ENGINEERING OF XENOGENEIC ISLETS WITH ANTI-APOPTIC GENES/CYTOKINE INHIBITORS

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Transplantation of islets from allogeneic or xenogeneic sources for the treatment of type I insulin dependent diabetes mellitus has encountered several problems including: primary non-function, rejection and recurrence of (autoimmune) disease. Based on available information, it appears that these problems are related in large part to apoptosis or programmed cell death of the Beta-cell as well as to the induction in Beta-cells of a series of genes that are NF-kB-dependent. Primary non- function, while poorly understood with respect to the underlying causes, involves apoptosis of Beta-cells. Apoptosis of the Beta-cell in this context, is probably caused by several factors including TNF and IL-1, but also stimulated by nitric oxide (NO) produced following induction in the Beta-cells of nitric oxide synthase, an NF-kB-dependent gene. Rejection of xenogeneic islets involves a cell mediated and most likely a humoral response; it also involves a non-specific inflammatory reaction supported, for instance, by the induced expression on the Beta- cells of adhesion molecules, the induction of which is NF-kB-dependent. Recurrence of disease involves IL-1, TNF and likely other factors; however, the end result is Beta-cell loss mainly by apoptosis. We propose to use recombinant adenoviruses to express in the Beta-cell one or more genes (A20, bcl-2 and/or bcl-xl) that were initially described based on their anti-apoptotic properties, but that we have shown have a novel additional function: inhibition of Nf-kB. Based on these two functions, we hypothesize that expression of these genes will "protect" the Beta-cell by preventing apoptosis and blocking the up- regulation of NF-kB-dependent genes, including NO synthase and the adhesion molecules, and thus ameliorate the problems listed above. The therapeutic efficacy of these genetic engineering strategies will be tested in a mouse model of streptozotocin-induced diabetes and/or in NOD mice. Based on these results, experiments will be undertaken in a preclinical model of streptozotocin-induced diabetes in a non-human primate. If expression of the anti-apoptotic genes is insufficient to achieve the preset goals, additional genetic engineering with a dominant negative human TNF receptor and/or an Il-1 receptor antagonist will be evaluated. Genes will be expressed either constitutively or in regulated fashion. Both adenovirus and lentivirus-mediated gene transfer will be assessed. As needed, we shall produce transgenic animals expressing protective genes in the Beta-cell using the insulin promoter or in bioengineered insulin-producing middle lobe pituitary cells in collaboration with project #2