Insulin-releasing pituitary cells as a model for somatic cell gene therapy in diabetes mellitus

Abstract

Insulin delivery by somatic cell gene therapy was evaluated using murine pituitary AtT20MtIns-1.4 cells. These cells have been stably transfected to release human insulin by the introduction of a recombinant plasmid bearing a human preproinsulin cDNA under the control of a zinc-sensitive metallothionein promoter. 6 x 10(7) AtT20MtIns-1.4 cells were implanted subcutaneously into streptozotocin-diabetic mice immunosuppressed with cyclosporin A. Release of human insulin was assessed using a specific plasma human C-peptide assay. On days 1 and 2 after implantation human C-peptide concentrations were about 0.02 pmol/ml. Consumption of zinc sulphate solution (500 mg/l) as drinking fluid for days 3-5 increased plasma human C-peptide concentrations to 0.11 +/- 0.01 pmol/ml (mean +/- S.E.M.), n = 11, P < 0.01, and concentrations declined when zinc was discontinued. The extent of hyperglycaemia was slightly lower (P < 0.05) than in a group implanted with non-transfected AtT20 cells. The study was terminated after 9 days, and tumour-like aggregations of implanted cells were identified at autopsy. These comprised a large necrotic core with insulin-containing cells at the periphery. The study provides support for the view that somatic cell gene therapy offers a potential approach to insulin delivery in diabetes mellitus.

Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells

“…However, these cell lines are abnormal or tumorgenic and may not be used directly for diabetes treatment. 24 Hepatocytes are non-neuroendocrine cells that naturally express Glut2 and GK, and can be successfully transduced in vitro and in vivo. Therefore, they represent a cell type potentially capable of glucose-regulated insulin expression.…”

Diabetes reversal via gene transfer: building on successes in animal models

“…68 Pituitary cells possess both proinsulin-processing enzymes and secretory granules, and have been modified to produce insulin via the transfer of a recombinant plasmid containing human preproinsulin cDNA. 15 Although these cells produced biologically active insulin, they lacked glucose responsiveness and therefore required further genetic modification via the transduction of GLUT2 and GK. Following the additional modifications, the modified cells became glucose-responsive, albeit at subphysiological levels.…”

“…13,14 In addition, gene transfer of pancreatic transcription factors and insulin for the production of "artificial" β-cells that are capable of synthesizing and secreting insulin in response to metabolic signals is a promising alternative. Two common methods of generating surrogate β-cells are the dedifferentiation and directed transdifferentiation of autologous or allogeneic cells ex vivo [15][16][17] followed by transplantation, and the in vivo [18][19][20] transdifferentiation of target tissue via gene transfer of transcription factors or insulin within viral vectors. Animal models have been used in T1D research since the discovery of insulin by Banting and Best in 1922 21 for studying the pathogenesis of the disease and its complications and for the discovery of new treatments.…”

Approaches for the Cure of Type 1 Diabetes by Cellular and Gene Therapy

“…A mouse corticotrophic cell line derived from the anterior pituitary, AtT20, was transfected with the insulin gene and expressed active insulin, but lacked glucose responsiveness [Moore et al, 1983;Stewart et al, 1994;Lipes et al, 1996]. Co-transfection with genes encoding the glucose transporter, Glut2, and glucokinase resulted in glucoseresponsive insulin secretion [Hughes et al, 1992;Hughes et al, 1993].…”