GENETIC MODIFICATION OF DENDRITIC CELLS FOR TOLERANCE

树突状细胞的基因修饰以获得耐受性

• 批准号: 6655228
• 负责人: David Terry Curiel
• 金额: $ 17.24万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6655228
• 关键词: Adenoviridae; CD40 molecule; Macaca mulatta; T lymphocyte; artificial immunosuppression; biological models; biotechnology; cell transplantation; cooperative study; dendritic cells; gene expression; gene targeting; gene therapy; genetic manipulation; genetic transduction; homologous transplantation; immune tolerance /unresponsiveness; immunogenetics; immunosuppressive; kidney transplantation; tissue /cell culture; transfection /expression vector; transforming growth factors; transplantation immunology

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.

该项目的长期目标是开发一种新颖有效的同种异体移植耐受诱导策略。 为了实现这一目标，我们将创建一种“工程否决细胞”，并将其应用于临床前非人类灵长类动物肾移植模型，以在移植周围耐受诱导期之后通过非免疫抑制治疗诱导持久耐受。 这项研究的独特之处在于它使用新颖的基因转移技术来利用否决效应来杀死或灭活受体同种反应细胞。 我们的耐受诱导方法将供体树突状细胞 (DC) 的基因修饰与短暂的免疫抑制治疗相结合，这种组合建立在我们之前在恒河猴模型中用供体骨髓进行耐受诱导的研究的基础上。 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-β1，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。 我们建议，当与抗胸腺细胞球蛋白的短期治疗结合使用时，TGF-β1 改造的 dDC 将有效地发挥“代用否决细胞”的作用，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，并且改造方式不会对其作为抗原呈递细胞 (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，可用的基因转移方法存在主要限制，因为当前一代载体通常效率低下和/或可能与DC的显着毒性相关。 为了成功移植到移植临床实践以及其他免疫学应用（例如自身免疫性疾病、癌症和疫苗），需要开发用于遗传修饰 DC 的改进载体系统。 在这方面，我们开发了改变腺病毒（Ad）载体趋向性的方法，作为增强其功效的手段。 我们已经证明，免疫重新靶向方法允许通过人类 DC 的 CD40 途径重新路由 Ad 载体，从而显着提高效率并对免疫呈递功能产生有益影响。 在拟议的研究中，Ad TGF-β1 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) 反应的体外降低进行优化。 最终目标是使用恒河猴肾移植模型在临床前环境中使用 TGF-β1 dDC 进行原理证明。 尽管我们期望这些研究将产生在肾移植背景下诱导耐受的新方法，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，所提出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使用项目 1 和 2 中描述的基于强效白喉的抗 CD3 免疫毒素治疗的接受者。最后，载体本文提出的开发和分析可能允许使用向性修饰剂腺病毒作为 DC 遗传修饰的基因递送方法，并将阐明体内针对 Ad 载体产生的免疫应答的关键方面。