CRISPR-mediated correction of skeletal muscle Ca2+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 2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca 2+ 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 2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca 2+ transients and mathematical modeling of Ca 2+ dynamics reveal significantly reduced cytosolic Ca 2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca 2+ 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 wild type PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca 2+ homeostasis in DMD and suggests that Ca 2+ 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.