Somatic Repeat Expansions as a Therapeutic Target for Trinucleotide Repeat Disorders - 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.
