GENE THERAPY OF X-LINKED CHRONIC GRANULOMATOUS DISEASE

X连锁慢性肉芽肿性疾病(X-CGD)源于 编码吞噬细胞特异性细胞色素b亚单位gp91/Phox的基因 这对呼吸爆发氧化酶的功能是必不可少的。受影响 患者缺乏主要的抗菌途径，并出现复发、严重 从儿童早期开始的感染。建议的目标是 研究是为了建立基因替换的实验基础 复制缺陷逆转录病毒治疗X-CGD的研究 Gp91/Phox的同源性。这一目标背后的中心假设是 逆转录病毒介导的gp91/Phox基因转移至X-CGD 造血干细胞将恢复呼吸爆发活性 成熟吞噬白细胞，纠正宿主防御缺陷。在……里面 建议的研究计划，含有gp91基因的逆转录病毒载体 将利用其他人之前展示的设计来准备授予 转移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 人X-CGD造血干/祖细胞中gp91/Phox的表达。在……里面 除骨髓细胞外，从 外周血将被评估为基因转移的目标，还 作为转导前体外扩增的候选基因。协议 为有效地转导骨髓细胞而开发的将被修改， 如有必要，可用于外周血细胞靶标。基因转移和 将首先使用体外克隆试验来研究表达情况 干细胞和祖细胞。一个人-羊异种移植模型将是 用于评估体内长期骨转导的靶细胞 骨髓增殖能力及其功能表达的研究 重组gp91/Phox在成熟吞噬细胞中的表达。概述的工作 在这个子项目中应有助于临床方案的制定 利用逆转录病毒介导的基因转移作为治疗X- CGD等单基因缺陷的造血干细胞。更多 总的来说，这些研究应该增加关于如何引入 在对造血干细胞进行特定的基因修饰时 保持自我更新和多潜能。

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.