Application of CRISPR/Cas9 strategy for gene therapy of Myotonic Dystrophy type 1

Introduction Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disease caused by the abnormal expansion of CTG-triplets in the 3' untranslated region of the DMPK gene. The recent advances in the CRISPR/Cas9 technology have revolutionized the field of genetics and have been exploited to correct the genetic basis of disease such as DM1. Cas9 endonuclease can be targeted to specific locations in the genome via a RNA-guided system to induce double-strand breaks in regions of interest. While therapeutic approaches that neutralize the toxic DMPK transcript provide only short-term effects, CRISPR/Cas9-mediated gene editing strategies can eliminate permanently the pathogenetic mutation. Results We have generated and transduced highly specific and inducible CRISPR/Cas9 components in myogenic cells derived from patients affected by DM1 and obtained the permanent removal of the pathogenetic CTG repeat expansion and the phenotypic reversion of edited cells. The occurrence of off-targets and on-target unintended editing events was evaluated. Transduction of the inducible CRISPR/Cas9 complex via Adeno-Associated Vectors in DMSXL transgenic mice, carrying a mutated human DMPK transgene, led to time-controlled CTGrepeat deletions in the skeletal muscles and in reversal of DM1-associated molecular alterations. Conclusion Differently from the available therapeutic approaches, CRISPR/Cas9-mediated therapeutic gene editing is a flexible and efficient technology for durable treatment of monogenic diseases such as DM1 and a detailed understanding of its therapeutic potential in preclinical models is crucial for a future application in DM1 patients. Tissue-specific and time-restricted Cas9-mediated deletion of CTG expansion is a promising tool for reducing the risk of unintended genomic effect.