Complete genetic correction of Duchenne Muscular Distrophy using Chromosome Transplantation in induced pluripotent stem cells

Duchenne Muscular dystrophy (DMD) is a degenerative neuromuscular X-linked disease affecting 1 in 3,500-5,000 new-borns, whose life span is about 25-30 years old. DMD is generally caused by out-of-frame mutations in dystrophin (DMD) gene, leading to the absence of dystrophin protein. Dystrophin is the core part of a complex structure, this so called “dystrophin glycoprotein complex” (DGC), involved in calcium (Ca2+) homeostasis, mitochondrial function, motor protein interaction and muscle specific gene expression. Dystrophin plays an essential role during muscle contraction and stretch. Loss of dystrophin protein leads to myocyte sarcolemma instability during contraction-relaxation cycle, making myocytes more susceptible to stretch-induced damage and necrosis. So, the absence of dystrophin protein negatively impacts on contractile apparatus. In fact, the major cause of death is DMD related cardiomyopathy, resulting in respiratory or cardiac failure. So far, there is no effective cure for DMD patients. In spite of the progress in gene editing, for many genetic human diseases, like DMD, associated to gross mutations such as deletions, duplications or inversions, conventional gene therapy is able to solve only partially the defect. In this study, we planned to fully correct the DMD gene by the validation of a novel approach of gene correction, called chromosome transplantation (CT), defined as the perfect replacement of an endogenous defective chromosome with an exogenous normal one, restoring a correct diploid karyotype. To achieve our purpose, we performed CT to correct DMD-induced pluripotent stem cells (iPSCs), which represent a powerful tool for several applications in biomedical research, as in vitro disease modelling and drug screening. Furthermore, human iPSCs can be generated from donor/patient-specific cells, being a potential source of autologous cells for transplantation. Corrected iPSCs were differentiated toward functional cardiomyocytes (CT-DMD-CMs) by two-dimensional (2D) cell culturing. In this way, we evaluated the molecular and functional rescue. In addition, it is known that DMD-iPSC-CMs display electrophysiological abnormalities. Common pathophysiological phenomena in DMD patients are supraventricular and ventricular arrhythmias and increased calcium influx into damaged cell, which leads to cell death. Based on these knowledge, for comprehensive electrophysiological analysis, we were able to demonstrate the restoration of the normal Ca2+ handling in CT-DMD-CMs. In conclusion, these findings show that CT is an innovative approach able to correct gross mutation in DMD patients. This advancement holds promise for potential autologous therapies not only for DMD patients, but also to every X-linked diseases.