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gp91Phox的基因，是吞噬细胞特异性细胞色素b的一个亚单位 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 Gp91Phox基因。这一目标背后的中心假设是 逆转录病毒介导的gp91Phox基因转入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 拟议的研究计划，逆转录病毒载体将利用 其他人展示的设计，以在体内长期表达 转移的基因序列。X-CGD的小鼠模型已经被 新近开发的基因打靶技术将被用于检测 逆转录病毒介导的gp91Phox表达与呼吸爆发氧化酶 体内功能及评价基因替代治疗的影响 关于X-CGD表型。有希望的载体也将被测试其 Gp91Phox在人X-CGD中的功能表达能力 造血干细胞和祖细胞。除了骨髓 从外周血中分离出的细胞、造血祖细胞 被评估为基因转移的目标，也是体外实验的候选对象 转导前的扩张。为高效而开发的协议 如有必要，骨髓细胞的转导将被修改，用于 外周血细胞靶标。除了体外菌落分析外， 将使用超过羊的人-异种移植模型来评估转导 靶细胞具有长期、体内、骨髓增殖的能力 重组gp91Phox在成熟期的功能表达 吞噬白细胞。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 移植作为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 了解如何将特定的基因修改引入 造血干细胞在维持自我更新和 多面性。

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.