This project proposes experiments to develop two independent strategies to block the pathogenic inflammatory events in the lesions of insulitis in an animal model (the NOD mouse) of insulin-dependent diabetes mellitus (IDDM). In NOD mice, IDDM has been demonstrated to be a T cell-mediated disease dependent upon the presence of CD4 (and CD8) T cells. Previous strategies aimed at removing or inactivating T cells did not specifically target T cells in the lesions of insulitis but targeted CD4 cells in general, thus leading to a potential therapeutic outcome of an acquired immune deficiency. The major goal of this project is to target CD4 cells in lesions of insulitis and inactivate these CD4 T cells by one of two independent therapeutic strategies. The first strategy is to use retroviral-mediated gene transfer to block signalling in CD4+ T cells in lesions of insulitis through the use of genes encoding dominant-negative NFkappaB or IkappaB. This model will be studied using CD4 cells bearing dominant-negative NFkappaB or IkappaB or mock transduced control T cells in co-adoptive transfer with CD8+ T cells from diabetic NOD mice into NOD- RAG-2 recipient mice. Then, we will apply targeted gene delivery to CD4+ OX-40+ T cells in lesions of insulitis using retroviral vectors similar to those described in Project I but engineered to produce dominant-negative NFkappaB or IkappaB which should incapacitate intracellular signalling and thus be incapable of generating proinflammatory cytokines, a prerequisite to the development of diabetes in NOD mice. The second therapeutic strategy is predicated on previous observations that one cytokine, TNFalpha, is predominantly involved in the early pathogenic lesions of insulitis in NOD mice. We will develop retroviral vectors containing a gene encoding a single chain anti-TNFalpha antibody which can be secreted by CD4 cells following a) transduction in vitro (of human and/or murine T cell clones) and b) targeted gene transfer to OX-40+ CD4+ cells in lesions of insulitis. Production of antibody in situ should lead to a locally effective target-specific amelioration of insulitis and IDDM.
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