Generating LynA-/- and LynB-/- mice by CRISPR/Cas9 genome editing

项目摘要/摘要 许多虚弱和昂贵的疾病与炎症有关，并由巨噬细胞信号驱动。 仅类风湿性关节炎(RA)每年的医疗费用就超过223亿美元，而且这是无法治愈的 疾病会严重影响患者的生活质量，包括工作能力。RA传导阻滞的现有治疗方法 巨噬细胞炎性细胞因子的释放或功能，从而也抑制抗菌素免疫； 因此，在感染患者中使用这些治疗方法是有问题的。因此，有一个 迫切需要针对导致类风湿关节炎的超敏巨噬细胞的新疗法 免疫功能。作为一名博士后研究员，我发现了巨噬细胞信号传递的一种机制，通过 LynA蛋白(但不是一种密切相关的蛋白LynB)，它只在炎症部位发挥作用，如在 RA患者的关节，不是抗菌免疫所必需的。这导致了一种假设，即抑制 Lyna信号可能是缓解RA和其他炎症症状的一种创新的新方法 在不抑制免疫力的情况下治疗疾病。Lyna和LynB被认为具有相反的功能，Lyna Pri- 主要是刺激细胞激活，而LynB主要是启动负调控。剖析他们的独立性 然而，角色一直很困难，因为现有的Lyn-/-小鼠既缺乏Lyna又缺乏LynB。我们计划克服 了解LynA和LynB在巨噬细胞信号转导和炎性反应中的真实功能的障碍 通过使用CRISPR/Cas9基因编辑技术产生Lyna-/-(仅LynB)和LynB-/-(仅Lyna-)小鼠而患上疾病 本体学。然后我们将进行实验，测试这些小鼠的巨噬细胞中LynA和LynB的功能， 在炎症环境中诱导巨噬细胞的抗菌信号和超敏信号 缺少Lyna或LynB。最后，我们将测试缺乏Lyna或LynB的小鼠炎性关节炎的进展情况。 我们预测，Lyna缺失将保护巨噬细胞免受炎症信号的影响，并保护小鼠免受去甲基化的影响。 发展为严重的炎症性关节炎，表明Lyna信号通路的抑制在 LynB的存在可能是治疗自身炎症性疾病的有效策略。通过建议的 我们的目标是在Lyna、超敏信号转导和炎症之间建立第一个明确的联系 关节炎，同时产生试剂和数据，以支持竞争性的R01应用。Lyna-/-和LynB-/-小鼠 将对机械信号转导和磷酸蛋白质组学实验有长期的效用，以确定其独特的作用 Lyna和LynB在免疫细胞信号中的作用。这些鼠标将是开始搜索的有用资源 针对类风湿性关节炎和其他自身免疫性疾病的Lyna通路的新疗法。因为这项创新 治疗策略预计不会削弱对病原体的天然免疫力，它可能会扩大治疗选择 对于患者，使长期抑制治疗更安全，并防止疾病复发。

Project Summary/Abstract Many debilitating and costly diseases are associated with inflammation and driven by macrophage signaling. Medical costs attributable to rheumatoid arthritis (RA) alone exceed $22.3 billion annually, and this incurable disease can severely impact patient quality of life, including the ability to work. Existing treatments for RA block macrophage inflammatory cytokine release or function and thereby also suppress antimicrobial immunity; the use of these treatments in patients suffering from infections is therefore problematic. Consequently, there is a pressing need for new therapies that target hypersensitive macrophages contributing to RA without impairing immune function. As a postdoctoral researcher I discovered a mechanism of macrophage signaling, through the protein LynA (but not a closely related protein, LynB), which functions only at sites of inflammation, as in the joints of RA patients, and is not required for antimicrobial immunity. This has led to the hypothesis that inhibition of LynA signaling could be an innovative new approach to relieving the symptoms of RA and other inflammatory diseases without suppressing immunity. LynA and LynB are thought to have opposing functions, with LynA pri- marily stimulating cell activation but LynB primarily initiating negative regulation. Dissecting their independent roles has been difficult, however, because existing Lyn-/- mice lack both LynA and LynB. We propose to overcome this obstacle to discerning the true functions of LynA and LynB in macrophage signaling and in inflammatory disease by generating LynA-/- (LynB-only) and LynB-/- (LynA-only) mice using CRISPR/Cas9 gene editing tech- nology. We will then perform experiments to test LynA and LynB function in macrophages from these mice, inducing antimicrobial signaling and hypersensitive signaling in an inflammatory environment in macrophages lacking LynA or LynB. Finally, we will test the progression of inflammatory arthritis in mice lacking LynA or LynB. We predict that LynA deletion will protect macrophages from inflammatory signaling and protect mice from de- veloping severe inflammatory arthritis, suggesting that inhibition of the LynA signaling pathway in the continued presence of LynB may be an effective strategy for treating autoinflammatory disease. Through the proposed approaches we aim to build the first definitive link between LynA, hypersensitive signaling, and inflammatory arthritis, while generating reagents and data to support a competitive R01 application. LynA-/- and LynB-/- mice will have long-term utility for mechanistic signaling and phosphoproteomics experiments to map unique roles of LynA and LynB in immune-cell signaling. These mice will be useful resources in which to begin the search for novel therapeutics targeting the LynA pathway in RA and other autoimmune diseases. Because this innovative therapeutic strategy is not expected to impair innate immunity to pathogens, it could expand treatment options for patients, make long-term suppressive treatment safer, and prevent disease recurrence.