Regulated secretion of proinsulin/insulin from human hepatoma cells transduced by recombinant adeno‐associated virus

Abstract

To employ hepatocytes as surrogate beta-cells for gene therapy of diabetes, a regulatory system was devised in this study by placing the human insulin cDNA under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, followed by the cytomegalovirus immediate early promoter-driven enhanced-green-fluorescent-protein open reading frame. The expression cassette was inserted into the adeno-associated virus vector between two inverted terminal repeats, and used to produce recombinant adeno-associated virus (rAAV). HepG2 human hepatoma cells were transduced by rAAV at the desired multiplicity of infection, followed by treatment with various concentrations of retinoic acid, dexamethasone, dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX). The cell-culture media were collected at 8, 16 and 24 h later. Proinsulin/insulin levels were determined with human proinsulin/insulin radioimmunoassay kits. Transduction of HepG2 cells by rAAV showed that green fluorescence was produced as early as 12 h after rAAV infection. Flow-cytometrical analysis demonstrated that transduction efficiency increased with the numbers of transducing rAAV particles used. The transduced hepatocytes were shown to secrete immunoreactive proinsulin/insulin, which were stimulated by the concentrations of retinoic acid, dexamethasone and dbcAMP in the culture medium. High conversion from proinsulin into insulin occurred when these cells were treated with dexamethasone and dbcAMP. The presence of IBMX enhanced the secretion of proinsulin/insulin from the dbcAMP-treated cells. We conclude that rAAV is a promising vector for gene therapy of diabetes. Regulated secretion of proinsulin/insulin can be obtained in the rAAV-transduced HepG2 cells conferred with the PEPCK promoter via rAAV-mediated gene transfer.

Gene- and cell-based therapeutics for type I diabetes mellitus

“…Injection of animals with a number of vectors like adenovirus [124][125][126][127][128][129] and adeno-associated virus [130][131][132][133] encoding proinsulin under the control of a number of promoters including CMV, insulin, phosphoenolpyruvate carboxykinase (PEPCK) and L-pyruvate kinase (LPK) has resulted in correction of hyperglycemia. In many instances, however, the effect appears to have been transient.…”

Incorporation of calcium phosphate enhances recombinant adeno‐associated virus‐mediated gene therapy in diabetic mice

“…The PEPCK promoter can be up-regulated by cyclic adenosine monophosphate (cAMP), glucagon, and down-regulated by insulin [32][33][34]. Regulated production of proinsulin/insulin has been demonstrated in rAAV-transduced human hepatoma cells [19]. Due to low yield and labor-intensive procedures of rAAV production which often limits the in vivo studies using this virus vector, improvement of rAAV-mediated gene transfer is thus desired.…”

“…Sustained expression of transgenes using rAAV has been illustrated in several studies [16,17]. This vector is presently being developed as a potential vector for gene therapy of diabetes mellitus [18][19][20].…”

New Advanced Technology to Improve Prediction & Prevention of Type 1 Diabetes

“…Injection of animals with a number of vectors like adenovirus [124][125][126][127][128][129] and adeno-associated virus [130][131][132][133] encoding proinsulin under the control of a number of promoters including CMV, insulin, phosphoenolpyruvate carboxykinase (PEPCK) and L-pyruvate kinase (LPK) has resulted in correction of hyperglycemia. In many instances, however, the effect appears to have been transient.…”

“…Additionally, in another transgenic mouse model deficient in GLUT-4, in which GLUT-4 was expressed under the control of the myosin light-chain promoter, the uptake of a synthetic glucose analogue and the actions of insulin were enhanced and restored to normal levels in vivo (132). In a heterozygous GLUT-4 knock-out mouse model, complementation of the gene in skeletal muscle was able to restore insulin sensitivity and prevented a diabetic phenotype (133).…”