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.

项目总结 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)引起的 吉恩。降低下游突变HTT蛋白的治疗方法显示出有限的临床成功。新证据 研究表明，HTT基因座的重复序列长度与疾病的发病/严重程度相关，而不是突变的HTT蛋白。 CAG重复长度是遗传的，但由于躯体不稳定而进一步扩大，这是导致HD的原因 进步。调节躯体扩张是治疗HD的关键途径。躯体扩张发生在非 当DNA重复转录后不对齐时，像神经元一样分裂细胞，形成一个滑移的环，激活 失配修复(MMR)。在MMR中，核酸酶复合体有助于识别打滑的环，并切割未打滑的环 链，以创建填充的间隙，以扩展重复。MMR复合体中的多态与HD有关 在HD模型中，MMR蛋白的起始、敲除或改变活性阻止扩张或诱导收缩。 然而，每种MMR蛋白对CAG扩张的贡献，以及它们的条件性中枢神经系统特异性的影响 HD结果的减少，未经测试。此外，更倾向于收缩而不是扩张的机制尚不清楚。 该项目寻求定义促进HTT CAG扩张/收缩的MMR复合体，使用二价小分子 干扰RNA(SiRNA)--可诱导有效的、中枢神经系统特异性的靶基因沉默--以及反义 寡核苷酸(ASO)--它可以破坏中枢神经系统中特定的蛋白质与核酸的结合。 目标1将使用二价siRNA来评估MMR沉默对HTT、CAG重复扩增和HD的影响 进步。针对每个MMR蛋白的siRNAs的有效性已经在人类和小鼠细胞中得到验证。 此外，这些siRNA中的一个被传递到HD小鼠模型BAC-CAG(携带人HTT)的中枢神经系统 120个正在扩张的CAG)，显示目标MMR沉默并阻止体细胞扩张2个月 后来。在目标1中，针对每个MMR酶的二价siRNA将被注射到BAC-CAG小鼠体内。目标静音 2个月后测量HTT CAG重复扩张率。顶端siRNA表示将重新进行区块扩展 注射到BAC-CAG小鼠体内，对运动行为、心室大小和HD病理的影响将是 探索了9个多月。AIM 2将开发HTT CAG靶向ASO诱导MMR介导的收缩 HD细胞和小鼠。针对HTT CAG重复序列的ASO最初小组在未转换的HD中进行了筛选 使用高通量格式的患者来源的成纤维细胞(HDPF)和增加收缩事件的ASO 都被确认了。为了提高收缩速度，ASO的化学成分和长度将进行优化和筛选 高密度脂蛋白使用相同的分析方法。HTT CAG重复长度/不稳定性将在40天内进行量化，以确定线索。 线索将与针对每个MMR蛋白的经过验证的siRNA结合在一起，交付给HDpF，以识别 MMR蛋白介导ASO诱导的收缩事件。同时，铅的体内有效性将得到证实。 在BAC-CAG小鼠中。这项工作将揭示躯体扩张/收缩机制，为HD治疗设计提供参考， 并为这位研究员提供治疗开发、神经生物学和生物信息学方面的关键培训。