Research Project
2733271
The human MDR1 gene encodes a multispecific drug transporter that prevents drug accumulation in resistant cells. Overexpression of MDR1 is sufficient for conferring multidrug resistance on otherwise normal cells. This feature has led to the proposal that MDR1 might be used in gene therapy to protect hematopoietic cells against chemotherapy-related myelotoxicity. Another possible application of MDR1 to gene therapy is to use it as an in vivo selectable marker to enhance the long-term expression of linked foreign genes in transduced cells. The feasibility of MDR1-mediated gene therapy and the rationale for work in this proposal are based on the observations that MDR1 is 1) a chemoprotective gene when expressed in hematopoietic cells of mice; 2) an in vivo selectable marker which confers a survival advantage on transduced progenitor cells in a mouse bone marrow transplantation setting; and 3) an in vitro selectable marker that can be used to overexpress heterologous genes transduced within the same vector construction as MDR1. We were the first to use MDR1 selection to overexpress foreign genes in tissue culture cell lines and we have further adapted this system for use with retroviruses. We now hypothesize that MDR1 can also serve as an in vivo selectable marker for the delivery and expression of foreign genes in human hematopoietic cells. Three aims are proposed to test this hypothesis: 1) Complete a quantitative analysis of MDR1-mediated foreign gene expression in cell lines. A quantitative reporter gene system will be developed for measuring MDR1-mediated foreign gene expression. Pseudotyped retroviruses will be used to increase the efficacy of gene transfer into hematopoietic cells. 2) Determine the ability of MDR1 to act as a selectable marker for foreign gene expression in human hematopoietic stem cells. The purpose of these experiments is to determine whether MDR1 selection works to achieve stable foreign gene expression in long-term repopulating hematopoietic cells. 3) Determine the effects of in vivo drug delivery on MDR1-mediated chemoprotection, selection, and expression of foreign genes in SCID-hu mice. Experiments will use the quantitative reporter gene system to study foreign gene expression in a relevant model of human hematopoiesis.
