Production of a functional human acid maltase in tobacco seeds; biochemical analysis, uptake by human GSDII cells and in vivo studies in GAA knockout mice.

摘要

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage\uddisease type II (GSDII) or Pompe’s disease. To investigate whether we could generate a\udfunctional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future\udenzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression\udplasmid-pBI101 under the control of the soybean β-conglycinin seed-specific promoter and\udbiochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that\udis more compatible with mammalian glycosylation−phosphorylation and processing. We\udfound the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts\udand in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the\udenzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration\udin GAA knockout (GAA−/−) mice. Additionally, we could purify the tobrhGAA since it\udbound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose.