I001 Cas9 nickase-mediated contraction of CAG repeats in Huntington’s disease

Huntington’s disease (HD) is a hereditary neurodegenerative disease characterized by debilitating cognitive and motor symptoms. It is caused by the expansion of a CAG repeat in the exon 1 of the huntingtin (HTT) gene, and it remains without an effective disease modifying treatment. Because the size of the repeat tract accounts for most of the variation in disease severity, preventing expansions or contracting them presents an attractive therapeutic avenue. Typical gene editing approaches aim to excise the mutant allele, or affect both alleles, both of which are error-prone processes which may have undesirable consequences. Additionally, allele-specific approaches based on single-nucleotide polymorphisms only currently benefit a minority of patients. To overcome these challenges, we employ a CRISPR-Cas9 nickase which targets the CAG repeat tract itself which leads to efficient contractions in HD patient-derived neurons and astrocytes (figure 1). Moreover, nickase system induce contraction in DMKP1 locus in DM1 patient-derived neurons. Using single-cell DNA sequencing, PCR-free whole genome sequencing, and targeted long-read sequencing of the HTT locus, we found no off-target mutations above background in neurons and astrocytes. Furthermore, we delivered the Cas9 nickase and sgRNA stereotactically to a mouse model of Huntington’s disease using adeno-associated viruses and found contractions accumulating in vivo in over half of the infected cells over a period of 5 months. Importantly, the nickase induced contractions in the expanded allele to non-pathological CAG repeat length. We also found that the Cas9 nickase was prone to silencing, further improving the safety of the approach. Our results provide the proof of concept for using the Cas9 nickase to contract the repeat tract safely in multiple cell types and diseases.