We describe two complementary strategies for the generation of genetically corrected myogenic stem cells from patients with Duchenne muscular dystrophy, one from muscle biopsy samples and the other from induced pluripotent stem (iPS) cells. We also describe the generation of a multifunctional DYS-HAC applicable in both strategies. Notably, these technologies are also being developed in genome-integration-free platforms.
Reversible immortalisation with lentivirally delivered excisable telomerase and BMI1 complementary DNAs allowed bypassing of replicative senescence and DYS-HAC transfer in muscle-derived cells. When isolation of tissue-derived progenitors proved challenging, we successfully derived skeletal myogenic cells from Duchenne muscular dystrophy iPS cells reprogrammed with non-integrating technologies and corrected them with the DYS-HAC. Finally we describe the engineering of a next-generation, multifunctional DYS-HAC that can provide complete genetic correction, enhanced proliferation, controllable cell death (as a safety system), and inducible myogenesis in both tissue-derived or iPS cell-derived stem cells.
This project provides the foundation for preclinical and clinical development of an autologous ex-vivo gene therapy protocol for Duchenne muscular dystrophy based on HACs and myogenic cells.
National Institute for Health Research, Medical Research Council, Takeda New Frontier Science, Muscular Dystrophy UK, Duchenne Children's Trust, Duchenne Research Fund, Duchenne Parent Project Onlus.