Genetic Modification of Human Hematopoietic Cells: Preclinical Optimization of Oncoretroviral-mediated Gene Transfer for Clinical Trials

  • Tulin Budak-Alpdogan
  • Isabelle Rivière
Part of the Methods In Molecular Biology™ book series (MIMB, volume 506)


This chapter provides information about the oncoretroviral transduction of human hematopoietic stem/ progenitor cells under clinically applicable conditions. We describe in detail a short −60 h transduction protocol which consistently yields transduction efficiencies in the range of 30–50% with five different oncoretroviral vectors. We discuss a number of parameters that affect transduction efficiency, including the oncoretroviral vector characteristics, the vector stock collection, the source of CD34+ cells and transduction conditions.

Key words

Retroviral gene transfer CD34+ cells Hematopoietic stem cells Transduction Oncoret-roviral vector Vector production RetroNectin Tissue culture bags Preclinical optimization 



The authors wish to thank Michel Sadelain for critical review of the manuscript. This work is supported by PO1 CA-033049, P30 CA-008748, PO1 CA-059350, by Lymphoma Research Foundation MCLI-05-020, and by Mr. William H. Goodwin and Mrs. Alice Goodwin, and the Commonwealth Cancer Foundation for Research & the Experimental Therapeutics Center of MSKCC.


