Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

• Published in: Nature communications 2025-01, Vol.16 (1), p.459-15, Article 459
• Main Authors: Kempthorne, Liam, Vaizoglu, Deniz, Cammack, Alexander J., Carcolé, Mireia, Roberts, Martha J., Mikheenko, Alla, Fisher, Alessia, Suklai, Pacharaporn, Muralidharan, Bhavana, Kroll, François, Moens, Thomas G., Yshii, Lidia, Verschoren, Stijn, Hölbling, Benedikt V., Moreira, Francisco C., Katona, Eszter, Coneys, Rachel, de Oliveira, Paula, Zhang, Yong-Jie, Jansen, Karen, Daughrity, Lillian M., McGown, Alexander, Ramesh, Tennore M., Van Den Bosch, Ludo, Lignani, Gabriele, Rahim, Ahad A., Coyne, Alyssa N., Petrucelli, Leonard, Rihel, Jason, Isaacs, Adrian M.
• Format: Article
• Language: English
• Subjects: 13; 13/44; 631/378/1689/1285; 631/378/1689/364; 64; 64/110; Amyotrophic lateral sclerosis; Amyotrophic Lateral Sclerosis - genetics; Amyotrophic Lateral Sclerosis - metabolism; Amyotrophic Lateral Sclerosis - therapy; Animal models; Animals; Antisense RNA; Antisense therapy; Biocompatibility; C9orf72 Protein - genetics; C9orf72 Protein - metabolism; Cell culture; Cell lines; CRISPR; CRISPR-Cas Systems; Dementia disorders; Disease Models, Animal; DNA Repeat Expansion - genetics; Excitotoxicity; Frontotemporal dementia; Frontotemporal Dementia - genetics; Frontotemporal Dementia - metabolism; Genetic Therapy - methods; HEK293 Cells; Humanities and Social Sciences; Humans; Induced Pluripotent Stem Cells - metabolism; Mice; multidisciplinary; Neurons - metabolism; Proteins; Ribonucleic acid; RNA; RNA, Antisense - genetics; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-55550-x

The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G 4 C 2 repeat expansion in C9orf72 . The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD. CRISPR-CasRx effectively reduces ALS- and FTD-causing C9orf72 sense and antisense repeat derived RNAs and proteins in cell lines, patient iPSC-neurons and two independent mouse models of C9orf72 repeat expansion.