MDR1 GENE THERAPY IN CD34+ CELLS AND SCID MICE

人类mdr1基因编码一种多特异性药物转运蛋白P-糖蛋白(Pgp)，可防止药物在耐药细胞中积累。MDR1的过度表达足以使正常细胞产生多药耐药。这提示MDR1可能用于基因治疗，以保护造血细胞免受化疗相关的骨髓毒性。在目前有效的164项与癌症相关的基因治疗试验中，9项纳入了造血细胞化学保护的概念；其中6项使用了mdr1基因。MDR1的另一个应用是将其用作体内可选择的标记，以增强转基因或病毒转导细胞中连接的外源基因的表达。小鼠实验表明，mdr1在体内可以具有化学保护性和选择性，但到目前为止，在人类基因治疗试验中将mdr1用作化学保护剂的尝试令人失望。有证据表明，mdr1在人类体内表现不佳的原因是mdr1是一种严格的可选择标记，需要非常高水平的mdr1基因产物P-糖蛋白来调节转导细胞的存活。事实上，mdr1基因治疗成功的最大障碍似乎是转导效率和基因表达--即可以转导的、能够表达足够高水平mdr1的细胞的数量，使其在选择中存活。我们假设，只有克服了严格的问题，mdr1才能作为体内有效的可选择标记或化学保护性基因。我们设计了四个特定的目标来验证这一假设，并开发出克服选择严格的最佳策略：1)确定是否可以通过使用最先进的基因治疗工具最大化基因转导效率和基因表达水平来克服MDR1选择严格。2)确定是否可以通过使用mdr1/pgp的突变版本作为选择标记来克服选择的严格性。3)确定是否可以使用两步选择策略来克服选择的严格性。4)确定多药耐药基因能否在体内赋予人造血细胞生存优势。在特定目标1-3的基础上，该目标将使用原代人类造血祖细胞在NOD/SCID和SCID-HU小鼠模型系统中研究MDR1转导细胞的存活。

The human MDR1 gene encodes a multispecific drug transporter, P- glycoprotein (Pgp), that prevents drug accumulation in resistant cells. Overexpression of MDR1 is sufficient for conferring multidrug resistance on otherwise normal cells. This suggests that MDR1 might be used in gene therapy to protect hematopoietic cells against chemotherapy-related myelotoxicity. Of 164 cancer- related gene therapy trials currently in force, nine incorporate the concept of hematopoietic cell chemoprotection; six of these use the MDR1 gene. Another application of MDR1 is to use it as an in vivo selectable marker to enhance the expression of linked foreign genes in transfected or virally transduced cells. Mouse experiments indicate that MDR1 can be chemoprotective and selectable in vivo, but attempts to use MDR1 as a chemoprotective agent in human gene therapy trials have, so far, been disappointing. Evidence will be provided suggesting that the reason for its poor in vivo performance in humans is that MDR1 is a stringent selectable marker that requires very high levels of P-glycoprotein, the MDR1 gene product, to mediate survival of transduced cells. Indeed, the most significant barriers to successful gene therapy with MDR1 appear to be transduction efficiency and gene expression--i.e., the number of cells that can be transduced and that can express high enough levels of MDR1 to survive selection. We hypothesize that MDR1 will serve as an effective in vivo selectable marker or chemoprotective gene only if the problem of stringency can be overcome. Four specific aims are designed to test this hypothesis and to develop the optimal strategy for overcoming selection stringency: 1) Determine if MDR1 selection stringency can be overcome by maximizing gene transduction efficiency and gene expression levels with state-of- the-art gene therapy tools. 2) Determine if selection stringency can be overcome by using mutant versions of MDR1/Pgp as the selectable marker. 3) Determine if selection stringency can be overcome by using a two-step selection strategy. 4) Determine if MDR1 can confer an in vivo survival advantage on human hematopoietic cells. Building on results in Specific Aims 1-3, this aim will use primary human hematopoietic progenitors to study the survival of MDR1-transduced cells in NOD/SCID and SCID- hu mouse model systems.