In Vitro and In Vivo Liposome-Mediated Gene Transfer Leads to Human MDR 1 Expression in Mouse Bone Marrow Progenitor Cells

Abstract

The ability to select bone marrow cells (BMC) expressing a selectable gene that confers resistance to anti-cancer drugs would be useful to protect bone marrow during chemotherapy. The human multidrug resistance (MDR1) gene encodes a 170-kD glycoprotein (P-gp), an ATP-dependent transmembrane efflux pump for many different cytotoxic drugs. In this work, we demonstrate efficient expression of the human MDR1 gene in mouse BMC after transfection with a liposomal delivery system (DLS-liposomes). The human MDR1 cDNA expression plasmid (pHaMDR1/A) was encapsulated in DLS-liposomes and delivered to mouse BMC using two approaches: (i) in vitro transfection of BMC followed by bone marrow transplantation and (ii) in vivo direct systemic gene transfer. After both the in vitro and the in vivo approaches, polymerase chain reaction (PCR) analysis confirmed that the human MDR1 gene was successfully transfected to bone marrow, spleen, and peripheral blood (PB) cells, with the human MDR1 gene detected in BMC for up to 30 days after bone marrow transplantation and 28 days after direct systemic administration. Efflux studies using rhodamine-123 demonstrated function of the MDR1 gene product in the in vitro-transfected BMC. Flow cytometry studies using the human MDR1-specific MRK16 monoclonal antibody confirmed the presence of P-gp in BMC after in vitro transfection, as well as in BMC from reconstituted or in vivo-transfected mice. Transgene expression in both lymphoid and myeloid subpopulations of BMC was demonstrated. Colony-forming units (CFU-Mix) were obtained after exposure of BMC to lethal doses of vincristine, demonstrating functional expression of the MDR1 gene in hematopoietic progenitor cells for up to 1 month.

Overview summary

Expression of the human MDR1 gene in bone marrow progenitor cells could help reduce toxicity to bone marrow cells (BMC) caused by anticancer drugs used in cancer patients undergoing chemotherapy. In addition, the MDR1 gene could be useful in vivo as a positive selectable marker to guarantee co-expression of nonselectable therapeutic genes. In this study, successful transfection of mouse BMC with the human MDR1 gene was followed by in vivo expression of a functional MDR1 gene product in hematopoietic cells, and particularly in hematopoietic progenitor cells. Facilitated by the use of a liposomal delivery system (DLS-liposomes), introduction into hematopoietic cells of an MDR1 gene containing plasmid was achieved either “directly” by intravenous administration into mice, or “indirectly” by adoptive transplantation of previously in vitro-transfected BMC. This study suggests that in vivo gene therapy using large, clinically important genes, such as the human MDR1 gene, could be achieved using a nonviral delivery system.