HIGH-LEVEL PRODUCTION OF RECOMBINANT HUMAN LYSOSOMAL ACID ALPHA-GLUCOSIDASE IN CHINESE-HAMSTER OVARY CELLS WHICH TARGETS TO HEART-MUSCLE AND CORRECTS GLYCOGEN ACCUMULATION IN FIBROBLASTS FROM PATIENTS WITH POMPE DISEASE

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 Chi nese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-gluc osidase was established. In 48 hr, the cells cultured in Iscove's medi um with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulat ed to 91 mu g . ml(-1) in the medium (activity, >22.6 mu mol . hr(-1). ml(-1)). This enzyme has a pH optimum similar to that of the mature f orm, but a lower V-max and K-m for 4-methylumbelliferyl-alpha-D-glucos ide. It is efficiently taken up by fibroblasts from Pompe patients, re storing normal levels of acid alpha-glucosidase and glycogen, The upta ke is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient producti on system now exists for recombinant human acid alpha-glucosidase targ eted to heart and capable of correcting fibroblasts from patients with Pompe disease.