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

Abstract

Diabetes is a common disorder worldwide, and exhaustive efforts have been made to cure this disease. Gene therapy has been considered as a potential curative method that has had more stability in comparison with other pharmaceutical methods. However, the application of gene therapy as a definitive treatment demands further investigation. This study is aimed to prepare a suitable high- performance vector for gene therapy in diabetes mellitus. The designed vector has had prominent characteristics, such as directed replacement, which makes it a suitable method for treating or preventing other genetic disorders. The whole rDNA sequence of the human genome was scanned. The 800 bp two homology arms were digested by EcoRI, 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 fragments were purified and transfected into Hela cell and then cultured. Afterward, the 300 µg/mL of glucose were added to the culture medium. Insulin expression in the transfected cells with 200 and 400 ng of the construct in comparison with negative control was detected using western blot and ELISA methods. Results have shown insulin expression in different glucose concentrates.