MDR1 Gene Expression in NOD/SCID Repopulating Cells after Retroviral Gene Transfer under Clinically Relevant Conditions

Abstract

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.

Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis

“…Thus, vectors capable of stable integration into a target cell's genome, such as the murine oncoretroviral-based vectors and lentiviral-based vectors, have been tested for their efficiency of transduction in primitive human hematopoietic cells from peripheral blood, bone marrow, and umbilical cord blood in both in vitro and in vivo assays. [1][2][3][4][5][6][7][8][9] The true HSC, defined by its capacity for long-term hematopoietic reconstitution by virtue of its high proliferative potential and its ability to self-renew, is, paradoxically, quiescent at most points in time. It is this property of the stem cell that has impeded vector-mediated gene therapy-based treatment of disease.…”

Hematopoietic stem cell gene therapy: progress toward therapeutic targets

“…Addition of thrombopoietin to the combination of stem cell factor and FLT-3 ligand during the transduction period results in an increase in the number of CD34+ progenitors and high levels of engraftment of the transduced human hematopoietic progenitors in immunocompromised mice, compared to previously used combinations. [22][23][24] The incorporation of both fibronectin fragment CH-296 and thrombopoietin have proved successful in clinical protocols.…”

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood progenitor cells using PCR with arbitrary primers

“…CD34 + cells were prepared as described. 22 Briefly, CD34 + cells were isolated from frozen PBPC samples by magnetic microbead selection using the ClinMACS system (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's description. Retroviral vector stocks were produced and stored as described.…”

“…23 Retroviral transduction was performed as described. 22 In brief, CD34 + cells were prestimulated for 16-20 h at a density of 1 Â 10 6 cells/ml X-VIVO-10 medium (BioWhittaker, Verviers, Belgiun), supplemented with interleukin 3 (20 ng/ml), interleukin 6 (10 ng/ml), stem cell factor (50 ng/ml), Flt-3 ligand (100 ng/ml) and thrombopoietin (20 ng/ml) (R & D Systems, Weisbaden, Germany). Following prestimulation, cells were incubated with retroviral supernatant, containing the hybrid vector SF1m, over two consecutive days.…”