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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]

The photoreceptor outer segment is a highly specialized primary cilium that is essential for phototransduction and vision. Biallelic pathogenic variants in the cilia-associated gene CEP290 cause non-syndromic Leber congenital amaurosis 10 (LCA10) and syndromic diseases, where the retina is also affected. While RNA antisense oligonucleotides and gene editing are potential treatment options for the common deep intronic variant c.2991+1655A>G in CEP290, there is a need for variant-independent approaches that could be applied to a broader spectrum of ciliopathies. Here, we generated several distinct human models of CEP290-related retinal disease and investigated the effects of the flavonoid eupatilin as a potential treatment. Eupatilin improved cilium formation and length in CEP290 LCA10 patient-derived fibroblasts, in gene-edited CEP290 knockout (CEP290 KO) RPE1 cells, and in both CEP290 LCA10 and CEP290 KO iPSCs-derived retinal organoids. Furthermore, eupatilin reduced rhodopsin retention in the outer nuclear layer of CEP290 LCA10 retinal organoids. Eupatilin altered gene transcription in retinal organoids by modulating the expression of rhodopsin and by targeting cilia and synaptic plasticity pathways. This work sheds light on the mechanism of action of eupatilin and supports its potential as a variant-independent approach for CEP290-associated ciliopathies. [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]

FDA-cleared ovarian cancer biomarkers are limited to CA-125 and HE4 for monitoring and recurrence and OVA1, a multivariate panel consisting of CA-125 and four additional biomarkers, for referring patients to a specialist. Due to relatively poor performance of these tests, more accurate and broadly applicable biomarkers are needed. We evaluated the dysregulation of 259 candidate cancer markers in serum samples from 499 patients. Sera were collected prospectively at 11 monitored sites under a single well-defined protocol. All stages of ovarian cancer and common benign gynecological conditions were represented. To ensure consistency and comparability of biomarker comparisons, all measurements were performed on a single platform, at a single site, using a panel of rigorously calibrated, qualified, high-throughput, multiplexed immunoassays and all analyses were conducted using the same software. Each marker was evaluated independently for its ability to differentiate ovarian cancer from benign conditions. A total of 175 markers were dysregulated in the cancer samples. HE4 (AUC = 0.933) and CA-125 (AUC = 0.907) were the most informative biomarkers, followed by IL-2 receptor α, α1-antitrypsin, C-reactive protein, YKL-40, cellular fibronectin, CA-72-4 and prostasin (AUC>0.800). To improve the discrimination between cancer and benign conditions, a simple multivariate combination of markers was explored using logistic regression. When combined into a single panel, the nine most informative individual biomarkers yielded an AUC value of 0.950, significantly higher than obtained when combining the markers in the OVA1 panel (AUC 0.912). Additionally, at a threshold sensitivity of 90%, the combination of the top 9 markers gave 88.9% specificity compared to 63.4% specificity for the OVA1 markers. Although a blinded validation study has not yet been performed, these results indicate that alternative biomarker combinations might lead to significant improvements in the detection of ovarian cancer. [ABSTRACT FROM AUTHOR]

Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER) of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate (Pi). This reaction depends on coupling the G6P transporter (G6PT) with glucose-6-phosphatase-α (G6Pase-α). Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a Pi-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:Pi exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, Pi-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis. [ABSTRACT FROM AUTHOR]

Background: Most women with a clinical presentation consistent with ovarian cancer have benign conditions. Therefore methods to distinguish women with ovarian cancer from those with benign conditions would be beneficial. We describe the development and preliminary evaluation of a serum-based multivariate assay for ovarian cancer. This hypothesis-driven study examined whether an informative pattern could be detected in stage I disease that persists through later stages. Methodology/Principal Findings: Sera, collected under uniform protocols from multiple institutions, representing 176 cases and 187 controls from women presenting for surgery were examined using high-throughput, multiplexed immunoassays. All stages and common subtypes of epithelial ovarian cancer, and the most common benign ovarian conditions were represented. A panel of 104 antigens, 44 autoimmune and 56 infectious disease markers were assayed and informative combinations identified. Using a training set of 91 stage I data sets, representing 61 individual samples, and an equivalent number of controls, an 11-analyte profile, composed of CA-125, CA 19-9, EGF-R, C-reactive protein, myoglobin, apolipoprotein A1, apolipoprotein CIII, MIP-1α, IL-6, IL-18 and tenascin C was identified and appears informative for all stages and common subtypes of ovarian cancer. Using a testing set of 245 samples, approximately twice the size of the model building set, the classifier had 91.3% sensitivity and 88.5% specificity. While these preliminary results are promising, further refinement and extensive validation of the classifier in a clinical trial is necessary to determine if the test has clinical value. Conclusions/Significance: We describe a blood-based assay using 11 analytes that can distinguish women with ovarian cancer from those with benign conditions. Preliminary evaluation of the classifier suggests it has the potential to offer approximately 90% sensitivity and 90% specificity. While promising, the performance needs to be assessed in a blinded clinical validation study. [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]

The islet-specific glucose-6-phosphatase-related protein (IGRP) has no known catalytic activity, but is of interest because it is the source of the peptide autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in non-obese diabetic mice. To better understand the potential roles of this protein in diabetes mellitus, we examine the subcellular localization and membrane topography of human IGRP. We show that IGRP is a glycoprotein, held in the endoplasmic reticulum by nine transmembrane domains, which is degraded in cells predominantly through the proteasome pathway that generates the major histocompatibility complex class I-presented peptides. [Copyright &y& Elsevier]

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Sequence alignments identify a signature motif shared by G6PT and a family of transporters of phosphorylated metabolites. Two null signature motif mutations have been identified in the G6PT gene of GSD-Ib patients. In this study, we characterize the activity of seven additional mutants within the motif. Five mutants lack microsomal G6P uptake activity and one retains residual activity, suggesting that in G6PT the signature motif is a functional element required for microsomal glucose-6-phosphate transport. [ABSTRACT FROM AUTHOR]