Abstract
A limitation of successful stem cell gene transfer to hematopoietic stem cells is low transduction efficiency. To overcome this hurdle and develop a gene transfer strategy that might be clinically feasible, retroviral vectors containing a drug resistance gene were utilized to transduce human CD34+-enriched cells and select gene-modified cells by drug administration. We constructed a high-titer retroviral vector containing a fusion gene (F/S-EGFP) consisting of a mutated dihydrofolate reductase (DHFR) (Leu22→Phe22, Phe31→Ser31; F/S) gene and enhanced green fluorescent protein (EGFP) cDNA. To test whether the fusion gene could function as a selectable marker, transduced CD34+ cells were assayed in long-term stromal co-cultures with and without addition of methotrexate (MTX). Without MTX exposure, the vector-transduced CD34+ cells generated 22–50% EGFP+ cobblestone area forming cells (CAFC) at week 5. By contrast, the vector-transduced cells cultured with MTX produced 96–100% EGFP+ CAFC in four separate experiments. These are the first investigations to demonstrate selection for transduced long-term culture initiating cells using MTX. The DHFR/MTX system holds promise for improving selection of gene-transduced hematopoietic progenitor cells in vivo.
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References
Brenner MK, Rill DR, Holladay MS et al. Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients Lancet 1993 342: 1134–1137
Dunbar CE, Cottler-Fox M, O'Shaughnessy JA et al. Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation Blood 1995 85: 3048–3057
Hesdorffer C, Ayello J, Ward M et al. Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation J Clin Oncol 1998 16: 165–172
Rahman Z, Kavanagh J, Champlin R et al. Chemotherapy immediately following autologous stem-cell transplantation in patients with advanced breast cancer Clin Cancer Res 1998 4: 2717–2721
Moore MA, MacKenzie KL . Optimizing conditions for gene transfer into human hematopoietic cells Prog Exp Tumor Res 1999 36: 20–49
Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells Nat Med 1996 2: 876–882
Abonour R, Williams DA, Einhorn L et al. Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells Nat Med 2000 6: 652–658
Dao MA, Hannum CH, Kohn DB et al. FLT3 ligand preserves the ability of human CD34+ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviral-mediated transduction Blood 1997 89: 446–456
Zhao SC, Li MX, Banerjee D et al. Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a'mutant dihydrofolate reductase Cancer Gene Ther 1994 1: 27–33
May C, Gunther R, McIvor RS . Protection of mice from lethal doses of methotrexate by transplantation with transgenic marrow expressing drug-resistant dihydrofolate reductase activity Blood 1995 86: 2439–2448
Flasshove M, Banerjee D, Mineishi S et al. Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer Blood 1995 85: 566–574
Allay JA, Persons DA, Galipeau J et al. In vivo selection of retrovirally transduced hematopoietic stem cells Nat Med 1998 4: 1136–1143
Allay JA, Davis BM, Gerson SL . Human alkyltransferase–transduced murine myeloid progenitors are enriched in vivo by BCNU treatment of transplanted mice Exp Hematol 1997 25: 1069–1076
Davis BM, Reese JS, Koc ON et al. Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea Cancer Res 1997 57: 5093–5099
Podda S, Ward M, Himelstein A et al. Transfer and expression of the human multiple drug resistance gene into live mice Proc Natl Acad Sci USA 1992 89: 9676–9680
Ercikan-Abali EA, Mineishi S, Tong Y et al. Active site-directed double mutants of dihydrofolate reductase Cancer Res 1996 56: 4142–4145
Persons DA, Allay JA, Allay ER et al. Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo Blood 1997 90: 1777–1786
van Hennik PB, Verstegen MM, Bierhuizen MF et al. Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice Blood 1998 92: 4013–4022
Ploemacher RE, van der Sluijs JP, Voerman JS et al. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse Blood 1989 74: 2755–2763
Sutherland HJ, Lansdorp PM, Henkelman DH et al. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers Proc Natl Acad Sci USA 1990 87: 3584–3588
Breems DA, Blokland EA, Neben S et al. Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay Leukemia 1994 8: 1095–1104
Breems DA, Van Driel EM, Hawley RG et al. Stroma-conditioned medium and sufficient prestimulation improve fibronectin fragment-mediated retroviral gene transfer into human primitive mobilized peripheral blood stem cells through effects on their recovery and transduction efficiency Leukemia 1998 12: 951–959
Banerjee D, Zhao SC, Li MX et al. Gene therapy utilizing drug resistance genes: a review Stem Cells 1994 12: 378–385
Markowitz D, Goff S, Bank A . A safe packaging line for gene transfer: separating viral genes on two different plasmids J Virol 1988 62: 1120–1124
Markowitz D, Goff S, Bank A . Construction and use of a safe and efficient amphotropic packaging cell line Virology 1988 167: 400–406
Riviere I, Sadelain M . Methods for the construction of retroviral vectors and the generation of high titer producers In: Robbins PD, Ed Gene Therapy Protocols 1st ed Totowa, NJ: Human Press 1997 59
Takebe N, Nakahara S, Zhao SC et al. Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors Cancer Gene Ther 2000 7: 910–919
Mineishi S, Nakahara S, Takebe N et al. Co-expression of the herpes simplex virus thymidine kinase gene potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene Gene Ther 1997 4: 570–576
Limon A, Briones J, Puig T et al. High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells Blood 1997 90: 3316–3321
Roehm NW, Rodgers GH, Hatfield SM et al. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT J Immunol Methods 1991 142: 257–265
Banerjee D, Schnieders B, Fu JZ et al. Role of E2F-1 in chemosensitivity Cancer Res 1998 58: 4292–4296
Gartner S, Kaplan HS . Long-term culture of human bone marrow cells Proc Natl Acad Sci USA 1980 77: 4756–4759
Xu LC, Kluerfel-Stahl S, Blanco M, Schiffmann R, Dunbar C, Karlsson S . Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients Blood 1995 86: 141–146
Itoh K, Tezuka H, Sakoda H et al. Reproducible establishment of hematopoietic supportive stromal cell lines from murine bone marrow Exp Hematol 1989 17: 145–153
Acknowledgements
We gratefully acknowledge the assistance of Diane Domingo for FACS analysis, Jessica Boklan for providing optimal conditions for colony PCR, and Emine Ercikan-Abali for providing the Pkt7 plasmid. Supported by CA59350, CA08748, and CA09512 (NT).
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Takebe, N., Xu, LC., MacKenzie, K. et al. Methotrexate selection of long-term culture initiating cells following transduction of CD34+ cells with a retrovirus containing a mutated human dihydrofolate reductase gene. Cancer Gene Ther 9, 308–320 (2002). https://doi.org/10.1038/sj.cgt.7700443
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DOI: https://doi.org/10.1038/sj.cgt.7700443
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