Glycogen Storage Disease Type Ia Mice Research Articles

Inhibition of Wnt/β-catenin signaling reduces renal fibrosis in murine glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in the enzyme glucose-6-phosphatase-α (G6Pase-α or G6PC) that is expressed primarily in the gluconeogenic organs, namely liver, kidney cortex, and intestine. Renal G6Pase-α deficiency in GSD-Ia is characterized by impaired gluconeogenesis, nephromegaly due to elevated glycogen accumulation, and nephropathy caused, in part, by renal fibrosis, mediated by activation of the renin-angiotensin system (RAS). The Wnt/β-catenin signaling regulates the expression of a variety of downstream mediators implicated in renal fibrosis, including multiple genes in the RAS. Sustained activation of Wnt/β-catenin signaling is associated with the development and progression of renal fibrotic lesions that can lead to chronic kidney disease. In this study, we examined the molecular mechanism underlying GSD-Ia nephropathy. Damage to the kidney proximal tubules is known to trigger acute kidney injury (AKI) that can, in turn, activate Wnt/β-catenin signaling. We show that GSD-Ia mice have AKI that leads to activation of the Wnt/β-catenin/RAS axis. Renal fibrosis was demonstrated by increased renal levels of Snail1, α-smooth muscle actin (α-SMA), and extracellular matrix proteins, including collagen-Iα1 and collagen-IV. Treating GSD-Ia mice with a CBP/β-catenin inhibitor, ICG-001, significantly decreased nuclear translocated active β-catenin and reduced renal levels of renin, Snail1, α-SMA, and collagen-IV. The results suggest that inhibition of Wnt/β-catenin signaling may be a promising therapeutic strategy for GSD-Ia nephropathy.

Yap signalling regulates ductular reactions in mice with CRISPR/Cas9-induced glycogen storage disease type Ia

ABSTRACT Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in the glucose-6-phosphatase (G6Pase, G6pc) enzyme, which catalyses the final step of gluconeogenesis and glycogenolysis. Accumulation of G6pc can lead to an increase in glycogen and development of fatty liver. Ductular reactions refer to the proliferation of cholangiocytes and hepatic progenitors, which worsen fatty liver progress. To date, however, ductular reactions in GSD-Ia remain poorly understood. Here, we studied the development and potential underlying mechanism of ductular reactions in GSD-Ia in mice. We first generated GSD-Ia mice using CRISPR/Cas9 to target the exon 3 region of the G6pc gene. The typical GSD-Ia phenotype in G6pc −/− mice was then analysed using biochemical and histological assays. Ductular reactions in G6pc −/− mice were tested based on the expression of cholangiocytic markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM). Yes-associated protein 1 (Yap) signalling activity was measured using western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Verteporfin was administered to the G6pc −/− mice to inhibit Yap signalling. The CRISPR/Cas9 system efficiently generated G6pc −/− mice, which exhibited typical GSD-Ia characteristics, including retarded growth, hypoglycaemia, and fatty liver disease. In addition, CK19- and EpCAM-positive cells as well as Yap signalling activity were increased in the livers of G6pc −/− mice. However, verteporfin treatment ameliorated ductular reactions and decreased Yap signalling activity. This study not only improves our understanding of GSD-Ia pathophysiology, but also highlights the potential of novel therapeutic approaches for GSD-Ia such as drug targeting of ductular reactions.

Downregulation of pathways implicated in liver inflammation and tumorigenesis of glycogen storage disease type Ia mice receiving gene therapy.

Glycogen storage disease type Ia (GSD-Ia) is characterized by impaired glucose homeostasis and long-term risks of hepatocellular adenoma (HCA) and carcinoma (HCC). We have shown that the non-tumor-bearing (NT), recombinant adeno-associated virus (rAAV) vector-treated GSD-Ia mice (AAV-NT mice) expressing a wide range (0.9-63%) of normal hepatic glucose-6-phosphatase-α activity maintain glucose homeostasis and display physiologic features mimicking animals living under calorie restriction (CR). We now show that in AAV-NT mice, the signaling pathways of the CR mediators, AMP-activated protein kinase (AMPK) and sirtuin-1 are activated. AMPK/sirtuin-1 inhibit the activity of STAT3 (signal transducer and activator of transcription 3) and NFκB (nuclear factor κB), the pro-inflammatory and cancer-promoting transcription factors. Sirtuin-1 also inhibits cancer metastasis via increasing the expression of E-cadherin, a tumor suppressor, and decreasing the expression of mesenchymal markers. Consistently, in AAV-NT mice, hepatic levels of active STAT3 and NFκB-p65 were reduced as were expression of mesenchymal markers, STAT3 targets, NFκB targets and β-catenin targets, all of which were consistent with the promotion of tumorigenesis. AAV-NT mice also expressed increased levels of E-cadherin and fibroblast growth factor 21 (FGF21), targets of sirtuin-1, and β-klotho, which can acts as a tumor suppressor. Importantly, treating AAV-NT mice with a sirtuin-1 inhibitor markedly reversed many of the observed anti-inflammatory/anti-tumorigenic signaling pathways. In summary, activation of hepatic AMPK/sirtuin-1 and FGF21/β-klotho signaling pathways combined with down-regulation of STAT3/NFκB-mediated inflammatory and tumorigenic signaling pathways can explain the absence of hepatic tumors in AAV-NT mice.

