BIOLOGY OF PERIPHERAL BLOOD PROGENITORS--A TARGET FOR GENE TRANSFER

外周血祖细胞的生物学——基因转移的目标

• 批准号: 3746626
• 负责人: H L MALECH
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3746626
• 关键词: Actinomycetales infection; Retroviridae; adolescence (12-20); cell differentiation; child (0-11); chronic granulomatous disease; colony stimulating factor; eosinophil; flow cytometry; gene therapy; hematopoietic stem cells; human subject; leukapheresis; monocyte; neutrophil; tissue /cell culture; transfection /expression vector; tuberculosis

This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy of inherited diseases affecting the function of human phagocytic cells, including neutrophils,eosinophils, and monocytes. A related goal of this project is development of novel cellular therapies based on genetically engineering human PBHP and their phagocytic cell progeny to endow them with new properties to augment host defense against chronic infections including tuberculosis and other mycobacteria infections. To achieve these goals it is necessary to define conditions for optimal harvest, purification and culture of the primitive human hematopoietic cells with a CD34 surface antigen phenotype. In a clinical study we determined that 5 ug/kg daily subcutaneous granulocyte colony stimulating factor administered for 5 or 6 daily doses is optimal for recruiting a 20 to 100 fold increase in circulating primitive CD34 progenitor cells. Using apheresis and magnetic bead purification technology over 100 million purified CD34 cells can be harvested from G-CSF recruited volunteers. These cells can be used as a target for gene transfer studies. The recruited cells are more receptive to retrovirus mediated gene transfer than similar CD34 cells from volunteers not treated with G-CSF. In related studies we find that the combination of the growth factors SCF, IL3 and IL6 used to culture these cells preserves the CD34+ primitive phenotype longest in culture. We developed specific culture conditions leading to predominantly neutrophils, eosinophils or monocyte/macrophages. This will be important for future studies aimed at engineering new characteristics into specific end stage phagocytes by gene transfer into progenitors. As part of our studies to achieve gene therapy for the inherited immunodeficiency, chronic granulomatous disease (CGD), a sensitive FACS assay of superoxide/hydrogen peroxide production by mature neutrophils was developed based on the oxidation induced increase in fluorescence of rhodamine-123. Using this assay it was possible to follow the maturation of functional oxidase as a marker of the appearance and number of maturing neutrophils over time in cultures of PBHP. This information is essential for planning and evaluating studies in which genes are transferred into CGD progenitors to correct NADPH oxidase activity as gene therapy for CGD.

该项目研究外周血造血祖细胞（PBHP） 作为影响该功能的遗传疾病基因治疗的靶标 人吞噬细胞，包括中性粒细胞，嗜酸性粒细胞和 单核细胞。 该项目的相关目标是开发新颖 基于基因工程人PBHP及其的细胞疗法 吞噬细胞的后代，以增强宿主的新特性赋予它们 防御慢性感染，包括结核病和其他 分枝杆菌感染。 为了实现这些目标，有必要 定义最佳收获，纯化和培养条件 原始的人类造血细胞具有CD34表面抗原 表型。 在一项临床研究中，我们确定每天5 ug/kg 施用5或 每天6剂对于招募20至100倍的增加是最佳的 循环原始CD34祖细胞。 使用放置和 磁珠纯化技术超过1亿纯CD34 可以从G-CSF招募的志愿者那里收集细胞。 这些细胞可以 用作基因转移研究的靶标。 招募的细胞是 比相似的CD34更接受逆转录病毒介导的基因转移 未用G-CSF处理的志愿者的细胞。 在相关研究中，我们发现 生长因子的组合SCF，IL3和IL6曾经 培养这些细胞保留CD34+原始表型最长 文化。 我们开发了特定的文化条件，导致 主要是中性粒细胞，嗜酸性粒细胞或单核细胞/巨噬细胞。 这 对于旨在工程新的研究将很重要 通过基因转移到特定的终端吞噬细胞中的特征 祖先。 作为我们实现基因疗法的研究的一部分 遗传性免疫缺陷，慢性肉芽肿性疾病（CGD），A 敏感的FACS测定超氧化物/过氧化氢的产生 根据氧化诱导的增加而开发成熟的中性粒细胞 在若丹明123的荧光中。 使用此测定法是可能的 遵循功能氧化酶作为外观标记的成熟 PBHP培养物随着时间的流逝，中性粒细胞的成熟数量。 这 信息对于计划和评估研究至关重要 将基因转移到CGD祖细胞中以纠正NADPH氧化酶 活性作为CGD的基因治疗。