Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.
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 (MDR1) 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 MDR1 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 MDR1-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.
Sugimoto
Yoshikazu
Y
Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan. ysugimot@jfcr.or.jp
Tsukahara
Satomi
S
Sato
Shigeo
S
Suzuki
Mutsumi
M
Nunoi
Hiroyuki
H
Malech
Harry L
HL
Gottesman
Michael M
MM
Tsuruo
Takashi
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D016026:Bone Marrow Transplantation; D002469:Cell Separation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D008040:Genetic Linkage; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006105:Granulomatous Disease, Chronic / Q000235:genetics* / Q000628:therapy; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D041681:NIH 3T3 Cells; D017239:Paclitaxel / Q000494:pharmacology; D010641:Phenotype; D012190:Retroviridae / Q000235:genetics*; D013481:Superoxides; D013997:Time Factors; D019076:Transgenes; D014750:Vincristine / Q000494:pharmacology; D014960:X Chromosome / Q000648:ultrastructure
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018952:Antigens, CD34; D000972:Antineoplastic Agents, Phytogenic; D008562:Membrane Glycoproteins; D013481:Superoxides; D014750:Vincristine; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D017239:Paclitaxel