Glycogen storage disease type Ia mice with less than 2% of normal hepatic glucose-6-phosphatase-α activity restored are at risk of developing hepatic tumors

Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3-63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring <2% of normal hepatic G6Pase-α activity are at risk of tumor development.

Mice expressing reduced levels of hepatic glucose-6-phosphatase-α activity do not develop age-related insulin resistance or obesity

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes.

The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia.

Oxidative stress mediates nephropathy in type Ia glycogen storage disease

Glycogen storage disease type Ia (GSD-Ia) patients, deficient in glucose-6-phosphatase-α, manifest disturbed glucose homeostasis with long-term renal disease. We have previously shown that renal fibrosis in GSD-Ia is mediated by the angiotensin/transforming growth factor-β1 (TGF-β1) pathway, which also elicits renal damage through oxidative stress. In this study, we further elucidate the mechanism of renal disease by showing that renal expression of Nox-2, p22phox, and p47phox, components of NADPH oxidase, are upregulated in GSD-Ia mice compared with controls. Akt/protein kinase B, a downstream mediator of angiotensin II and TGF-β1, is also activated, leading to phosphorylation and inactivation of the Forkhead box O family of transcription factors. This in turn triggers downregulation of superoxide dismutase and catalase (CAT) activities that have essential roles in oxidative detoxification in mammals. Renal oxidative stress in GSD-Ia mice is shown by increased oxidation of dihydroethidium and by oxidative damage of DNA. Importantly, renal dysfunction, reflected by elevated serum levels of blood urea nitrogen, reduced renal CAT activity, and increased renal fibrosis, is improved in GSD-Ia mice treated with the antioxidant drug tempol. These data provide the first evidence that oxidative stress is one mechanism that underlies GSD-Ia nephropathy.

AAV Vector-mediated Reversal of Hypoglycemia in Canine and Murine Glycogen Storage Disease Type Ia

Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia.

Necrotic foci, elevated chemokines and infiltrating neutrophils in the liver of glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD-Ia) patients manifest the long-term complication of hepatocellular adenoma (HCA) of unknown etiology. We showed previously that GSD-Ia mice exhibit neutrophilia and elevated serum cytokine levels. This study was conducted to evaluate whether human GSD-Ia patients exhibit analogous increases and whether in GSD-Ia mice a correlation exists between immune abnormalities and, biochemical and histological alterations in the liver. Differential leukocyte counts and cytokine levels were investigated in GSD-Ia patients. Hepatic chemokine production, neutrophil infiltration, and histological abnormalities were investigated in GSD-Ia mice. We show that GSD-Ia patients exhibit increased peripheral neutrophil counts and serum interleukin-8 (IL-8). Compared to normal subjects, HCA-bearing GSD-Ia patients have a 2.8-fold higher serum IL-8 concentration, while GSD-Ia patients without HCA have a 1.4-fold higher concentration. Hepatic injury in GSD-Ia mice is evidenced by necrotic foci, markedly elevated infiltrating neutrophils, and increased hepatic production of chemokines. Peripheral neutrophilia and elevated serum chemokines are characteristic of GSD-Ia with HCA-bearing GSD-Ia patients having the highest serum IL-8. In GSD-Ia mice these elevations correlate with elevated hepatic chemokine levels, neutrophil infiltration, and necrosis. Taken together, peripheral neutrophilia and increased serum chemokines may indicate hepatic injuries in GSD-Ia.

