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

• 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
• 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

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 亨廷顿病（HD）是由亨廷顿蛋白（HTT）外显子1的三核苷酸重复序列（CAG）扩增引起的 基因降低下游突变HTT蛋白的疗法显示出有限的临床成功。新证据 揭示了HTT基因座中的重复序列长度（而不是突变HTT蛋白）与疾病发作/严重程度相关。 CAG重复长度是遗传的，但由于体细胞不稳定而进一步扩展，这有助于HD 进展调节体细胞扩张是治疗HD的关键途径。体细胞扩张发生在非 当DNA重复序列在转录后错位时，分裂的细胞就像神经元一样，形成一个滑动的环， 错配修复（MMR）。在MMR中，核酸酶复合物帮助识别滑动环并切割非滑动环。 链以创建被填充以扩展重复的间隙。MMR复合物中的多态性与HD有关 发病，以及敲除或改变MMR蛋白的活性阻断HD模型中的扩张或诱导收缩。 然而，每种MMR蛋白对CAG扩增的贡献，以及其条件性中枢神经系统特异性的作用， 减少HD结局，未经检验。此外，有利于收缩而非扩张的机制尚不清楚。 该项目旨在定义MMR复合物，其使用二价小分子来促进HTT CAG扩张/收缩。 干扰RNA（siRNA）-诱导有效的，CNS特异性沉默的靶基因-和反义 寡核苷酸（ASO）-其可以破坏CNS中的特异性蛋白质-核酸结合。 目的1：利用二价siRNA技术研究MMR沉默对HTT CAG重复扩增和HD的影响。 进展靶向每种MMR蛋白的siRNA的效力已经在人和小鼠细胞中得到验证。 此外，将这些siRNA之一递送至HD小鼠模型BAC-CAG（携带人HTT）的CNS 具有120个经历扩增的CAG），显示靶MMR沉默和阻断的体细胞扩增2个月 后在目标1中，将靶向每种MMR酶的二价siRNA注射到BAC-CAG小鼠中。靶沉默 2个月后测量HTT CAG重复扩增。阻止扩增的顶级siRNA将被重新命名。 注射到BAC-CAG小鼠，对运动行为，心室大小和HD病理学的影响将被评估。 探索了9个多月。目的2将开发HTT CAG靶向ASO以诱导MMR介导的收缩， HD细胞和小鼠。在未转化的HD细胞中筛选靶向HTT CAG重复序列的一组初始ASO。 使用高通量形式的患者来源的成纤维细胞（HDpF），以及增加收缩事件的ASO 被确认了身份为了提高收缩率，将优化和筛选阿索的化学成分和长度， HDpF使用相同的测定。将在40天内定量HTT CAG重复长度/不稳定性，以识别导联。 导线将与靶向每种MMR蛋白的经验证的siRNA组合递送至HDpF，以鉴定 MMR蛋白介导ASO诱导的收缩事件。同时，将确认电极导线的体内有效性 BAC-CAG小鼠。这项工作将揭示躯体扩张/收缩机制，为HD治疗设计提供信息， 并为研究员提供治疗开发、神经生物学和生物信息学方面的重要培训。