Dose- and genotype-dependent cardiac arrhythmia and sudden death in rats following microdystrophin gene therapy.

Recombinant adeno-associated virus (rAAV) vectors encoding microdystrophin (MD) are a promising treatment for Duchenne muscular dystrophy (DMD). GNT0004, an rAAV2/8 vector expressing MD1, is currently being tested in patients with DMD. Here, we explored supra-optimal intravenous doses of GNT0004 (2.1 × 1014 and 4.2 × 1014 vg/kg, up to 14 times the therapeutic dose) in wild-type (WT) and DMDmdx rats. In all cohorts, robust MD1 protein expression was observed. In DMD animals, creatine kinase levels were normalized, and skeletal muscle and heart histology and functions were improved. However, unexpected sudden deaths occurred at the highest dose. In WT animals, deaths were observed at both doses and were associated with increased arrhythmic events, which may promote structural and functional heart issues. Immunohistological analysis suggested that overexpression of MD1 may disrupt the dystrophin-associated protein complex, increasing the risk of arrhythmias and sudden death. In DMDmdx rats, the 2.1 × 1014 vg/kg dose was well tolerated, but some deaths occurred at 4.2 × 1014 vg/kg, for which a causal link to GNT0004 cannot be excluded. At this dose, increased arrhythmic risk and cardiac pathological remodeling were observed. These observations highlight the potential risk of MD overexpression in the heart and suggest a need for careful monitoring of patients with DMD treated with gene therapy.