Bioengineered AAV9 and Optimised Microdystrophin Vectors Augment Phenotypic Rescue in a Murine Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder without an effective cure. Adeno-associated virus (AAV) based gene therapy has improved dystrophin function, with sub-optimal clinical outcomes. We reasoned that a combination of rational engineering of AAV9 capsids modified at the post-translational modification sites, optimal promoter selection, and codon-optimisation of the microdystrophin (μDys) can enhance the AAV9 vector functionality. Our initial promoter screening demonstrated improved dystrophin expression in muscle fibres with a ubiquitous CAG promoter (1.61-fold in CAG vs. MHCK7, p < 0.0001) in mdx mice. We then evaluated two engineered AAV9 capsids (N57Q, K51Q) containing CAG-μDys intramuscularly in vivo, which demonstrated a significant improvement in grip strength 18 weeks after gene therapy. Subsequent evaluation of a codon-optimised microdystrophin transgene under the control of the optimal CAG promoter and capsid (AAV9K51Q) by intramuscular administration revealed enhanced muscle grip strength and dystrophin-glycoprotein complex restoration up to 4 months after gene therapy. Based on the improved performance of AAV9K51Q vectors during intramuscular gene transfer, we performed a systemic administration of these vectors alone, and a comparison with the control group revealed a significantly increased muscle contraction force by 1.6-1.7 fold and a 30%-60% dystrophin restoration in skeletal and cardiac muscles, up to 14 months after gene therapy. Collectively, our study underscores the therapeutic potential of engineered AAV9 vectors for potential clinical application in patients with DMD.