Reversal of Diabetes in Mice by Implantation of Human Fibroblasts Genetically Engineered to Release Mature Human Insulin

Abstract

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Regulated hepatic insulin gene therapy of STZ-diabetic rats

“…Functional insulin genes can be transferred to multiple tissues, 4,7,9 and the capacity of non-␤-cells to secrete biologically active transgenic insulin in sufficient quantities to affect metabolism is well established. 4,[7][8][9]13 However, an inability to coordinate transgenic insulin secretion with fluctuating demands for insulin action has limited the efficacy of insulin gene therapy models in vivo. Recently, we demonstrated the utility of combining a metabolically responsive promoter with a modified insulin expression sequence to create a transcriptionally regulated system that couples human insulin production to glucose exposure in rat hepatocytes.…”

“…Multiple investigators have demonstrated functional insulin gene transfer both in vitro and in vivo. [2][3][4] However, attempts to regulate transgenic insulin production have proven inadequate. 5,6 Consequently, secretion of transgenic insulin has been either insufficient to normalize blood glucose, 4,[6][7][8][9] or has produced lethal hypoglycemia.…”

“…[2][3][4] However, attempts to regulate transgenic insulin production have proven inadequate. 5,6 Consequently, secretion of transgenic insulin has been either insufficient to normalize blood glucose, 4,[6][7][8][9] or has produced lethal hypoglycemia. 3,4,7 We have designed a system of insulin gene therapy that utilizes transcription to regulate hepatic production of transgenic insulin.…”

“…5,6 Consequently, secretion of transgenic insulin has been either insufficient to normalize blood glucose, 4,[6][7][8][9] or has produced lethal hypoglycemia. 3,4,7 We have designed a system of insulin gene therapy that utilizes transcription to regulate hepatic production of transgenic insulin. 10 Transgene expression is regulated by a promoter composed of three copies of a stimulatory glucose responsive element (GlRE) from the rat liver pyruvate kinase (rL-PK) gene inserted directly upstream of an inhibitory insulin response element of the insulin-treated animals (n = 8) remained elevated, and animals failed to gain weight (n = 4), or died (n = 4).…”

Engineering physiologically regulated insulin secretion in non-β cells by expressing glucagon-like peptide 1 receptor

“…Furthermore, these cells secrete insulin via the constitutive secretory pathway and thus will secrete some insulin regardless of the extracellular glucose concentrations. [38][39][40] Insulin secretion independent of the glucose concentration is a serious limitation of such approaches with non-b cells. Thus, most non-b cells must be engineered to not only sense the extracellular glucose concentration, but also to express a large number of proteins required for regulated secretion via secretory vesicles.…”