Targeting Dystroglycanopathies using Pluripotent-derived Myogenic Progenitors - Summary The biochemical hallmark of FKRP-associated dystroglycanopathies is the hypoglycosylation of α-dystroglycan (α-DG), which leads to disruption in the interaction of α-DG with extracellular matrix proteins, ultimately leading to muscle wasting. Recessive mutations in FKRP are associated with a heterogeneous spectrum of muscle disorders, ranging from severe early-onset to mild late-onset limb-girdle muscular dystrophy (LGMD2I) to several forms of congenital muscular dystrophy (MDC1C), including severe Walker-Warburg Syndrome. Respiratory impairment due to loss of diaphragm function is a prominent complication of both LGMD2I and MDC1C. No approved therapy currently exists for dystroglycanopathies. There has been tremendous excitement for the therapeutic potential of reprogrammed induced pluripotent stem (iPS) cells in treating genetic diseases. The premise of this project is that stem cell-based therapy consisting of human skeletal myogenic progenitors derived from iPSCs will replenish diseased muscle with normal functional muscle fibers as well as muscle stem cells, which have the potential to provide long-term therapeutic effect in dystroglycanopathies. We have developed and extensively validated a method to generate engraftable skeletal myogenic progenitors from pluripotent stem cells through conditional expression of Pax3 or Pax7. This approach results in highly efficient generation of therapeutic myogenic progenitors, which when transplanted into dystrophic mice locally or systemically produce large quantities of functional skeletal muscle tissue that incorporates normally into the host muscle. Importantly, a fraction of transplanted cells remains mononuclear, and displays key features of skeletal muscle stem cells, including satellite cell localization, response to re-injury, and contribution to muscle regeneration in secondary transplantation assays. Therefore, our technology comprises a cell therapy to rebuild functional skeletal muscle, robust to future damage, in hosts with muscular dystrophy. We have recently shown that mouse and human PSC-derived myogenic progenitors contribute to significant myofiber and satellite cell repopulation in the immunodeficient FKRPP448L-NSG mouse model that we generated. Of therapeutic relevance, we have evidence of successful delivery of these myogenic progenitors directly into the diaphragm of FKRP mice. In addition, we have developed a universal gene correction strategy for FKRP, applied this to patient-specific WWS and LGMD2I iPSCs, and demonstrated in vitro and in vivo rescue of functional α-DG glycosylation. In this application, we propose studies that are critical for the development of successful therapeutic approaches for dystroglycanopathies, including understanding 1) the effect of the environment on the engraftment of transplanted cells and 2) the long-term functionality and molecular characteristics of human gene edited WWS and unaffected iPSC-derived myogenic progenitors, important for both autologous and allogeneic future therapeutic applications, respectively.
