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: 6666378
• 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/6666378
• 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; leukocyte depletion therapy; 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.

该项目的远程目标是制定一种新颖有效的同种异体耐受性诱导策略。 为了实现这一目标，我们将创建一个“工程化的否决细胞”，并将其应用于肾脏移植的临床前非人类灵长类动物模型，以诱导耐用的耐受性，并在腹膜耐受耐受性诱导期之外使用非免疫抑制治疗。 这项研究的独特之处在于它使用新型基因转移技术来利用否决效应来杀死或失活同种反应性细胞。 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，这种结合是基于我们先前在恒河类模型中与供体骨髓的耐受性诱导研究的研究。 该研究计划将开发并利用自定义的基因转移方法来组成性地表达供体树突状细胞（DDC）的TGF-BETA1，以便在T细胞-DDC交叉谈话的微环境中传递，这是对响应的供体特异性T细胞的致命信号。 我们建议，当与抗胸腺细胞球蛋白短暂处理中，TGF-BETA1工程DDC将有效地充当“ Ersatz veto细胞”，以诱导克隆缺失和特定的耐受性。 实现我们的目标的必要条件是DDC具有高效率的遗传修饰，并且以其作为抗原呈现细胞（APC）的功能不利影响的方式不利。 已经探索了各种向量方法，以实现直流的有效转导，以表达与该策略和其他策略相关的异源基因。 但是，可用的基因转移方法代表了一个主要局限性，因为当前一代向量通常具有低效率和/或可能与DC的显着毒性有关。 为了成功地移植移植中的临床实践以及其他免疫应用（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基因修改DC。 在这方面，我们开发了改变腺病毒（AD）载体的最向量的方法，以此来增强其功效概况。 我们已经证明，免疫学重新目标方法允许通过人类DC的CD40途径重新整理AD向量，并具有巨大的效率增强，并且对免疫表现功能具有有益的效果。 在拟议的研究中，将首先优化AD TGF-BETA1 DC细胞构建体，以在猴子模型中体外减少同种毒性T细胞（CTL）反应。 最终的目标是使用恒河猕猴中建立的肾脏移植模型在临床前的临床前环境中使用TGF-BETA1 DDC的本金证明。 尽管我们预计这些研究将在肾脏移植的背景下产生诱导耐受性的新方法，但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，提出的耐受策略也将适用于那些受体，他们通过高白喉敏化或其他原因，并不是候选基于有效的白喉的治疗方法。 DC，并将阐明体内对AD载体产生的免疫反应的关键方面。