Binding of Epstein Barr Virus EBNA2 Unifies Multiple Sclerosis Genetic Mechanisms

多发性硬化症（MS）是一种中枢神经系统的毁灭性疾病，影响超过230万人 世界各地的人们。目前的MS疗法充其量只是部分有效，并且没有治愈方法。改进 对MS疾病机制的理解将导致更好的诊断、治疗和预防。MS是 由遗传和环境风险因素引起。EB病毒（EBV）是最一致的 复制环境因素。越来越多的证据表明，EB病毒感染的B细胞及其下游细胞， 免疫学效应是MS疾病过程的关键驱动因素。我们小组和其他人最近的研究表明， EB病毒编码的EBNA 2基因调控蛋白在机制上几乎占已建立的MS遗传学的一半。 危险位点在这个建议中，我们将测试EBNA 2驱动的等位基因依赖性改变对人类基因组的影响的假设。 基因表达导致不同的B和T细胞表型，直接导致MS的疾病过程。 目标1。多发性MS患者中EBV特异性人基因表达的定量 人口统计学我们将扩大我们的队列，包括欧洲，非洲和亚洲的男性和女性 祖先我们将在这些队列中测量MS和EBV特异性人类基因表达。 目标二。全球发现EBV和MS基因型依赖性基因调控机制。我们 将使用大规模平行报告基因测定（MPRAs）系统地识别EBV和风险等位基因依赖性 在有和没有EBV感染的原代B细胞中所有MS风险变体的基因表达。我们将识别人类 在这些基因座上与EBNA 2、EBNA 3C和/或EBNA-LP作用的转录调节因子，并确认它们的等位基因- MS衍生的B细胞中的依赖性作用。我们将验证 这些依赖于EBV和基因型的基因调控机制使用基于CRISPR的基因组编辑， 患者来源的原代B细胞在CD 37、CD 58、ZMIZ 1和其他MS风险基因座。 目标3：发现基于等位基因EBV的机制改变MS中的细胞行为。 EBV和特异性MS变体（例如：CD 37、CD 58、ZMIZ 1）对B细胞功能的必要性和充分性， CRISPR编辑的B细胞。我们将测量对B细胞受体信号传导，细胞因子产生， 增殖和凋亡。我们将使用诱导性多能干细胞衍生的血脑屏障（BBB） 内皮模型来测量EBV和MS风险等位基因对B细胞-BB B相互作用的影响。 疾病过程可能受病毒控制的等位基因调节蛋白影响的概念 复合物是高度创新的，以前从未被证明过。我们的工作首次提供了 通过统一的基因环境模型，对EBV在MS中的既定作用的机制性见解。 全面的编目，解剖和理解遗传机制的下游效应 EB病毒的影响将是重要的，因为它将提供强有力的理由来开发治疗， 这些机制，甚至是预防EBV感染的疫苗，以治愈MS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.