G-CSF in Human Severe Congenital Neutropenia

G-CSF 在人类严重先天性中性粒细胞减少症中的应用

• 批准号: 9099909
• 负责人: Jose A Cancelas
• 金额: $ 30.81万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099909
• 关键词: Agranulocytosis; Alternative Therapies; Animal Model; Biology; CSF3 gene; Cell Death; Cell Differentiation process; Cell Line; Cell model; Cessation of life; Characteristics; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Colony-Stimulating Factor Therapy; Constitutional; Cytokine Signaling; Data; Development; Disease; Disease model; Dose; Down-Regulation; Endoplasmic Reticulum; Failure; Gene Targeting; Genotype; Goals; Granulocyte Colony-Stimulating Factor; Granulocyte Colony-Stimulating Factor Receptors; Granulopoiesis; Health; Hematopoietic; Heterozygote; Human; Impairment; In Vitro; Inflammatory; Knock-in; Leukocyte Elastase; Ligands; Macrophage Colony-Stimulating Factor; Malignant Neoplasms; Mediating; Methods; Modeling; Molecular; Monocytosis; Mutation; Myelogenous; Myeloid Cells; Myelopoiesis; Neutropenia; North America; Pathogenesis; Patients; Phagocytes; Phenotype; Phosphotransferases; Point Mutation; Population; Progranulocytes; Proteins; Protocols documentation; Publishing; Receptor Signaling; Role; Severities; Signal Pathway; Signal Transduction; Subgroup; System; Therapeutic Intervention; Up-Regulation; bactericide; base; biological adaptation to stress; cytokine; granulocyte; human disease; improved; in vitro Model; induced pluripotent stem cell; insight; monocyte; mutant; neutrophil; neutrophil elastase inhibitor; novel; outcome forecast; progenitor; receptor binding; repaired; research study; response; small molecule; stem cell technology; technology development; tool

 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.

 描述（由适用提供）：严重的先天性中层（SCN）的特征是由于宪法遗传缺陷而导致慢性中部减少症。超过50％的SCN病例与通过Elane编码的嗜中性粒细胞弹性酶（NE）突变有关，其特征是具有临床细胞停滞和单核细胞增多症的肿瘤细胞增多症。浮力突变的严重程度和预后是可变的，但如果未经治疗，预后通常很差。 G-CSF（粒细胞菌落刺激因子）已大大改善了SCN患者亚组的治疗，但无法降低恶性率或恢复生存吞噬细胞的正常细菌活性。缺乏足够的人脊髓病的体外模型，SCN患者的原发性造血物质的稀缺性以及缺乏再现SCN生物学的相关动物模型阻碍了我们对这种疾病的理解。已经提出了诱导多能干细胞（IPSC）技术作为在培养皿中和特定方案中建模人类疾病的替代方法，这将概括髓粉的大多数细胞和分子特征。根据已发表和初步数据，我们假设通过损害G-CSF/CSF/CSF3R粒状信号传导，Elane突变是必要和足够的，足以诱导SCN，我们将利用人类髓细胞系统来通过哪种善良的突变来通过哪种质虫突变来分析机制。我们将首先分析Elane突变是否足够和/或必要，以通过最先进的CRISPR/CAS9技术以及ISEGENIC IPSC线的开发以及敲入/沉默的表达方法以及使用标准化方法来生成患者和控制IPSC的体外骨髓疫粒。其次，我们将使用细胞和分子信号分析和救援实验对髓样祖细胞和前体中的G-CSF/CSF3R信号通路的影响分析突变体对G-CSF/CSF3R信号通路的影响。最后，我们将分析烯烃突变对髓样祖细胞对非细胞自主炎症信号传导的后果，以探索单核细胞增多症的潜在基础。该提案不仅将提供有关烯烃突变如何导致功能障碍粒状和SCN的关键见解，而且还揭示了控制正常人颗粒的分子机制。这些研究还将确定疾病相关的信号通路，以预测SCN对G-CSF疗法的反应性，并为SCN的替代疗法提供概念概念。