Glycogen storage disease type 1 (GSD-1) is a group of autosomal recessive disorders caused by a deficiency in the endoplasmic reticulum-bound glucose-6-phosphatase (G6Pase) system. The disease presents with both clinical and biochemical heterogeneity consistent with the existence of two major subgroups, GSD-1a and GSD-1b. To evaluate the feasibility of gene therapy for GSD-1a, we have generated G6Pase-deficient (G6Pase-/-) mice that manifest symptoms characteristic of humans GSD-1a. We demonstrate that infusion of an adenoviral vector containing the murine G6Pase gene (Ad- mG6Pase) into G6Pase-/- mice has greatly increased the survival rate of G6Pase-/- mice, restored G6Pase activity in the liver which was accompanied by a concomitant increase in microsomal glucose-6-phosphate (G6P) uptake mediated by the G6P transporter (G6PT). Ad-mG6Pase infusion also greatly improved growth of G6Pase-/- mice, normalized plasma glucose, cholesterol, triglyceride, and uric acid profiles, and alleviated liver and kidney enlargement with near normal levels of glycogen depositions in both organs. Our data demonstrate that a single administration of a recombinant adenovirus vector can alleviate the clinical and pathological manifestations of GSD-1a in mice, suggesting that this disorder in humans can potentially be corrected by gene therapy. GSD-1b is proposed to be caused by a deficiency in microsomal G6P transport. To understand the biology and pathogenesis of this disorder, we mapped the GSD-1b locus to chromosome 11q23, characterized the G6PT cDNA and the gene. Further, we identified mutations in the G6PT gene that segregate with the GSD-1b disorder, developed a functional assay for the recombinant G6P transporter, and demonstrated that mutations uncovered in GSD-1b patients disrupt G6P transport. Our results, for the first time, define a molecular basis for functional G6Pase deficiency in GSD-1b. To broaden our understanding of the effects of mutations that cause GSD- 1b, we have characterized the transmembrane topology of the G6PT protein. Protease protection assays showed that both amino- and carboxyl termini of G6PT face the cytoplasm. This is consistent with ten and twelve transmembrane domain models for G6PT predicted by hydropathy analyses. Glycosylation scanning analysis confirmed that G6PT is anchored in the ER membrane by ten transmembrane helices. - glycogen storage disease type 1, glucose-6-phosphatase, G6P transporter, gene therapy, methionine adenosyltransferase I/III deficiency
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