Neutrophilia and elevated serum cytokines are implicated in glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α manifest a disturbed glucose homeostasis. We hypothesized that disturbed glucose homeostasis might affect myeloid functions. Here, we show that GSD-Ia mice exhibit normal neutrophil activities but have elevated myeloid progenitor cells in the bone marrow and spleen. Interestingly, GSD-Ia mice exhibit a persistent increase in peripheral blood neutrophil counts along with elevated serum levels of granulocyte colony stimulating factor and cytokine-induced neutrophil chemoattractant. Taken together, our results suggest that a loss of glucose homeostasis can compromise the immune system, resulting in neutrophilia. This may explain some of the unexpected clinical manifestations seen in GSD-Ia.

Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia

The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n = 9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth-retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD-Ia, including hypoglycemia, hyperlipidemia and growth failure.

487. Liver-Targeted Gene Therapy in Glycogen Storage Disease Type Ia (GSD-Ia) Requires Widespread, Regulated Glucose-6-phosphatase

The glucose-6-phosphatase (G6Pase) deficiency of GSD-Ia prevents the conversion of glycogen to glucose in the liver. G6Pase overexpression was associated with blood glucose elevations; therefore, unregulated G6Pase expression should be avoided when considering strategies for gene therapy for GSD-Ia. A minimal G6Pase promoter containing insulin-responsive sequence elements was placed upstream of the human G6Pase cDNA in AAV-GRPhG6PpA. This vector was pseudotyped as AAV2/8 and administered to two week-old affected GSD-Ia mice (3 × 1011 DNase-resistant vector particles; 6 × 1013 particles/kg). An AAV2/8 vector containing the universally active CB promoter, AAV-CBcG6PpA, was also administered to affected GSD-Ia mice; however, the CB promoter is NOT glucose-responsive. Importantly, G6Pase expression with the glucose-responsive promoter (GRP) was as effective as the CB promoter/enhancer at correcting hypoglycemia. AAV-GRPhG6PpA significantly corrected hypoglycemia in fasting, affected GSD-Ia mice at 4 weeks of age. Pseudotyping AAV-GRPhG6PpA as AAV2/8 or AAV2/1 corrected hypoglycemia equivalently in GSD-Ia mice. Resolution of hypoglycemia was associated with the correction of other biochemical abnormalities, including hypercholesterolemia and G6Pase deficiency/glycogen storage in the liver. Furthermore, AAV- GRPhG6pA promoted normal growth and prolonged the survival of GSD-Ia mice by greater than 6 months, in comparison to untreated, affected mice which failed to grow and did not survive weaning. AAV-GRPhG6PpA was modified by deleting the D sequence from the terminal repeat to package scAAV-GRPhG6PpA, thereby increasing the efficiency of liver transduction. When scAAV- GRPhG6PpA, pseudotyped as AAV2/8, was administered to 2 week- old affected G6Pase-KO mice (3 × 1011 vector particles), blood glucose was significantly increased (90 +/- 16 mg/dl versus 31 +/- 19 mg/dL for untreated, affected mice; p<0.005). Furthermore, the number of scAAV vector particles administered could be reduced 10-fold compared to the analogous non-scAAV vector, while still correcting hypoglycemia and prolonging survival. An AAV vector encoding a liver-specific promoter (AAV-LSPhG6PpA) to drive unregulated G6Pase expression was administered to unaffected (wild-type) G6Pase-KO mice at 2 weeks of age and a glucose tolerance test was performed at 1 month old. The glucose tolerance test detected elevated glucose, in comparison to untreated wild-type mice, which reflected glucose intolerance related to unregulated G6Pase expression driven by the LSP. In contrast, AAV-GRPhG6PpA administration did not cause glucose intolerance in affected or wild-type mice. Hence, an scAAV vector efficaciously delivered a regulated transgene to liver without causing glucose intolerance in GSD-Ia. The glucose-6-phosphatase (G6Pase) deficiency of GSD-Ia prevents the conversion of glycogen to glucose in the liver. G6Pase overexpression was associated with blood glucose elevations; therefore, unregulated G6Pase expression should be avoided when considering strategies for gene therapy for GSD-Ia. A minimal G6Pase promoter containing insulin-responsive sequence elements was placed upstream of the human G6Pase cDNA in AAV-GRPhG6PpA. This vector was pseudotyped as AAV2/8 and administered to two week-old affected GSD-Ia mice (3 × 1011 DNase-resistant vector particles; 6 × 1013 particles/kg). An AAV2/8 vector containing the universally active CB promoter, AAV-CBcG6PpA, was also administered to affected GSD-Ia mice; however, the CB promoter is NOT glucose-responsive. Importantly, G6Pase expression with the glucose-responsive promoter (GRP) was as effective as the CB promoter/enhancer at correcting hypoglycemia. AAV-GRPhG6PpA significantly corrected hypoglycemia in fasting, affected GSD-Ia mice at 4 weeks of age. Pseudotyping AAV-GRPhG6PpA as AAV2/8 or AAV2/1 corrected hypoglycemia equivalently in GSD-Ia mice. Resolution of hypoglycemia was associated with the correction of other biochemical abnormalities, including hypercholesterolemia and G6Pase deficiency/glycogen storage in the liver. Furthermore, AAV- GRPhG6pA promoted normal growth and prolonged the survival of GSD-Ia mice by greater than 6 months, in comparison to untreated, affected mice which failed to grow and did not survive weaning. AAV-GRPhG6PpA was modified by deleting the D sequence from the terminal repeat to package scAAV-GRPhG6PpA, thereby increasing the efficiency of liver transduction. When scAAV- GRPhG6PpA, pseudotyped as AAV2/8, was administered to 2 week- old affected G6Pase-KO mice (3 × 1011 vector particles), blood glucose was significantly increased (90 +/- 16 mg/dl versus 31 +/- 19 mg/dL for untreated, affected mice; p<0.005). Furthermore, the number of scAAV vector particles administered could be reduced 10-fold compared to the analogous non-scAAV vector, while still correcting hypoglycemia and prolonging survival. An AAV vector encoding a liver-specific promoter (AAV-LSPhG6PpA) to drive unregulated G6Pase expression was administered to unaffected (wild-type) G6Pase-KO mice at 2 weeks of age and a glucose tolerance test was performed at 1 month old. The glucose tolerance test detected elevated glucose, in comparison to untreated wild-type mice, which reflected glucose intolerance related to unregulated G6Pase expression driven by the LSP. In contrast, AAV-GRPhG6PpA administration did not cause glucose intolerance in affected or wild-type mice. Hence, an scAAV vector efficaciously delivered a regulated transgene to liver without causing glucose intolerance in GSD-Ia.

Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-alpha (G6Pase-alpha), a nine-transmembrane domain, endoplasmic reticulum-associated protein expressed primarily in the liver and kidney. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase-alpha (AAV2-G6Pase-alpha) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. We now show that neonatal infusion of GSD-Ia mice with an AAV serotype 1-G6Pase-alpha (AAV1-G6Pase-alpha) or AAV serotype 8-G6Pase-alpha (AAV8-G6Pase-alpha) results in hepatic expression of the G6Pase-alpha transgene and markedly improves the survival of the mice. However, only AAV1-G6Pase-alpha can achieve significant renal transgene expression. A more effective strategy, in which a neonatal AAV1-G6Pase-alpha infusion is followed by a second infusion at age one week, provides sustained expression of a complete, functional, G6Pase-alpha system in both the liver and kidney and corrects the metabolic abnormalities in GSD-Ia mice for the 57 week length of the study. This effective use of gene therapy to correct metabolic imbalances and disease progression in GSD-Ia mice holds promise for the future of gene therapy in humans.

Increased cellular cholesterol efflux in glycogen storage disease type Ia mice: A potential mechanism that protects against premature atherosclerosis

Glycogen storage disease type Ia (GSD-Ia) patients manifest a pro-atherogenic lipid profile but are not at elevated risk for developing atherosclerosis. Serum phospholipid, which correlates positively with the scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux, and apolipoprotein A-IV and E, acceptors for ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol transport, are increased in GSD-Ia mice. Importantly, sera from GSD-Ia mice are more efficient than sera from control littermates in promoting SR-BI- and ABCA1-mediated cholesterol effluxes. As the first step in reverse cholesterol transport, essential for cholesterol homeostasis, these observations provide one explanation why GSD-Ia patients are apparently protected against premature atherosclerosis.

Sustained hepatic and renal glucose-6-phosphatase expression corrects glycogen storage disease type Ia in mice.

Deficiency of glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, causes glycogen storage disease type Ia (GSD-Ia), an autosomal recessive disorder characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. G6Pase is an endoplasmic reticulum-associated transmembrane protein expressed primarily in the liver and the kidney. Therefore, enzyme replacement therapy is not feasible using current strategies, but somatic gene therapy, targeting G6Pase to the liver and the kidney, is an attractive possibility. Previously, we reported the development of a mouse model of G6Pase deficiency that closely mimics human GSD-Ia. Using neonatal GSD-Ia mice, we now demonstrate that a combined adeno virus and adeno-associated virus vector-mediated gene transfer leads to sustained G6Pase expression in both the liver and the kidney and corrects the murine GSD-Ia disease for at least 12 months. Our results suggest that human GSD-Ia would be treatable by gene therapy.