Perfect genomic correction of Human iPSCs from Duchenneand Becker Muscular Dystrphy through Chromosome Transplantation and generation of functional cardiomyocytes

Background: Dystrophinopathies are a group of X-linked disorders primarily caused by large mutations in the DMD gene, which encodes the dystrophin protein. These mutations are responsible for more than 80% of cases. The disorders are characterized by progressive loss of muscle strength, affecting both respiratory and cardiac muscles, as well as nerve tissue integrity. This spectrum includes Duchenne muscular dystrophy (DMD) typically caused by out-of-frame mutations leading to the absence of the dystrophin protein and Becker muscular dystrophy (BMD) where in-frame mutations result in a partially functional but abnormal protein. The lack of dystrophin in DMD makes myocytes more susceptible to stretch-induced damage and necrosis due to myocyte sarcolemma instability during contraction-relaxation cycle, while BMD, represents a milder phenotype. Despite recent advances, current gene editing techniques face challenges in correcting the large size mutations of the DMD gene. In our laboratory, we developed chromosome transplantation as an innovative genomic tool to fully restore genomic defects in induced pluripotent stem cells derived from patients affected by DMD and BMD. This technique involves replacing the defective endogenous chromosome with a normal exogenous one, achieving an euploid karyotype in the corrected cells. Methods: Chromosome transplantation was performed on induced pluripotent stem cells derived from patients affected by Duchenne and Becker muscular dystrophies. The process involved the transfer of a normal X chromosome (donor) into target cells through the RETRO-microcell mediated chromosome transfer (RETRO-MMCT) approach. To select the X-transferred cells, the HPRT gene of the target cells was knocked out using CRISPR-based technology. After transplantation, resistant HPRT-positive clones were isolated and analyzed for genomic stability (karyotype, M-FISH, whole-exome sequencing), stemness, and pluripotency (immunofluorescence and RT-PCR). The corrected cells were differentiated into cardiomyocytes for functional analysis. Dystrophin expression was evaluated through RT-PCR, immunofluorescence and Western blot. Finally, functional recovery was electrophysiologically assessed via Ion Optix and Patch Clamp. Results: Chromosome transplantation was successfully performed, generating corrected DMD and BMD cell lines containing a normal X chromosome. After CRISPR-based inactivation, HPRT-defective DMD and BMD cells were used as recipients in the RETRO-MMCT experiments. Following HPRT-based selection, 47,XXY-transferred cells were isolated. We then identified 46,XY chromosome-transplanted clones that had spontaneously lost the endogenous mutated X chromosome, achieving complete substitution. Genomic stability, stemness, and pluripotency of the transplanted clones were confirmed through detailed molecular analysis, including M-FISH and whole-exome sequencing, which demonstrated a normal 46,XY karyotype and the absence of unintended genetic variations. The corrected and parental induced pluripotent stem cells were subsequently differentiated into cardiomyocytes. The restored dystrophin expression was confirmed at multiple levels. We observed correct gene expression and protein localization, and electrophysiological evaluation confirmed functional recovery of the corrected cells compared to the mutated ones. Conclusions: We successfully established an in vitro gene therapy model for DMD and BMD based on induced pluripotent stem cells. This approach allowed us to obtain fully genetically and functionally corrected cells, thereby validating the model's ability to restore all types of the mutations responsible for muscular dystrophies. Extensive analyses are ongoing to further validate the approach, which represents a potential advancement in the field of regenerative medicine for Duchenne and Becker muscular dystrophies.