416. Glucose-Dependent Insulin Production by Liver-Specific, Glucose-Regulatable Synthetic Promoters Results in the Cure of Diabetes

Abstract

Top of pageAbstract Type 1 diabetes is caused by insulin deficiency. Insulin gene therapy has been attempted to cure diabetes; however, there is no satisfactory system that results in the tight regulation of insulin production in response to physiological changes in glucose levels. To develop a liver-specific, glucose-responsive synthetic promoter that can tightly regulate insulin production in response to blood glucose levels, we employed 3 kinds of transcription factor-binding cis-elements: hepatocyte nuclear factor-1, CAAT/enhancer binding protein response element, and glucose-response element. We generated a library of synthetic promoters that contained 9 of these cis-elements in random combinations and screened each promoter for transcriptional activity. Among 821 promoters tested, we selected 3 with the highest transcriptional activity for further testing. To determine the glucose responsiveness of these promoters in vivo, we generated recombinant adenoviruses expressing furin-cleavable rat insulin under the control of selected synthetic promoters (rAd-SP-rINSfur) and the cytomegalovirus promoter (rAd-CMV-rINSfur) as a control for ubiquitous strong activity. rAd-SP-rINSfur (1010 viral particles) or rAd-CMV-rINSfur (109 viral particles) was administered into streptozotocin-induced diabetic NOD.scid mice (n=6/group) via the tail vein, and blood glucose levels were examined. We found that diabetic mice treated with rAd-SP-rINSfur became normoglycemic a few days after treatment, and normoglycemia was maintained for more than 1 month, whereas mice treated with rAd-CMV-rINSfur were dead within 10 days due to uncontrolled constitutive expression of the insulin gene. Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice produced insulin in response to glucose and cleared exogenous glucose from the blood in a manner similar to non-diabetic control mice. Insulin expression in rAd-SP-rINSfur-treated mice was observed only in the liver, not in other tissues or organs, whereas insulin expression in rAd-CMV-rINSfur-treated mice was present in all tissues tested, indicating that the synthetic promoters were liver-specific. On the basis of these observations, we conclude that our newly constructed, liver-specific synthetic promoters can tightly regulate insulin production in response to glucose and may have therapeutic potential for the cure of diabetes in humans by liver-directed insulin gene therapy.