LONG–TERM REVERSAL OF TYPE 1 DIABETES IN RATS AFTER IN VIVO DELIVERY OF THE HUMAN INSULIN GENE

Abstract

O197 Aims: Attempts to replace the β-cells lost in type 1 diabetes include pancreas and islet transplantation, derivation of β-cells from stem cells, and gene therapy. Despite these varied approaches, permanent reversal of type 1 diabetes is yet to be achieved. The aim of this study was to reverse streptozotocin (STZ)-induced diabetes in Wistar rats. Methods: The human proinsulin gene was cloned into a viral vector. Transduction efficiency was assessed by flow cytometry. We introduced the viral vector which expressed the insulin gene into male Wistar rats (250-300 g in body weight) via the portal circulation. Prior to vector delivery, diabetes was induced by the intraperitoneal injection of STZ (75 mg/ kg body weight). Following delivery of the insulin gene or empty vector, rats were monitored for changes in body weight and blood glucose levels. An intravenous glucose tolerance test was performed after a 14 h fast in untreated (n = 3), empty vector-transduced (n = 4) and insulin vector-trransduced (n=6) rats. At various times (0, 2, 5, 10, 15, 30, 60, 120 min) after the infusion of glucose (0.5 g /kg body weight), blood samples were collected from the tail vein and assayed for glucose and insulin. Two months after vector infusion, liver, pancreas, kidney, spleen and lung were harvested. Expression of insulin was determined by RT-PCR and immunohistochemistry. Results: The ideal amount of vector for successful delivery was 4 X 106 transduction units. Rats treated with the insulin-expressing vector, exhibited normalization of blood glucose levels within 48 h and normoglycaemia was maintained for over 11 months. Insulin was expressed only in the liver of insulin-transduced rats. Glucose tolerance tests revealed that insulin-transduced rats showed similar responses to normal rats. In contrast, diabetic rats that received the empty vector remained hyperglycaemic (blood glucose > 24mmol/L) and failed to increase body weight. Conclusions: This study shows that hepatic insulin gene therapy can induce the long-term reversal of diabetes. Importantly, hepatocytes transfected with transgenic insulin are sufficiently responsive to cure STZ–induced diabetes without inducing hypoglycemia.