GENETIC MODIFICATION OF DENDRITIC CELLS FOR TOLERANCE

该项目的长期目标是开发一种新颖有效的诱导同种异体移植物耐受的策略。为了实现这一目标，我们将创造一种“工程否决细胞”，并将其应用于临床前非人类灵长类肾脏移植模型，以诱导移植前后耐受诱导期以外的非免疫抑制治疗的持久耐受。这项研究的独特之处在于它使用了新的基因转移技术来利用否决效应来杀死或灭活受体同种异体反应细胞。我们的耐受性诱导方法结合了供体树突状细胞（DC）的遗传修饰和短暂的免疫抑制治疗，这种组合建立在我们之前在恒河猴模型中使用供体骨髓诱导耐受性的研究基础上。该研究计划将开发和利用定制的基因转移方法，在供体树突状细胞（dDC）中组成性地表达tgf - β 1，以便在T细胞-dDC串音的微环境中向响应的供体特异性T细胞传递致命信号。我们提出，当与抗胸腺细胞球蛋白短期治疗联合使用时，tgf - β 1工程的dDC将有效地作为“替代否决细胞”，诱导克隆缺失和特异性耐受。实现我们目标的必要条件是对dDC进行高效的基因改造，并且不会对其作为抗原提呈细胞（APC）的功能产生不利影响。人们已经探索了多种载体方法来实现DC的有效转导，以表达与该策略和其他策略相关的异源基因。然而，现有的基因转移方法有很大的局限性，因为目前的载体通常效率低，并且/或者可能与DC的显著毒性有关。为了成功地移植到移植的临床实践中，以及其他免疫学应用（例如，自身免疫性疾病、癌症和疫苗），需要开发改良的载体系统，用于基因修饰DC。在这方面，我们已经开发了改变腺病毒（Ad）载体的向性的方法，作为提高其疗效的一种手段。我们已经证明，免疫重靶方法允许通过人类DC的CD40途径重新路由Ad载体，从而显著提高效率并对免疫呈递功能产生有益影响。在本研究中，将首先优化Ad tgf - β 1 DC细胞结构，用于体外减少猴模型中的同种异体细胞毒性T细胞（CTL）反应。最终目标是在恒河猴成熟的肾脏移植模型中，在临床前环境中证明tgf - β 1 dDC的主要应用。虽然我们期望这些研究将产生新的方法来诱导肾移植耐受，但我们的总体策略也可能扩展到其他器官和组织的移植。此外，建议的耐受策略也将适用于那些由于白喉高致敏或其他原因而不适合使用项目1和2中描述的基于白喉的抗cd3免疫毒素治疗的受体。最后，本文提出的载体发展和分析可能允许使用嗜性修饰腺病毒作为DC基因修饰的基因传递方法，并将阐明针对Ad载体在体内产生的免疫反应的关键方面。

The long-range goal of this project is to develop a novel and effective strategy for allograft tolerance induction. To accomplish this goal, we will create an "engineered veto cell" and apply it in a preclinical non-human primate model of kidney transplant to induce durable tolerance with non immunosuppressive treatment beyond the peritransplant tolerance induction period. This study is unique in that it uses novel gene transfer technology to exploit the veto effect to kill or inactivate recipient alloreactive cells. Our approach to tolerance induction combines genetic modification of donor dendritic cells (DC) with brief immunosuppressive treatment, a combination that builds upon our previous studies of tolerance induction with donor bone marrow in the rhesus model. The research plan will develop and utilize customized gene transfer methods to constitutively express TGF-beta1 in donor dendritic cells (dDC) in order to deliver within the microenvironment of T cell-dDC cross talk, a lethal signal to the responding donor-specific T cells. We propose that when used in conjunction with a short treatment with anti-thymocyte globulin, TGF-beta1 engineered dDC will effectively function as "ersatz veto cells" to induce clonal deletion and specific tolerance. A requisite condition for achieving our goals is the genetic modification of dDC with high efficacy and in a manner that does not impact unfavorably in their functionality as antigen presenting cells (APC). A variety of vector approaches have been explored to achieve effective transduction of DC to express heterologous genes relevant to this strategy and others. Available gene transfer methods, however, have represented a major limitation, as current generation vectors are generally of low efficiency and/or may be associated with significant toxicity for DC. For successful transplantation to clinical practice in transplantation and also for other immunological applications (e.g., autoimmune disease, cancer, and vaccines), there is a need to develop improved vector systems for genetically modifying DC. In this regard, we have developed methods to alter the tropism of adenoviral (Ad) vectors as a means to enhance their efficacy profile. We have demonstrated that an immunologic re-target approach allows re-routing of Ad vectors via the CD40 pathway of human DC with dramatic enhancements of efficiency and with beneficial effects in immune presentation function. In the proposed studies, the Ad TGF-beta1 DC cellular construct will be first optimized for in vitro reduction of allospecific cytotoxic T cell (CTL) responses in the monkey model. The final goal is proof of principal for the employment of TGF-beta1 dDC in a preclinical setting using the well-established kidney transplant model in rhesus macaques. Although we expect that these studies will yield novel approaches for induction of tolerance in the context of kidney transplantation, our general strategy might also be extended to the transplantation of other organs and tissues. In addition, the propose tolerance strategy will also be applicable to those recipients, who by virtue of high diphtheria sensitization or other causes, are not candidates for treatment with the potent diphtheria based anti-CD3 immunotoxin described in Projects 1 and 2. Finally, vector developments and analyses proposed herein may allow the use of tropism-modifier adenovirus as a gene delivery method for genetic modification of DC, and will elucidate key aspects of the immune response generated in vivo against Ad vectors.