Recombinant Human Acid α-Glucosidase: High Level Production in Mouse Milk, Biochemical Characteristics, Correction of Enzyme Deficiency in GSDII KO Mice

Glycogen storage disease type II (GSDII) is caused by lysosomal acid α-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective production of recombinant human acid α-glucosidase in the milk of transgenic animals, we have fused the human acid α-glucosidase gene to 6.3 kb of the bovine αS1-casein gene promoter and have tested the performance of this transgene in mice. The highest production level reached was 2 mg/ml. The major fraction of the purified recombinant enzyme has a molecular mass of 110 kDa and resembles the natural acid α-glucosidase precursor from human urine and the recombinant precursor secreted by CHO cells, with respect to pH optimum, Km, Vmax, N-terminal amino acid sequence and glycosylation pattern. The therapeutic potential of the recombinant enzyme produced in milk is demonstrated in vitro and in vivo. The precursor is taken up in a mannose 6-phosphate receptor-dependent manner by cultured fibroblasts, is converted to mature enzyme of 76 kDa and depletes the glycogen deposit in fibroblasts of patients. When injected intravenously, the milk enzyme corrects the acid α-glucosidase deficiency in heart and skeletal muscle of GSDII knockout mice.