TY - JOUR T1 - CRISPR/Cas9-Mediated Excision of ALS/FTD-Causing Hexanucleotide Repeat Expansion in <em>C9ORF72</em> rescues major disease mechanisms <em>in vivo</em> and <em>in vitro</em> JF - bioRxiv DO - 10.1101/2022.05.17.492303 SP - 2022.05.17.492303 AU - Katharina E. Meijboom AU - Abbas Abdallah AU - Nicholas P. Fordham AU - Hiroko Nagase AU - Tomás Rodriguez AU - Carolyn Kraus AU - Tania F. Gendron AU - Gopinath Krishnan AU - Rustam Esanov AU - Nadja S. Andrade AU - Matthew J. Rybin AU - Melina Ramic AU - Zachary D. Stephens AU - Alireza Edraki AU - Meghan T. Blackwood AU - Aydan Kahriman AU - Nils Henninger AU - Jean-Pierre A. Kocher AU - Michael Benatar AU - Michael H. Brodsky AU - Leonard Petrucelli AU - Fen-Biao Gao AU - Erik J. Sontheimer AU - Robert H. Brown AU - Zane Zeier AU - Christian Mueller Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/05/17/2022.05.17.492303.abstract N2 - A hexanucleotide repeat expansion (HRE) consisting of GGGGCC24+ in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are fatal neurodegenerative diseases with no current approved treatments that significantly slow disease progression or extend life expectancy. Several hypotheses have emerged to explain how this HRE causes neuronal death, including C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. In the present study we used a CRISPR/Cas9 gene-editing approach to remove the HRE from the C9ORF72 genomic locus, designing guide RNAs (gRNAs) flanking the HRE, and delivered Cas9 and gRNAs using adeno-associated virus serotype 9 (AAV9) vectors. Here, we demonstrate successful excision of the HRE in C9ORF72 in primary cortical neurons and brains of three mouse models containing the C9ORF72 expanded HRE (ranging from 500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, the major hallmarks of C9-ALS/FTD, making this an extremely attractive therapeutic approach to these diseases.Competing Interest StatementChristian Mueller is inventor on the patent but has no further competing interests ER -