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Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision

ABSTRACT: Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived iPSC line and a WT line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 FTD/ALS in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding SNP in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using ddPCR, we determined that transcription from the mutant allele is upregulated at least ten-fold, and that sense transcription is independently regulated from each allele whereas antisense transcription is regulated by the opposite allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all DPRs, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including ASOs and CRISPR gene editing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252200 | GEO | 2024/01/02

REPOSITORIES: GEO

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

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Project description:G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/Igf signalling is reduced in fly models of C9orf72 repeat expansion using RNA-sequencing of adult brain. We further demonstrate that activation of insulin/Igf signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/Igf signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/Igf signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

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