Gene therapy using a novel G6PC-S298C variant enhances the long-term efficacy for treating glycogen storage disease type Ia.

The current phase I/II clinical trial for human glycogen storage disease type-Ia (GSD-Ia) (NCT03517085) uses a recombinant adeno-associated virus (rAAV) vector expressing a codon-optimized human glucose-6-phosphatase-α (G6Pase-α or G6PC). DNA sequence changes introduced by codon-optimization can negatively impact gene expression. We therefore generated a novel variant in which a single amino acid change, S298C, is introduced into the native human G6PC sequence. Short term gene transfer study in G6pc-/- mice showed that the rAAV-G6PC-S298C vector is 3-fold more efficacious than the native rAAV-G6PC vector. We have shown previously that restoring 3% of normal hepatic G6Pase-α activity in G6pc-/- mice prevents hepatocellular adenoma/carcinoma (HCA/HCC) development and that mice harboring <3% of normal hepatic G6Pase-α activity are at risk of tumor development. We have also shown that G6Pase-α deficiency leads to hepatic autophagy impairment that can contribute to hepatocarcinogenesis. We now undertake a long-term (66-week) preclinical characterization of the rAAV-G6PC-S298C vector in GSD-Ia gene therapy. We show that the increased efficacy of rAAV-G6PC-S298C has enabled the G6pc-/- mice treated with a lower dose of this vector to survive long-term. We further show that mice expressing ≥3% of normal hepatic G6Pase-α activity do not develop hepatic tumors or autophagy impairment but mice expressing <3% of normal hepatic G6Pase-α activity display impaired hepatic autophagy with one developing HCA/HCC nodules. Our study shows that the rAAV-G6PC-S298C vector provides equal or greater efficacy to the codon optimization approach, offering a valuable alternative vector for clinical translation in human GSD-Ia.

Liver-directed gene therapy for murine glycogen storage disease type Ib.

Glycogen storage disease type-Ib (GSD-Ib), deficient in the glucose-6-phosphate transporter (G6PT), is characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenoma (HCA). We examined the efficacy of G6PT gene therapy in G6pt-/- mice using recombinant adeno-associated virus (rAAV) vectors, directed by either the G6PC or the G6PT promoter/enhancer. Both vectors corrected hepatic G6PT deficiency in murine GSD-Ib but the G6PC promoter/enhancer was more efficacious. Over a 78-week study, using dose titration of the rAAV vectors, we showed that G6pt-/- mice expressing 3-62% of normal hepatic G6PT activity exhibited a normalized liver phenotype. Two of the 12 mice expressing < 6% of normal hepatic G6PT activity developed HCA. All treated mice were leaner and more sensitive to insulin than wild-type mice. Mice expressing 3-22% of normal hepatic G6PT activity exhibited higher insulin sensitivity than mice expressing 44-62%. The levels of insulin sensitivity correlated with the magnitudes of hepatic carbohydrate response element binding protein signaling activation. In summary, we established the threshold of hepatic G6PT activity required to prevent tumor formation and showed that mice expressing 3-62% of normal hepatic G6PT activity maintained glucose homeostasis and were protected against age-related obesity and insulin resistance.

Downregulation of SIRT1 signaling underlies hepatic autophagy impairment in glycogen storage disease type Ia.

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 ß-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.

Rapamycin treatment of healthy pigs subjected to acute myocardial ischemia-reperfusion injury attenuates cardiac functions and increases myocardial necrosis.

BACKGROUND: The mammalian target of rapamycin (mTOR) pathway is a major regulator of cell immunity and metabolism. mTOR is a well-known suppressor of tissue rejection in organ transplantation. However, it has other nonimmune functions: in the cardiovascular system, it is a regulator of heart hypertrophy and locally, in coated vascular stents, it inhibits vascular wall cell growth and hence neointimal formation/restenosis. Because the mTOR pathway plays major roles in normal cell growth, metabolism, and survival, we hypothesized that inhibiting it with rapamycin before an acute myocardial ischemia-reperfusion injury (IRI) would confer cardioprotection by virtue of slowing down cardiac function and metabolism. METHODS: Yorkshire pigs received either placebo or 4 mg/d rapamycin orally for 7 days before the IRI. All animals underwent median sternotomy, and the mid-left anterior descending coronary artery was occluded for 60 minutes followed by 120 minutes of reperfusion. Left ventricular pressure-volume data were collected throughout the operation. The ischemic and infarcted areas were determined by monastral blue and triphenyltetrazolium chloride staining, respectively, and plasma cardiac troponin I concentration. mTOR kinase activities were monitored in remote cardiac tissue by Western blotting with specific antibodies against mTOR substrates phosphorylating sites. RESULTS: Rapamycin before treatment impaired endothelial-dependent vasorelaxation, attenuated cardiac function during IRI, and increased myocardial necrosis. Western blotting confirmed effective inhibition of myocardial mTOR kinase activities. CONCLUSIONS: Acute myocardial IRI, in healthy pigs treated with rapamycin, is associated with decreased cardiac function and higher myocardial necrosis.

Optical sensors based on III-nitride photodetectors for flame sensing and combustion monitoring.

Combustion control requires visible photodetectors to sense the CH* CL emission at 430 nm that combined with a visible-blind UV photodetector allows us to obtain the OH*/CH* ratio. UV-visible P-InGaN/GaN multiple quantum well-N photodiodes with 15-18 mm2 areas were fabricated to conduct OH* (308 nm) and CH* CL detection without external filters. Bandpass detectors at 230-390 nm and 360-450 nm presented linear responses over five decades and rejection ratios >10(3) at 430 and 308 nm, respectively. A full optical sensor system was built and detectors operated at 120 degrees C in a combustion chamber, showing linear responses within the dynamic range, maximum signal-to-noise ratios of 103 and response times of <1 s. An exponential association dependence between the optical OH*/CH* CL signals and the gas/air ratios was found.