CRISPR-mediated correction of skeletal muscle Ca 2+ handling in a novel DMD patient-derived pluripotent stem cell model.

Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca handling is a pathological feature of DMD, mechanisms underlying defective Ca homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca transients and mathematical modeling of Ca dynamics reveal significantly reduced cytosolic Ca clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca homeostasis in DMD and suggests that Ca signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.