Inducible CRISPR/Cas9 strategy mediates efficient gene editing of trinucleotide repeat expansion in DMPK locus

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder, characterized by progressive myopathy, myotonia and multiorgan involvement, for which no cure is yet available. The pathogenic mechanism of the disease involves a CTG-repeats expansion in the 3' untranslated region of the DMPK (dystrophia myotonica protein kinase) gene. The mRNAs transcribed from the expanded allele are trapped in cell nuclei, where they form abnormal hairpin-like structures that accumulate as foci in the nucleus and bind with high affinity proteins of the Muscleblind-like (MBNL) family. The loss of function of MBNL proteins leads to aberrant alternative splicing of many transcripts, resulting in a fetal-like splicing pattern in patient with DM1. To identify a therapeutic strategy aimed at eliminating the pathogenic mutation, we have developed a gene modification strategy using a drug-inducible and tissue-specific CRISPR/Cas9 system to delete CTG repeats in the human DMPK locus. We have demonstrated that this strategy determines time-limited gene editing in proliferating and post-mitotic myogenic cell models generated by fibroblasts derived from patients with DM1. Removal of CTG expansion is accompanied by reduction of ribonuclear foci and partial recovery of normal splicing. Furthermore, we have evaluated this approach in a well-characterized DM1 mouse model carrying a mutated human DMPK gene with >1,000 CTG repeats. We demonstrated that a single intramuscular injection of recombinant AAV9 vectors expressing CRISPR-Cas9 components into the tibialis anterior muscle of DM1 mice was sufficient to achieve inducible gene editing in vivo. Given that this treatment results in mutation removal from the genome, reduces the accumulation of expanded transcripts and minimizes the potential occurrence of unintended events in off-target genomic loci, it could open the way for future gene therapy application in humans.