Gene Therapeutic Approach for Tolerance Induction

描述（由申请人提供）：调节针对中枢神经系统抗原的免疫反应，如髓鞘碱性蛋白（MBP）和髓鞘少突胶质细胞糖蛋白（MOG），是多发性硬化症治疗干预的目标。我们实验室的重点是开发诱导抗原特异性耐受性的新方法，该方法可用于预防或逆转不良免疫反应，例如，在ms等自身免疫性疾病中。在过去十年中，我们创建了一个平台技术，可用于基因治疗，诱导对多个表位的耐受性。在我们的模型中，lps激活的B细胞通过逆转录病毒载体转导，表达具有目标表位的Ig融合蛋白。四种实验性自身免疫模型的数据表明，B细胞表达自身抗原Ig融合蛋白可以预防和逆转自身免疫反应。重要的是，我们发现CD25+， FoxP3+调节性T细胞在该系统中诱导和维持耐受性都是必需的。我们还报道了转导的B细胞的激活模式是B细胞传递基因治疗耐受成功的关键因素，这一观察结果对潜在的翻译很重要。我们假设不同的B细胞激活剂导致不同的趋化因子和细胞因子的产生和/或刺激耐受性（边缘区）与非耐受性B细胞。我们的假设是，转导的抗原呈递B细胞（APC），特别是边缘区B细胞，将加工过的表位呈递给效应T细胞和/或treg，通过调节细胞因子抑制反应性。此外，我们认为产生耐受性的B细胞直接通过CTLA-4募集Tregs诱导耐受性。为了检验这些假设，并将我们的努力转化为临床潜在的未来治疗MS，我们将在这次更新中关注以下三个具体目标。(1)基于观察LPS和cpg激活的B细胞产生不同的细胞因子和趋化因子，我们将使用敲除小鼠，确定在这些和其他激活条件下产生的特异性可溶性介质的作用。我们还将分析转导B细胞的寿命和表型，以及它们向淋巴组织和中枢神经系统的迁移。我们将验证抗cd20治疗导致B细胞部分耗竭，保留耐受性MZ细胞的假设。(2)我们将确定TCR转基因T细胞在B细胞传递基因治疗中的迁移和命运，并将使用双光子显微镜分析它们与耐受性或非耐受性B细胞相互作用的动力学。(3)在我们的最终目标中，我们将使用“人源化”系统，通过分析HLA DR2转基因小鼠和体外人类T细胞的反应，包括通过基因治疗激活Tregs，开始将这些研究转化为人类疾病。我们还将研究非整合载体（例如，无肠腺病毒）在后期系统和EAE模型中用于b细胞传递基因治疗的功效。这些研究将提供人类T细胞在临床相关模型中的原理证明，将建立B细胞传递基因治疗耐受性的机制，并将该项目推进到临床转化，作为多发性硬化症的潜在治疗方法。来自多发性硬化症患者的T细胞已被证明对多种中枢神经系统抗原有反应，如髓鞘少突胶质细胞糖蛋白（MOG）、髓鞘碱性蛋白（MBP）和磷脂（PLP）；因此，调节这些免疫反应是ms治疗干预的目标。我们实验室的重点是开发一种新的b细胞传递基因治疗方法来诱导耐受性，特别是在ms等自身免疫性疾病中。该平台已成功应用于ms的小鼠模型。然后我们将开始将这些研究转化为临床，使用MS患者的T细胞暴露于体外用MBP-Ig结构转导的B细胞。这些研究将在临床相关模型中建立人类T细胞耐受性的新方法，并将继续将该项目转化为临床多发性硬化症的潜在治疗方法。

DESCRIPTION (provided by applicant): Modulation of immune responses against CNS antigens, such as myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), is a goal for therapeutic intervention in multiple sclerosis. The focus of our lab has been to develop novel approaches for the induction of antigen-specific tolerance that can be applied to the prevention or reversal of undesirable immune responses, e.g., in autoimmune diseases like MS. During the last decade, we created a platform technology that can be used for gene therapy to induce tolerance to multiple epitopes. In our model, LPS-activated B cells are transduced via retroviral vectors to express an Ig fusion protein with the targeted epitopes. Data in four experimental autoimmune models have demonstrated clinical efficacy in that expression of Ig fusion proteins of autoantigens by B cells can both prevent and reverse autoimmune responsiveness. Importantly, we found that CD25+, FoxP3+ regulatory T cells were required for both the induction and maintenance of tolerance in this system. We also reported that the mode of activation of the transduced B cells was a critical factor for the success of B cell-delivered gene therapy for tolerance, an observation that is important for potential translation. We hypothesize that different B cell activators lead to the production of distinct sets of chemokines and cytokines and/or the stimulation of tolerogenic (marginal zone) versus non-tolerogenic B cells. Our hypothesis is that transduced antigen-presenting B cells (APC), in particular marginal zone B cells, present the processed epitopes to effector T cells and/or Tregs to inhibit responsiveness via regulatory cytokines. Further, we suggest that the tolerogenic B cells directly recruit Tregs via CTLA-4 to induce tolerance. In order to test these hypotheses and to translate our efforts to the clinic for potential future treatment of MS, we will focus in this renewal on the following three specific aims. (1) Based on the observation that LPS- and CpG-activated B cells produce different cytokines and chemokines, using knockout mice, we will define the roles of specific soluble mediators generated under these and other activation conditions. We will also analyze the lifespan and phenotype of transduced B cells, as well as their migration to lymphoid tissues and the CNS. We will test the hypothesis that anti-CD20 treatments leads to partial depletion of B cells, sparing the tolerogenic MZ cells. (2) We will determine the migration and fate of the TCR transgenic T cells in B cell delivered gene therapy, and will analyze the kinetics of their interaction with tolerogenic or non-tolerogenic B cells using two photon microscopy. (3) In our final aim, we will use "humanized" systems to begin to translate these studies to human disease by analyzing responses of HLA DR2 transgenic mice and of human T cells in vitro, including the activation of Tregs by gene therapy. We will also examine the efficacy of non-integrating vectors (e.g., gutless adenovirus) for B-cell delivered gene therapy in both the later system and in EAE models. These studies will provide proof of principle with human T cells in a clinically relevant model, will establish the mechanisms of B cell-delivered gene therapy for tolerance, and will move this project forward to translation in the clinic as a potential therapy for multiple sclerosis. PUBLIC HEALTH RELEVANCE T cells from multiple sclerosis patients have been shown to respond to a variety of CNS antigens, such as myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) and phospholipoprotein (PLP); hence, modulation of these immune responses is a goal for therapeutic intervention in MS. The focus of our lab has been to develop a novel B-cell delivered gene therapy method for the induction of tolerance, particularly in autoimmune diseases like MS. This platform has been successfully applied in murine models for MS. Herein, we will investigate the mechanism of B-cell delivered tolerance in animal models, and then we will begin to translate these studies to the clinic using T cells from MS patients exposed to B cells transduced with MBP-Ig constructs in vitro. These studies will establish a novel approach for tolerance with human T cells in a clinically relevant model, and will continue the translation of this project forward to the clinic as a potential therapy for multiple sclerosis.