GENE THERAPY OF X-LINKED CHRONIC GRANULOMATOUS DISEASE

X-连锁慢性肉芽肿病（X-CGD）是由于 编码gp 91 phox的基因，gp 91 phox是吞噬细胞特异性细胞色素B的亚单位 这是呼吸爆发氧化酶功能所必需的。 影响 患者缺乏主要的抗微生物途径， 从幼儿期开始感染。 建议的目标 研究是为基因替换建立实验基础 使用复制缺陷型逆转录病毒进行表达的X-CGD治疗 GP 91 PHOX的 这一目标的核心假设是， 逆转录病毒介导的gp 91 phox基因转移到X-CGD中 造血干细胞将恢复呼吸爆发活性， 成熟的吞噬白细胞和纠正宿主防御的缺陷。 在 根据拟议的研究计划，将利用 其他人所示的设计赋予在体内的长期表达， 转移的基因序列。 X-CGD的小鼠模型， 最近开发的基因靶向技术将用于检查 逆转录病毒介导的gp 91 phox表达和呼吸爆发氧化酶 功能，并评估基因替代疗法的影响 对X-CGD表型的影响 有希望的载体也将被测试其 在人X-CGD中赋予gp 91 phox功能性表达的能力 造血干细胞和祖细胞。 除了骨髓 细胞，从外周血中分离的造血前体将被 作为基因转移的靶标以及作为离体免疫治疗的候选物进行评估。 在转导之前进行扩增。 为高效的 如有必要，将修改骨髓细胞的转导， 外周血细胞靶点。 除了体外集落测定外， 将使用人多于绵羊的异种移植物模型来评估转导的 靶细胞用于长期体内骨髓增殖能力 重组gp 91 phox在成熟大肠杆菌中的功能性表达 吞噬性白细胞 本子项目中概述的工作应有助于 在使用逆转录病毒介导的基因的临床方案的开发中， 转移作为X-CGD和其他单基因缺陷的治疗策略 造血干细胞 更广泛地说，这些研究应该增加 如何将特定的遗传修饰引入 造血干细胞，同时保持自我更新， 多潜能性

X-linked chronic granulomatous disease (X-CGD) arises from defects in the gene encoding gp91phox, a subunit of a phagocyte-specific cytochrome b that is essential for respiratory burst oxidase function. Affected patients lack a major antimicrobial pathway and develop recurrent, severe infections beginning in early childhood. The objective of the proposed research is to establish an experimental basis for gene replacement therapy of X-CGD using replication-defective retroviruses for expression of gp91phox. The central hypothesis underlying this objective is that retroviral-mediated gene transfer of gp91phox cDNA into X-CGD hematopoietic stem cells will restore respiratory burst activity in mature phagocytic leukocytes and correct the defect in host defense. In the proposed research plan, retroviral vectors will be prepared utilizing designs shown by others to confer long-term expression in vivo of transferred gene sequences. A mouse model of X-CGD that has been recently developed by gene targeting will be utilized to examine retroviral-mediated gp91phox expression and respiratory burst oxidase function in vivo and to evaluate the impact of gene replacement therapy on the X-CGD phenotype. Promising vectors will also be tested for their ability to confer functional expression of gp91phox in human X-CGD hematopoietic stem and progenitor cells. In addition to bone marrow cells, hematopoietic precursors isolated from peripheral blood will be evaluated as targets for gene transfer and also as candidates for ex vivo expansion prior to transduction. Protocols developed for efficient transduction of marrow cells will be modified, if necessary, for peripheral blood cell targets. In addition to in vitro colony assays, a human-more than sheep xenograft model will be used to assess transduced target cells for long term, in vivo, bone marrow populating capabilities and the functional expression of recombinant gp91phox in mature phagocytic leukocytes. The work outlined in this subproject should aid in the development of clinical protocols using retroviral-mediated gene transfer as a therapeutic strategy in X-CGD and other single-gene defects of hematopoietic stem cells. More broadly, these studies should add to knowledge of how to introduce specific genetic modifications into hematopoietic stem cells while maintaining self-renewal and multipotentiality.