G-CSF in Human Severe Congenital Neutropenia

 描述（由申请人提供）：重度先天性中性粒细胞减少症（SCN）的特征是由于体质遗传缺陷导致的慢性中性粒细胞减少症。超过50%的SCN病例与ELANE编码的中性粒细胞弹性蛋白酶（NE）突变相关，并以粒细胞缺乏伴早幼粒细胞停滞和单核细胞增多为特征。ELANE突变的严重程度和预后是可变的，但如果不治疗，预后通常很差。G-CSF（粒细胞集落刺激因子）已极大地改善了SCN患者亚组的管理，但无法降低恶性肿瘤发生率或恢复存活吞噬细胞的正常杀菌活性。缺乏足够的体外模型的人骨髓细胞生成，缺乏初级造血材料的SCN患者，并缺乏相关的动物模型，复制SCN的生物学阻碍了我们对这种疾病的理解。诱导多能干细胞（iPSC）技术已被提出作为在培养皿中和在特定方案中模拟人类疾病的替代方法，其将概括骨髓生成的大多数细胞和分子特征。基于已发表的和初步的数据，我们假设ELANE突变是通过损害G-CSF/CSF 3R粒细胞生成信号传导导致祖细胞/前体细胞存活和粒细胞分化受损来诱导SCN所必需的和足够的。在这个项目中，我们将利用人类骨髓细胞系统来分析ELANE突变导致粒细胞缺乏症和单核细胞增多症的机制。我们将首先分析ELANE突变是否足以和/或必需通过最先进的CRISPR/Cas9技术诱导中性粒细胞减少症，并沿着敲入/沉默表达方法开发等基因iPSC系，并使用标准化方法从患者和对照iPSC产生体外骨髓生成。第二，我们将使用细胞和分子信号传导分析和对离体产生的iPSC衍生的髓样祖细胞的拯救实验，分析突变NE对髓样祖细胞和前体细胞中的G-CSF/CSF 3R信号传导途径的影响。最后，我们将分析ELANE突变对髓系祖细胞非细胞自主炎症信号传导的影响，以探索单核细胞增多症的潜在基础。这一提议不仅将为ELANE突变如何导致功能障碍性粒细胞生成和SCN提供关键见解，而且还将揭示控制正常人类粒细胞生成的分子机制。这些研究还将确定预测SCN对G-CSF治疗反应性的疾病相关信号通路，并提供SCN替代疗法的概念验证。

 DESCRIPTION (provided by applicant): Severe congenital neutropenia (SCN) is characterized by chronic neutropenia due to a constitutional genetic defect. Over 50% of SCN cases are associated with mutations in neutrophil elastase (NE) coded by ELANE, and are characterized by agranulocytosis with promyelocytic arrest and monocytosis. The severity and prognosis of ELANE mutations is variable but if untreated, the prognosis is generally poor. G-CSF (granulocyte colony-stimulating factor) has vastly improved the management of a subgroup of SCN patients, but has been unable to reduce malignancy rates or restore normal bactericidal activity of surviving phagocytes. The absence of adequate in vitro models of human myelopoiesis, scarcity of primary hematopoietic material from SCN patients, and lack of relevant animal models that reproduce the biology of SCN have hampered progress in our understanding of this disease. Induced pluripotent stem cells (iPSC) technologies have been proposed as an alternative method to model human disease in the culture dish and in specific protocols, which would recapitulate the majority of the cellular and molecular characteristics of myelopoiesis. Based on published and preliminary data, we hypothesize that ELANE mutations are necessary and sufficient to induce SCN through impairment of the G-CSF/CSF3R granulopoietic signaling resulting in impaired progenitor/precursor survival and granulocytic differentiation. In this project, we will exploit human myeloid cell systems to analyze mechanisms by which ELANE mutations result in agranulocytosis and monocytosis. We will first analyze whether ELANE mutations are sufficient and/or necessary to induce neutropenia through state-of-the-art CRISPR/Cas9 technology and development of isogenic iPSC lines along with knock-in/silencing expression methods and use of standardized methods to generate in vitro myelopoiesis from patient and control iPSC. Second, we will analyze the impact of mutant NE upon G-CSF/CSF3R signaling pathways in myeloid progenitors and precursors, using cellular and molecular signaling analysis and rescue experiments on ex vivo generated iPSC-derived myeloid progenitors. Finally, we will analyze the consequences of ELANE mutations on non-cell-autonomous inflammatory signaling on myeloid progenitors to explore the underlying basis of monocytosis. This proposal will not only provide key insights into how ELANE mutations result in dysfunctional granulopoiesis and SCN, but also reveal molecular mechanisms controlling normal human granulopoiesis. These studies will additionally identify disease relevant signaling pathways that predict responsiveness of SCN to G-CSF therapy and provide proof-of-concept on alternative therapies for SCN.