A heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene is the most frequent known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal and irreversible neurodegenerative diseases. Given that there are no effective treatments for FTD (an Alzheimer?s-related dementia) and ALS, novel therapeutic strategies are urgently needed. Targeting the C9orf72 gene itself by CRISPR/Cas9 gene editing may provide a curative intervention. This work proposes novel applications of CRISPR gene editing technology to edit or silence the pathogenic C9orf72 disease gene in FTD/ALS patient derived induced pluripotent stem cells (iPSCs). Three editing strategies will be evaluated for ability to correct the pathologic C9orf72 repeat expansion, including: (A) bi- allelic excision of the repeat expansion, (B) SNP-based allele-specific inactivation of the mutant allele leaving the normal allele intact (Aim 1) and (C) disruption of a regulatory promotor region to silence gene expression (Aim 2). We will examine the ability of these editing strategies to correct disease pathology in cell types relevant to disease ? human cortical and motor neurons. We have developed rapid, robust methods to generate neurons from human iPSCs derived from controls and patients. Analysis of edited control cell lines will allow us to screen for unanticipated effects of precise gene edits on normal cellular function and fitness. Our findings will not only advance our understanding of potential therapeutic approaches, but will also inform our understanding C9orf72 biology, including C9orf72 gene regulation and potential mechanisms of disease.
The Alzheimer?s-related disease frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two fatal and incurable neurodegenerative diseases linked by a shared genetic cause ? a heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene. The goal of this work is to develop novel CRISPR based therapeutic gene editing technologies and test whether gene editing can reverse the cellular pathology caused by this repeat expansion in patient derived cells. The gene editing strategies we are developing are applicable to other repeat expansion and single gene disorders, potentially including genetic cases of Alzheimer?s disease and other dementias.
