Somatic Repeat Expansions as a Therapeutic Target for Trinucleotide Repeat Disorders

体细胞重复扩增作为三核苷酸重复疾病的治疗靶点

• 批准号: 10605261
• 负责人: Ricardo Mouro Pinto
• 金额: $ 41.28万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605261
• 关键词: Address; Affect; Alleles; Antisense Oligonucleotides; Biological Markers; Biopsy; Brain; CAG repeat; CRISPR therapeutics; Candidate Disease Gene; Cell model; Cells; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Corpus striatum structure; DNA Repair Gene; DNA Repair Pathway; DNA Repeat Expansion; DNA Sequence Alteration; Development; Disease; Disease Progression; Drug Targeting; Endonuclease I; Exhibits; Expanded DNA Repeat; Fibroblasts; Friedreich Ataxia; Genes; Genetic; Goals; Heart; Human; Huntington Disease; Investigation; Knock-out; Knowledge; Length; Liver; Mendelian disorder; Methodology; Methods; Modeling; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Diseases; Neurons; Oligonucleotides; Onset of illness; Parents; Pathogenicity; Pathway interactions; Patients; Phenotype; Process; RNA Splicing; Reagent; Reporting; Research; Role; Sampling; Spinal Ganglia; System; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Translating; Trinucleotide Repeat Expansion; Trinucleotide Repeats; Validation; base editor; candidate validation; clinical phenotype; effective therapy; efficacy testing; endonuclease; experimental study; genome wide association study; in vivo; insight; intergenerational; minimally invasive; mouse model; new therapeutic target; novel; novel strategies; novel therapeutics; potential biomarker; promoter; stool sample; therapeutic target; tool

ABSTRACT Huntington’s disease (HD) and Friedreich ataxia (FA) are rare neurodegenerative diseases caused by expanded trinucleotide repeats (CAG and GAA, respectively) in the HTT and FXN genes, respectively, with larger alleles being associated with earlier disease onset and more severe clinical phenotypes. Despite these being single gene disorders, where the respective underlying genetic mutations have been known for over 20 years, there remains no cure or disease-modifying therapies, indicating that novel approaches are critical. A hallmark of most repeat expansion disorders is that the repeats are highly unstable, both intergenerationally (parent to child) and in somatic tissues, where the repeat expands progressively over time in a cell-/tissue-specific manner. Notably, in HD, medium-spiny neurons of the striatum, which succumb most severely to the effects of the HTT mutation, exhibit the most dramatic CAG expansions. Similarly, larger GAA repeat expansions have been reported in the heart and dorsal root ganglia of FA patients, where such tissues are most severely affected. These observations, together with growing evidence from GWAS and candidate gene association studies in HD patients, support the hypothesis that progressive repeat length increases in somatic tissues contribute to the pathogenic process. Thus, understanding the roles of disease modifiers in somatic repeat expansion may provide novel targets for therapeutic intervention directed at the repeat mutation itself. To that end, we have leveraged a CRISPR-based in vivo system, recently developed by us, to screen a number of candidate DNA repair genes and determine their role as potential modifiers of somatic CAG repeat instability in HD. Remarkably, this has resulted in the identification of novel genes, which when knocked out in the liver of HD mice, reduced CAG expansions and promoted contractions. We hereby propose a set of experiments aimed at: 1) Identifying non-invasive samples to study CAG repeat instability as a potential biomarker of disease, as well as developing novel long-read sequencing-based methodologies to more accurately size and quantify repeat instability; 2) Validating candidate modifier genes in a new model of CAG expansions using HD patient-derived fibroblasts, recently developed by us, as well as understand the potential adverse implications associated with inactivating such DNA repair genes. We also propose to investigate if such genes are equally involved in FA GAA expansions, using the same in vivo CRISPR platform and patient derived cellular models; 3) Development and testing of novel antisense oligonucleotide- and CRISPR-based therapeutic approaches targeting the repeat expansion process to suppress repeat expansions or actually promote contractions. This will lead to a better understanding of shared mechanisms across these diseases and potentially result in novel therapeutics that can be used in all repeat expansion disorders.

摘要 亨廷顿病(HD)和Friedreich共济失调(FA)是一种罕见的神经退行性疾病 通过HTT和FXN基因中的扩展三核苷酸重复(分别为CAG和GAA)， 更大的等位基因分别与更早的疾病发病和更严重的临床相关 表型。尽管这些都是单基因疾病，但各自的潜在基因 突变已经被发现超过20年了，仍然没有治愈或改变疾病的疗法， 这表明新的方法是至关重要的。大多数重复扩张性疾病的一个特点是 重复序列是高度不稳定的，无论是代际(父母对孩子)还是在体细胞组织中，在那里 Repeat以特定于细胞/组织的方式随时间逐渐扩展。值得注意的是，在高清中，中刺 纹状体的神经元是受HTT突变影响最严重的，表现出 最戏剧性的CAG扩张。同样，更大的GAA重复扩张也已在 FA患者的心脏和背根神经节，这些组织受影响最严重。这些 观察结果，以及来自GWAS和候选基因关联研究的越来越多的证据 HD患者支持躯体组织渐进性重复长度增加的假设 在致病过程中起作用。因此，了解疾病修饰物在体细胞中的作用 重复扩增可能为针对重复突变的治疗干预提供新的靶点 它本身。为此，我们利用我们最近开发的基于CRISPR的体内系统来 筛选一些候选DNA修复基因，并确定它们作为潜在修饰物的作用 HD患者体细胞CAG重复序列不稳定。值得注意的是，这导致了新基因的识别， 当该基因在HD小鼠的肝脏中被敲除时，减少了CAG的扩张并促进了收缩。 我们在此提出了一套实验，旨在：1)识别非侵入性样本进行研究 CAG重复序列不稳定可作为疾病的潜在生物标志物，以及开发新的长期阅读的 基于测序的方法，以更准确地确定重复不稳定性的大小和数量；2)验证 利用HD患者来源的成纤维细胞进行CAG扩张的新模型中的候选修饰基因， 以及了解与以下内容相关的潜在不利影响 使这种DNA修复基因失活。我们还建议调查这些基因是否同样涉及 在FA GAA扩增中，使用相同的体内CRISPR平台和患者来源的细胞模型； 3)基于CRISPR和反义寡核苷酸的新型治疗药物的开发和检测 以重复扩张过程为目标的方法，以抑制重复扩张或实际上促进 宫缩。这将有助于更好地理解这些疾病的共同机制，并 可能导致新的治疗方法，可用于所有重复扩张性疾病。