A myotropic AAV vector combined with skeletal muscle cis-regulatory elements improve glycogen clearance in mouse models of Pompe disease.

Pompe disease is a glycogen storage disorder caused by mutations in the acid α-glucosidase (GAA) gene, leading to reduced GAA activity and glycogen accumulation in heart and skeletal muscles. Enzyme replacement therapy with recombinant GAA, the standard of care for Pompe disease, is limited by poor skeletal muscle distribution and immune responses after repeated administrations. The expression of GAA in muscle with adeno-associated virus (AAV) vectors has shown limitations, mainly the low targeting efficiency and immune responses to the transgene. To address these issues, we developed AAV capsids with improved skeletal muscle targeting and reduced off-targeting. These capsids combined with codon optimization, muscle-specific cis-regulatory elements, and a synthetic promoter demonstrated a strong skeletal muscle tropism, reduced liver targeting, and enhanced GAA transgene expression and reduced glycogen accumulation in a Gaa (-/-) mouse model. However, increased muscle-specific expression led to a robust anti-hGAA immune response. To circumvent this, the AAVMYO2 capsid was tested in immunodeficient Gaa (-/-) Cd4 (-/-) mice and compared to AAV9 at different doses. The combination of AAVMYO2 with an optimized transgene expression cassette provided a dose-dependent advantage for glycogen reduction in skeletal muscles of Gaa (-/-) Cd4 (-/-) mice. These findings support the potential of muscle-specific AAV gene therapy for Pompe disease at lower doses with greater specificity.