Here we demonstrate that AT2220 has multiple modes of action during the synthesis and maturation of mutant GAA, including increased catalytic activity prior to proteolytic processing in lysosomes, facilitated export from the ER with subsequent trafficking and processing through the secretory Manidipine dihydrochloride pathway to lysosomes, and stabilization of the mature isoforms in lysosomes. Furthermore, to study the in vivo effects of AT2220, a new mouse model of Pompe disease was Terbinafine created. These mice express a human transgene of mutant P545L GAA on a Gaa knockout background, and show low GAA activity and elevated glycogen levels in disease-relevant tissues including heart, diaphragm, multiple skeletal muscles, and brain. Daily oral administration of AT2220 to hP545L GAA Tg/KO mice resulted in significant and dose-dependent increases in GAA activity with concomitant reduction in tissue glycogen levels; less-frequent AT2220 administration regimens resulted in even greater glycogen reduction. Collectively, these results provide support for the continued evaluation of AT2220 as a potential treatment for Pompe disease. Furthermore, unlike pure Gaa knockout mice, the P545L GAA Tg/KO mice were tolerant to repeat administrations of rhGAA, potentially providing a useful model for long-term preclinical ERT studies. Previous in vitro studies have shown that the pharmacological chaperone AT2220 directly binds and stabilizes wild-type GAA, and increases the stability, trafficking, and maturation of different mutant forms of GAA in transfected cells and Pompe patientderived fibroblasts, thereby leading to increased cellular levels of GAA activity. However, the precise mechanism of action of AT2220 on mutant GAA remains unclear. Here we show that AT2220 has multiple modes of action during GAA synthesis and maturation that ultimately lead to increased delivery of active GAA to the lysosome and greater reduction of its natural substrate, glycogen, in vivo. In our cell-based studies, a clear increase in the specific activity of AT2220-responsive mutant forms of GAA was observed using the artificial substrate 4-MUG, as well as glycogen.