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的过表达足以使正常细胞产生多药耐药。这表明MDR1可能用于基因治疗，以保护造血细胞免受化疗相关的髓毒性。目前正在进行的164项癌症相关基因治疗试验中，有9项纳入了造血细胞化学保护的概念；其中6种使用MDR1基因。MDR1的另一个应用是将其作为体内选择性标记物，在转染或病毒转导的细胞中增强连接外源基因的表达。小鼠实验表明，MDR1在体内具有化学保护作用和选择性，但迄今为止，在人类基因治疗试验中使用MDR1作为化学保护剂的尝试令人失望。将有证据表明，其在人体内表现不佳的原因是MDR1是一种严格的可选择标记物，需要非常高水平的p糖蛋白（MDR1基因产物）来介导转导细胞的存活。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。即能够转导并能够表达足够高水平的MDR1以在选择中存活下来的细胞数量。我们假设，只有克服严格性的问题，MDR1才能成为有效的体内选择标记或化学保护基因。设计了四个特定目标来验证这一假设并制定克服选择严格性的最佳策略：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选择严格性。2)确定使用MDR1/Pgp突变体作为选择标记是否可以克服选择严格性。3)确定是否可以通过使用两步选择策略来克服选择严格性。4)确定MDR1是否能赋予人造血细胞体内生存优势。在Specific Aims 1-3的基础上，本目标将使用人类造血祖细胞研究mdr1转导细胞在NOD/SCID和SCID- hu小鼠模型系统中的存活情况。

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.