Targeting Dystroglycanopathies using Pluripotent-derived Myogenic Progenitors

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Summary Dystroglycanopathies are a common and heterogeneous subset of muscular dystrophies associated with abnormal O-glycosylation of alpha-dystroglycan (α-DG). They are frequently caused by mutations in fukutin- related protein (FKRP), which encodes a putative Golgi-based glycosyltransferase, resulting in a broad spectrum of MD phenotypes; with most cases being classified as congenital MD type 1 (MDC1C) or limb-girdle MD type 2I (LGMD2I). In the case of LGMD2I, clinical presentation varies from severe early-onset to mild late- onset MD. The biochemical hallmark of FKRP-associated diseases is the hypoglycosylation of α-DG, which results in dysfunction of this critical component of the dystrophin glycoprotein complex (DGC), and consequently leads to cell membrane damage and apoptosis of muscle cells, resulting in chronic tissue degeneration and impaired muscle contractility. Although no effective treatment is available at present for this or any other type of Dystroglycanopathy, one attractive therapeutic approach is to use cell based therapies to promote muscle regeneration. Because pluripotent stem cells can be expanded indefinitely, while maintaining differentiation potential, they represent an advantageous option for therapeutic application. In the case of muscular dystrophies, either allogeneic or autologous cell transplantations have the potential to lead to an effective treatment. For allogeneic transplantation, one would utilize iPS-derived myogenic progenitors obtained from a healthy HLA-matched donor, which following transplantation would give rise to new healthy myofibers. The autologous approach would require ex vivo genetic correction of dystrophic iPS cells prior to transplantation. Our group has pioneered methods to derive skeletal myogenic cells from mouse and human pluripotent ES /iPS cells through transient induction of Pax3 or Pax7 during early mesoderm development. Transplantation of these cells in mouse models for Duchenne Muscular Dystrophy (DMD), results in myofiber and satellite cell engraftment that is accompanied by improvement in muscle force generation. Here we propose to investigate for the first time a pluripotent stem cell-based therapy approach for muscle diseases associated with FKRP mutations. Exciting preliminary data, using a LGMD2I mouse model, reveal the ability of mouse pluripotent-derived myogenic progenitors to engraft the severely affected diaphragm of these mice upon systemic delivery. In Aim 1, we will investigate diaphragm engraftment in further detail. In Aim 2, we propose to establish gene editing tools to correct FKRP mutations of patient-specific iPS cells using the CRISPR/Cas9 system, which will be validated in vitro and in vivo. Finally, to begin laying the groundwork for clinical development of pluripotent stem cells for muscle diseases, in Aim 3 will focus on the scalability, purification, and safety of pluripotent-derived myogenic progenitors.