Low-dose MyoAAV 2A-mediated delivery of engineered micro-utrophin achieves pan- muscle tissue distribution with...

The pathogenesis of Duchenne muscular dystrophy (DMD) is driven by a deficiency of functional dystrophin. Although gene therapy is a promising strategy, current approaches face substantial challenges in balancing therapeutic efficacy with toxicity, largely owing to the requirement for high-dose adeno-associated virus (AAV) administration.

We employed a low-dose strategy in neonatal mdx mice, using a muscle-targeted MyoAAV 2 A capsid to deliver a rationally designed, codon-optimized micro-utrophin construct (ΔR4-R22/ΔC + H3/H4) at 1 × 10¹² vg/kg via intraperitoneal injection. Four weeks post-injection, micro-utrophin expression and localization, as well as γ- and α-sarcoglycan recruitment, were assessed by western blotting and immunofluorescence. Vector genome distribution was quantified by real-time quantitative PCR. Tissue histology was evaluated using Evans blue dye uptake, H&E staining, and Masson's trichrome staining. Muscle function was measured by grip strength and treadmill exercise tests. Serum biomarkers of cardiac injury, heart failure, and systemic inflammation were detected by ELISA.

Robust micro-utrophin expression was detected in skeletal muscles, diaphragm, and myocardium, with minimal off-target expression in the liver. Sarcolemmal localization and functional recruitment of γ- and α-sarcoglycan confirmed restoration of the dystrophin-glycoprotein complex. In treated animals, serum creatine kinase levels, Evans blue dye uptake, and central nucleation in multiple muscles were significantly reduced, accompanied by improvements in relative grip strength, running time, and distance to exhaustion. No cardiac or hepatic toxicity was observed, as indicated by unchanged cTnI, NT-proBNP, ALT, and AST levels. Additionally, serum IL-6 and C5a concentrations were significantly decreased, and fibrosis and inflammation in the diaphragm and myocardium were markedly attenuated.

These findings demonstrate that combining a functional micro-utrophin construct with an advanced muscle-tropic MyoAAV 2 A capsid constitutes a promising strategy to overcome the efficacy-toxicity trade-off that has hindered the clinical translation of DMD gene therapies.