The hexanucleotide repeat expansion (HRE) mutation within C9orf72 gene is the most
common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia
(FTD). Several hypotheses have been proposed for how the mutation contributes to
pathogenicity, including the loss of C9orf72 gene function, RNA-mediate toxicity and
the formation of toxic dipeptides by repeat-associated non-ATG (RAN) translation.
Patient-specific iPSCs provide a promising tool for the study of the cellular and molecular
mechanisms of human diseases in relevant cell types and discovering potential therapies.
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9-mediated
homology directed repair (HDR) system represents an attractive approach for disease
modelling and development of therapeutic strategies. In this thesis, iPSCs derived from
ALS/FTD patient carrying the HRE mutation were generated and subsequently gene
edited to remove a massive repeat expansion from the patient cells and replace it with the
wild-type size of the repeats using HDR and a plasmid donor template. The successful
genotypic correction of the mutation resulted in the normalization of the C9orf72 gene
promoter methylation level and the gene variants RNA expression level. Removal of the
mutation also resulted in abolition of sense and antisense RNA foci formation and
reduction of DPRs accumulation. Furthermore, the repeat size correction also rescued the
susceptibility of cells to Glutamate excitotoxicity, decreased the apoptotic cell death and
stress granules formation under the baseline and stress conditions. This work provides a
proof-of-principle that removal of the HRE can rescue ALS disease phenotypes and
provides an evidence that HRE mutation is an attractive target for therapeutic strategies
and drug screening, to block the underlying disease mechanisms.</p