RECOMBINANT HUMAN ACID ALPHA-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 a lpha-glucosidase deficiency. Patients have a rapidly fatal or slowly p rogressive impairment of muscle function. Enzyme replacement therapy i s under investigation, For large-scale, cost-effective production of r ecombinant human acid alpha-glucosidase in the milk of transgenic anim als, we have fused the human acid alpha-glucosidase gene to 6.3 kb of the bovine alpha(S1)-casein gene promoter and have tested the performa nce of this transgene in mice. The highest production level reached wa s 2 mg/ml, The major fraction of the purified recombinant enzyme has a molecular mass of 110 kDa and resembles the natural acid alpha-glucos idase precursor from human urine and the recombinant precursor secrete d by CHO cells, with respect to pH optimum, K-m, V-max, N-terminal ami no acid sequence and glycosylation pattern. The therapeutic potential of the recombinant enzyme produced in milk is demonstrated in vitro an d in vivo, The precursor is taken up in a mannose 6-phosphate receptor -dependent manner by cultured fibroblasts, is converted to mature enzy me of 76 kDa and depletes the glycogen deposit in fibroblasts of patie nts. When injected intravenously, the milk enzyme corrects the acid al pha-glucosidase deficiency in heart and skeletal muscle of GSDII knock out mice.