Janice Y. Chou, PhD, Head, Section on Cellular Differentiation
Shih-Yin Chen, PhD, Postdoctoral Fellow
So Youn Kim, PhD, Postdoctoral Fellow
Wai Han Yiu, PhD, Postdoctoral Fellow
Wentao Peng, PhD, Staff Scientist
Brian C. Mansfield, PhD, Guest Researcher
Chi-Jiunn Pan, BS, Senior Research Assistant
Yuk Yin Cheung, MS, Graduate Student
Mohammad Allamarvdasht, BS, Technical Training Fellow
Robert A. Ruef, BS, Technical Training Fellow

Glycogen storage disease type I (GSD-I) is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha) complex that consists of a glucose-6-phosphate transporter (G6PT) and a G6Pase-alpha catalytic unit. G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum, and G6Pase-alpha hydrolyzes 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. There is no cure for GSD-I, and current dietary therapy cannot prevent the development of long-term complications in adult patients. The recent development of animal disease models now provides the opportunity to delineate the disease more precisely and to develop therapies targeting the underlying disease process. GSD-Ib patients exhibit neutrophil dysfunctions. The most noticeable difference between GSD-Ia and GSD-Ib that might explain the dysfunctions is their expression patterns. G6Pase-alpha expression is restricted to the liver, kidney, and intestine while G6PT is expressed ubiquitously. Recently, a second ubiquitously expressed G6Pase activity, G6Pase-beta, was reported, suggesting that the G6Pase-beta-G6PT complex might be functional in neutrophils and that the myeloid defects in GSD-Ib result from the loss of activity of that complex.

Recombinant adeno-associated virus-mediated gene therapy for murine GSD-Ia

Ghosh, ¹ Allamarvdasht, Pan, Sun, ² Mansfield, Chou; in collaboration with Byrne

G6Pase-alpha is a highly hydrophobic protein anchored to the ER by nine-transmembrane helices. The protein cannot 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 kidney, is an attractive possibility. Using G6Pase-alpha-/- mice (Chen L-Y et al., Hum Mol Genet 2003;12:2547), we evaluated two AAV serotypes, AAV serotype 1 (AAV1) and AAV serotype 8 (AAV8), which reportedly 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, holds promise for the future of gene therapy in humans.

The islet-specific G6Pase-related protein

Shieh, ³ Pan, Mansfield, Chou

The islet-specific G6Pase-related protein (IGRP) is devoid of phosphohydrolase activity. Recently, amino acids 206-214 in IGRP were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. This finding suggests that amino acids 206-214 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 and that the low hydrolytic activity in IGRP is attributable to the combination of several independent mutations. We also showed that G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, thereby preventing the targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells dictate susceptibilities to diabetes.

Increased cellular cholesterol efflux in GSD-Ia

Nguyen, ⁴ Pan, Shieh, ³ Chou; in collaboration with Weinstein

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 they are not at elevated risk for developing atherosclerosis. We investigated cellular cholesterol efflux (the first step in reverse cholesterol transport) and antioxidant capacity, which both protect against atherosclerosis in GSD-Ia patients. We showed that sera from these 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 higher total antioxidant capacity than controls; the higher capacity 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.

G6PT is required for normal myeloid functions

Kim, Nguyen,4 Mansfield, Chou; in collaboration with Gao, Murphy

In addition to disturbed glucose homeostasis, 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 issue, we transferred bone marrow 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 the 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 number of myeloid progenitor cells is elevated while the serum reveals 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.

Gene therapy for murine GSD-Ib

Yiu, Pan, Allamarvdasht, Kim, Chou

G6PT is a hydrophobic protein anchored to the ER by 10-transmembrane helices. The protein cannot 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.

¹ Abhijit Ghosh, PhD, former Postdoctoral Fellow
² Mao-Sen Sun, MD, former Postdoctoral Fellow
³ Jeng-Jer Shieh, PhD, former Postdoctoral Fellow
⁴ Andres Nguyen, MS, former Technical Training Fellow

COLLABORATORS

Barry J. Byrne, MD, University of Florida, Gainesville, FL
Ji-Liang Gao, PhD, Laboratory of Molecular Immunology, NIAID, Bethesda, MD
Philip M. Murphy, MD, Laboratory of Molecular Immunology, NIAID, Bethesda, MD
David A. Weinstein, MD, University of Florida College of Medicine, Gainesville, FL

For further information, contact chouja@mail.nih.gov.