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Glycogen storage disease type‐Ia (GSD‐Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC). Using the G6pc‐R83C mouse model of GSD‐Ia, we explored a CRISPR/Cas9‐based double‐strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end‐joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon‐2. The strategy is based on the insertion of a short dsODN into G6pc exon‐2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild‐type mRNA encoding the native G6Pase‐α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP‐dsODN at age 4 weeks or with two doses of LNP‐dsODN at age 2 and 4 weeks. The G6pc‐R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase‐α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD‐Ia phenotype. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type‐Ia (GSD‐Ia), caused by a deficiency in glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC), is characterized by impaired glucose homeostasis with a hallmark hypoglycemia, following a short fast. We have shown that G6pc‐deficient (G6pc−/−) mice treated with recombinant adeno‐associated virus (rAAV) vectors expressing either wild‐type (WT) (rAAV‐hG6PC‐WT) or codon‐optimized (co) (rAAV‐co‐hG6PC) human (h) G6Pase‐α maintain glucose homeostasis if they restore ≥3% of normal hepatic G6Pase‐α activity. The co vector, which has a higher potency, is currently being used in a phase I/II clinical trial for human GSD‐Ia (NCT 03517085). While routinely used in clinical therapies, co vectors may not always be optimal. Codon‐optimization can impact RNA secondary structure, change RNA/DNA protein‐binding sites, affect protein conformation and function, and alter posttranscriptional modifications that may reduce potency or efficacy. We therefore sought to develop alternative approaches to increase the potency of the G6PC gene transfer vectors. Using an evolutionary sequence analysis, we identified a Ser‐298 to Cys‐298 substitution naturally found in canine, mouse, rat, and several primate G6Pase‐α isozymes, that when incorporated into the WT hG6Pase‐α sequence, markedly enhanced enzymatic activity. Using G6pc−/− mice, we show that the efficacy of the rAAV‐hG6PC‐S298C vector was 3‐fold higher than that of the rAAV‐hG6PC‐WT vector. The rAAV‐hG6PC‐S298C vector with increased efficacy, that minimizes the potential problems associated with codon‐optimization, offers a valuable vector for clinical translation in human GSD‐Ia. [ABSTRACT FROM AUTHOR]

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long‐term complication of glycogen storage disease type‐Ia (GSD‐Ia), which is caused by a deficiency in glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC), a key enzyme in gluconeogenesis. Currently, there is no therapy to address HCA/HCC in GSD‐Ia. We have previously shown that a recombinant adeno‐associated virus (rAAV) vector‐mediated G6PC gene transfer to 2‐week‐old G6pc−/− mice prevents HCA development. However, it remains unclear whether G6PC gene transfer at the tumor developing stage of GSD‐Ia can prevent tumor initiation or abrogate the pre‐existing tumors. Using liver‐specific G6pc‐knockout (L‐G6pc−/−) mice that develop HCA/HCC, we now show that treating the mice at the tumor‐developing stage with rAAV‐G6PC restores hepatic G6Pase‐α expression, normalizes glucose homeostasis, and prevents de novo HCA/HCC development. The rAAV‐G6PC treatment also normalizes defective hepatic autophagy and corrects metabolic abnormalities in the nontumor liver tissues of both tumor‐free and tumor‐bearing mice. However, gene therapy cannot restore G6Pase‐α expression in the HCA/HCC lesions and fails to abrogate any pre‐existing tumors. We show that the expression of 11 β‐hydroxysteroid dehydrogenase type‐1 that mediates local glucocorticoid activation is downregulated in HCA/HCC lesions, leading to impairment in glucocorticoid signaling critical for gluconeogenesis activation. This suggests that local glucocorticoid action downregulation in the HCA/HCC lesions may suppress gene therapy mediated G6Pase‐α restoration. Collectively, our data show that rAAV‐mediated gene therapy can prevent de novo HCA/HCC development in L‐G6pc−/− mice at the tumor developing stage, but it cannot reduce any pre‐existing tumor burden. [ABSTRACT FROM AUTHOR]

Abstract: Glycogen storage disease type Ib (GSD‐Ib) is caused by a deficiency in the ubiquitously expressed glucose‐6‐phosphate (G6P) transporter (G6PT or SLC37A4). The primary function of G6PT is to translocate G6P from the cytoplasm into the lumen of the endoplasmic reticulum (ER). Inside the ER, G6P is hydrolyzed to glucose and phosphate by either the liver/kidney/intestine‐restricted glucose‐6‐phosphatase‐α (G6Pase‐α) or the ubiquitously expressed G6Pase‐β. A deficiency in G6Pase‐α causes GSD type Ia (GSD‐Ia) and a deficiency in G6Pase‐β causes GSD‐I‐related syndrome (GSD‐Irs). In gluconeogenic organs, functional coupling of G6PT and G6Pase‐α is required to maintain interprandial blood glucose homeostasis. In myeloid tissues, functional coupling of G6PT and G6Pase‐β is required to maintain neutrophil homeostasis. Accordingly, GSD‐Ib is a metabolic and immune disorder, manifesting impaired glucose homeostasis, neutropenia, and neutrophil dysfunction. A G6pt knockout mouse model is being exploited to delineate the pathophysiology of GSD‐Ib and develop new clinical treatment options, including gene therapy. The safety and efficacy of several G6PT‐expressing recombinant adeno‐associated virus pseudotype 2/8 vectors have been examined in murine GSD‐Ib. The results demonstrate that the liver‐directed gene transfer and expression safely corrects metabolic abnormalities and prevents hepatocellular adenoma (HCA) development. However, a second vector system may be required to correct myeloid and renal dysfunction in GSD‐Ib. These findings are paving the way to a safe and efficacious gene therapy for entering clinical trials. [ABSTRACT FROM AUTHOR]

