Infantile Pompe disease is a fatal genetic muscle disorder caused by a 
deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We 
constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase 
cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside 
phosphotransferase and dihydrofolate reductase genes. Following transfection in 
dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with 
Geneticin, and amplification with methotrexate, a cell line producing high 
levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in 
Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that 
accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 
mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature 
form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is 
efficiently taken up by fibroblasts from Pompe patients, restoring normal levels 
of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 
6-phosphate. Following intravenous injection, high enzyme levels are seen in 
heart and liver. An efficient production system now exists for recombinant human 
acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts 
from patients with Pompe disease.