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

具有抗凋亡基因/细胞因子抑制剂的异种胰岛工程

基本信息

批准号：
6301189
负责人：
CHRISTIANE FERRAN
金额：
$ 12.01万
依托单位：
JOSLIN DIABETES CENTER
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-12-15 至 2000-11-30
项目状态：
已结题

项目摘要

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

从同种异体或异种来源的胰岛移植 I型胰岛素依赖性糖尿病的治疗已遇到几个问题，包括：主要非功能，拒绝和（自身免疫性）疾病的复发。基于可用信息，看来这些问题在很大程度上与凋亡或 Beta细胞的程序性细胞死亡以及有关诱导依赖NF-KB的一系列基因的β细胞。主要非功能，虽然对基本原因的理解很少，但涉及β细胞的凋亡。 β细胞的凋亡上下文，可能是由TNF和IL-1在内的几个因素引起的，但也受到诱导后产生的一氧化氮（NO）的刺激在一氧化氮合酶的β细胞中，NF-KB依赖性基因。异构胰岛的排斥涉及介导的细胞，很可能一种体液反应；它还涉及非特异性炎症例如，反应由β-的诱导表达支持粘附分子的细胞，其诱导是NF-KB依赖性的。疾病的复发涉及IL-1，TNF以及可能的其他因素；但是，最终结果是β细胞损失主要是由于细胞凋亡。我们建议使用重组腺病毒在β细胞中表达最初是一个或多个基因（A20，Bcl-2和/或BCl-XL）根据它们的抗凋亡特性进行描述，但我们有显示的具有新的附加功能：抑制NF-KB。基于这两个功能，我们假设这些基因的表达将通过防止细胞凋亡并阻止上UP-来“保护”β细胞 NF-KB依赖性基因的调节，包括NO合酶和粘附分子，从而改善上述问题。这些基因工程策略的治疗功效将在链蛋白酶诱导的糖尿病和/或中的小鼠模型中进行测试点头小鼠。基于这些结果，将在非人类链蛋白酶诱导的糖尿病的临床前模型灵长类动物。如果抗凋亡基因的表达不足实现预设目标，额外的基因工程负人TNF受体和/或IL-1受体拮抗剂将是评估。基因将被组成或在受监管的时尚。腺病毒和慢病毒介导的基因都转移将进行评估。根据需要，我们将产生转基因使用胰岛素在β细胞中表达保护基因的动物启动子或生物工程胰岛素产生的中叶垂体与项目2合作的单元格