Modulation of Somatic Repeat Expansion as a Therapeutic Approach to Huntington's Disease

调节体细胞重复扩增作为亨廷顿病的治疗方法

基本信息

批准号：
10678016
负责人：
Jillian Belgrad
金额：
$ 3.25万
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：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10678016
关键词：
Adverse effects Antisense Oligonucleotides Bacterial Artificial Chromosomes Binding Binding Proteins Bioinformatics Biological Assay CAG repeat Cells Chemistry Clinical Complex Corpus striatum structure DNA DNA-Protein Interaction Defect Disease Disease Outcome Disease Progression Disease model Event Exons Fibroblasts Frequencies Gait Genes Genetic Genetic Polymorphism Genetic Transcription Hand Strength Harvest Histology Human Huntington Disease Huntington gene Immunohistochemistry Impaired cognition Inherited Injections Interphase Cell Knock-out Lead Length Link MLH1 gene MSH2 gene MSH3 gene Measures Mediating Mediator Mental Depression Messenger RNA Mismatch Repair Motor Mus Mutation Nerve Degeneration Neurobiology Neurodegenerative Disorders Neurons Nucleotides Oligonucleotides Onset of illness PMS1 gene PMS2 gene Pathogenicity Pathology Patients Pharmaceutical Preparations Process Proteins Radiology Specialty Repair Complex Severities Severity of illness Small Interfering RNA Small RNA Symptoms System Therapeutic Time Training Trinucleotide Repeat Expansion Ventricular Walking Work autosome clinically relevant curative treatments design disease phenotype early onset efficacy validation endonuclease gene repair improved in vivo in vivo Model motor behavior motor disorder mouse model mutant nervous system disorder novel nuclease nucleic acid binding protein pre-clinical prevent rational design recruit repair enzyme success therapeutic development therapy design transcriptome sequencing

项目摘要

PROJECT SUMMARY Huntington’s disease (HD) is caused by expanded trinucleotide repeats (CAG) in exon 1 of the huntingtin (HTT) gene. Therapies lowering the downstream mutant HTT protein show limited clinical success. New evidence reveals that repeat tract length in the HTT locus, not mutant HTT protein, correlates to disease onset/severity. CAG repeat length is inherited, but further expands due to somatic instability, which contributes to HD progression. Modulating somatic expansion is a key path toward treating HD. Somatic expansion occurs in non- dividing cells like neurons when DNA repeats misalign after transcription, forming a slipped loop that activates mismatch repair (MMR). In MMR, nuclease complexes help recognize the slipped loop and cut the non-slipped strand to create a gap that is filled to expand the repeat. Polymorphisms in MMR complexes are linked to HD onset, and knocking out or altering activity of MMR proteins block expansion or induce contraction in HD models. Yet, the contribution of each MMR protein to CAG expansion, and the effect of their conditional CNS-specific reduction on HD outcomes, is untested. Also, mechanisms favoring contraction over expansion are unknown. This project seeks to define MMR complexes facilitating HTT CAG expansion/contraction using divalent small interfering RNA (siRNA)—which induce potent, CNS-specific silencing of target genes—and antisense oligonucleotides (ASOs)—which can disrupt specific protein-nucleic acid binding in the CNS. Aim 1 will use divalent siRNA to evaluate the effects of MMR silencing on HTT CAG repeat expansion and HD progression. Efficacies of siRNAs targeting each MMR protein have been validated in human and mouse cells. Furthermore, one of these siRNAs was delivered to CNS of an HD mouse model, BAC-CAG (carries human HTT with 120 CAG that undergo expansion), showing target MMR silencing and blocked somatic expansion 2 months later. In Aim 1, divalent siRNA targeting each MMR enzyme will be injected into BAC-CAG mice. Target silencing and HTT CAG repeat expansion will be measured 2 months later. Top siRNA that block expansion will be re- injected into BAC-CAG mice, and the impact on motor behavior, ventricular size, and HD pathology will be explored over 9 months. Aim 2 will develop HTT CAG-targeting ASOs to induce MMR-mediated contraction in HD cells and mice. An initial panel of ASOs targeting HTT CAG repeats was screened in non-transformed HD patient-derived fibroblasts (HDpFs) using a high-throughput format, and ASOs that increase contraction events were identified. To improve contraction rates, ASO chemistries and lengths will be optimized and screened in HDpFs using the same assay. HTT CAG repeat length/instability will be quantified over 40 days to identify leads. Leads will be delivered to HDpFs, in combination with validated siRNA targeting each MMR protein, to identify MMR proteins mediating ASO-induced contraction events. In parallel, in vivo efficacy of leads will be confirmed in BAC-CAG mice. This work will reveal somatic expansion/contraction mechanisms, inform HD therapy design, and provide the fellow with crucial training in therapeutic development, neurobiology, and bioinformatics.

项目摘要亨廷顿氏病（HD）是由亨廷顿（Huntingtin 基因。降低下游突变体HTT蛋白的疗法显示有限的临床成功。新证据揭示了HTT基因座中的重复道长，而不是突变的HTT蛋白与疾病的发作/严重程度有关。 CAG重复长度是继承的，但由于躯体不稳定性而进一步扩展，这有助于HD 进展。调节体膨胀是治疗高清的关键途径。体细胞扩张发生在非 - 当DNA在转录后重复不一致时，将细胞等细胞分开，形成一个切片的环，激活不匹配维修（MMR）。在MMR中，核酸酶配合物有助于识别切片环并切割未扫描的循环链创建一个填充的差距以扩大重复。 MMR复合物中的多态性与HD有关发作，敲除或改变MMR蛋白的活性阻止HD模型中的缩小或影响合同。然而，每个MMR蛋白对CAG扩展的贡献及其条件CNS特异性的影响降低高清结果，未经测试。同样，有利于收缩而不是扩展的机制尚不清楚。该项目旨在定义MMR综合体，支持HTT CAG扩展/收缩使用二价小干扰RNA（siRNA） - 诱导靶基因的电势，CNS特异性沉默 - 和反义寡核苷酸（ASO） - 可能破坏CNS中特定蛋白核酸的结合。 AIM 1将使用二价siRNA评估MMR沉默对HTT CAG重复膨胀和HD的影响进展。靶向每种MMR蛋白的siRNA的疗效已在人和小鼠细胞中得到验证。此外，这些siRNA之一被输送到HD小鼠模型的CNS，BAC-CAG（携带人htt 带有120个CAG进行膨胀），显示目标MMR沉默并阻止体细胞扩张2个月之后。在AIM 1中，靶向每种MMR酶的二价siRNA将被注入BAC-CAG小鼠。目标沉默和HTT CAG重复扩展将在2个月后测量。最高的sirna将重新膨胀注射到BAC-CAG小鼠中，对运动行为，心室大小和HD病理的影响将是探索了9个月以上。 AIM 2将发展HTT CAG靶向ASO，以诱导MMR介导的收缩 HD细胞和小鼠。在非转化的HD中筛选了靶向HTT CAG重复序列的ASOS的初始面板使用高通量格式的患者衍生的成纤维细胞（HDPF）和增加收缩事件的ASO 被确定。为了提高收缩率，ASO化学和长度将被优化和筛选使用相同测定的HDPF。 HTT CAG重复长度/不稳定性将在40天内量化以识别导线。铅将与靶向每个MMR蛋白的经过验证的siRNA结合使用，以识别 MMR蛋白介导ASO诱导的收缩事件。同时，将确认铅的体内效率在BAC-CAG小鼠中。这项工作将揭示躯体扩张/收缩机制，告知高清治疗设计，并为研究员提供热发展，神经生物学和生物信息学的关键培训。