DEVELOPMENT OF OPTIMAL MDR RETROVIRAL VECTORS FOR BONE MARROW GENE THERAPY

这种RFA的目的是转移和表达人类的多种药物 慢性粒细胞白血病患者CD34+细胞耐药(MDR)基因 使用安全有效的逆转录病毒治疗晚期乳腺癌。这个 这种治疗的预计结果是，这些转导MDR的细胞将 改善后续抗癌化疗的清髓作用 使用依赖MDR活性来灭活它们的药物，例如 蒽环类、长春花碱、依托泊苷和紫杉醇。正常造血 祖细胞具有低水平的MDR活性，因此， 对这些药物优先敏感。在计划的研究中，CD34+ 将从患者的骨髓和外周血中分离出细胞 将接受大剂量的自体骨髓化疗 移植(ABMT)。在最初的临床方案中，部分患者 骨髓细胞将被MDR逆转录病毒转导，然后返回到 病人。转导细胞数、多药耐药基因的表达 随着时间的推移，这些细胞以及该程序的毒性将得到评估。 随后，紫杉醇对多药耐药细胞的增敏作用 转导的骨髓细胞将被确定。这个项目的目标是 为(1)开发更高效的新型MDR逆转录病毒载体 MDR的转导和表达，以及(2)建立最佳的体外实验 培养条件将允许最有效的转导和 多药耐药基因在造血祖细胞中的表达更多 具体来说，较小的MDR逆转录病毒载体可能导致病毒与 更高的效价将被评估。此外，发起人 逆转录病毒长末端重复序列(LTR)将与MDR基因在 尝试获得更高水平的转移MDR基因的表达。 添加磷酸甘油酸激酶(PGK)启动子的效果将是 先学习了一下。其他促进剂和增强剂将在下列情况下随后试用 这是必要的。这些研究的目标也是利用不同的EX 体内培养条件可以有效地扩增转导细胞 有可能使这些细胞的最佳数量可以用于骨髓 重建。建立更高效的矢量设计，并实现最优 MDR转导和表达条件可能允许较少的CD34+细胞 具有更大的耐多药耐药性，将用于临床试验。

The goals of this RFA are to transfer and express the human multiple drug resistance (MDR) gene in CD34+ hematopoietic cells of patients with advanced breast cancer using a safe and efficient retrovirus. The projected result of this treatment is that these MDR-transduced cells will ameliorate the myeloablative effects of subsequent anti-cancer chemotherapy with drugs that depend on MDR activity for their inactivation, such as the anthracyclines, vinca alkaloids, etoposide and taxol. Normal hematopoietic progenitor cells have low levels of MDR activity and are, therefore, preferentially sensitive to these drugs. In the projected studies, CD34+ cells will be isolated from the marrow and peripheral blood of patients who will undergo high dose chemotherapy with autologous bone marrow transplantation (ABMT). In initial clinical protocols, a portion of the marrow cells will be transduced with MDR retrovirus and then returned to the patient. The number of cells transduced, the expression of MDR in these cells over time and the toxicity of the procedure will be assessed. Subsequently, the effect of taxol administration in enriching for MDR- transduced marrow cells will be determined. The goals of this project are to (1) develop new MDR retroviral vectors capable of more efficient transduction and expression of MDR, and (2) establish optimal ex vivo culture conditions which will permit the most efficient transduction and expression of the MDR gene in hematopoietic progenitors. More specifically, smaller MDR retroviral vectors which may lead to viruses with higher titers will be evaluated. In addition, promoters other than the retroviral long terminal repeat (LTR) will be fused to the MDR cDNA in an attempt to obtain higher levels of expression of the transferred MDR gene. The effect of adding the phosphoglycerate kinase (PGK) promoter will be studied first. other promoters and enhancers will be tried subsequently if necessary. These studies will also have the goal of utilizing different ex vivo culture conditions to expand transduced cells as effectively as possible so that optimal numbers of these cells can be used for marrow reconstitution. Establishment of more efficient vector design, and optimal conditions of MDR transduction and expression may permit less CD34+ cells with greater MDR drug resistance to be used in clinical trials.