DEVELOPMENT OF OPTIMAL MDR RETROVIRAL VECTORS FOR BONE MARROW GENE THERAPY

该RFA的目标是转移和表达人类多种药物， 耐药（MDR）基因在CD 34+造血细胞的患者 使用安全有效的逆转录病毒治疗晚期乳腺癌。 的 这种治疗的预期结果是，这些MDR转导的细胞将 改善后续抗癌化疗的清髓作用 与依赖MDR活性来使其失活的药物，例如 蒽环类、长春花生物碱、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， 对这些药物敏感。 在计划的研究中，CD 34 + 将从患者的骨髓和外周血中分离细胞， 将接受高剂量自体骨髓化疗 移植（ABMT）。 在最初的临床方案中， 骨髓细胞将用MDR逆转录病毒转导，然后返回至 病人 转染细胞数、MDR表达、细胞凋亡率 将评估这些细胞随时间的变化以及该过程的毒性。 随后，研究了紫杉醇给药在富集MDR-1中的作用。 将测定转导的骨髓细胞。 该项目的目标是 （1）开发新的MDR逆转录病毒载体， MDR的转导和表达，以及（2）建立最佳的离体 允许最有效转导的培养条件， MDR基因在造血祖细胞中的表达。 更 特别是，较小的MDR逆转录病毒载体可能导致病毒与 将评价更高的滴度。 除此之外， 逆转录病毒长末端重复序列（LTR）将与MDR cDNA融合， 试图获得更高水平的转移MDR基因的表达。 加入磷酸甘油酸激酶（PGK）启动子的效果将是 首先学习。 随后将尝试其它启动子和增强子， 必要 这些研究的目标也将是利用不同的基因， 在体内培养条件下， 因此，这些细胞的最佳数量可以用于骨髓移植。 重组 建立更高效的载体设计，并优化 MDR转导和表达的条件可能允许较少的CD 34+细胞 具有更高的MDR耐药性，可用于临床试验。

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.