CRISPR/Cas9-Mediated Excision of ALS/FTD-Causing Hexanucleotide Repeat Expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. Human

A GGGGCC24 hexanucleotide repeat expansion HRE in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis ALS and frontotemporal dementia FTD fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency sequestration of RNAbinding proteins in the nucleus and production of dipeptide repeat proteins Here we used an adenoassociated viral vector system to deliver CRISPRCas9 geneediting machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion 500600 repeats as well as in patientderived iPSC motor neurons and brain organoids 450 repeats This resulted in a reduction of RNA foci polydipeptides and haploinsufficiency major hallmarks of C9ALSFTD making this a promising therapeutic approach to these diseases