Perfect genomic correction of human iPSCs from Becker Muscular Dystrophy through chromosome transplantation and generation of functional cardiomyocytes

Dystrophinopathy is a group of X-inherited disorders caused by mutations in the DMD gene (Xp21.2 chromosome) encoding for the dystrophin protein. It features loss of respiratory and cardiac muscle strength and the destruction of nerve tissue. Within this spectrum, there are Duchenne (DMD) and Becker muscular dystrophies (BMD)1,2. The worldwide prevalence of BMD is around 1:19,000 and it is results in progressive muscle degeneration and proximal muscle weakness and in 70% of the cases also cardiomyopathy, the leading cause of death. Dystrophin mutations in BMD typically exhibit an in-frame pattern, leading to the production of misfolded or abnormal protein with reduced functionality. Due to the extensive size of this gene and the typical gross mutations associated with the disease, conventional gene therapy approaches are unable to correct the defects. Thus, we propose to correct the molecular defect of the dystrophin gene by using a novel genomic tool previously developed in our lab, the chromosome transplantation (CT) approach, in induced pluripotent stem cells (iPSCs)3. CT consists of the perfect substitution of an endogenous defective chromosome with an exogenous normal one, resulting in a normal euploid karyotype with a complete resolution of the gross mutation. After CT, we conducted genomic stability validation and assessed pluripotency. BMD iPSCs and corrected iPSC clones will undergo differentiation into cardiomyocytes (CMs) to assess protein restoration at various levels: transcription and expression (via RT-PCR and WB), localization (via immunofluorescence) and functional recovery (electrophysiological properties using Ion Optix and Patch Clamp, as well as metabolomics and RNAseq profile analysis). This study provides an opportunity to fully correct not only this pathology but also, in the future, other X-linked genomic diseases.