Complete Genetic Correction of Duchenne and Becker Muscular Dystrophy Using Chromosome Transplantation in Induced Pluripotent Stem Cells

Duchenne (DMD) and Becker Muscular Dystrophies (BMD) are X-linked disorders caused by mutations in the dystrophin gene (Xp21.2) encoding the dystrophin protein and leading to a progressive and irreversible muscle deterioration. DMD is determined by the complete loss of the dystrophin protein, while BMD, carrying in-frame mutations that lead to the production of a shorter form of protein with reduced functionality, exhibits a milder phenotype. Due to the huge size of dystrophin gene and the typical large genomic mutations associated with both clinical forms, conventional gene therapy approaches are unable to completely correct these genetic defects. To date, the only available therapeutic strategy aims at converting DMD into BMD with only a partial rescue of the disease phenotype. Here, we propose a novel genomic approach previously developed in our lab, namely Chromosome Transplantation (CT), to fully correct the molecular defect of the dystrophin gene in induced pluripotent stem cells (iPSCs). CT consists in 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. To achieve our purpose, we exploited CT to correct both DMD- and BMD-iPSCs, and verified the functional recovery of dystrophin protein in cardiomyocytes (CMs) differentiated from both disease and corrected iPSC lines. CT is a multi-step protocol; the first step consists in the transfer of an entire normal exogenous X chromosome to DMD/BMD patient-derived iPSCs. The second step entails the selection of cells containing the exogenous normal X chromosome. For this purpose, we took advantage of a selection system based on the X-linked HPRT gene. By CRISPR-Cas9 technology, we successfully inactivated the HPRT gene in our targeted cells (both parental BMD and DMD cells) and produced a donor cell line containing a normal X chromosome. For chromosome transfer, we used an improved microcell-mediated chromosome transfer (RETRO-MMCT) approach. Cells containing wild-type HPRT/dystrophin X chromosome were selected, and clones that spontaneously lost the endogenous X chromosome, resulting in a perfect substitution of the defective X chromosome, were identified. We analysed the isolated chromosome transplanted iPSCs (CT-iPSCs) for genomic stability, stemness and pluripotency capability. To confirm the success of the CT, we performed whole exome sequencing (WES), which showed the absence of unexpected different variations (DV) due to manipulation, and the presence of the expected DV linked to the transplanted X chromosome. In addition, CMs differentiated from CT-iPSCs showed the expression of the complete form of dystrophin, both at the transcriptional and protein levels, further indicating the successful correction of the genomic defect. Finally, in order to demonstrate the functional rescue, we performed a comprehensive electrophysiological analysis on CMs differentiated from both pathological and CT-iPSCs. This analysis showed the restoration of normal contraction/relaxation and Ca2+ dynamics in the corrected CMs. In conclusion, these findings demonstrate that CT is an innovative approach capable of correcting dystrophin gross mutations, opening the opportunity for its potential application to other X-linked genomic diseases and, using a different selection technology, to autosomal diseases as well.