MDR1 SELECTION IN CD34+ CELLS AND SCID-HU MICE

人类多药耐药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累。Mdr1基因的过表达 足以使原本正常的人产生多药耐药性 细胞。这一功能导致了MDR1可能被用在 基因治疗对化疗相关造血细胞的保护作用 骨髓毒性。MDR1在基因治疗中的另一个可能的应用是 将其作为体内可选择的标志物，以增强长期 连锁外源基因在转导细胞中的表达。可行性 MDR1介导的基因治疗的基本原理和该提案中工作的基本原理 是基于观察到mdr1是1)化学保护性基因，当 在小鼠的造血细胞中表达；2)体内可选择的标记 这为转导的祖细胞提供了生存优势 小鼠骨髓移植环境；3)体外可选择的 可用于过表达转导的外源基因的标记 在与mdr1相同的载体结构中。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MDR1选择 我们还进一步调整了这一系统，使其适用于逆转录病毒。我们 现在假设mdr1也可以作为体内可选择的标记 用于外源基因在人造血系统中的传递和表达 细胞。为了检验这一假设，我们提出了三个目标： 1)完成mdr1介导的外源基因的定量分析 在细胞系中表达。一个定量的报告基因系统将是 专为测量MDR1介导的外源基因表达而开发。 伪型逆转录病毒将用于提高基因的疗效 转移到造血细胞。2)确定MDR1的能力 作为外源基因在人类中表达的可选择标记 造血干细胞。这些实验的目的是 确定MDR1选择是否有效以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 体内给药对MDR1介导的化学保护的影响 外源基因的筛选及其在SCID-HU小鼠中的表达。实验 将利用定量报告基因系统研究外源基因 在人类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.