MDR1 SELECTION IN CD34+ CELLS AND SCID-HU MICE

人MDR 1基因编码一种多特异性药物转运蛋白， 防止药物在耐药细胞中积聚。 MDR 1过表达 足以使原本正常的人产生多药耐药性 细胞 这一特点导致了MDR 1可能被用于 基因治疗保护造血细胞免受化疗相关 骨髓毒性 MDR 1在基因治疗中的另一个可能应用是 将其用作体内选择性标记，以增强 转导细胞中连接的外源基因的表达。 可行性 MDR 1介导的基因治疗和本提案中工作的基本原理 基于以下观察：MDR 1是1）当 在小鼠的造血细胞中表达; 2）体内选择标记 其赋予转导的祖细胞以存活优势， 小鼠骨髓移植环境;和3）体外可选择的 可用于过表达转导的异源基因的标记 在与MDR 1相同的载体构建中。 我们是第一个使用 在组织培养细胞系中过表达外源基因的MDR 1选择 我们进一步调整了这个系统，使之适用于逆转录病毒。 我们 现在假设MDR 1也可以作为体内选择标记 用于在人造血细胞中递送和表达外源基因 细胞 提出了三个目标来检验这一假设： 1)完成MDR 1介导的外源基因定量分析 在细胞系中表达。 定量报告基因系统将是 用于测量MDR 1介导的外源基因表达。 假型逆转录病毒将用于提高基因治疗的功效。 转移到造血细胞中。 2)确定MDR 1的能力 作为外源基因在人体内表达的选择标记 造血干细胞 这些实验的目的是 确定MDR 1选择是否能获得稳定的外源基因 在长期再生的造血细胞中表达。 3)确定 体内药物递送对MDR 1介导的化学保护作用的影响， 选择和在SCID-hu小鼠中表达外源基因。 实验 将利用定量报告基因系统研究外源基因 在人造血的相关模型中表达。

The human MDR1 gene encodes a multispecific drug transporter that prevents drug accumulation in resistant cells. Overexpression of MDR1 is sufficient for conferring multidrug resistance on otherwise normal cells. This feature has led to the proposal that MDR1 might be used in gene therapy to protect hematopoietic cells against chemotherapy-related myelotoxicity. Another possible application of MDR1 to gene therapy is to use it as an in vivo selectable marker to enhance the long-term expression of linked foreign genes in transduced cells. The feasibility of MDR1-mediated gene therapy and the rationale for work in this proposal are based on the observations that MDR1 is 1) a chemoprotective gene when expressed in hematopoietic cells of mice; 2) an in vivo selectable marker which confers a survival advantage on transduced progenitor cells in a mouse bone marrow transplantation setting; and 3) an in vitro selectable marker that can be used to overexpress heterologous genes transduced within the same vector construction as MDR1. We were the first to use MDR1 selection to overexpress foreign genes in tissue culture cell lines and we have further adapted this system for use with retroviruses. We now hypothesize that MDR1 can also serve as an in vivo selectable marker for the delivery and expression of foreign genes in human hematopoietic cells. Three aims are proposed to test this hypothesis: 1) Complete a quantitative analysis of MDR1-mediated foreign gene expression in cell lines. A quantitative reporter gene system will be developed for measuring MDR1-mediated foreign gene expression. Pseudotyped retroviruses will be used to increase the efficacy of gene transfer into hematopoietic cells. 2) Determine the ability of MDR1 to act as a selectable marker for foreign gene expression in human hematopoietic stem cells. The purpose of these experiments is to determine whether MDR1 selection works to achieve stable foreign gene expression in long-term repopulating hematopoietic cells. 3) Determine the effects of in vivo drug delivery on MDR1-mediated chemoprotection, selection, and expression of foreign genes in SCID-hu mice. Experiments will use the quantitative reporter gene system to study foreign gene expression in a relevant model of human hematopoiesis.