MDR1 GENE THERAPY IN CD34+ CELLS AND SCID MICE

CD34 细胞和 SCID 小鼠中的 MDR1 基因治疗

基本信息

批准号：
6513024
负责人：
JAMES H DOROSHOW
金额：
$ 30.1万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-07-01 至 2003-12-31
项目状态：
已结题

项目摘要

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.

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蓄积。 MDR1 的过度表达足以赋予其他正常细胞多重耐药性。这表明 MDR1 可用于基因治疗，以保护造血细胞免受化疗相关的骨髓毒性。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 个使用 MDR1 基因。 MDR1 的另一个应用是将其用作体内选择标记，以增强转染或病毒转导细胞中连锁外源基因的表达。小鼠实验表明，MDR1 在体内具有化学保护性和选择性，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。将提供证据表明其在人类体内表现不佳的原因是MDR1是严格的选择标记，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，成功利用 MDR1 进行基因治疗的最大障碍似乎是转导效率和基因表达，即可以转导并且可以表达足够高水平的 MDR1 以在选择中生存的细胞数量。我们推测，只有克服严格性问题，MDR1 才能作为有效的体内选择标记或化学保护基因。设计了四个具体目标来检验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是否可以赋予人类造血细胞体内生存优势。基于具体目标 1-3 的结果，该目标将使用原代人类造血祖细胞来研究 MDR1 转导细胞在 NOD/SCID 和 SCID-hu 小鼠模型系统中的存活情况。