Gene Therapeutic Approach for Tolerance Induction

描述（由申请人提供）：调节针对CNS抗原（如髓磷脂碱性蛋白（MBP）和髓磷脂少突胶质细胞糖蛋白（MOG））的免疫应答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用于诱导抗原特异性耐受的新方法，其可应用于预防或逆转不期望的免疫应答，例如，在过去的十年中，我们创造了一种平台技术，可用于基因治疗，以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以表达具有靶向表位的IG融合蛋白。四种实验性自身免疫模型中的数据已经证明了临床功效，即通过B细胞表达自身抗原的IG融合蛋白可以预防和逆转自身免疫应答。重要的是，我们发现CD 25+，FoxP 3+调节性T细胞是诱导和维持该系统中耐受性所必需的。我们还报道了转导的B细胞的激活模式是B细胞递送的耐受性基因治疗成功的关键因素，这一观察对于潜在的翻译是重要的。我们假设不同的B细胞活化剂导致不同的趋化因子和细胞因子组的产生和/或致耐受性（边缘区）与非致耐受性B细胞的刺激。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，将加工的表位呈递给效应T细胞和/或T细胞，以通过调节细胞因子抑制应答。此外，我们认为致耐受性B细胞通过CTLA-4直接募集TGF 1 α以诱导耐受。为了验证这些假设，并将我们的努力转化为临床，以用于MS的潜在未来治疗，我们将在本次更新中重点关注以下三个具体目标。(1)基于观察到LPS和CpG激活的B细胞产生不同的细胞因子和趋化因子，使用基因敲除小鼠，我们将定义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。我们还将分析转导的B细胞的寿命和表型，以及它们向淋巴组织和CNS的迁移。我们将检验抗CD 20治疗导致部分B细胞耗竭，保留致耐受性MZ细胞的假设。(2)我们将确定TCR转基因T细胞在B细胞递送的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致耐受性或非致耐受性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”系统开始通过分析HLA DR 2转基因小鼠和体外人T细胞的反应，包括通过基因治疗激活T细胞，将这些研究转化为人类疾病。我们还将检查非整合载体的功效（例如，无肠腺病毒）在后者系统和EAE模型中用于B细胞递送的基因治疗。这些研究将提供在临床相关模型中使用人类T细胞的原理证明，将建立B细胞递送的耐受性基因治疗的机制，并将推动该项目在临床上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细胞已显示对多种CNS抗原应答，如髓磷脂少突胶质细胞糖蛋白（MOG）、髓磷脂碱性蛋白（MBP）和磷脂蛋白（PLP）;因此，在本发明中，这些免疫应答的调节是MS治疗干预的目标。我们实验室的重点是开发一种新的B细胞递送基因治疗方法用于诱导耐受性，特别是在自身免疫性疾病如MS中。该平台已成功应用于MS的鼠模型中。在此，我们将研究动物模型中B细胞递送耐受性的机制，然后我们将开始使用来自MS患者的T细胞将这些研究转化到临床，所述患者暴露于体外用MBP-Ig构建体转导的B细胞。这些研究将在临床相关模型中建立一种新的人类T细胞耐受方法，并将继续将该项目转化为临床，作为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.