Complete correction of acid α-glucosidase deficiency in Pompe disease fibroblasts in vitro, and lysosomally targeted expression in neonatal rat cardiac and skeletal muscle

The enzyme acid alpha-glucosidase catalyzes the breakdown of lysosomal glycogen. Absence of this enzyme results in infantile Pompe disease, characterized by hypertrophic cardiomyopathy, skeletal muscle weakness and fatal heart failure by 2 years of age. We have examined the possibility of gene replacement therapy for this disease, by constructing an E1-deleted recombinant adenovirus encoding human acid alpha-glucosidase (Ad-GAA). The dose-response in fibroblasts from patients with Pompe disease transduced with this vector is linear over the range tested (one to 2000 plaque forming units (p.f.u.) of Ad-GAA per cell), and acid alpha-glucosidase activity comparable to that of normal fibroblasts is achieved at 100 p.f.u. per cell. Targeting of the recombinant protein to the lysosomal compartment was confirmed by immunocytochemistry. In vivo expression was examined by injecting Ad-GAA into newborn rats; intracardiac administration produced 10 times the normal level of acid alpha-glucosidase activity in whole heart lysates while a hind-limb i.m. injection increased activity in that muscle to six times the normal level. Western blotting of these tissues detected species at 76 kDa consistent with the size of processed lysosomal enzyme and levels of expression as high as 1.0 mg recombinant protein per gram of tissue wet weight were produced. These data demonstrate high-level, lysosomal expression of recombinant acid alpha-glucosidase in treated target tissues and support the feasibility of gene replacement strategies for Pompe disease.