Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice.
Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)
Schiedlmeier
B
B
German Cancer Research Center, Department D 0200, Heidelberg, Germany.
Kühlcke
K
K
Eckert
H G
HG
Baum
C
C
Zeller
W J
WJ
Fruehauf
S
S
D017667:Adipocytes / Q000166:cytology*; D000818:Animals; D018952:Antigens, CD34; D001854:Bone Marrow Cells / Q000166:cytology; D002460:Cell Line; D019450:Genes, MDR; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology / Q000502:physiology; D006801:Humans; D017493:Leukocyte Common Antigens / Q000276:immunology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009369:Neoplasms / Q000097:blood; D016133:Polymerase Chain Reaction; D012190:Retroviridae; D017154:Stromal Cells / Q000166:cytology; D014162:Transfection / Q000379:methods*; D014183:Transplantation, Heterologous / Q000276:immunology*
D018952:Antigens, CD34; D017493:Leukocyte Common Antigens