MDR1 SELECTION OF GENE MODIFIED HEMATOPOIETIC CELLS

基因修饰造血细胞的 MDR1 选择

• 批准号: 2684027
• 负责人: Kevin T. McDonagh
• 金额: $ 10.65万
• 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/2684027
• 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.

这项研究计划将集中在人类MDR 1基因作为一个模型， 最大化人造血细胞中的基因表达， 逆转录病毒基因转移 MDR 1基因最终可能被用作 显性标记，以在体内富集基因修饰的细胞，或保护 骨髓细胞免受化疗的毒性。 这两个应用程序 依赖于MDR 1在原始细胞中的高水平表达 干细胞及其定向后代。 动物模型研究表明 MDR逆转录病毒载体赋予细胞耐药性， 移植后造血，但表达减弱的隐性 在载体内拼接。 我们已经开发了第二代载体， 旨在最大化造血细胞中的表达，并将 表征它们在人长期骨髓培养物中的表达 系统 建议的具体目标是：（1）使用现场指导 MDR 1cDNA的突变以使细胞耐药性最大化 逆转录病毒基因转移后;（2）制定一项方案， 原始人造血细胞的可重复和有效的转导 使用骨髓基质支持的逆转录病毒载体的细胞;（3） 建立人类长期骨髓培养模型， MDR逆转录病毒载体在造血祖细胞中的表达;和（4） 确定基于Harvey小鼠的替代MDR逆转录病毒载体是否 肉瘤病毒（HaMSV）和骨髓增生肉瘤病毒（MPSV） 主链将赋予人类更高水平的耐药性， 造血祖细胞在长期骨髓培养。 的 这些研究中获得的信息也将具有更普遍的价值， 设计逆转录病毒载体， 这种特殊的细胞群。