Abstract|Motor Neuron Disease 1| Volume 357, SUPPLEMENT 1, e48, October 15, 2015

Modelling amyotrophic lateral sclerosis (ALS) using mutant and CAS9/CRISPR-corrected motor neurons from patients with C9ORF72 mutations reveals disease-specific cellular phenotypes

      Background: The C9orf72 hexanucleotide expansion is the commonest genetic cause of ALS and Frontotemporal Dementia (FTD). In addition to cytoplasmic aggregation of phospho-TDP-43, pathological features include RNA foci and aggregations of dipeptide protein. The relative contribution of these pathologies to the disease remains unresolved.
      Objective: To use human motor neurons from patients with ALS, and correction with gene editing, to resolve the key pathological features of ALS.
      Methods: Induced pluripotent stem cell (iPSC) lines were generated from four ALS patients carrying the C9ORF72 repeat expansion. One line was corrected by genome editing to serve as an isogenic control. Cells were characterized functionally and pathologically.
      Results: ALS/FTD iPSC line OXC9-02-02 was successfully used to target the expanded G4C2 repeat using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated homologous recombination, in the presence of plasmid DNA donor template containing a positive selection cassette. In C9orf72 iPSC-derived motor neurons, dysfunction in Ca2+ homeostasis and endoplasmic reticulum tER) stress correlated with decreased cellular survival and reduced levels of the anti-apoptotic protein Bcl-2. Furthermore, the C9orf72 motor neurons showed evidence of abnormal protein aggregation and stress granule formation in the absence of external stress. These phenotypes were corrected by excision of the mutation by gene editing.
      Conclusions: We have demonstrated that genome editing can be used to validate an ALS/FTD model system. The identification of a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signalling and altered proteostasis demonstrates the value of iPSC-derived motor neurons as a cellular model for the investigation of neurodegeneration.