Our studies also indicate that AT2220 increases the stability of mutant GAA precursors that are secreted into the Clonidine hydrochloride culture media, as well as the mature GAA isoforms in lysosomes. For the GAA precursors that were secreted from transfected COS-7 cells, AT2220 increased the Methocarbamol specific activity nearly ten-fold. This large change in specific activity results from more than just increased catalytic activity due to improved folding, as it was substantially greater than the changes seen for the intracellular forms of each respective mutant. Our previous study demonstrated that AT2220 confers increased thermal stability to wild-type GAA that is timetemperature-, and pH-dependent, with low stability seen at neutral pH. Taken together, these results suggest that AT2220 stabilizes mutant GAA precursors that are secreted into the cell culture medium, protecting them from denaturation. In addition, AT2220 markedly increased the cellular half-life of the mature lysosomal forms of mutant GAA, suggesting a chaperone-mediated protection against degradation by lysosomal proteases, and indicating an additional mode of action even after maturation of the enzyme. Stabilization of mutant protein by chaperone in the lysosome may allow for a buildup of enzyme levels in the target organelle that are sufficient to reduce glycogen after the chaperone has been cleared. The studies in hP545L Tg/KO mice support this hypothesis. The in vivo effects of AT2220 on P545L GAA were investigated using a newly generated mouse model of Pompe disease. This model expresses a human transgene of mutant P545L GAA on a Gaa knock-out background, and shows low tissue GAA activity and elevated glycogen levels in diseaserelevant tissues including heart, diaphragm, multiple skeletal muscles, and brain. While assessments of muscle function and strength have not been made in these mice and represent key areas of future experimentation, the hP545L GAA Tg/KO mice do represent an excellent biochemical model of Pompe disease.To this end, daily oral administration of AT2220 for four weeks resulted in dose-dependent and significant increases in GAA activity in disease-relevant tissues. As also seen in our cell-based studies, AT2220 increased the levels of the 110 kDa precursor form of P545L GAA, and led to the appearance of the mature 76 kDa lysosomal form, in these tissues.