An Experimental Study Of Therapy For Type â…  Diabetes By Genetically Engineered K Cells

Background/Aim. Type I or insulin-dependent diabetes mellitus is resulted from autoimmune destruction of the pancreatic β cells. Due to severe deficiency of insulin, type I diabetic patients depend on insulin replacement therapy. However, chronic hyperglycemia due to failure to maintain proper glycemic control leads to microvascular, macrovascular and neurological complications. The functional restoration provided by allogeneic β-cell transplantation is limited by adverse effects of immunosuppression. A gene therapy based treatment of type I diabetes mellitus requires the development of a surrogate β cell that can synthesize and secrete functionally active insulin in response to physiologically relevant changes in ambient glucose levels. To pursue an insulin replacement therapy for type I diabetes mellutis based on autologous engineered K cells, a vector encoding glucose-responsive promoters driving human insulin gene was generated to provide a glucose-regulated secretion of insulin.Methods. Transient expression assays were performed using glucose-dependent insulinotropic polypeptide (GIP) promoter reporter plasmids (pGL3-Basic-GIP) constructed by inserting a 1160-base pair fragment of this promoter into luciferase reporter vector pGL3-Basic. Glucose could stimulate transcriptional activity of GIP promoter. A insulin expression vector pcDNA3-GIP-hIns was constructed by placing human insulin gene linked to 5'-regulatory region of GIP. A tumor-derived K-cell line STC-1 was transfected with the pGIP/ins encoding neomycin phosphotransferase, driven by the GIP promoter-only cells in which the GIP promoter was active survived genetic selection. Stable clones expressing pGIP/ins were isolated, which produced mature insulin. The regulating effect of glucose in culture medium on the expression...