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] w ith respect to clinical requirements such as large-volume vector stock gene ration, adequate cell source, high cell numbers, and serum-free conditions. We present data on transduction efficacy and expression of the multidrug r esistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral bl ood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retrovi ral vector. Using a 1-day cytokine-mediated prestimulation, consisting of h uman interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), a nd thrombopoietin (TPO), followed by a 3-day transduction procedure, we wer e able to detect up to 51% CD34(+) cells expressing MDR1. Xenotransplantati on 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 i n the bone marrow of chimeric mice contained the MDR1 cDNA. Furthermore, en hanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population. Our data suggest that NOD/SCID repopu lating cells derived from mobilized PB can be transduced efficiently with e xisting retroviral vector systems under clinically applicable conditions.