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Third-party-funded project

Title:
Reduction of immunogenicity of human insulin producing pancreatic β-cells by specific suppression of autoantigenic glutamic acid decarboxylase (GAD) expression

Project management at the University of Würzburg:

Participating scientists:

Abstract:
The enzyme glutamic acid decarboxylase (GAD) represents an autoantigen, which plays a key role during the initial immunopathogenesis of diabetes mellitus type 1. Recent results suggest that GAD initiates the specific T-cell response leading to so called "insulitis" of pancreatic islets. Transplantation of pancreatic islets for treatment of type 1 diabetes is an experimental therapeutic procedure associated with immunological complications. In addition to HLA-dependent initiation of acute and chronic graft rejection, long-term transplant dysfunction is largely due to reintroduction of T-cell dependent insulitis within the graft that is dependent on GAD autoantigen exposure by the transplanted islets. Thus, experimental approaches seeking targeted suppression of GAD autoantigen presentation by pancreatic β-cells represent key steps in the development of strategies for immunoprotection of transplanted pancreatic islets. Based on this scientific background we are exploiting genetic engineering procedures for targeted suppression of GAD expression in pancreatic beta-cell lines and animal models in the currently funded project C-15. Using specific antisense oligonucleotides or transfection of antisense plasmid vectors targeted suppression of GAD expression in pancreatic β-cell lines was achieved. Ongoing experiments include the analysis of insulin biosynthesis and secretion, β-cell specific gene expression and immunogenicity of β-cell lines with suppressed GAD expression as outlined in the work plan of project C-15.
The current application is for a one year extension, during which the scientific findings in cell lines and animal models yielding from the presently funded IZKF-project C-15 will be transferred to human pancreatic β-cells. Major aim of the project during the extension period will be the targeted suppression of GAD autoantigen expression in primary human pancreatic islet β-cells. For this approach a novel gene transfer strategy based upon enzyme activated expression vectors will be exploited. We propose to investigate two specific aims:
a) Development of gene transfer strategies based on enzyme activated targeting vectors for specific suppression of GAD expression in primary human pancreatic β-cells. Enzyme activated vectors will be constructed that will allow tetracycline-inducible and β-cell specific expression of GAD-65 and GAD-67 antisense mRNA under the control of the human insulin promoter. Enzyme activation of targeting vectors yields highly reactive DNA-nucleoprotein intermediates bearing extensive recombination potential. From this strategy gene transfer into primary human pancreatic β-cells with high efficiency can be expected that aims at inducible and β-cell specific suppression of endogenous GAD expression in primary human pancreatic islets ("GAD-less islets").
b) Functional and immunological characterization of human pancreatic islet β-cells with suppressed GAD expression. Glucose-dependent insulin secretion from human pancreatic islets with suppressed GAD expression will be investigated. In collaboration with the Department of Virology and Immunobiology (Prof. Hünig) the autoantigenic potential of GAD-suppressed human pancreatic islets will be tested in co-culture experiments exploiting autoreactive T-cell clones.
The proposed strategies will yield important insights into the immunopathogenesis of diabetes mellitus type 1, which may be transferred to clinical pancreatic islet transplantation.
The proposed extension period project integrates into main focus no. I (Misdirected Immune Reactions), specifically project area C (Autoimmunity) of the IZKF Würzburg and receives major input from interdisciplinary collaboration with other groups within this focus.

Key words:
    diabetes mellitus type 1
    autoimmunity
    GAD
    pancreas
    beta-cell
    islet

Projekt period: from 07.2000 to 06.2003

Funding institution:
Landeshaushalt Wissenschaftsministerium
Bund

Publications:

Links:
Department Homepage
IZKF-Wuerzburg, Project C-15