Molecular Pathogenesis of Severe Congential Neutropenia

严重先天性中性粒细胞减少症的分子发病机制

• 批准号: 7473927
• 负责人: Daniel C Link
• 金额: $ 32.64万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7473927
• 关键词: Acute leukemia; Allogeneic Bone Marrow Transplantation; Amino Acids; Animal Model; Arts; Attenuated; Bacterial Artificial Chromosomes; Bone Marrow; Bone Marrow Transplantation; CSF3 gene; Cell Line; Cells; Chronic; Clinical; Count; Cultured Cells; Data; Differentiation and Growth; Disease; Gene Expression; Genes; Genetic; Granulopoiesis; Hematopoiesis; Human; Infection; Inherited; Knock-in Mouse; Lead; Leukocyte Elastase; Mediating; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Myelogenous; Myeloid Cells; Neutropenia; Pathogenesis; Patients; Phenotype; Physiological; Predisposition; Proteins; RNA Interference; RNAi vector; Recurrence; Research Personnel; Sampling; Series; Syndrome; Testing; Transgenic Mice; Transgenic Organisms; Xenograft procedure; disulfide bond; gain of function; immortalized cell; in vivo; leukemia; mortality; mouse model; mutant; neutrophil; novel; novel strategies; prevent; progenitor; protein expression; research study; tool

DESCRIPTION (provided by applicant): Severe congenital neutropenia (SCN) is a rare inherited syndrome manifested by impaired granulopoiesis and a marked predisposition to develop acute leukemia. G-CSF treatment, while effective in increasing neutrophil counts in most patients, does not prevent progression to leukemia. Allogeneic bone marrow transplantation, while potentially curative, is associated with significant mortality and morbidity. Thus, there is a clear need for more effective therapies for SCN. There is compelling genetic evidence implicating mutations of the ELA2 gene, encoding neutrophil elastase (NE), as the cause of most cases of SCN. Studies of the molecular mechanisms by which ELA2 mutants induce a block in granulocytic differentiation have been limited by the rarity of SCN and the resulting difficulty in obtaining clinical samples. Instead, most recent studies have focused on ectopic NE expression in immortalized cell lines. However, these cell lines have a limited capacity for differentiation and the level of NE expression may not be physiological. Thus, an accurate animal model of SCN would be an invaluable tool to investigators in this field. We propose to generate transgenic mice in which mutant NE is expressed under the regulatory control of the endogenous ELA2 locus. These mice will be used to explore mechanisms of disease pathogenesis and test novel therapies. A second major objective of this application is to explore the feasibility of RNAi-mediated inhibition of ELA2 gene expression to rescue the block in granulocytic differentiation in primary progenitors from patients with SCN. These studies may ultimately lead to a novel molecularly targeted therapy for SCN. The following specific aims are proposed. 1. We will determine whether expression of mutant NE under the regulatory control of the ELA2 gene locus is sufficient to induce an SCN phenotype in mice. Two complementary state-of-the-art transgenic approaches will be used to generate a mouse model of SCN. 2. We will characterize the effect of RNAi-mediated inhibition of ELA2 gene expression on the growth and differentiation of primary myeloid progenitors. Preliminary data show the feasibility of RNAi to attenuate NE protein expression in cell lines. The efficacy of lentiviral RNAi vectors to inhibit NE protein expression and revert the block in granulocytic differentiation in primary progenitors from patients with SCN will be determined. A novel xenotransplantation model of SCN will be developed to study hematopoiesis in vivo.

描述（由申请人提供）： 严重的先天性中性粒细胞减少症（SCN）是一种罕见的遗传综合征，表现为受损的粒状疾病，并且明显地倾向于发展急性白血病。 G-CSF的治疗虽然有效地增加了大多数患者的中性粒细胞计数，但并不能阻止血清血症发展。 同种异体骨髓移植虽然可能治愈，但与显着的死亡率和发病率有关。 因此，显然需要对SCN进行更有效的疗法。 有令人信服的遗传证据，涉及ELA2基因的突变，编码中性粒细胞弹性酶（NE）是大多数SCN病例的原因。 ELA2突变体在粒细胞分化中诱导块的分子机制的研究受到SCN的罕见性和获得临床样本的困难的限制。 取而代之的是，最近的研究集中在永生细胞系中的异位NE表达。 但是，这些细胞系的分化能力有限，NE表达水平可能不是生理的。因此，SCN的精确动物模型对于该领域的研究者来说将是一个宝贵的工具。 我们建议产生转基因小鼠，其中突变体在内源性ELA2基因座的调节控制下表达。 这些小鼠将用于探索疾病发病机理和测试新疗法的机制。 该应用的第二个主要目的是探索RNAi介导的ELA2基因表达抑制的可行性，以挽救SCN患者一级祖细胞中粒细胞分化中的阻滞。 这些研究最终可能导致一种新型的SCN分子靶向疗法。 提出了以下特定目标。 1。我们将确定在ELA2基因基因座的调节控制下突变NE的表达是否足以诱导小鼠的SCN表型。 将使用两种互补的最先进的转基因方法来生成SCN的鼠标模型。 2。我们将表征RNAi介导的ELA2基因表达抑制对原发性髓样祖细胞生长和分化的影响。初步数据表明RNAi可以减弱细胞系中NE蛋白表达的可行性。将确定慢病毒RNAi载体抑制NE蛋白表达并恢复SCN患者一级祖细胞粒细胞分化的阻滞的功效。 将开发出一种新型SCN的异种移植模型来研究体内造血。