GENETIC MODIFICATION OF DENDRITIC CELLS FOR TOLERANCE

该项目的长期目标是开发一种新的、有效的诱导同种异体移植耐受的策略。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的非人类灵长类肾脏移植模型，以诱导移植周耐受诱导期后的非免疫抑制治疗，以诱导持久耐受。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权效应来杀死或灭活受体同种异体反应细胞。我们的耐受诱导方法结合了供体树突状细胞(DC)的基因修饰和短暂的免疫抑制治疗，这一组合建立在我们之前在恒河猴模型中用供体骨髓诱导耐受的研究基础上。该研究计划将开发和利用定制的基因转移方法，在供者树突状细胞(DDC)中结构性地表达转化生长因子-β1，以便在T细胞-DDC串扰的微环境中向反应的供者特异性T细胞传递致命信号。我们认为，当联合抗胸腺细胞球蛋白的短期治疗时，转化生长因子-β1工程的DDC将有效地发挥“假否决细胞”的作用，诱导克隆性缺失和特异性耐受。实现我们目标的一个必要条件是对DDC进行高效的、不会对其作为抗原提呈细胞(APC)的功能产生不利影响的基因修饰。人们已经探索了多种载体方法来实现DC的有效转导，以表达与这一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存在着很大的局限性，因为当前一代的载体通常效率较低和/或可能与DC的显著毒性有关。为了将移植成功地应用于临床移植以及其他免疫学应用(例如，自身免疫性疾病、癌症和疫苗)，需要开发改进的载体系统来对DC进行基因修饰。在这方面，我们已经开发了改变腺病毒(Ad)载体的趋向性的方法，以此作为提高其疗效的一种手段。我们已经证明，免疫学重靶向方法允许通过人DC的CD40途径重新路由Ad载体，从而显著提高效率并对免疫递呈功能产生有益影响。在拟议的研究中，将首先优化Ad TGF-beta1 DC细胞结构，以在体外减少猴子模型中同种异体细胞毒性T细胞(CTL)的反应。最终目标是证明在恒河猴肾脏移植模型的基础上，在临床前环境中使用转化生长因子-β1 DDC的原理。虽然我们预计这些研究将为诱导肾移植耐受提供新的方法，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，拟议的耐受策略也将适用于那些由于白喉高度致敏或其他原因而不适合接受项目1和2中所述的基于白喉的高效抗CD3免疫毒素治疗的受者。最后，本文提出的载体开发和分析可能允许使用趋向性修饰性腺病毒作为DC基因修饰的基因输送方法，并将阐明体内产生的免疫应答的关键方面。

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.