MDR1 GENE THERAPY IN CD34+ CELLS AND SCID MICE

CD34 细胞和 SCID 小鼠中的 MDR1 基因治疗

• 批准号: 2911359
• 负责人: SUSAN E KANE
• 金额: $ 27.55万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2911359
• 关键词: CD34 molecule; P glycoprotein; SCID mouse; breast neoplasms; chemoprevention; cytoprotection; female; gene expression; gene therapy; genetic markers; genetic transduction; hematopoietic stem cells; human tissue; multidrug resistance; neoplasm /cancer therapy; ovary neoplasms; technology /technique development; tissue /cell culture; transfection /expression vector

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MDR 1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。 MDR 1的过表达足以使其他正常细胞产生多药耐药性。 这表明，MDR 1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项使用了MDR 1基因。 MDR 1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接的外源基因的表达。 小鼠实验表明，MDR 1可以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MDR 1作为化学保护剂的尝试令人失望。 将提供的证据表明，其在人体内表现不佳的原因是，MDR 1是一种严格的选择性标记，需要非常高水平的P-糖蛋白，MDR 1基因产物，介导转导细胞的存活。 事实上，用MDR 1成功进行基因治疗的最重要障碍似乎是转导效率和基因表达--即，可以被转导并且可以表达足够高水平的MDR 1以在选择中存活的细胞的数量。 我们假设，MDR 1将作为一个有效的体内选择标记或化学保护基因，只有当严格的问题可以克服。 设计了四个具体目标来测试该假设并开发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MDR 1选择严格性。 2)确定是否可以通过使用MDR 1/Pgp突变体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克服选择严格性。 4)确定MDR 1是否可以赋予人造血细胞体内存活优势。 基于特异性目的1-3的结果，该目的将使用原代人造血祖细胞来研究MDR 1转导的细胞在NOD/SCID和SCID-hu小鼠模型系统中的存活。