Alternative Splicing and Development of Small Molecule Therapeutics in CAG Expansion Spinocerebellar Ataxias - Abstract The spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of rare, dominantly inherited neurological disorders characterized by progressive ataxia. Although the genetic causes of SCAs are diverse, multiple SCAs (1,2,3,6,7 & 12) are caused by CAG expansion mutations and they share the production of CAG expansion RNAs and in most cases, the expression of toxic polyglutamine containing proteins. Despite these shared features, there are no cross-disease therapeutic approaches for these disorders, and we lack a clear understanding of the precise molecular mechanisms responsible for disease pathogenesis. Our central hypothesis is that targeting CAG expansions with small molecules will provide therapeutic efficacy across multiple SCAs. To test this hypothesis, we leveraged our extensive experience from developing small molecules that target CUG expansion RNAs in myotonic dystrophy (DM). We generated a CAG expansion reporter cell line with an integrated (CAG)60 lacking genomic context of any SCA gene. Using this cell line, we have identified small molecules that selectively reduce levels of CAG transcripts across SCA1, SCA3 and SCA7 patient-derived fibroblasts and SCA1 mice providing proof-of-concept for a cross-disease small molecule approach for CAG expansion SCAs. In this study, we will leverage our CAG screening system and established pipeline to identify FDA-approved small molecules and natural products with therapeutic efficacy across CAG SCAs. Interestingly, transcriptomic characterization of our CAG reporter line identified gene expression signatures that are also found in SCA mouse models, as well as widespread alternative splicing dysregulation. Changes in alternative splicing are a molecular feature for many repeat expansion diseases including DM, suggesting alternative splicing may be a novel CAG-dependent transcriptomic hallmark of SCAs. Indeed, we identified widespread alternative splicing in SCA patient-derived fibroblasts and published RNA-Seq data from across CAG SCA mouse models, including presymptomatic animals. In DM, specific mis-spliced events are directly connected with disease outcomes and can be used as target engagement biomarkers for lead candidate prioritization in preclinical therapy studies. We propose that dysregulation of alternative splicing provides a missing link between presymptomatic transcriptional changes and dysregulation of key cellular pathways in CAG expansion SCAs. To address this, we propose to identify alternative splicing events that contribute to disease onset and neuronal dysfunction in the human neuronal context and to assess the feasibility of measuring alternative splicing dysregulation and rescue to aid in cross CAG SCA lead compound prioritization. Together these studies will identify novel, clinic-ready CAG selective compounds and will help to accelerate these compounds towards clinical testing as well as identifying novel therapeutic targets that may serve as the basis for future investigations. This work will have broad implications for therapeutic development and our understanding of disease across CAG SCAs, non-CAG SCAs, CAG repeat diseases and the broad class of microsatellite expansion disorders.
