MDR1 gene expression in NOD/SCID repopulating cells after retroviral gene transfer under clinically relevant conditions.
We have adapted a recently published protocol for retroviral gene transfer into hematopoietic cells [A. J. Schilz et al. (1998) Blood 92: 3163-3171] with respect to clinical requirements such as large-volume vector stock generation, adequate cell source, high cell numbers, and serum-free conditions. We present data on transduction efficacy and expression of the multidrug resistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral blood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector. Using a 1-day cytokine-mediated prestimulation, consisting of human interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO), followed by a 3-day transduction procedure, we were able to detect up to 51% CD34(+) cells expressing MDR1. Xenotransplantation of transduced cells into NOD/LtSz-scid/scid (NOD/SCID) mice resulted in a mean engraftment level of 23% (0.1 to 87%). As shown by quantitative PCR analysis, a mean of 12.7% (range 0.3 to 55%) of the engrafted human cells in the bone marrow of chimeric mice contained the MDR1 cDNA. Furthermore, enhanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population. Our data suggest that NOD/SCID repopulating cells derived from mobilized PB can be transduced efficiently with existing retroviral vector systems under clinically applicable conditions.
Schilz
A J
AJ
EUFETS GmbH, Idar-Oberstein, D0200, Germany.
Schiedlmeier
B
B
Kühlcke
K
K
Fruehauf
S
S
Lindemann
C
C
Zeller
W J
WJ
Grez
M
M
Fauser
A A
AA
Baum
C
C
Eckert
H G
HG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D001483:Base Sequence; D002455:Cell Division; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D012190:Retroviridae / Q000235:genetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D017931:DNA Primers