In vitro and in vivo liposome-mediated gene transfer leads to human MDR1 expression in mouse bone marrow progenitor cells.
The ability to select bone marrow cells (BMC) expressing a selectable gene that confers resistance to anticancer 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.
Aksentijevich
I
I
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Pastan
I
I
Lunardi-Iskandar
Y
Y
Gallo
R C
RC
Gottesman
M M
MM
Thierry
A R
AR
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000818:Animals; D017690:Cell Transplantation; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004337:Drug Carriers; D005786:Gene Expression Regulation / Q000502:physiology*; D018014:Gene Transfer Techniques; D019450:Genes, MDR; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D051379:Mice; D008810:Mice, Inbred C57BL; D012091:Repetitive Sequences, Nucleic Acid; D009053:Sarcoma Viruses, Murine / Q000235:genetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary; D004279:DNA, Viral; D004337:Drug Carriers; D008081:Liposomes