FROM CELLULAR PHENOTYPES TO CRISPR-BASED GENOME EDITING IN A MOUSE MODEL OF SCA1

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder characterized by cerebellar atrophy and motor dysfunction, caused by an expanded CAG repeat in the ATXN1 gene, leading to a polyglutamine-expanded protein. Despite the understanding of the genetic basis, no therapeutic intervention exists. This project aims to elucidate the cellular mechanisms underlying SCA1 pathogenesis and to develop the CRISPR-based gene editing tool to ameliorate disease-associated cellular phenotypes.
The in vitro models, such as primary fibroblasts, embryonic neural stem cells (eNSCs), and eNSC-derived neurons, were generated from the SCA1^154Q/2Q mice and wild-type (WT) mice. Upon investigation of disease-relevant cellular phenotype, SCA1 fibroblasts exhibited reduced viability and mitochondrial dysfunction. In addition, primary cilia, a non-motile sensory organelle that serves as a key cellular signalling hub, were also studied as a complementary phenotype potentially linked to metabolic impairment in SCA1. This revealed a significant reduction in primary cilia in SCA1 cells - a novel finding.
For the CRISPR-based CAG repeat editing, single-guide RNAs (sgRNAs) were designed and tested in silico, and validated in vitro by chemical transfection and electroporation protocols. The preliminary results suggest that two sgRNAs induce a double-strand break in the CAG repeat. These two sgRNAs will next be tested in combination to excise the expanded CAG tract and potentially rescue the disease-associated cellular phenotypes, followed by their in vivo validation for their functional efficacy assessment.
The project was supported by GAUK: 70124, the Ministry of Health of the Czech Republic (grant No. NW26-08-00119), and Cooperatio (research area NEUR), SVV: 260774.