MDR1 SELECTION OF GENE MODIFIED HEMATOPOIETIC CELLS

基因修饰造血细胞的 MDR1 选择

• 批准号: 2900072
• 负责人: Kevin T. McDonagh
• 金额: $ 11.96万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900072
• 关键词: Retroviridae; bone marrow; gene expression; genes; genetic transduction; hematopoietic stem cells; human tissue; mouse sarcoma virus; multidrug resistance; site directed mutagenesis; tissue /cell culture

This research proposal will focus on the human MDR 1 gene as a model for maximizing gene expression in human hematopoietic cells following retroviral gene transfer. The MDR 1 gene may ultimately be used as a dominant marker to enrich for gene modified cells in vivo, or to protect bone marrow cells from the toxicity of chemotherapy. Both applications are dependent on achieving high level expression of MDR 1 in primitive stem cells and their committed progeny. Studies in animal models indicate that MDR retroviral vectors confer drug resistance to cells reconstituting hematopoiesis after transplant, but expression is attenuated by cryptic splicing within the vector. We have developed second generation vectors, designed to maximize expression in hematopoietic cells, and will characterize their expression in a human long-term bone marrow culture system. The specific aims of the proposal are: (1) to use site directed mutagenesis of the MDR 1 cDNA to maximize cellular drug resistance following retroviral gene transfer; (2) to develop a protocol for reproducible and efficient transduction of primitive human hematopoietic cells with retroviral vectors using bone marrow stromal support; (3) to develop a human long-term bone marrow culture model to examine expression of MDR retroviral vectors in hematopoietic progenitors; and (4) to determine if alternative MDR retroviral vectors based on Harvey murine sarcoma virus (HaMSV) and myeloproliferative sarcoma virus (MPSV) backbones will confer higher levels of drug resistance to human hematopoietic progenitors in long-term bone marrow culture. The information gained in these studies will also have more general value in designing retroviral vectors that direct high level gene expression in this specialized cell population.

这项研究计划将重点放在人类MDR-1基因上，作为 基因在人造血细胞中的最大表达 逆转录病毒基因转移。MDR-1基因最终可能被用作 显性标记在体内为基因修饰细胞丰富，还是为了保护 骨髓细胞的毒性来自于化疗。两个应用程序 依赖于在原始细胞中实现MDR-1的高水平表达 干细胞及其承诺的后代。对动物模型的研究表明 多药耐药逆转录病毒载体对重组细胞产生耐药性 移植后的造血，但表达被隐蔽的减弱 在矢量中拼接。我们已经开发了第二代载体， 旨在最大化在造血细胞中的表达，并将 鉴定它们在人长期骨髓培养中的表达 系统。该提案的具体目标是：(1)使用定向站点 诱变MDR-1基因以最大限度提高细胞耐药性 在逆转录病毒基因转移后；(2)制定一项方案，用于 原始人类造血细胞的可重复性和高效转导 使用骨髓基质支持的逆转录病毒载体的细胞； 建立人骨髓长期培养模型以检测其表达 多药耐药逆转录病毒载体在造血祖细胞中的表达；及 确定基于Harvey小鼠的替代MDR逆转录病毒载体 肉瘤病毒(HaMSV)和骨髓增生性肉瘤病毒(MPSV) 脊椎骨将赋予人类更高水平的抗药性 骨髓长期培养中的造血祖细胞。这个 从这些研究中获得的信息也将在 设计逆转录病毒载体，指导基因的高水平表达 这种特殊的细胞群体。