Liver-directed gene therapy of diabetic rats using an HVJ-E vector containing EBV plasmids expressing insulin and GLUT 2 transporter.
ABSTRACT Insulin gene therapy in clinical medicine is currently hampered by the inability to regulate insulin secretion in a physiological manner, the inefficiency with which the gene is delivered, and the short duration of gene expression. To address these issues, we injected the liver of streptozotocin-induced diabetic rats with hemagglutinating virus of Japan-envelope (HVJ-E) vectors containing Epstein-Barr virus (EBV) plasmids encoding the genes for insulin and the GLUT 2 transporter. Efficient delivery of the genes was achieved with the HVJ-E vector, and the use of the EBV replicon vector led to prolonged hepatic gene expression. Blood glucose levels were normalized for at least 3 weeks as a result of the gene therapy. Cotransfection of GLUT 2 with insulin permitted the diabetic rats to regulate their blood glucose levels upon exogenous glucose loading in a physiologically appropriate manner and improved postprandial glucose levels. Moreover, cotransfection with insulin and GLUT 2 genes led to in vitro glucose-stimulated insulin secretion that involved the closure of K(ATP) channels. The present study represents a new way to efficiently deliver insulin gene in vivo that is regulated by ambient glucose level with prolonged gene expression. This may provide a basis to overcome limitations of insulin gene therapy in humans.
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ABSTRACT: Diabetes mellitus has long been targeted, as yet unsuccessfully, as being curable with gene therapy. The main hurdles have not only been vector-related toxicity but also the lack of physiological regulation of the expressed insulin. Recent advances in understanding the developmental biology of beta-cells and the transcriptional cascade that drives it have enabled both in vivo and ex vivo gene therapy combined with cell therapy to be used in animal models of diabetes with success. The associated developments in the stem cell biology and immunology have opened up further opportunities for gene therapy to be applied to target autoimmune diabetes.Gene Therapy 02/2005; 12(2):101-7. · 4.32 Impact Factor
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ABSTRACT: Previous studies demonstrating the efficacy of insulin gene therapy have mostly involved use of adenoviral vectors or naked DNA to deliver the insulin gene. However, this procedure may not guarantee long-term insulin production. To improve the performance, we prepared recombinant adeno-associated viral vectors (rAAV) harboring the gene encoding a furin-modified human insulin under the cytomegalovirus (CMV) promoter [rAAV-hPPI(F12)]. Streptozotocin (STZ)-induced diabetic Sprague-Dawley rats were used as a diabetic animal model. The levels of blood glucose, insulin, and HbA1c were measured to test the effect. An intraperitoneal glucose tolerance test was performed to test the capability of blood glucose disposal. Immunohistochemical staining and Northern blot analyses were performed to survey the expression pattern of the therapeutic insulin gene. STZ-induced diabetic Sprague-Dawley rats infused via the portal vein with rAAV-hPPI(F12) produced human insulin and after a 6-h fast were normoglycemic for over 90 days post-treatment, whereas diabetic rats treated with recombinant adenoviral vector harboring the hPPI(F12) gene [rAV-hPPI(F12)] were normoglycemic only for days 3 to 13 post-treatment. Insulin mRNA was detected mainly in the liver of the rAAV-hPPI(F12)-treated diabetic rats. The glucose tolerance capability of the rAAV-hPPI(F12)-treated diabetic rats was comparable to that of non-diabetic rats, even without injection of recombinant insulin. Furthermore, blood HbA1c concentrations in rAAV-hPPI(F12)-treated diabetic rats were reduced to almost the normal level. Importantly, studies of rAV or rAAV vector-dependent side effects on the targeted liver strongly suggested that only rAAV treatment caused no side effects. These results demonstrate that our rAAV-mediated in vivo insulin gene therapy provides safer maintenance of the insulin gene expression required for long-term and thus more effective blood glycemic control.The Journal of Gene Medicine 06/2005; 7(5):621-9. · 2.16 Impact Factor
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ABSTRACT: As part of our research into the liver-directed gene therapy of Type I diabetes, we have engineered a human hepatoma cell line (HEPG2ins/g cells) to store and secrete insulin to a glucose stimulus. The aim of the present study was to determine whether HEPG2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels (KATP). Using patch-clamp electrophysiology with symmetrical KCl solutions, the single-channel conductance of KATP was 61pS. KATP was inhibited by ATP (1 mM) or cAMP (50 microM) applied to the cytosolic side of the membrane. Single KATP channels and macroscopic whole-cell currents were inhibited by glucose (20 mM) and glibenclamide (20 microM) and were activated by diazoxide (150 microM). Immunoprecipitation and Western blot analysis confirmed the presence of Kir6.2 KATP channel subunit protein in HEPG2ins/g and HEPG2ins cells. Using radioimmunoassay techniques, we report that exposure of the cells to tolbutamide (100 microM) resulted in an increase in insulin secretion from 0.3 +/- 0.05 to 1.8 +/- 0.2 pmol insulin/10(6) cells and glibenclamide (20 microM) from 0.4 +/- 0.06 to 2.1 +/- 0.3 (n=4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide (150 microM) completely inhibited glucose-stimulated insulin release. Glucose 20 mM and glibenclamide 100 microM increased intracellular Ca2+ level in the HEPG2ins/g cells. However, glucose 20 mM did not stimulate a rise in intracellular Ca2+ in the un-transfected parent cell-line HEPG2. We used confocal microscopy to confirm that glucose (20 mM) stimulated the release of insulin from the fluorescently labeled secretion granules in the cells. Furthermore, glibenclamide (20 microM) also stimulated the release of insulin from fluorescently labeled secretion granules, and diazoxide (150 microM) blocked that stimulated release of insulin. Our results suggest that HEPG2ins/g cells respond to glucose via signaling pathways that depend on KATP, similar to a normal pancreatic beta cell.The FASEB Journal 10/2003; 17(12):1682-4. · 5.70 Impact Factor