Integrated Functional Genomics Analysis of Druggable T Cell-Activating Genes

所有主要基因组的完整测序为全基因组提供了对基因的洞察 序列和结构。然而，我们对如此大的基因集进行功能分析的能力仍然是 有限的。为了开发更有效的功能基因注释的新技术，我们提出了 一种多学科的方法，使用T细胞受体(TCR)信号作为 生理和病理上重要的细胞过程仍不完全清楚， 但只要有可用的数据，就可以进行集成的大规模分析，以最大限度地提高新颖性和活体 关联性。如果成功，我们的“系统”方法在许多领域的广泛适用性 生物学承诺通过采用生物知识来加速整个生物学知识的产生 其他的，深刻地影响着大的科学和患者社区。作为以下关键组件 适应性免疫系统，T细胞是我们防御病原体，癌症的关键媒介 细胞和移植排斥反应。T细胞功能受损可导致 免疫缺陷，促进感染或癌症。T细胞过度活跃或放松调节是主要的 导致过敏、自身免疫性疾病或神经退行性疾病。这些严重的疾病 导致个人残疾、生活质量下降、无法自立、过早死亡 给社会和医疗保健系统带来了很高的经济负担。通过识别新基因 调节T细胞活化并分析其分子功能，我们的研究将因此 促进我们对T细胞功能和疾病中的功能障碍以及信号的理解 一般情况下，转导。通过专注于功能可以调节的可用药基因 通过小分子或生物疗法，我们的目标是加速开发新的， 针对严重免疫紊乱或癌症的改进和更具成本效益的治疗方法 庞大的患者群体。设计并验证我们的创新、集成、多学科 基因发现和分析的方法，我们将首先在一个 5,000靶向阵列化shRNA文库T细胞高通量RNA干扰筛选 可下药的基因。我们将使用慢病毒shRNA传递来克服 非附着者 对传统机器人siRNA递送方法的生长和相对T细胞抵抗力， 以及新颖的屏幕设计和分析策略，以最大限度地提高敏感度并最大限度地减少错误 积极的比率。接下来我们将结合基因表达谱，磷酸酪氨酸-蛋白质组学， 单倍型关联定位、数量性状基因座定位、全基因组关联定位 和基因表达QTL数据与基因/蛋白质网络和路径分析，以确定优先顺序 这些命中结合了最高的新颖性和最大的活体相关性，以进行详细的随访。

The completed sequencing of all major genomes provided genome-wide insight into gene sequence and structure. However, our ability to functionally analyze such large gene sets is still limited. To develop novel technologies for more efficient functional gene annotation, we propose a multidisciplinary approach, using T cell receptor (TCR) signaling as an example of a physiologically and pathologically important cellular process that is still incompletely understood, but where available data enable integrated large-scale analysis to maximize novelty and in vivo relevance. If successful, the broad applicability of our "systems" approach to many areas of biology promises to accelerate generation of biological knowledge at large through its adoption by others, profoundly impacting large scientific and patient communities. As critical components of the adaptive immune system, T cells are key mediators of our defense against pathogens, cancer cells and, undesired, of transplant rejection. Impaired T cell function can result in immunodeficiency, promoting infections or cancer. T cell hyperactivity or deregulation is a main cause for allergies, autoimmune or neurodegenerative diseases. These serious disorders contribute to individual disability, loss of quality of life, inability to self-sustain, premature death and high economic burden for society and health care system. By identifying novel genes mediating T cell activation and analyzing their molecular functions, our proposed studies will thus promote our understanding of T cell function and malfunction in disease, and of signal transduction in general. By focusing on druggable genes whose functions can be modulated through small molecule or biological therapeutics, we aim to accelerate the development of new, improved and more cost-effective therapies for severe immune disorders or cancer that affect large patient populations. To devise and validate our innovative, integrated, multidisciplinary approach for gene discovery and analysis, we will first identify novel TCR signaling genes in a high-throughput RNA interference screen in T cells of an arrayed shRNA library that targets 5,000 druggable genes. We will use lentiviral shRNA delivery to overcome the challenges of nonadherent growth and relative T cell resistance to conventional robotic siRNA delivery methods, and novel screen designs and analysis strategies to maximize sensitivity and minimize false positive rates. We will next combine gene expression profiling, phosphotyrosine-proteomics, haplotype association mapping, quantitative trait locus (QTL), genome-wide associtation mapping and gene expression QTL data with gene/protein network and pathway analyses to prioritize those hits which combine highest novelty with maximal in vivo relevance for detailed follow-up.