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

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology
• RNA Therapeutics

Background:

• The C9orf72 G4C2 repeat expansion is the most frequent genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS).
• This expansion leads to toxic sense and antisense repeat RNAs and dipeptide repeat proteins (DPRs), contributing to disease pathology.
• Targeting both RNA and protein species derived from both repeat strands is a potential therapeutic strategy.

Purpose of the Study:

• To evaluate the efficacy of a CRISPR-Cas13d variant, CasRx, in reducing C9orf72 repeat transcripts and DPRs.
• To assess the therapeutic potential of CasRx in cellular and animal models of C9orf72-associated ALS/FTD.

Main Methods:

• Utilized CRISPR-Cas13d variant CasRx to target C9orf72 sense and antisense repeat transcripts.
• Tested CasRx in HEK cells with overexpressed C9orf72 repeats, patient-derived iPSC-neuron lines, and C9orf72 repeat mouse models.
• Assessed reduction of repeat RNAs and DPRs, and protection against excitotoxicity.

Main Results:

• CasRx effectively reduced both sense and antisense C9orf72 repeat transcripts and DPRs in HEK cells.
• In patient-derived neurons, CasRx decreased endogenous repeat RNAs and DPRs, mitigating glutamate-induced excitotoxicity.
• AAV-mediated delivery of CasRx significantly reduced repeat-containing transcripts in two C9orf72 mouse models.

Conclusions:

• CRISPR-Cas13d system CasRx demonstrates potent activity against C9orf72 repeat transcripts and associated toxic proteins.
• RNA-targeting CRISPR systems represent a promising therapeutic avenue for C9orf72-linked amyotrophic lateral sclerosis and frontotemporal dementia.
• CasRx offers a potential strategy for simultaneously targeting multiple pathogenic species in C9orf72 ALS/FTD.