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Drug‐selected co‐expression of P‐glycoprotein and gp91 in vivo from an MDR 1‐bicistronic retrovirus vector Ha‐MDR‐IRES‐gp91
Author(s) -
Sugimoto Yoshikazu,
Tsukahara Satomi,
Sato Shigeo,
Suzuki Mutsumi,
Nunoi Hiroyuki,
Malech Harry L.,
Gottesman Michael M.,
Tsuruo Takashi
Publication year - 2003
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.362
Subject(s) - transduction (biophysics) , retrovirus , microbiology and biotechnology , biology , genetic enhancement , cytotoxic t cell , internal ribosome entry site , p glycoprotein , viral vector , virology , multiple drug resistance , in vitro , gene , virus , translation (biology) , messenger rna , drug resistance , recombinant dna , biochemistry
Background Retroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols. Co‐expression of a human multidrug resistance gene ( MDR 1) with a therapeutic gene affords selectable growth advantage to genetically modified cells. Methods A bicistronic retrovirus vector, Ha‐MDR‐IRES‐gp91, was constructed for the co‐expression of MDR 1 and gp91, a gene responsible for X‐linked chronic granulomatous disease (X‐CGD). Drug‐selected co‐expression of P‐glycoprotein and gp91 was evaluated in transduced cells. Results Epstein‐Barr virus‐transformed B cells from X‐CGD patients transduced with Ha‐MDR‐IRES‐gp91 co‐expressed human P‐glycoprotein and gp91, and acquired superoxide‐generating activity. Human CD34‐positive cells from an X‐CGD patient were transduced with Ha‐MDR‐IRES‐gp91 and subsequently treated with 2 ng/ml vincristine. After 13 days, 20% of Ha‐MDR‐IRES‐gp91‐transduced cells were P‐glycoprotein‐ and gp91‐positive by FACS analysis. The superoxide‐generating activity of the transduced population was 27% of that of normal cells. Mice transplanted with Ha‐MDR‐IRES‐gp91‐transduced bone marrow cells showed co‐expression of P‐glycoprotein and gp91 in peripheral blood mononuclear cells. By administering paclitaxel, the proportions of P‐glycoprotein‐ and gp91‐positive cells were increased in all the four mice examined. When mice transplanted with Ha‐MDR‐IRES‐gp91‐transduced cells were repeatedly administered paclitaxel, the ratios of P‐glycoprotein‐ and gp91‐positive cells were maintained for over 1 year. Conclusions These results suggest that MDR 1‐bicistronic vectors may be useful to select the transduced hematopoietic cells in vivo . This may lead to the sustained expression of transgenes in the blood cells of patients treated with stem cell gene therapy. Copyright © 2003 John Wiley & Sons, Ltd.