Time-controlled and muscle-specific CRISPR/Cas9-mediated deletion of CTG-repeat expansion in the DMPK gene

CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 30 untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcriptcontaining ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific proguide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo.

Automated summary

CRISPR/Cas9-mediated therapeutic gene editing shows promise in treating monogenic diseases like myotonic dystrophies. Recent studies have successfully applied this technology to delete the pathogenic CTG-repeat expansion in the DMPK gene, leading to beneficial effects on disease hallmarks. An approach using specific proguide RNAs and a dual-vector system has enabled efficient and inducible gene editing in cells and tissues relevant to myotonic dystrophy.