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

项目总结/摘要 许多使人衰弱和代价高昂的疾病与炎症有关，并由巨噬细胞信号传导驱动。 仅类风湿性关节炎（RA）的医疗费用每年就超过223亿美元，而这种无法治愈的疾病 疾病会严重影响患者的生活质量，包括工作能力。RA阻滞的现有治疗方法 巨噬细胞炎性细胞因子释放或功能，从而也抑制抗微生物免疫; 因此，在感染患者中使用这些治疗是有问题的。因此，有一个 迫切需要新的治疗方法，靶向过敏性巨噬细胞有助于RA，而不损害 免疫功能作为一名博士后研究员，我发现了一种巨噬细胞信号传导机制， 蛋白质LynA（但不是密切相关的蛋白质LynB），其仅在炎症部位起作用，如在 RA患者的关节，并且不需要抗微生物免疫。这导致了一个假设， LynA信号传导可能是一种创新的新方法，可以缓解RA和其他炎症性疾病的症状。 疾病而不抑制免疫力。LynA和LynB被认为具有相反的功能，LynA优先于LynB。 LynB刺激细胞活化，但LynB主要启动负调节。剖析他们的独立性 然而，由于现有的林恩-/-小鼠缺乏LynA和LynB，因此很难发挥作用。我们建议克服 这一障碍阻碍了识别LynA和LynB在巨噬细胞信号传导和炎症反应中的真正功能。 通过使用CRISPR/Cas9基因编辑技术产生LynA-/-（仅LynB）和LynB-/-（仅LynA）小鼠来治疗疾病。 nology.然后，我们将进行实验以测试来自这些小鼠的巨噬细胞中的LynA和LynB功能， 在巨噬细胞的炎症环境中诱导抗微生物信号传导和过敏信号传导 缺少LynA或LynB。最后，我们将在缺乏LynA或LynB的小鼠中测试炎症性关节炎的进展。 我们预测，LynA缺失将保护巨噬细胞免受炎症信号的影响，并保护小鼠免受去炎性信号的影响。 抑制严重的炎症性关节炎，这表明抑制LynA信号通路在持续的炎症反应中起作用。 LynB的存在可能是治疗自身炎性疾病的有效策略。通过拟议的 我们的目标是建立LynA，超敏信号和炎症之间的第一个明确的联系。 关节炎，同时生成试剂和数据，以支持竞争R 01应用。LynA-/-和LynB-/-小鼠 将长期用于机械信号传导和磷酸化蛋白质组学实验，以绘制 LynA和LynB在免疫细胞信号传导中的作用这些老鼠将是开始寻找 在RA和其他自身免疫性疾病中靶向LynA通路的新疗法。因为这种创新 治疗策略预计不会损害对病原体的先天免疫，它可以扩大治疗选择 对患者而言，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.