Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO-DHFRneg cell line

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid ma ltase (GAA) results in the autosomal recessive glycogen storage disease typ e II (GSD II), To investigate whether we could generate a functional recomb inant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cD NAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFRneg Chi nese hamster ovary cells and established a stable cotransformant that expre ssed rhGAA. We cultured the recombinant cells in media with progressively i ncreasing concentrations of methotrexate and found that human GAA enzyme ac tivity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein b y rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activ ity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulte d in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-ph osphate in the media blocked uptake. GAA -/- mice were treated with the rhG AA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, mos t notably skeletal muscle, heart and diaphragm after two infusions, More im portantly, after multiple infusions, hind, and fore-limb muscle weakness wa s reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II. (C) 2000 Academic Press.