CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities

Abstract

CRISPR/Cas technology holds promise for the development of therapies to treat inherited diseases. Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder with a variable multisystemic character for which no cure is yet available. Here, we review CRISPR/Cas-mediated approaches that target the unstable (CTG•CAG)n repeat in the DMPK/DM1-AS gene pair, the autosomal dominant mutation that causes DM1. Expansion of the repeat results in a complex constellation of toxicity at the DNA level, an altered transcriptome and a disturbed proteome. To restore cellular homeostasis and ameliorate DM1 disease symptoms, CRISPR/Cas approaches were directed at the causative mutation in the DNA and the RNA. Specifically, the triplet repeat has been excised from the genome by several laboratories via dual CRISPR/Cas9 cleavage, while one group prevented transcription of the (CTG)n repeat through homology-directed insertion of a polyadenylation signal in DMPK. Independently, catalytically deficient Cas9 (dCas9) was recruited to the (CTG)n repeat to block progression of RNA polymerase II and a dCas9-RNase fusion was shown to degrade expanded (CUG)n RNA. We compare these promising developments in DM1 with those in other microsatellite instability diseases. Finally, we look at hurdles that must be taken to make CRISPR/Cas-mediated editing a therapeutic reality in patients.

Recovery in the Myogenic Program of Congenital Myotonic Dystrophy Myoblasts after Excision of the Expanded (CTG)n Repeat

“…Here, we are the first to report a comprehensive study on the myogenic properties of a unique series of myoblasts, which are near-isogenic and only differ in the presence of a cDM-length (CTG)2600 repeat in the DM1 locus. Our data confirm and extend the initial findings published by our own group and others on gene-edited cells in DM1 (reviewed in [27]).…”

“…To investigate how the presence of a large-scale (CTG) n repeat in the mutant DMPK allele of a cDM muscle progenitor cell (referred to as parental DM11 myoblasts) influences myoblast-to-myotube formation along the path of terminal differentiation, we generated a panel of eight isogenic myoblast lines (Figure S1A). The lines were initially generated for a study of repeat instability upon the induction of dsDNA (double strand DNA) breaks up- and downstream of the (CTG) n expansion by CRISPR/Cas9 genome editing [26,27].…”

Trinucleotide Repeat-Targeting dCas9 as a Therapeutic Strategy for Fuchs’ Endothelial Corneal Dystrophy

“… 36 Previous studies have demonstrated the feasibility of this approach in excising the causal CTG DNA repeat expansion in DM1 cell lines with CRISPR-Cas9 and a pair of flanking gRNAs followed by repair of the two DSBs with nonhomologous end joining albeit with variable cutting and repair efficiencies. 37 It remains to be seen whether this approach of excision of a pathogenic genomic DNA repeat is feasible in terminally differentiated cells such as corneal endothelium. Additionally, there is a risk of permanent and off-target genome editing with this approach.…”

248th ENMC International Workshop: Myotonic dystrophies: Molecular approaches for clinical purposes, framing a European molecular research network, Hoofddorp, the Netherlands, 11–13 October 2019

“…Finally, results were presented showing that the CTG •CAG repeat can be excised by CRISPR/Cas9-mediated gene editing. After excision, corrected cDM myoblasts do show recovery in their myogenic program, but CpG hypermethylation is not reversed [24][25][26][27][28][29] .…”