Duchenne muscular dystrophy (DMD) is a severe, progressive inherited disorder affecting approximately one in 5,000 boys. It results from mutations in the DMD gene that lead to the absence of dystrophin, a structural protein of the sarcolemma. Deprived of this protein, the membrane of muscle fibers becomes highly vulnerable to contraction-induced injury, triggering cycles of degeneration and regeneration, inflammation, and the subsequent development of fibrosis and adipose tissue. Although respiratory impairment was long the leading cause of death, improved patient management has led to cardiomyopathy emerging as a major cause of mortality in patients receiving assisted ventilation. Gene therapy based on micro-dystrophin transfer, which is applicable regardless of the underlying mutation and capable of targeting both skeletal muscle and the heart, is currently under clinical investigation. However, cardiac adverse events recently observed during these trials have raised serious concerns.
To assess the long-term effects of this strategy, the authors treated double knock-out (dKO) mice, deficient in both dystrophin and utrophin, with an adeno-associated viral vector AAV2/9 carrying micro-dystrophin under the control of a striated muscle-specific promoter. This model faithfully reproduces the human dystrophic pathology, including cardiomyopathy associated with premature death. The team also tested the administration of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO), as both pretreatment and post-treatment, and included wild-type mice treated with micro-dystrophin in order to distinguish protein-related effects from those of the dystrophic background.
After one year of treatment, micro-dystrophin markedly improved survival and restored normal cardiac function. However, the analyses revealed thickening of the interventricular septum and posterior wall, associated with inflammation. Genome-wide transcriptomic analysis identified inflammatory signaling pathways, and an increased presence of immune cells (B lymphocytes, macrophages, granulocytes) was confirmed in the hearts of both dKO and wild-type mice treated with micro-dystrophin, compared with untreated animals or those receiving the vector without the transgene. The same cardiac remodeling observed in wild-type mice indicates that these alterations are independent of the dystrophic phenotype and constitute, according to the authors, a signature of micro-dystrophin overexpression rather than the natural progression of the disease.
PPMO post-treatment, combined with pretreatment, improved the histology and function of skeletal muscle without cardiac benefit, and even appeared to be associated with increased inflammation and fibrosis of the diaphragm. The authors acknowledge as the main limitation the severity of the dKO model, in which death occurs before the development of cardiomyopathy, as well as the small number of surviving control animals. They emphasize the need to compare these observations with clinical data from patients and to explore targeted immune modulators with a view to optimizing micro-dystrophin therapy.