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（吞噬细胞特异性细胞色素B的亚单位）的基因 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 GP91/PHOX 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 提出的研究计划，逆转录病毒载体含有gp 91 cDNA 将利用他人先前展示的设计进行准备， 转移的基因序列在体内的长期表达。 这些 将测试载体赋予功能表达的能力 gp 91/phox在人X-CGD造血干细胞和祖细胞中的表达。 在 除了骨髓细胞外，从骨髓中分离出的造血前体细胞 外周血将被评估为基因转移的靶， 作为转导前离体扩增的候选物。 协议 将对用于有效转导骨髓细胞的开发进行修改， 如果需要，用于外周血细胞靶。 基因转移和 表达将首先使用体外克隆形成测定来研究 干细胞和祖细胞。 人-羊异种移植模型将 用于评估转导的靶细胞的长期体内骨 骨髓增殖能力和功能表达 重组gp 91/phox在成熟吞噬白细胞中的表达。 概述的工作 应有助于临床方案的制定 使用逆转录病毒介导的基因转移作为X- CGD等造血干细胞单基因缺陷。 更 总的来说，这些研究应该增加如何引入 造血干细胞的基因修饰， 保持自我更新和多潜能性。

X-linked chronic granulomatous disease (X-CGD) arises from defects in the gene encoding gp91/phox, 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 gp91/phox. The central hypothesis underlying this objective is that retroviral-mediated gene transfer of gp91/phox 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 containing the gp91 cDNA will be prepared utilizing designs previously shown by others to confer long-term expression in vivo of transferred gene sequences. These vectors will be tested for their ability to confer functional expression of gp91/phox 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 of ex vivo expansion prior to transduction. Protocols developed for efficient transduction of marrow cells will be modified, if necessary, for peripheral blood cell targets. Gene transfer and expression will be first investigated using in vitro clonogenic assays of stem and progenitor cells. A human- 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 gp91/phox 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.