Binding of Epstein Barr Virus EBNA2 Unifies Multiple Sclerosis Genetic Mechanisms

多发性硬化症(MS)是一种毁灭性的中枢神经系统疾病，影响着230多万人 世界各地的人们。目前治疗多发性硬化症的方法充其量只有部分有效，而且没有治愈的方法。改进 了解多发性硬化症的发病机制将有助于更好地诊断、治疗和预防。MS IS 这是由遗传和环境风险因素造成的。爱泼斯坦-巴尔病毒(EBV)是最稳定的 重复的环境因素。越来越多的证据表明，EBV感染的B细胞及其下游 免疫效应，是MS疾病过程的关键驱动因素。我们团队和其他人最近的工作表明 EBV编码的EBNA2基因调控蛋白在几乎一半已建立的MS遗传机制中 风险基因。在这个提议中，我们将检验EBNA2驱动的等位基因依赖于人类的改变的假设 基因表达导致不同的B和T细胞表型，直接参与MS的疾病过程。 目的1.定量检测多发性硬化症患者中EB病毒特异性基因的表达 人口统计数据。我们将扩大我们的队列，将欧洲、非洲和亚洲的男性和女性包括在内 祖先。我们将测量MS和EBV特异的人类基因在这些队列中的表达。 目的2.全球发现依赖EBV和MS基因型的基因调控机制。我们 将使用大规模平行报告分析(MPRA)系统地识别EBV和风险等位基因依赖 在有和没有EBV感染的原代B细胞中，所有多发性硬化症的基因表达都是危险变量。我们会确认人类的身份 在这些基因座上与EBNA2、EBNA3C和/或EBNA-LP共同作用的转录调控因子，并确认它们的等位基因-- 利用尖端功能基因组学技术研究多发性硬化症患者B细胞的依赖行为。我们将验证 使用基于CRISPR的基因组编辑来实现这些依赖于EBV和基因型的基因调控机制 CD37、CD58、ZMIZ1和其他MS危险基因的患者来源的原始B细胞。 目的3.发现基于EBV的等位基因机制改变多发性硬化症的细胞行为 EB病毒和MS特异性变异体(如CD37、CD58、ZMIZ1)对B细胞功能影响的必要性和充分性 CRISPR编辑的B细胞。我们将测量对B细胞受体信号、细胞因子产生的影响， 增殖和细胞凋亡。我们将使用可诱导的多能干细胞来源的血脑屏障(Bbb)。 内皮模型，以衡量EBV和MS风险等位基因对B细胞-BBB相互作用的影响。 疾病进程可能受病毒控制的等位基因调节蛋白影响的概念 复合体是高度创新的，以前从未被展示过。我们的工作首次提供了 通过一个统一的基因与环境模型，对EBV在多发性硬化症中确立的作用进行机械性的洞察。 对遗传机制的下游效应进行全面编目、剖析和理解 受EBV的影响将是重大的，因为它将为开发干扰 通过这些机制，甚至是预防EBV感染的疫苗来治愈多发性硬化症和其他EBV相关疾病。

Multiple sclerosis (MS) is a devastating disease of the central nervous system that affects over 2.3 million people worldwide. Current therapies for MS are at best only partially effective, and there is no cure. Improved understanding of MS disease mechanisms would lead to better diagnosis, treatment, and prevention. MS is caused by both genetic and environmental risk factors. Epstein-Barr virus (EBV) is the most consistently replicated environmental factor. Mounting evidence indicates that EBV-infected B cells, and their downstream immunological effects, are key drivers of MS disease processes. Recent work by our group and others implicates the EBV-encoded EBNA2 gene regulatory protein in mechanisms at almost half of the established MS genetic risk loci. In this proposal, we will test the hypothesis that EBNA2-driven allele-dependent alterations to human gene expression lead to distinct B and T cell phenotypes directly contributing to disease processes in MS. Aim 1. Quantification of EBV-specific human gene expression in multiple MS patient demographics. We will expand our cohort to include males and females of European, African, and Asian ancestries. We will measure MS- and EBV-specific human gene expression in these cohorts. Aim 2. Global discovery of EBV- and MS genotype-dependent gene regulatory mechanisms. We will use Massively Parallel Reporter Assays (MPRAs) to systematically identify EBV- and risk allele-dependent gene expression at all MS risk variants in primary B cells with and without EBV infection. We will identify human transcriptional regulators acting with EBNA2, EBNA3C, and/or EBNA-LP at these loci and confirm their allele- dependent actions in MS-derived B cells using cutting-edge functional genomics technologies. We will validate these EBV- and genotype-dependent gene regulatory mechanisms using CRISPR-based genome editing of patient-derived primary B cells at CD37, CD58, ZMIZ1, and other MS risk loci. Aim 3. Discovery of allelic EBV-based mechanisms altering cellular behavior in MS. We will gauge the necessity and sufficiency of EBV and specific MS variants (e.g: CD37, CD58, ZMIZ1) on B cell function using the CRISPR-edited B cells. We will measure impact on B cell receptor signaling, cytokine production, proliferation, and apoptosis. We will use an inducible pluripotent stem cell-derived blood brain barrier (BBB) endothelium model to measure the impact of EBV and MS risk alleles on B cell - BBB interactions. The concept that disease processes might be influenced by virus-controlled, allelic regulatory protein complexes is highly innovative and has never before been demonstrated. Our work for the first time provides mechanistic insight into the established role of EBV in MS through a unified gene by environment model. Comprehensive cataloging, dissection, and understanding of the downstream effects of genetic mechanisms impacted by EBV will be significant because it will provide strong rationale to develop therapies that interfere with these mechanisms, or even vaccines that prevent EBV infection to cure MS and other EBV-related diseases.