An Experimental Study Of Therapy For Type Ⅰ Diabetes By Recombinant Adenovirus Harboring The GIP-Human Insulin Gene

Background/Aim. In recent years, the worldwide prevalence of diabetes mellitus has risen dramatically. Diabetes has become the third important chronic noninfectious diseases after malignant tumors, cerebrovascular and cardiovascular diseases in China. However, all the treatment choices have the certain insufficiency and the limitation. With the rapid development of molecular biology technique, gene therapy has become a new strategy in treating diabetes. Insulin gene therapy is divided into reproductive cell gene therapy and somatic cell gene therapy, the later uses one of two gene delivery methods: ex vivo and in vivo. Reproductive cell gene therapy is only used for experiments because of the bar of technique and ethnics, which can not be used for human. Ex vivo gene therapy has been used to expand cell lines transduced with a therapeutic gene in vitro for transplantation. When reimplanted back in the animals, cells will form transplantation tumor, which is the limitation in the clinical management of diabetes. In vivo gene therapy is the optimal delivery method. Now, it is not difficult to introduce an insulin-producing gene into none β cells and process proinsulin to mature insulin in the transfected cells using an effective insulin gene transfer system. The most challenging part of insulin gene therapy is to confer glucose responsiveness to expression of the insulin transgene. On the other side, the development of safe and effective vectors for in vivo gene therapy is demanding. To date, there are two kinds of vector systems including nonviral or viral vectors. Nonviral methods are considered safe, cost-effective, simple to use and do not induce an immune response, but generally have a lower gene transfer efficacy. However, the modified viral vectors have a higher gene transfer efficacy, is most widely used for insulin gene delivery. Recombinant adenoviruses vector has some advantages including transducing nondividing cells with high efficiency, a relatively high titer of virus produced, and the transferred genes not integrated into the host genome. It has been used more and more for gene delivery because of the advantages. To pursue an insulin replacement therapy for type I diabetes mellutis based on autologous engineered K cells, a recombinant adenoviruses vector encoding glucose-responsive promoters driving human insulin gene was constructed to provide a glucose-regulatedsecretion of insulin.Methods. A insulin expression non-replicating adenoviral vector (Ad.GIP-hIns) was constructed by placing human insulin gene linked to 5'-regulatory region of GIP. Transduction of STC-1 cells was performed using a multiplicity of infection (MOI) of different infectious units per cell to detect the viral ability of infection. The regulating effect of glucose and cell-specificity of GIP promoter in culture medium on the expression of insulin gene was estimated at 100 MOI by radioimmunoassay. The insulin mRNA was confirmed by RT-PCR. Ad.GIP-hlns transduced STC-1 cells were examined by immunochemical staining and double immunofiuorescence for GIP and insulin expression. Administration of adenoviral vectors to streptozotocin-induced diabetic rats was carried out by injection through superior mesenteric artery. Glucose levels, body weight and insulin levels of treated rats were assayed.Results. Transfection of STC-1 cells with Ad.GIP-hlns resulted in a correctly processed human preproinsulin mRNA transcript. At 100 MOI, the highest insulin was produced and secreted into media. When the adenoviral vector was transfected into different cell lines, human preproinsulin mRNA was detectable in STC-1 cells. Data shows a typical result of insulin expression in cells 3 days after transduction and continue until 7 days. Glucose had no statistically significant effect on insulin secretion. Insulin expression was confirmed by RT-PCR and immunochemical staining. The recombinant advenoviruses were injected into diabetic rats through superior mesenteric artery and transduced intestinal K cells sucessfully, the blood glucose levels of diabetic rats were reduced to normal. Intraperitoneal glucose tolerance tests (IPGTTs) showed the characteristic decline in blood glucose of normal rats after glucose administration. In contrast, diabetic rats received control implants showed high levels of glucose and did not show significant clearances of blood glucose.Conclusion. Our data suggest that the recombinant advenovirus Ad.GIP-hlns can transduce K cells in vitro or in vivo, and genetically engineered K cells secrete mature insulin. The blood glucose levels of diabetic rats were reduced nearly to normal in period of time. These results lend support to the study of gene therapy in vivo in diabetes mellitus.