Induction of Immunological Tolerance for Gene Therapy

描述（由申请人提供）： 基因治疗的理论基础是通过将功能基因转导到受体组织中来纠正遗传缺陷。现在存在多种技术，用于将遗传物质越来越有效地引入基因治疗接受者。然而，受体免疫系统对转导基因的排斥仍然是稳定长期表达的持续障碍。在这项研究中，我们提出了一种策略，利用否决细胞为基础的方法来选择性地诱导免疫耐受的治疗基因，以规避免疫排斥的障碍。CDS* 否决细胞作为缺失性抗原呈递细胞发挥功能，其消除识别CDS* 否决细胞的MHC I类呈递的抗原的T细胞。因此，为了工程改造将删除能够识别治疗基因产物的T细胞的受体否决细胞，必须从受体产生CD 8+否决细胞并将治疗基因插入CD 8+否决细胞。该授权申请的中心假设是，用治疗性基因转导的受体来源的CDS* 否决细胞可用于选择性地使受体免疫系统耐受治疗性基因。以这种方式，将消除作为基因治疗障碍的基于免疫的对转导基因的排斥。具体目标1-产生表达转导基因的受体来源的CDS* 否决细胞。在这个目标中，我们将优化所需的条件，以快速，有效地从受体组织中分离大量的基因转导的CD 8+否决细胞。为了进行尽可能复杂的分析，我们将利用一个模型转基因，其中存在大量的工具来分析免疫反应（鸡卵清蛋白（OVA））。这种优化将包括最大化CDS* veto细胞的扩增以优化产量和最大化基因转导、表达和呈递MHC I类分子的效率。具体目标2-评估基因治疗转导的CDS* 否决细胞诱导对治疗基因的抗原特异性耐受的能力。在这个目的中，我们将测试受体来源的表达CDS* 的OVA否决细胞耐受抗OVA T细胞应答的能力。这将通过在体外研究OVA特异性否决活性和通过进行体内耐受化实验来实现。通过将更简化的体外系统与更生理的体内系统相结合，我们将能够评估机制决定和生理功效。

DESCRIPTION (provided by applicant): The theoretical basis of gene therapy is the correction of a genetic defect through the transduction of a functional gene into the tissues of the recipient. A variety of technologies now exist for the increasingly efficient introduction of genetic material into a gene therapy recipient. However, rejection of the transduced gene by the recipient immune system remains a persistent obstacle to stable long-term expression. In this grant, we propose a strategy that circumvents the obstacle of immune rejection by utilizing a veto cell based approach to selectively induce immunological tolerance to the therapeutic gene. CDS* veto cells function as deletional antigen presenting cells, which eliminate T cells that recognize antigens presented by the MHC class I of the CDS* veto cell. Thus, to engineer a recipient veto cell that will delete T cells capable of recognizing the product of a therapeutic gene, one must both generate CD8+ veto cells from the recipient and insert the therapeutic gene into the CDS* veto cells. The central hypothesis of this grant application is that recipient derived CDS* veto cells, which are transduced with the therapeutic gene, can be used to selectively tolerize the recipient immune system to the therapeutic gene. In this way, immune based rejection of the transduced gene will be eliminated as an obstacle to gene therapy. Specific Aim 1- Generation of recipient derived CDS* veto cells expressing a transduced gene. In this aim, we will optimize the conditions required to rapidly and efficiently isolate large numbers of gene transduced CD8+ veto cells from recipient tissues. To perform as sophisticated an analysis as possible, we will utilize a model transgene for which a large arsenal of tools exist to analyze immune responses (chicken ovalbumin (OVA)). This optimization will include maximizing expansion of CDS* veto cells to optimize yields and maximizing the efficiency of gene transduction, expression and presentation on MHC class I molecules. Specific Aim 2- Assess the ability of gene therapy transduced CDS* veto cells to induce antigenspecific tolerance to the therapeutic gene. In this aim, we will test the ability of the recipient derived OVA expressing CDS* veto cells to tolerize anti- OVA T cell responses. This will be accomplished by both studying OVA specific veto activity in vitro and by performing in vivo tolerization experiments. By combining the more reductionist in vitro systems with the more physiological in vivo systems, we will be able to assess both mechanistic determinations and physiological efficacy.