默复合物（RISC），以靶向并降解引起疾病的基因。和可以在整个中央交付二价（DI）-SiRNA化学体系结构的开发啮齿动物和非人类隐私的神经系统（CNS）。为了确保中枢神经系统的稳定性，di-sirnas需要每种核苷酸的化学修饰。但是，化学修饰会影响siRNA活性和细胞定位 - 限制CNS中二核NAS的效用和灵活性。 siRNA中最常见的核苷酸修饰用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，在某些核苷酸位置合并了2'OME和2'f的材料可能阻碍siRNA加载到RISC或RISC的目标结合/裂解中，并可能改变核对胞质 sirnas的本地化。然而，这项工作的有限范围使得难以识别一般设计有效，室特异性siRNA的参数。在Drs的指导下。 Anastasia Khvorova（siRNA）化学），Neil Aronin（HD），Phillip Zamore（RNA生物化学）和Athma Pai（RNA测序），此提案将系统地评估修饰模式对siRNA效率和细胞定位的影响在中枢神经系统中，将siRNA作为HD疗法和研究工具优化，用于剖析HD病理。 AIM 1将表征siRNA化学修饰如何影响RISC在体内和目标结合和目标结合以及体外切割。为了衡量修改如何改变RISC加载，将差异化的siRNA池被注射到小鼠的中枢神经系统中，RISC将被拉下来，并将加载siRNA。确定 siRNA化学修饰对RISC-TARGET相互作用，目标结合和裂解动力学的影响将使用单分子的总内反射荧光对一组改良的siRNA进行测量显微镜。关于修饰如何影响CNS siRNA效率的机械洞察力将提供设计优化的siRNA以治疗高清和其他中枢神经系统疾病的框架。 AIM 2将使用相同的 AIM 1的修改siRNA池以确定最佳的修饰模式，以增强核定位的核定位置中枢神经系统中的siRNA。这些数据将用于设计靶向核的有效化学修饰的Di-SiRNA 或仅细胞质的HTT RNA。然后，这些二核NA将被注入Yac128 HD小鼠，并对运动定义，神经变性和纹状体mRNA表达将被评估。这些结果将提供对HD生物学的有价值的见解，并确定涉及核RNA的siRNA的潜力具有潜在RNA毒性的重复扩张障碍的治疗范式。