DEVELOPMENT OF OPTIMAL MDR RETROVIRAL VECTORS FOR BONE MARROW GENE THERAPY

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.