Improved methods of insulin delivery are required for the treatment of insulin-dependent diabetes mellitus (IDDM) to achieve a more physiological profile of glucose homeostasis. Somatic cell gene therapy offers the prospect that insulin could be delivered by an autologous cell implant, engineered to secrete insulin in response to glucose.
This study explores the feasibility of manipulating somatic cells to behave as a surrogate insulin-secreting β-cells.
Initial studies were conducted using mouse pituitary AtT20 cells as a model, since these cells possess an endogenous complement of enzymes capable of processing proinsulin to mature insulin.
Glucose sensitive insulin secretion was conferred to these cells by transfection with plasmids containing the human preproinsulin gene (hppI-1) and the GLUT2 gene for the glucose transporter isoform 2. Insulin secretion was responsive to changes in the glucose concentration up to about 50μM. Further studies to up-rate this glucose sensitivity into the mM range will require manipulation of the hexokinase and glucokinase enzymes.
Intraperitoneal implantation of the manipulated AtT20 cells into athymic nude mice with streptozotocin-induced diabetes resulted in decreased plasma glucose concentrations. The cells formed vascularised tumours in vivo which were shown to contain insulin-secreting cells.
To achieve proinsulin processing in non-endocrine cells, co-transfection with a suitable enzyme, or mutagenesis of the proinsulin itself are necessary. The mutation of the human preproinsulin gene to the consensus sequence for cleavage by the subtilisin-like serine protease, furin, was carried out. Co-transfection of human fibroblasts with wild-type proinsulin and furin resulted in 58% conversion to mature insulin by these cells.
Intraperitoneal implantation of the mature-insulin secreting human fibroblasts into the diabetic nude mouse animal model gave less encouraging results than the AtT20 cells, apparently due to poor vascularisation. Cell aggregations removed from the mice at autopsy were shown to contain insulin secreting cells only at the periphery.
This thesis provides evidence that it is possible to construct, by cellular engineering, a glucose-sensitive insulin-secreting surrogate β-cell. Therefore, somatic cell gene therapy offers a feasible alternative for insulin delivery in IDDM patients.
|Date of Award||Sept 1996|
|Supervisor||Clifford Bailey (Supervisor) & K. Docherty (Supervisor)|
- insulin secretion
- proinsulin processing
- human fibroblasts
- |AtT20 cells