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

## TL;DR

High-dose microdystrophin gene therapy in rats improves muscle disease but can cause dangerous heart issues and sudden death.

## Contribution

Reveals dose- and genotype-dependent cardiac toxicity of microdystrophin gene therapy in DMD models.

## Key findings

- High doses of GNT0004 caused sudden deaths and arrhythmias in both DMDmdx and wild-type rats.
- MD1 overexpression may disrupt heart proteins, increasing arrhythmic risk and cardiac remodeling.
- Lower doses were well tolerated in DMDmdx rats, showing muscle and heart improvements without toxicity.

## Abstract

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.

High-dose microdystrophin gene therapy improves muscle pathology in DMDmdx rats but can induce dose-dependent cardiac toxicity, including arrhythmias and sudden death, in both DMDmdx and wild-type rats. These findings reveal potential cardiac risks of microdystrophin overexpression, emphasizing the need for caution and cardiac monitoring in DMD gene therapy trials.