Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early onset, severe and rapidly progressive form of muscular dystrophy affecting equally girls and boys. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and the heart. We isolated human muscle stem cells from two donors with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of two co-regulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with >90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base edited patient cells regenerated muscle and contributed to the Pax7 positive satellite cell compartment in vivo in mouse xenografts. We hereby provide the first evidence that autologous gene repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.
Helena Escobar, Anne Krause, Sandra Keiper, Janine Kieshauer, Stefanie Müthel, Manuel García de Paredes, Eric Metzler, Ralf Kühn, Florian Heyd, Simone Spuler