GENETIC MODIFICATION OF DENDRITIC CELLS FOR TOLERANCE

本项目的长期目标是开发一种新的和有效的策略诱导同种异体移植耐受。 为了实现这一目标，我们将创建一个“工程否决细胞”，并将其应用于临床前非人灵长类动物模型的肾移植，以诱导持久的耐受性与非免疫抑制治疗超过围移植耐受诱导期。 这项研究的独特之处在于它使用了新的基因转移技术，利用否决效应杀死或抑制受体同种异体反应性细胞。 我们的耐受诱导方法将供体树突状细胞（DC）的遗传修饰与短暂的免疫抑制治疗相结合，这种结合建立在我们先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，在供体树突状细胞（dDC）中组成型表达TGF-β 1，以便在T细胞-dDC串扰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的短期治疗结合使用时，TGF-β 1工程化的dDC将有效地发挥“免疫否决细胞”的功能，以诱导克隆缺失和特异性耐受。 实现我们的目标的必要条件是以高效率和不会不利地影响其作为抗原呈递细胞（APC）的功能的方式对DDC进行遗传修饰。 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，可用的基因转移方法已经表现出主要的局限性，因为当前一代载体通常效率低和/或可能与DC的显著毒性相关。 对于成功移植到移植的临床实践以及对于其他免疫学应用（例如，自身免疫性疾病、癌症和疫苗），需要开发用于遗传修饰DC的改进的载体系统。 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，作为增强其功效概况的手段。 我们已经证明，免疫重新靶向方法允许通过人DC的CD 40途径重新路由Ad载体，具有显著增强的效率和在免疫呈递功能中的有益效果。 在所提出的研究中，Ad TGF-β 1 DC细胞构建体将首先针对猴模型中同种异体特异性细胞毒性T细胞（CTL）应答的体外降低进行优化。 最终的目标是证明在临床前环境中使用充分建立的恒河猴肾移植模型的TGF-β 1 dDC的原理。 虽然我们预计这些研究将产生新的方法诱导耐受的背景下，肾移植，我们的一般策略也可能扩展到其他器官和组织的移植。 此外，拟定的耐受性策略也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中描述的基于白喉的强效抗CD 3免疫毒素治疗的受试者。 最后，本文提出的载体开发和分析可以允许使用向性修饰腺病毒作为DC遗传修饰的基因递送方法，并且将阐明体内产生的针对Ad载体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.