Assessing outcomes and therapeutic potential of targeted C9ORF72 methylation

C9FTD/ALS is the major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). It is characterized by expansion of a G4C2 repeat in the first intron of the C9ORF72 gene. Multiple factors emerging from the mutant C9ORF72 chromatin locus contribute to molecular and cellular pathology, including production of harmful RNA transcript variants (containing the repeat), loss of transcripts encoding C9ORF72 protein, and aberrations at the chromatin level due to unusual structure, modification, or protein interactions. Although the genomic context and transcript landscape at the C9ORF72 locus are complex and influenced by the repeat expansion, the locus may lend well to gene-targeted approaches that can modulate the epigenetic state. A body of literature supports a positive role for DNA methylation at the C9ORF72 locus, such as reducing hallmarks of molecular disease and even improving patient outcomes. Methylation of the repeat expansion sequence, flanking regions, and the promoter has been correlated with reduced production of repeat-containing transcript variants and poly-dipeptide repeats (poly-DPRs) translated from them. Deletion of the C9ORF72 promoter was shown to eliminate detection of a harmful transcript variant, as well as poly-DPRs, and surprisingly preserve most of the expression of the primary C9ORF72 protein coding transcript. We propose to induce methylation of the C9ORF72 promoter and the repeat expansion sequence using CRISPRoff, a CRISPR-based dCas9-methyltransferase fusion system. CRISPRoff can deposit CpG methylation for targeted and heritable gene silencing with little or no reported off-target methylation. CRISPRoff does not induce DNA cleavage and can be delivered transiently. Methylation of the repeat sequence may be allele-selective for the expansion since G4C2 repeat tracts are relatively rare in the human genome. Thus, the potential safety profile of sequence-specific epigenetic editors like CRISPRoff could be favorable for C9FTD/ALS. Site-specific methylation at the C9ORF72 locus will enable investigation of gene expression and regulatory mechanisms to help determine whether epigenetic modulation is a potential candidate for therapeutic intervention. To assess the impact of methylation at the C9ORF72 locus via CRISPRoff, we will employ model patient-derived cell lines and quantify gene expression from the C9ORF72 locus, characterize DNA methylation status with nanopore sequencing, and evaluate the ability of CRISPRoff to correct disease- associated cellular defects, including global gene expression, repeat RNA foci, and mitochondrial dysfunction. If successful, this study will offer new insight into epigenetic regulation of C9ORF72 as well as a potential new therapeutic direction for C9FTD/ALS. Project Number: 1R21NS145112-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Keith Gagnon | Institution: WAKE FOREST UNIVERSITY HEALTH SCIENCES, WINSTON-SALEM, NC | Award Amount: $406,640 | Activity Code: R21 | Study Section: Cellular and Molecular Biology of Neurodegeneration Study Section[CMND] View on NIH RePORTER: https://reporter.nih.gov/project-details/11222818