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. However, we need to
learn about the biology of the C9orf72 gene in order to employ gene editing strategies.
This work proposes novel applications of CRISPR gene editing technology to edit or silence the pathogenic
C9orf72 disease allele in FTD/ALS patient derived iPSC. With the completion of these aims, we will have
systematically evaluated three complementary methods for silencing a deleterious repeat expansion in the
C9orf72 gene: (1) bi-allelic excision of non-coding DNA harboring only the repeat expansion (Aim 1), (2) allele-
specific excision of the mutant allele containing the repeat expansion (Aim 1), (3) regulatory region disruption
to selectively silence the C9orf72 repeat expansion (Aim 3). 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 fast and robust methods to generate neurons from human induced pluripotent stem cells
(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. This and our future studies
will develop a pipeline for systematically evaluating editing strategies that are potentially curative.