Pompe disease (PD) is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), which leads to progressive glycogen accumulation, resulting in severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs early in life due to a cardiorespiratory failure. There is no cure for PD. Here, using CRISPR/Cas9 technology, we have generated the first rat model recapitulating key features of human IOPD. This rat model was used to assess a muscle-directed gene therapy based on the highly myotropic adeno-associated viral vector capsid AAVMYO3 and an optimized muscle-specific promoter, leading to widespread Gaa expression only in skeletal and cardiac muscles. Treatment of young PD rats with AAVMYO3-Gaa vectors mediated long-term correction of glycogen storage, prevented severe skeletal and cardiac muscle pathology, increased grip strength and rescued survival. This study provides a rationale for future clinical translation of this approach to treat PD.