GENE EDITING IN MYOTONIC DYSTROPHY TYPE 1: ASSESSMENT OF EFFICIENCY, SAFETY AND THERAPEUTIC EFFECT OF CTG-REPEAT DELETION IN A MOUSE MODEL OF DISEASE

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 and accumulation of the toxic mutated transcripts into ribonuclear foci, leading to a generalized alteration of gene expression. The recent advances in the CRISPR/Cas9 technology have been exploited to correct the genetic basis of diseases such as DM1. Cas9 endonuclease can be targeted to specific locations in the genome via an RNA-guided system to induce double-strand breaks in regions of interest and eliminate permanently the pathogenetic mutation. To achieve this goal, we have generated and transduced tissue-specific and inducible CRISPR/Cas9 components in myogenic cells derived from patients affected by DM1 and obtained the removal of the pathogenetic CTG-repeat expansion and the phenotypic reversion of edited cells. The occurrence of off-target and on-target unintended editing was carefully evaluated. The Cas9 and RNA guides previously tested in DM1 patient-derived cells were then inserted in the backbone of Adeno-Associated Vectors (AAVs) for in vivo administration. Local and systemic transduction of the CRISPR/Cas9 molecules in DMSXL mice, a DM1 mouse model carrying a mutated human transgene from a DM1 patient, resulted in CTG-repeat deletions in the skeletal muscles and in the heart. Although editing efficiency was variable among individuals, partial reversal of DM1-associated molecular alterations in edited tissues was obtained. We have recently optimized editing efficiency by using AAVs showing enhanced tropism for skeletal muscles. Systemic transduction by these myotropic AAVs resulted in much higher expression of CRISPR/Cas9 components in muscles and reduction of ribonuclear foci in the heart of all treated animals. Importantly, recovery of molecular alterations was paralleled by a significant improvement of body weight, that is much reduced in DMSXL mice. Differently from the available therapeutic approaches, CRISPR/Cas9-mediated gene editing is a flexible and efficient technology for durable treatment of DM1 and a detailed understanding of its therapeutic potential in preclinical models is crucial for future application in DM1 patients.