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 Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.
Van Hove
J L
JL
Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA.
Yang
H W
HW
Wu
J Y
JY
Brady
R O
RO
Chen
Y T
YT
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism*; D006009:Glycogen Storage Disease Type II / Q000201:enzymology* / Q000628:therapy; D006801:Humans; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D000520:alpha-Glucosidases / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D006003:Glycogen; D000520:alpha-Glucosidases