Abstract: Glycogen storage disease type Ia (GSD‐Ia) deficient in glucose‐6‐phosphatase‐α (G6Pase‐α) is a metabolic disorder characterized by impaired glucose homeostasis and a long‐term complication of hepatocellular adenoma/carcinoma (HCA/HCC). Mitochondrial dysfunction has been implicated in GSD‐Ia but the underlying mechanism and its contribution to HCA/HCC development remain unclear. We have shown that hepatic G6Pase‐α deficiency leads to downregulation of sirtuin 1 (SIRT1) signaling that underlies defective hepatic autophagy in GSD‐Ia. SIRT1 is a NAD+‐dependent deacetylase that can deacetylate and activate peroxisome proliferator‐activated receptor‐γ coactivator 1α (PGC‐1α), a master regulator of mitochondrial integrity, biogenesis, and function. We hypothesized that downregulation of hepatic SIRT1 signaling in G6Pase‐α‐deficient livers impairs PGC‐1α activity, leading to mitochondrial dysfunction. Here we show that the G6Pase‐α‐deficient livers display defective PGC‐1α signaling, reduced numbers of functional mitochondria, and impaired oxidative phosphorylation. Overexpression of hepatic SIRT1 restores PGC‐1α activity, normalizes the expression of electron transport chain components, and increases mitochondrial complex IV activity. We have previously shown that restoration of hepatic G6Pase‐α expression normalized SIRT1 signaling. We now show that restoration of hepatic G6Pase‐α expression also restores PGC‐1α activity and mitochondrial function. Finally, we show that HCA/HCC lesions found in G6Pase‐α‐deficient livers contain marked mitochondrial and oxidative DNA damage. Taken together, our study shows that downregulation of hepatic SIRT1/PGC‐1α signaling underlies mitochondrial dysfunction and that oxidative DNA damage incurred by damaged mitochondria may contribute to HCA/HCC development in GSD‐Ia. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type Ia (GSD-Ia) deficient in glucose-6-phosphatase-α (G6Pase-α) is a metabolic disorder characterized by impaired glucose homeostasis and a long-term complication of hepatocellular adenoma/carcinoma (HCA/HCC). Mitochondrial dysfunction has been implicated in GSD-Ia but the underlying mechanism and its contribution to HCA/HCC development remain unclear. We have shown that hepatic G6Pase-α deficiency leads to downregulation of sirtuin 1 (SIRT1) signaling that underlies defective hepatic autophagy in GSD-Ia. SIRT1 is a NAD+-dependent deacetylase that can deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a master regulator of mitochondrial integrity, biogenesis, and function. We hypothesized that downregulation of hepatic SIRT1 signaling in G6Pase-α-deficient livers impairs PGC-1α activity, leading to mitochondrial dysfunction. Here we show that the G6Pase-α-deficient livers display defective PGC-1α signaling, reduced numbers of functional mitochondria, and impaired oxidative phosphorylation. Overexpression of hepatic SIRT1 restores PGC-1α activity, normalizes the expression of electron transport chain components, and increases mitochondrial complex IV activity. We have previously shown that restoration of hepatic G6Pase-α expression normalized SIRT1 signaling. We now show that restoration of hepatic G6Pase-α expression also restores PGC-1α activity and mitochondrial function. Finally, we show that HCA/HCC lesions found in G6Pase-α-deficient livers contain marked mitochondrial and oxidative DNA damage. Taken together, our study shows that downregulation of hepatic SIRT1/PGC-1α signaling underlies mitochondrial dysfunction and that oxidative DNA damage incurred by damaged mitochondria may contribute to HCA/HCC development in GSD-Ia. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the ubiquitously expressed glucose-6-phosphate (G6P) transporter (G6PT or SLC37A4). The primary function of G6PT is to translocate G6P from the cytoplasm into the lumen of the endoplasmic reticulum (ER). Inside the ER, G6P is hydrolyzed to glucose and phosphate by either the liver/kidney/intestine-restricted glucose-6-phosphatase-α (G6Pase-α) or the ubiquitously expressed G6Pase-β. A deficiency in G6Pase-α causes GSD type Ia (GSD-Ia) and a deficiency in G6Pase-β causes GSD-I-related syndrome (GSD-Irs). In gluconeogenic organs, functional coupling of G6PT and G6Pase-α is required to maintain interprandial blood glucose homeostasis. In myeloid tissues, functional coupling of G6PT and G6Pase-β is required to maintain neutrophil homeostasis. Accordingly, GSD-Ib is a metabolic and immune disorder, manifesting impaired glucose homeostasis, neutropenia, and neutrophil dysfunction. A G6pt knockout mouse model is being exploited to delineate the pathophysiology of GSD-Ib and develop new clinical treatment options, including gene therapy. The safety and efficacy of several G6PT-expressing recombinant adeno-associated virus pseudotype 2/8 vectors have been examined in murine GSD-Ib. The results demonstrate that the liver-directed gene transfer and expression safely corrects metabolic abnormalities and prevents hepatocellular adenoma (HCA) development. However, a second vector system may be required to correct myeloid and renal dysfunction in GSD-Ib. These findings are paving the way to a safe and efficacious gene therapy for entering clinical trials. [ABSTRACT FROM AUTHOR]

