Molecular Genetics Of Heritable Human Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The Section on Cellular Differentiation conducts research to understand the biology and pathogenesis of GSD-Ia, GSD-Ib, and GSD-Irs (G6Pase-beta deficiency) and to develop novel therapeutic approaches for these disorders. GSD-Ia patients, deficient in G6Pase-alpha, manifest impaired glucose homeostasis. While dietary therapies are sufficiently successful to enable patients to attain near normal growth and pubertal development, long-term complications including hepatocellular adenoma (HCA) with malignant potential still persist in GSD-Ia patients. We examined the efficacy of liver G6Pase-alpha delivery mediated by rAAV8-GPE, a recombinant AAV pseudotype 2/8 vector expressing human G6Pase-alpha directed by the 2864-bp of human G6PC promoter/enhancer (GPE). A recent study showed that HCA develops in 100% of liver-specific G6pc-null mice 78 weeks after gene deletion. Therefore we examined the disease risk for hepatic neoplasia in a long-term dose-ranging study using G6pc-/- mice. We showed that rAAV8-GPE-mediated gene transfer, deliberately titrated down to determine the minimum therapeutic dose, showed that restoring 3-128% of normal hepatic G6Pase-alpha activity and producing 61-90% of normal endogenous hepatic glucose in G6pc-/- mice, was sufficient to maintain glucose homeostasis. The treated mice displayed normal hepatic fat storage, normal blood metabolite and glucose tolerance profiles, reduced fasting blood insulin levels, and had no evidence of hepatic abnormalities or HCA. Fasting hypoglycemia is the hallmark of GSD-Ia. It is promising that the rAAV8-GPE-treated mice were able to sustain a 24-hour fast, which is a stress test of the ability of the liver to maintain blood normoglycemia through glycogenolysis and gluconeogenesis catalyzed by the G6Pase-alpha/G6PT complex in the absence of dietary glucose. This correlated with an increase in hepatic G6PT mRNA expression and a corresponding increase in microsomal G6P uptake activity, leading to the production of low but sufficient glucose to maintain interprandial glucose homeostasis. Our results suggest that G6Pase-alpha gene transfer may offer a therapeutic approach to the management of human GSD-Ia. The most efficacious gene therapy vectors for GSD-Ia reported to date are rAAV8-GPE and rAAV8-miGPE, the latter is a rAAV8 vector expressing human G6Pase-alpha directed by a shorter 382-bp minimal (m) GPE. 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 that the rAAV8-GPE vector directs significantly higher levels of hepatic G6Pase-alpha expression, achieves greater reduction in hepatic glycogen accumulation, and leads to a better tolerance of fasting, than the rAAV8-miGPE vector, suggesting that the rAAV8-GPE vector is the best vector to take forward into clinical trials. The underlying cause of GSD-Ib neutropenia is an enhanced neutrophil apoptosis but patients also manifest neutrophil dysfunction of unknown etiology. Previously, we showed G6PT interacts with the enzyme G6Pase-beta to regulate the availability of G6P/glucose in neutrophils. A deficiency in G6Pase-beta activity in neutrophils impairs both their energy homeostasis and functionality. We now show that G6PT-deficient neutrophils from human GSD-Ib patients are similarly impaired. Their energy impairment is characterized by decreased glucose uptake and reduced levels of intracellular G6P, lactate, ATP, and NADPH, while functional impairment is reflected in reduced neutrophil respiratory burst, chemotaxis, and calcium mobilization. We have also shown that the expression and membrane translocation of the NADPH oxidase subunit p47phox is downregulated in G6PT-deficient neutrophils, explaining why respiratory burst activity is impaired. Finally, we show that the hypoxia-inducible factor-1alpha (HIF-1alpha)/peroxisome proliferators-activated receptor-gamma (PPAR-gamma) pathway that directly impacts neutrophil respiratory burst, chemotaxis, and calcium mobilization is activated in G6PT-deficient neutrophils. Taken together, our results demonstrate that the underlying cause of neutrophil dysfunction in GSD-Ib arises from impaired neutrophil energy homeostasis and activation of the HIF-1alpha/PPAR-gamma pathway. The insight into the etiology of neutrophil dysfunction in GSD-Ib should facilitate the development of novel therapies for this disorder. G6PT belongs to the SLC37 family of sugar-phosphate exchangers that consists of four members, A1, A2, A3, and A4, which are anchored in the ER membrane. The best characterized family member is SLC37A4, better known as the G6PT. A deficiency in G6PT causes GSD-Ib. SLC37A1, SLC37A2, and G6PT function as phosphate (Pi)-linked G6P antiporters catalyzing G6P:Pi and Pi:Pi exchanges. The activity of SLC37A3 is unknown. The primary in vivo function of the G6PT protein is to translocate G6P from the cytoplasm into the ER lumen where it couples with either the liver/kidney/intestine-restricted G6Pase-α or the ubiquitously expressed G6Pase-β to hydrolyze G6P to glucose and Pi. The G6PT/G6Pase-α complex maintains interprandial glucose homeostasis and the G6PT/G6Pase-β complex maintains neutrophil energy homeostasis and functionality. G6PT is highly selective for G6P and is competitively inhibited by cholorogenic acid and its derivatives. Neither SLC37A1 nor SLC37A2 can couple functionally with G6Pase-α or G6Pase-β and the antiporter activities of SLC37A1 or SLC37A2 are not inhibited by cholorogenic acid. There are no known disease associations for SLC37A1, A2 or A3. Since only G6PT matches the characteristics of the physiological ER G6P transporter involved in blood glucose homeostasis and neutrophil energy metabolism, the biological roles for the other SLC37 proteins remain to be determined. In a long-term (70-90 weeks) study, we have shown that that rAAV-GPE-mediated gene therapy completely normalizes hepatic G6Pase-alpha deficiency in G6pc-/- mice. Interestingly, the old (age 70-90 weeks) rAAV-GPE-treated G6pc-/- mice expressing 3-63% of normal hepatic G6Pase-alpha activity (AAV-L/M mice) exhibit fasting blood insulin levels of young adult mice and a leaner phenotype, compared to their control littermates. Hepatic levels of endogenous glucose produced in the old AAV-L/M mice averaged 61-68% of those of control littermates, suggesting that AAV-L/M mice lived under chronically energy stress mimicking calorie restriction. We now show that the AAV-L/M mice are protected against age-induced obesity and insulin resistance, in contrast to the old wild type mice that develop age-related obesity and decrease in peripheral insulin sensitivity. Hepatic overexpression of the carbohydrate response element binding protein (ChREBP) is shown to improve glucose tolerance and insulin signaling. Several longevity factors in the calorie restriction pathway have been identified, including: the NADH shuttle systems; NAD+ concentrations that are decreased in both obese and aged; and the peroxisome proliferator-activated receptor-γ coactivator 1alpha (PGC-1alpha), a master of mitochondria biogenesis that is activated either by the AMP-activated protein kinase (AMPK) or sirtuin 1 (SIRT1). Here we show that the underlying mechanisms responsible for protecting the AAV-L/M mice against age-related insulin resistance and obesity correlate with activation of ChREBP signaling; increases in the expression of both malate-aspartate and glycerol-3-phosphate shuttle systems; increases in NAD+ concentrations; and activation of the AMPK/SIRT1/PGC-1alpha pathway in the livers of AAV-L/M mice. Taken together, the results demonstrate that reduced hepatic G6Pase-alpha expression exerts beneficial effects on insulin sensitivity