Molecular Genetics of Heritable Human Disorders? ? Glycogen storage disease type I (GSD-I) is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha) complex that is consisted of a glucose-6-phosphate transporter (G6PT) that translocates G6P from cytoplasm to the lumen of the endoplasmic reticulum (ER) and G6Pase-alpha that hydrolyses G6P to glucose. Together, they maintain interprandial glucose homeostasis. Deficiencies in G6Pase-alpha cause GSD-Ia and deficiencies in G6PT1 cause GSD-Ib. Both manifest the symptoms of disturbed glucose homeostasis. GSD-Ib patients also suffer from myeloid dysfunctions. There is no cure for GSD-I and the current dietary therapy can not prevent the development of long-term complications in adult patients. Recent development of animal disease models now opens the opportunity to delineate the disease more precisely and develop therapies targeting the underlying disease process.? ? Until recently, G6Pase activity was considered confined to the liver, kidney and intestine, the only tissues known to contain the G6Pase-alpha catalytic unit. However, several studies suggested that GSD-Ia patients are still capable of producing endogenous glucose even when the G6Pase-alpha complex is disrupted. This led to our discovery of a second G6P hydrolase, G6Pase-beta that couples with G6PT to form an active G6Pase complex in the same way as G6Pase-alpha. Our findings challenge the current dogma that only liver, kidney and intestine can contribute to blood glucose homeostasis.? ? G6Pase-alpha is a highly hydrophobic protein anchored to the ER by 9-transmembrane helices. The protein can not be expressed in a soluble form, therefore, enzyme replacement therapy is not an option for the treatment of GSD-Ia but somatic gene therapy, targeting a G6Pase-alpha gene to the liver and the kidney, is an attractive possibility for treating GSD-Ia. Using G6Pase-alpha-/- mice we evaluated two AAV serotypes, AAV serotype 1 (AAV1) and AAV serotype 8 (AAV8), reported to direct efficient hepatic gene transfer to develop gene replacement therapies for GSD-Ia. We showed that neonatal infusion of G6Pase-alpha-/- mice with the AAV1-G6Pase-alpha or AAV8-G6Pase-alpha resulted in hepatic expression of the G6Pase-alpha transgene and markedly improved the survival of the mice. However, only the AAV1-G6Pase-alpha could achieve significant renal transgene expression. We show that neonatal AAV1-G6Pase-alpha infusion followed by a second infusion at age one week provided sustained expression of a complete, functional, G6Pase-alpha system in both the liver and kidney and corrected the murine GSD-Ia disorder for the full 57 weeks of the study. This type of approach, which is effective in correcting the metabolic imbalances and disease progression in GSD-Ia mice hold promise for the future of gene therapy in humans.? ? Amino acids 206 to 214 in the islet-specific G6Pase-related protein (IGRP) were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. This suggests that amino acids 206 to 214 in IGRP confer functional specificity to IGRP. We therefore investigated the molecular events that inactivate IGRP activity and the effects of the beta cell antigen sequence on the stability and enzymatic activity of G6Pase-alpha. We showed that the residues responsible for G6Pase-alpha activity are more extensive than previously recognized. We also showed that G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, which prevents the targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells dictate susceptibilities to diabetes.? ? GSD-Ia patients manifest a pro-atherogenic lipid profile characterized by hypercholesterolemia, hypertriglyceridemia, reduced cholesterol in HDL, and increased cholesterol in LDL and VLDL fractions but are not at elevated risk for developing atherosclerosis. We investigate cellular cholesterol efflux, the first step in reverse cholesterol transport, and antioxidant capacity, both are protective against atherosclerosis, in the sera of GSD-Ia patients. We show that sera from GSD-Ia patients are more efficient than sera from control subjects in promoting the scavenger receptor class B type I (SR-BI)-mediated cellular cholesterol efflux which correlates with the increase in phospholipid and the ratio of HDL-phospholipid to HDL in the sera of GSD-Ia patients. Moreover, sera from GSD-Ia patients have increased total antioxidant capacity compared to controls and this increase correlates with elevated levels of uric acid, a powerful plasma antioxidant. Taken together, the results suggest that the increase in SR-BI-mediated cellular cholesterol efflux and antioxidant capacity in the sera of GSD-Ia patients may contribute to protection against premature atherosclerosis.? ? GSD-Ib patients have defects in the neutrophil respiratory burst, chemotaxis, and calcium flux, and manifest neutropenia. However, whether G6PT deficiency in the bone marrow underlies myeloid dysfunctions in GSD-Ib remains controversial. To investigate this, we transferred bone marrows from G6PT-deficient (G6PT-/-) mice to wild-type mice to generate chimeric mice (BM-G6PT-/-). While wild-type mice have normal myeloid functions, BM-G6PT-/- mice manifest myeloid abnormalities characteristic of G6PT-/- mice. Both have impairments in their neutrophil respiratory burst, chemotaxis response, and calcium flux activities and exhibit neutropenia. In the bone marrow of G6PT-BM-/- and G6PT-/- mice, the numbers of myeloid progenitor cells are increased, while in the serum there is an increase in granulocyte colony stimulating factor and chemokine KC levels. Moreover, in an experimental model of peritoneal inflammation, local production of KC and the related chemokine macrophage inflammatory protein-2 is depressed in both BM-G6PT-/- and G6PT-/- mice along with depressed peritoneal neutrophil accumulation. These findings demonstrate that myeloid dysfunctions in GSD-Ib are intrinsically linked to G6PT deficiency in the bone marrow and neutrophils. ? ? G6PT is a hydrophobic protein anchored to the ER by ten-transmembrane helices. The protein can not be expressed in a soluble form, and to be functional, G6PT must embed correctly in the ER membrane. Therefore, protein replacement therapy is not an option for the treatment of GSD-Ib but somatic gene therapy, targeting the G6PT gene to the gluconeogenic and myeloid tissues is an attractive possibility. To evaluate the feasibility of gene replacement therapy for GSD-Ib, we have infused adenoviral (Ad) vector containing human G6PT (Ad-hG6PT) into G6PT-/- mice. Ad-hG6PT-infusion restores significant levels of G6PT mRNA expression in the liver, bone marrow, and spleen and corrects metabolic as well as myeloid abnormalities in G6PT-/- mice. The G6PT-/- mice receiving gene therapy exhibit improved growth; normalized serum profiles for glucose, cholesterol, triglyceride, uric acid, and lactic acid; and reduced hepatic glycogen deposition. The therapy also corrects neutropenia and lowers the elevated serum levels of granulocyte colony stimulating factor. The development of bone and spleen in the infused G6PT-/- mice is improved and accompanied by increased cellularity and normalized myeloid progenitor cell frequencies in both tissues. This effective use of gene therapy to correct metabolic imbalances and myeloid dysfunctions in GSD-Ib mice holds promise for the future of gene therapy in humans.
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