A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid β-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia. [ABSTRACT FROM AUTHOR]

A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-α. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including "lactic acidosis", larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type Ib (GSD-Ib) is caused by deficiencies in the glucose-6-phosphate (G6P) transporter (G6PT) that have been well characterized. Interestingly, deleterious mutations in the G6PT gene were identified in clinical cases of GSD type Ic (GSD-Ic) proposed to be deficient in an inorganic phosphate (Pi) transporter. We hypothesized that G6PT is both the G6P and PPi transporter. Using reconstituted proteoliposomes we show that both G6P and PPi are efficiently taken up into PPi-loaded G6PT-proteoliposomes. The G6P uptake activity decreases as the internal:external PPi ratio decreases and the PPi uptake activity decreases in the presence of external G6P. Moreover, G6P or PPi uptake activity is not detectable in PPi-loaded proteoliposomes containing the p.R28H G6PT null mutant. The G6PT-proteoliposome-mediated G6P or PPi uptake is inhibited by cholorgenic acid and vanadate, both specific G6PT inhibitors. Glucose-6-phosphatase-α (G6Pase-α), which facilitates microsomal G6P uptake by G6PT, fails to stimulate G6P uptake in PPi-loaded G6PT-proteoliposomes, suggesting that the G6Pase-α-mediated stimulation is caused by decreasing G6P and increasing PPi concentrations in microsomes. Taken together, our results suggest that G6PT has a dual role as a G6P and a PPi transporter and that GSD-Ib and GSD-Ic are deficient in the same G6PT gene.--Chen, S.-Y., Pan, C.-.J, Nandigama, K., Mansfield, B., Ambudkar, S., Chou, J. The glucose-6-phosphate transporter is a phosphate-linked antiporter deficient in glycogen storage disease type Ib and and Ic. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphatase-α (G6PC) is a key enzyme in glucose homeostasis that catalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis. Mutations in the G6PC gene, located on chromosome 17q21, result in glycogen storage disease type Ia (GSD-Ia), an autosomal recessive metabolic disorder. GSD-Ia patients manifest a disturbed glucose homeostasis, characterized by fasting hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, lactic acidemia, and growth retardation. G6PC is a highly hydrophobic glycoprotein, anchored in the membrane of the endoplasmic reticulum with the active center facing into the lumen. To date, 54 missense, 10 nonsense, 17 insertion/deletion, and three splicing mutations in the G6PC gene have been identified in more than 550 patients. Of these, 50 missense, two nonsense, and two insertion/deletion mutations have been functionally characterized for their effects on enzymatic activity and stability. While GSD-Ia is not more prevalent in any ethnic group, mutations unique to Caucasian, Oriental, and Jewish populations have been described. Despite this, GSD-Ia patients exhibit phenotypic heterogeneity and a stringent genotype-phenotype relationship does not exist. Hum Mutat 29(7), 921-930, 2008. Published 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The pancreatic secretory phospholipase A[sub 2] (sPLA[sub 2]IB) is considered to be a digestive enzyme, although it has several important receptor-mediated functions. In this study, using the newly isolated murine sPLA[sub 2]IB cDNA clone as a probe, we demonstrate that in addition to the pancreas, the sPLA[sub 2]IB mRNA was expressed in extrapancreatic organs such as the liver, spleen, duodenum, colon, and lungs. We also demonstrate that sPLA[sub 2]IB mRNA expression was detectable from the 17[sup th] day of gestation in the developing mouse fetus, coinciding with the time of completion of differentiation of the pancreas. Furthermore, the mRNA expression pattern of sPLA[sub 2]IB was distinct from those of sPLA[sub 2]IIA and cPLA[sub 2] in various tissues examined. The murine sPLA[sub 2]IB gene structure is well conserved, consistent with findings in other mammalian species, and this gene mapped to the region of mouse chromosome 5F1-G1.1. Taken together, our results suggest that sPLA[sub 2]IB plays important roles both in the pancreas and in extrapancreatic tissues and that in the mouse, its expression is developmentally regulated. [ABSTRACT FROM AUTHOR]