Summary Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed for evaluation of dystrophin in disease relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. HPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Utilizing RNA-SEQ, we identified cell surface receptors ERBB3 and NGFR that demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of TGF-β signaling during differentiation improved fusion efficiency, ultrastructural organization, and expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibers after engraftment of CRISPR/Cas9-corrected Duchenne muscular dystrophy hiPSC-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels equal to directly-isolated human fetal muscle cells.

Highlights d Human atlas of limb skeletal muscle in embryonic, fetal, and adult tissues d Human limb skeletal muscle populations and supportive cells vary across development d PAX7 muscle progenitor and stem cells are not identical across developmental states d hPSC-PAX7 cells align to the embryonic-to-fetal transition in human development