Designing an Engineered Construct Gene Sensitive to Carbohydrate In-vitro and Candidate for Human Insulin Gene Therapy In-vivo

Diabetes is a common disorder worldwide, and exhaustive efforts have been made to cure this disease. Gene therapy has considered as a potential curative method that has more stability in comparison with the other pharmaceutical methods. However, the application of gene therapy as a definitive treatment demands further investigation. This study aim is to prepare a suitable high- performance vector for gene therapy in diabetes mellitus. The designed vector has prominent characteristics, such as directed replacement, which makes it a suitable method for treating or preventing other genetic disorders. Whole rDNA sequence of the human genome was scanned. The 800 bp two homology arms were digested by EcoRI and synthesized and cloned into the pGEM-B1 plasmid (prokaryotic moiety). The carbohydrate sensitive promoter, L-pyruvate kinase, and insulin gene were sub-cloned between homologous arms (eukaryotic moiety). The PGEM-B1 plasmid was digested by EcoRI, and the eukaryotic fragment was purified and transfected into Hela cell and cultured. The 300 µg/ ml of glucose was added to the culture medium. Insulin expression in transfected cells with 200 and 400 ng of the construct, in compare with negative control was detected using western blot and ELISA. Results demonstrated that insulin was expressed toward glucose concentrates.