Tausif Alam1, Hans W Sollinger. 1. Division of Transplantation, Department of Surgery, University of Wisconsin, Madison, WI 53792, USA. alam@surgery.wisc.edu.
Abstract
BACKGROUND: A gene-therapy-based treatment of type 1 diabetes mellitus requires the development of a surrogate beta cell that can synthesize and secrete functionally active insulin in response to physiologically relevant changes in ambient glucose levels. Failure to duplicate the storage-granule-based mechanism of insulin secretion of beta cells, however, has made it difficult to develop a surrogate beta cell. The authors' strategy for achieving glucose-dependent insulin secretion relies on glucose-responsive transcription of insulin mRNA and the constitutive secretory pathway of liver cells. METHODS: Insulin gene constructs containing three S14-based glucose-inducible regulatory elements, the liver-specific albumin promoter, and the human insulin cDNA modified for furin cleavage compatibility, were prepared and evaluated for biologic function in hepatocytes, in vitro, and in diabetic rats in vivo. RESULTS: The authors' insulin gene constructs induced insulin expression in hepatocytes. All detectable insulin produced by transduced hepatocytes in vitro was secreted, and the amount was dependent on the concentration of glucose and the duration of glucose stimulation. Analysis of in vivo functional efficacy of insulin gene therapy in streptozotocin-treated diabetic rats revealed the following: (1) fasting blood glucose levels were reduced to normal; (2) blood glucose levels of rats fed ad libitum were significantly reduced; and (3) peak blood glucose levels during oral glucose tolerance tests were significantly reduced. CONCLUSIONS: These studies demonstrate in vivo glucose-regulated insulin secretion from an autologous non-beta cell leading to fasting euglycemia and an improved glucose tolerance, thereby supporting the feasibility of hepatocyte-based insulin gene-therapy for treatment of type 1 diabetes mellitus.
BACKGROUND: A gene-therapy-based treatment of type 1 diabetes mellitus requires the development of a surrogate beta cell that can synthesize and secrete functionally active insulin in response to physiologically relevant changes in ambient glucose levels. Failure to duplicate the storage-granule-based mechanism of insulin secretion of beta cells, however, has made it difficult to develop a surrogate beta cell. The authors' strategy for achieving glucose-dependent insulin secretion relies on glucose-responsive transcription of insulin mRNA and the constitutive secretory pathway of liver cells. METHODS:Insulin gene constructs containing three S14-based glucose-inducible regulatory elements, the liver-specific albumin promoter, and the humaninsulin cDNA modified for furin cleavage compatibility, were prepared and evaluated for biologic function in hepatocytes, in vitro, and in diabeticrats in vivo. RESULTS: The authors' insulin gene constructs induced insulin expression in hepatocytes. All detectable insulin produced by transduced hepatocytes in vitro was secreted, and the amount was dependent on the concentration of glucose and the duration of glucose stimulation. Analysis of in vivo functional efficacy of insulin gene therapy in streptozotocin-treated diabeticrats revealed the following: (1) fasting blood glucose levels were reduced to normal; (2) blood glucose levels of rats fed ad libitum were significantly reduced; and (3) peak blood glucose levels during oral glucose tolerance tests were significantly reduced. CONCLUSIONS: These studies demonstrate in vivo glucose-regulated insulin secretion from an autologous non-beta cell leading to fasting euglycemia and an improved glucose tolerance, thereby supporting the feasibility of hepatocyte-based insulin gene-therapy for treatment of type 1 diabetes mellitus.
Authors: S Goverdhana; M Puntel; W Xiong; J M Zirger; C Barcia; J F Curtin; E B Soffer; S Mondkar; G D King; J Hu; S A Sciascia; M Candolfi; D S Greengold; P R Lowenstein; M G Castro Journal: Mol Ther Date: 2005-08 Impact factor: 11.454