GENE THERAPY OF X-LINKED CHRONIC GRANULOMATOUS DISEASE

X连锁慢性肉芽肿性疾病的基因治疗

• 批准号: 6105676
• 负责人: Mary C Dinauer
• 金额: $ 12.08万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-15 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6105676
• 关键词: Retroviridae; biomarker; bone marrow; chronic granulomatous disease; gene expression; gene therapy; genetic transduction; hematopoietic stem cells; hematopoietic tissue transplantation; human tissue; polymerase chain reaction; recombinant proteins; tissue /cell culture; tissue mosaicism; transfection /expression vector; xenotransplantation

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.

X连锁慢性肉芽肿病（X-CGD）是由细胞缺陷引起的 编码 gp91/phox 的基因，吞噬细胞特异性细胞色素 b 的一个亚基 这对于呼吸爆发氧化酶功能至关重要。 做作的 患者缺乏主要的抗菌途径并出现复发性、严重的 感染始于儿童早期。 拟议的目标 研究目的是为基因替代建立实验基础 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 gp91/phox cDNA 基因转移至 X-CGD 造血干细胞将恢复呼吸爆发活性 成熟的吞噬白细胞并纠正宿主防御的缺陷。 在 拟议的研究计划，含有 gp91 cDNA 的逆转录病毒载体 将利用其他人之前展示的设计来准备 转移基因序列的体内长期表达。 这些 将测试载体赋予功能表达的能力 人 X-CGD 造血干细胞和祖细胞中的 gp91/phox。 在 除了骨髓细胞外，还分离出造血前体细胞 外周血将被评估为基因转移的目标，并且 作为转导前离体扩增的候选者。 协议 为有效转导骨髓细胞而开发的技术将被修改， 如有必要，外周血细胞目标。 基因转移和 首先将使用体外克隆实验研究表达 干细胞和祖细胞。 人羊异种移植模型将 用于评估转导靶细胞的长期、体内、骨 骨髓填充能力和功能表达 成熟吞噬白细胞中的重组 gp91/phox。 工作概述 在这个子项目中应该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- 的治疗策略 CGD和其他造血干细胞的单基因缺陷。 更多的 从广义上讲，这些研究应该增加如何引入 对造血干细胞进行特定的基因修饰，同时 保持自我更新和多重潜力。