Somatic Repeat Expansions as a Therapeutic Target for Trinucleotide Repeat Disorders

摘要 亨廷顿氏病（HD）和弗里德赖希共济失调（FA）是罕见的神经退行性疾病， 通过HTT和FXN基因中扩展的三核苷酸重复序列（分别为CAG和GAA）， 更大的等位基因与更早的疾病发作和更严重的临床症状相关。 表型尽管这些都是单基因疾病，其中各自的潜在遗传 已知突变已经超过20年，仍然没有治愈或疾病修饰疗法， 这表明新的方法至关重要。大多数重复扩张障碍的一个标志是， 重复是高度不稳定的，无论是代际（父母到孩子）和在体细胞组织，其中 重复以细胞/组织特异性的方式随着时间的推移逐渐扩大。值得注意的是，在HD中，中等多刺 纹状体的神经元，最严重地屈服于HTT突变的影响，表现出 最引人注目的CAG扩张。类似地，较大的GAA重复扩增已在文献中报道。 FA患者的心脏和背根神经节，这些组织受到最严重的影响。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，支持体细胞组织中进行性重复长度增加的假设 有助于致病过程。因此，了解疾病修饰因子在体细胞免疫中的作用， 重复扩增可以为针对重复突变的治疗干预提供新的靶点 本身为此，我们利用了我们最近开发的基于CRISPR的体内系统， 筛选一些候选DNA修复基因，并确定它们作为潜在修饰剂的作用， HD患者体细胞CAG重复序列不稳定性值得注意的是，这导致了新基因的鉴定， 当在HD小鼠的肝脏中敲除时，减少CAG扩张并促进收缩。 我们在此提出了一组实验，旨在：1）识别非侵入性样品进行研究 CAG重复不稳定性作为疾病的潜在生物标志物，以及开发新的长读 基于测序的方法，以更准确地确定重复不稳定性的大小和量化; 2）验证 在使用HD患者来源的成纤维细胞的CAG扩增的新模型中的候选修饰基因， 我们最近开发的，以及了解潜在的不利影响， 使这些DNA修复基因失活。我们还建议调查这些基因是否同样参与 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)基于新型反义寡核苷酸和CRISPR的治疗剂的开发和测试 靶向重复扩增过程以抑制重复扩增或实际上促进 宫缩这将有助于更好地了解这些疾病的共同机制， 潜在地产生可用于所有重复扩增病症的新治疗剂。

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.