Development of a liver-specific Tet-off AAV8 vector for improved safety of insulin gene therapy for diabetes

Background Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno-associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)-induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long-acting insulin therapy. Methods We have developed a single AAV8 vector with a Tet-Off regulatable system as a safety mechanism to turn off insulin secretion should hypoglycaemia develop in vector-treated diabetic mice. We first transfected HepG2 cells or freshly isolated rat hepatocytes in vitro with the Tet-Off system (pAAV-Tetoff(bidir)-Alb-luc) regulating a luciferase reporter gene. We subsequently incorporated a furin-cleavable codon-optimised human proinsulin cDNA into pAAV-Tetoff(bidir) backbone to form the doxycycline inducible pAAV-Tetoff(bidir)-Alb-hINSco. Results Using STZ-induced diabetic mice, we were able to switch off insulin secretion repeatedly with doxycycline administration, and showed full restoration of insulin secretion on withdrawing doxycycline. Conclusions The present study provides proof of concept that, under circumstances when inappropriate basal insulin secretion is a safety concern, insulin secretion from AAV8 gene therapy can be turned off reversibly with doxycycline.