Summary This project aims to establish a reliable platform to use patient-specific induced pluripotent stem (iPS) cells to model Duchenne Muscular Dystrophy (DMD) and Becker MD (BMD) in the Petri dish, allowing not only for better insight on the pathogenesis of dystrophin-associated disorders, but specially for the development of a relevant system for drug screening. Reprogramming technology provides an unprecedented opportunity to generate large numbers of patient-specific cell types for in vitro disease modeling and drug discovery. Accordingly, iPS cells have been used extensively for these purposes for several disorders, including amyotrophic lateral sclerosis, long-QT syndrome, and spinal muscular atrophy, among others. However to date, there has been scarce literature on the use of this approach to model skeletal muscle disorders, and none studying both skeletal and cardiac muscle in the context of DMD and BMD. Our research group has pioneered methods to derive large quantities of skeletal myogenic progenitor cells from mouse and human pluripotent stem cells, and validated these in vitro and in vivo. Specifically relevant for this proposal, we have recently published a paper focusing on the in vitro maturation of iPS cells towards the skeletal muscle lineage (eLIFE, 2019), which is critical for proper disease modeling. For this project, we have samples from ten DMD/BMD patients, encoding various distinct mutations and displaying mild to severe phenotypes. iPS cells from these samples will be used to produce large numbers of DMD/BMD skeletal myotubes and cardiomyocytes. The hypothesis behind this project is that we will be able to recapitulate disease phenotypes in vitro, including membrane fragility, calcium handling, contraction using three-dimensional myobundles, and electrophysiology measurements. We foresee that molecular/biological signatures resulting from this work will not only enhance our understanding of the pathophysiology behind DMD/BMD, but may also serve as in vitro screening for potential treatments. To validate this hypothesis, we will have control cohort samples, in which we genetically introduce selected DMD mutations in unaffected iPS cells and/or correct selected DMD mutations. The work we propose here, of not only generating skeletal and cardiac muscle from iPS cells but also studying the underlying biology, holds tremendous potential for exploring phenotypes of different mutations, in vitro disease modeling in general, and drug discovery.

Narrative This proposal takes advantage of the ability of pluripotent stem cells to uniquely produce differentiated somatic cell types in large quantities. This allows the generation of specific cell populations for disease modeling and regenerative medicine. Our investigations will focus on establishing a reliable platform to model both skeletal and cardiac muscle for DMD and BMD in vitro, which is key for better insight on the pathogenesis of dystrophin- associated disorders and for the development of a relevant system for drug screening.