ATF3 and iNOS in Islet Distruction and Graft Rejection

描述(申请人提供)：目标和假设：本方案的长期目标是提高胰岛移植的效率。尽管朝着这一目标(如埃德蒙顿协议)取得了巨大的进步，但有两个关键的限制阻碍了胰岛移植在临床上的广泛应用：(1)需要大量的免疫抑制，(2)要求每个受者有大量的胰岛。这项建议将通过测试应激诱导的细胞凋亡在胰岛移植过程中8细胞破坏中起重要作用的假设来解决这些局限性。两个压力诱导的促凋亡基因将是研究的重点：ATF3和iNOS。目的1：验证ATF3在死亡受体介导的途径中不起主要作用的假设。Caspase8是从死亡受体Fas和TNFR传递细胞凋亡信息的关键分子。将努力确定ATF3/iNOS介导的途径是否有别于死亡受体介导的途径。如果是这样的话，抑制caspase 8应该会进一步增强ATFS/iNOS基因敲除的胰岛抵抗应激诱导的细胞凋亡的能力。目的2：检测ATF3和/或iNOS缺陷的胰岛是否能减少移植排斥反应。三种实验性胰岛移植模型将被用来确定ATF3和/或iNOS的缺乏是否减轻了胰岛移植物存活的任何主要障碍(S)：原发无功能(同种模型)、同种异体免疫(异体模型)和自身免疫(自体免疫模型)。目的：以ATF3和iNOS为靶基因，通过RNA干扰(RNAi)检测胰岛基因沉默的可行性。在U6启动子的控制下，将产生表达短发夹状RNA的DMA构建体，以靶向ATF3或/和iNOS mRNA的降解。他们的效率将首先在产生胰岛素的MIN6R细胞中进行测试，然后在初级胰岛中进行测试。如果它们奏效，野生型胰岛将被RNAi“敲除”ATF3和/或iNOS，以确定它们是否比没有这些促凋亡基因的胰岛存活得更好。意义：这项建议将8细胞死亡的机制研究与基因沉默技术的发展相结合，目的是改善胰岛移植。如果成功，这项拟议的研究不仅将增强我们对胰岛破坏的理解，还将增强我们设计具有更高生存能力的胰岛的能力。反过来，这将使每个接收者能够以较低的数量移植胰岛。此外，由于胰岛不那么脆弱，它们可以耐受轻度免疫抑制条件下残留的免疫攻击，从而避免了移植中常用的重度免疫抑制的有害影响。

DESCRIPTION (provided by applicant): Goal and Hypothesis: The long-term goal of this proposal is to improve the efficiency of islet transplantation. While tremendous progress has been made toward this goal (such as the Edmonton Protocol), two key limitations prevent islet transplantation from being a widespread clinical reality: (1) the need for heavy immunosuppression, and (2) the requirement of large numbers of islets per recipient. This proposal will address these limitations by testing the hypothesis that stress-induced apoptosis plays an important role in 8 cell destruction during islet transplantation. Two stress-inducible, pro-apoptotic genes will be the focus of the studies: ATF3 and iNOS. Aim 1: To test the hypothesis that the ATF3 does not play a major role in the death receptor-mediated pathway. Caspase 8 is a key molecule to transmit the apoptotic information from the death receptors: Fas and TNFR. Efforts will be made to determine whether ATF3/iNOS-mediated pathway is distinct from death receptor mediated pathway. If they are, inhibition of caspase 8 should further enhance the ability of the ATFS/iNOS knockout islets to resist to stress-induced apoptosis. Aim 2: To test whether islets deficient in ATF3 and/or iNOS have reduced graft rejection. Three experimental islet transplant models will be used to determine whether the lack of ATF3 and/or iNOS alleviate(s) any of the main obstacles for islet graft survival: primary non-function (by syngeneic model), allo-immunity (by allogeneic model) and auto-immunity (by auto-immune model). Aim 3: To test gene silencing in the islets by RNA interference (RNAi) - feasibility test using ATF3 and iNOS as target genes. DMA constructs expressing short hairpin RNAs under the control of the U6 promoter will be generated to target the degradation of ATF3 or/and iNOS mRNA. Their efficiency will be tested in the insulin-producing MIN6 R cells first then in primary islets. If they work, wild type islets with "knockdown" of ATF3 and/or iNOS by RNAi will be tested to determine whether they survive better than islets without the knockdown of these pro-apoptotic genes. Significance: This proposal combines mechanistic studies of 8 cell death with technological development of gene silencing, with the objective to improve islet transplantation. If successful, the proposed research will enhance not only our understanding of islet destruction but also our ability to engineer islets with improved survivability. This, in turn, will enable islets to be grafted at lower numbers per recipient. In addition, because the islets are less vulnerable, they may tolerate the immune attacks remained under the condition of mild immunosuppression, thus avoiding the deleterious effects of heavy immunosuppression commonly used in transplantation.