CRISPR/CAS9 GENE EDITING AND TARGETED LONG-READ SEQUENCING FOR SPINOCEREBELLAR ATAXIA TYPE 2 THERAPY

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in ATXN2. Neuronal dysfunction and atrophy results from this toxic gain-of-function mutation. There are currently no effective treatments for SCA2. We hypothesize that CRISPR/Cas9 editing of ATXN2 will reduce mutant ATXN2 gene products and provide therapeutic benefit in a SCA2 mouse model. Here, we investigate an indel knockout strategy with a single guide RNA (gRNA) and a CAG repeat deletion strategy with dual-gRNAs. Both strategies resulted in indels and reduced ATXN2 protein levels in vitro. By leveraging the rules of nonsense mediated mRNA decay (NMD), we designed gRNAs that reduced all ATXN2 mRNA isoform levels, and a gRNA that more specifically reduced levels of isoform 1, which harbors the CAG repeat. We further hypothesize that specifically reducing isoform 1, compared to reducing all isoforms, will provide greater therapeutic benefit because isoforms 2 and 3, which also contain conserved functional domains, will compensate for reducing isoform 1. Adeno-associated viral (AAV) vector delivery of CRISPR machinery into the cerebellum of SCA2 mice excised the CAG repeat with dual-gRNAs and reduced mutant ATXN2 protein levels in vivo. However, this approach did not prevent behavior deficits and end-stage disease readouts, therefore requiring further refinement. Because advancing CRISPR-based therapies requires fully defining in vivo editing outcomes, we developed and tested a PCR-free, targeted long-read nanopore sequencing method to evaluate AAV-CRISPR editing in the brain of SCA2 mice. Unbiased high sequencing coverage showed 10-25% editing. Along with intended edits there was AAV integration, 1-2% of which contained the entire AAV genome and were largely unmethylated. Greater than 150 kb deletions at target loci and rearrangements of the transgenic allele were also found. In contrast, PCR-based nanopore sequencing showed bias for AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. In summary, the work presented in this thesis advances our knowledge for the future development of CRISPR-based therapies for SCA2 and other neurodegenerative disorders.