Glucose-Modulated Transgene Expression Via Recombinant Adeno-Associated Virus

Purpose. The objective of this study was to examine glucose- modulated reporter gene expression via recombinant adeno- associated viral vectors both in vitro and in vivo. Methods. Huh7 human hepatoma cells were transduced by recombinant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluorescence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin secretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection. Results. At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post-transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to millimolars of glucose. The addition of phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous insulin up to 10(-)7 M inhibited luciferase gene expression in rAAV-transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin- induced diabetic animals. Conclusion. rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene expression in rAAV -transduced hepatoma cells, suggesting that conditions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells conferred with insulin gene promoter. Results obtained from in vivo experiments demonstrated that glucose modulated transgene expression can be obtained in rAAV- treated diabetic C57BL/6J mice