Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

Abstract

CRISPR gene editing holds promise to cure or arrest genetic disease, if we can find and implement curative edits reliably, safely and effectively. Expansion of a hexanucleotide repeat in C9orf72 is the leading known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We evaluated three approaches to editing the mutant C9orf72 gene for their ability to correct pathology in neurons derived from patient iPSCs: excision of the repeat region, excision of the mutant allele, and excision of regulatory region exon 1A. All three approaches normalized RNA abnormalities and TDP-43 pathology, but only repeat excision and mutant allele excision completely eliminated pathologic dipeptide repeats. Our work sheds light on the complex regulation of the C9orf72 gene and suggests that because of sense and anti-sense transcription, silencing a single regulatory region may not reverse all pathology. Our work also provides a roadmap for evaluating CRISPR gene correction using patient iPSCs.

Validated assays for the quantification of C9orf72 human pathology

“…When exposed to doxycycline for three days, the iPSCs harboring the hNIL cassette turn on 3 human transcription factors; NGN2, ISL1, and LHX3, that drive them to differentiate into lower motor neurons (iPSC-MNs). The neurons express high levels of motor neuron markers HB9 and ChAT compared to iPSCs at the time point investigated in our expression studies (2 weeks of age) 27 , 29 .…”

Reviewing the Potential Links between Viral Infections and TDP-43 Proteinopathies

“…In the future, some of these compounds could be tested in cellular/animal models of viral infections to test their therapeutic functionality. In parallel, siRNA molecules and CRISPR/Cas9 therapeutic approaches aimed against specific TDP-43-viral protein interactors could also be tested to assess their ability to revert aberrant TDP-43 pathology during viral infections, as has been tried for neurodegenerative processes [ 150 , 151 ]. Finally, different antisense oligonucleotides (ASOs) have also been designed to target RBPs or their modifiers looking at neurodegenerative disorders and cancer.…”

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

“…This gap in understanding hinders research into many diseases such as neurodegeneration and aging, and also limits our control over CRISPR editing outcomes. Many neurodegenerative diseases are caused by dominant genetic mutations, making them strong candidates for CRISPR-based gene inactivation [11][12][13][14][15][16] . Cas9-induced double strand breaks (DSBs) can disrupt these mutant alleles and reverse disease phenotypes.…”