Genetically engineered insulin mediated by glucose transporter-2 (GLUT2) promoter for the biosynthesis of insulin in rat hepatocytes

Abstract

The expression of insulin in hepatocytes by hepatic gene therapy is a promising treatment for diabetes. The conversion of immature proinsulin to mature insulin occurs only in cells that contain the enzymes responsible for the cleavage of proinsulin to insulin. For successful hepatic insulin gene therapy, insulin production must be tightly coupled to glucose concentration. Hepatocytes are excellent target cells for insulin gene therapy since, they are similar to pancreatic beta cells, they have the ability to rapidly adapt to blood glucose concentrations as they possess glucose-sensing components, such as Glucose Transporter-2 (GLUT2). So, we engineered rat proinsulin with the sites of cleavage (Furin Cleavable Sites) using site-directed mutagenesis for the removal of C-peptide to form the two chains A and B for mature insulin production. This engineered proinsulin was constructed into a non-viral expressing vector and regulated by glucose transporter-2 promoter (GLUT2 promoter) to control the amount of mature insulin expressed and to modulate the amount of glucose found in hepatocytes. The mature, active, and regulated expressed insulin was secreted according to the amount of glucose-regulated by the glucose transporter-2 (GLUT2) promoter.