C9orf72 frontotemporal dementia (FTD) and amyotrophic lateral sclerosis(ALS): using patient cells and CRISPR to reveal therapeutic approaches

项目总结/文摘

PROJECT SUMMARY/ABSTRACT 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.