  1. 1.
    Abonour, R., Williams, D.A., Einhorn, L., Hall, K.M., Chen, J., Coffman, J., et al. (2000) Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat. Med. 6, 652–658CrossRefPubMedGoogle Scholar
  2. 2.
    Cornetta, K., Croop, J., Dropcho, E., Abon- our, R., Kieran, M.W., Kreissman, S., et al. (2006) A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-meth-ylguanine DNA methyltransferase. Cancer Gene Ther. 13, 886–895CrossRefPubMedGoogle Scholar
  3. 3.
    Cowan, K.H., Moscow, J.A., Huang, H., Zujewski, J.A., O'Shaughnessy, J., Sorren-tino, B., et al. (1999) Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multid-rug resistance complementary DNA (MDR1) in metastatic breast cancer patients. Clin. Cancer Res. 5, 1619–1628PubMedGoogle Scholar
  4. 4.
    Hanania, E.G., Giles, R.E., Kavanagh, J., Fu, S.Q., Ellerson, D., Zu, Z., et al. (1996) Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy. Proc. Natl. Acad. Sci. U S A 93, 15346–15351CrossRefPubMedGoogle Scholar
  5. 5.
    Herrera, C., Sanchez, J., Torres, A., Bellido, C., Rueda, A., Alvarez, M.A. (2001) Early-acting cytokine-driven ex vivo expansion of mobilized peripheral blood CD34+ cells generates post-mitotic offspring with preserved engraftment ability in non-obese diabetic/ severe combined immunodeficient mice. Br. J. Haematol. 114, 920–930CrossRefPubMedGoogle Scholar
  6. 6.
    Hesdorffer, C., Ayello, J., Ward, M., Kaubisch, A., Vahdat, L., Balmaceda, C., et al. (1998) 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. 16, 165–172PubMedGoogle Scholar
  7. 7.
    Kohn, D.B., Bauer, G., Rice, C.R., Rothschild, J.C., Carbonaro, D.A., Valdez, P., et al. (1999) A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34(+) cells from the bone marrow of human immunodeficiency virus-1-infected children. Blood 94, 368–371PubMedGoogle Scholar
  8. 8.
    McGuckin, C.P., Forraz, N., Pettengell, R., Thompson, A. (2004) Thrombopoietin, flt3-ligand and c-kit-ligand modulate HOX gene expression in expanding cord blood CD133 cells. Cell Prolif. 37, 295–306CrossRefPubMedGoogle Scholar
  9. 9.
    Passegue, E., Wagers, A.J., Giuriato, S., Anderson, W.C., Weissman, I.L. (2005) Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J. Exp. Med. 202, 1599–1611CrossRefPubMedGoogle Scholar
  10. 10.
    Piacibello, W., Gammaitoni, L., Bruno, S., Gunetti, M., Fagioli, F., Cavalloni, G., et al. (2000) Negative influence of IL3 on the expansion of human cord blood in vivo long-term repopulating stem cells. J. Hema-tother. Stem Cell Res. 9, 945–956CrossRefGoogle Scholar
  11. 11.
    Rahman, Z., Kavanagh, J., Champlin, R., Giles, R., Hanania, E., Fu, S., et al. (1998) Chemotherapy immediately following autol-ogous stem-cell transplantation in patients with advanced breast cancer. Clin. Cancer Res. 4, 2717–2721PubMedGoogle Scholar
  12. 12.
    Roe, T., Reynolds, T.C., Yu, G., Brown, P.O. (1993) Integration of murine leukemia virus DNA depends on mitosis. Embo J. 12, 2099–2108PubMedGoogle Scholar
  13. 13.
    Murray, L.J., Young, J.C., Osborne, L.J., Luens, K.M., Scollay, R., Hill, B.L. (1999) Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division. Exp. Hematol. 27, 1019–1028CrossRefPubMedGoogle Scholar
  14. 14.
    Murray, L., Luens, K., Tushinski, R., Jin, L., Burton, M., Chen, J., et al. (1999) Optimization of retroviral gene transduction of mobilized primitive hematopoietic progenitors by using thrombopoietin, Flt3, and Kit ligands and RetroNectin culture. Hum. Gene Ther. 10, 1743–1752CrossRefPubMedGoogle Scholar
  15. 15.
    Barrette, S., Douglas, J., Orlic, D., Anderson, S.M., Seidel, N.E., Miller, A.D., et al. (2000) Superior transduction of mouse hematopoietic stem cells with 10A1 and VSV-G pseu-dotyped retrovirus vectors. Mol. Ther. 1, 330–338CrossRefPubMedGoogle Scholar
  16. 16.
    Orlic, D., Girard, L.J., Anderson, S.M., Barrette, S., Broxmeyer, H.E., Bodine, D.M. (1999) Amphotropic retrovirus transduction of hematopoietic stem cells. Ann. N. Y. Acad. Sci. 872, 115–123CrossRefPubMedGoogle Scholar
  17. 17.
    Orlic, D., Girard, L.J., Anderson, S.M., Pyle, L.C., Yoder, M.C., Broxmeyer, H.E., et al. (1998) Identification of human and mouse hematopoietic stem cell populations expressing high levels of mRNA encoding retrovirus receptors. Blood 91, 3247–3254PubMedGoogle Scholar
  18. 18.
    Orlic, D., Girard, L.J., Jordan, C.T., Anderson, S.M., Cline, A.P., Bodine, D.M. (1996) The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc. Natl. Acad. Sci. U S A 93, 11097–11102CrossRefPubMedGoogle Scholar
  19. 19.
    Sabatino, D.E., Do, B.Q., Pyle, L.C., Seidel, N.E., Girard, L.J., Spratt, S.K., et al. (1997) Amphotropic or gibbon ape leukemia virus retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines. Blood Cells Mol. Dis. 23, 422–433CrossRefPubMedGoogle Scholar
  20. 20.
    Zielske, S.P., Gerson, S.L. (2003) Cytokines, including stem cell factor alone, enhance lentiviral transduction in nondividing human LTCIC and NOD/SCID repopulating cells. Mol. Ther. 7, 325–333CrossRefPubMedGoogle Scholar
  21. 21.
    Dorrell, C., Gan, O.I., Pereira, D.S., Hawley, R.G., Dick, J.E. (2000) Expansion of human cord blood CD34(+)CD38(−) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulat-ing cell (SRC) frequency: dissociation of SRC phenotype and function. Blood 95, 102–110PubMedGoogle Scholar
  22. 22.
    Gothot, A., van der Loo, J.C., Clapp, D.W., Srour, E.F. (1998) Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34(+) cells in non-obese diabetic/severe combined immune-deficient mice. Blood 92, 2641–2649PubMedGoogle Scholar
  23. 23.
    Mazurier, F., Gan, O.I., McKenzie, J.L., Doedens, M., Dick, J.E. (2004) Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood 103, 545–552CrossRefPubMedGoogle Scholar
  24. 24.
    Tisdale, J.F., Hanazono, Y., Sellers, S.E., Agricola, B.A., Metzger, M.E., Donahue, R.E., et al. (1998) Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 92, 1131–1141PubMedGoogle Scholar
  25. 25.
    Kurre, P., Morris, J., Miller, A.D., Kiem, H.P. (2001) Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34(+) cells emphasize limited transduction at low receptor levels. Gene Ther. 8, 593–599CrossRefPubMedGoogle Scholar
  26. 26.
    Hennemann, B., Conneally, E., Pawliuk, R., Leboulch, P., Rose-John, S., Reid, D., et al. (1999) Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables. Exp. Hematol. 27, 817–825CrossRefPubMedGoogle Scholar
  27. 27.
    Budak-Alpdogan, T., Przybylowski, M., Gonen, M., Sadelain, M., Bertino, J., Riviere, I. (2006) Functional assessment of the engraftment potential of gammaretrovirus-modified CD34+ cells, using a short serum-free transduction protocol. Hum. Gene Ther. 17, 780–794CrossRefPubMedGoogle Scholar
  28. 28.
    Demaison, C., Brouns, G., Blundell, M.P., Goldman, J.P., Levinsky, R.J., Grez, M., et al. (2000) A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector. Hum. Gene Ther. 11, 91–100CrossRefPubMedGoogle Scholar
  29. 29.
    Deola, S., Scaramuzza, S., Birolo, R.S., Carballido-Perrig, N., Ficara, F., Mocchetti, C., et al. (2004) Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice. Hum. Gene Ther. 15, 305–311CrossRefPubMedGoogle Scholar
  30. 30.
    Gaspar, H.B., Parsley, K.L., Howe, S., King, D., Gilmour, K.C., Sinclair, J., et al. (2004) Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaret-roviral vector. Lancet 364, 2181–2187CrossRefPubMedGoogle Scholar
  31. 31.
    Ott, M.G., Merget-Millitzer, H., Ottmann, O.G., Martin, H., Bruggenolte, N., Bialek, H., et al. (2002) Mobilization and transduction of CD34(+) peripheral blood stem cells in patients with X-linked chronic granuloma-tous disease. J. Hematother. Stem Cell Res. 11, 683–694CrossRefPubMedGoogle Scholar
  32. 32.
    Relander, T., Karlsson, S., Richter, J. (2002) Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes. J. Gene Med. 4, 122–132CrossRefPubMedGoogle Scholar
  33. 33.
    Trarbach, T., Greifenberg, S., Bardenheuer, W., Elmaagacli, A., Hirche, H., Flasshove, M., et al. (2000) Optimized retroviral trans-duction protocol for human progenitor cells utilizing fibronectin fragments. Cytotherapy 2, 429–438CrossRefPubMedGoogle Scholar
  34. 34.
    van der Loo, J.C., Liu, B.L., Goldman, A.I., Buckley, S.M., Chrudimsky, K.S. (2002) Optimization of gene transfer into primitive human hematopoietic cells of granulocyte-colony stimulating factor-mobilized peripheral blood using low-dose cytokines and comparison of a gibbon ape leukemia virus versus an RD114-pseudotyped retroviral vector. Hum. Gene Ther. 13, 1317–1330CrossRefPubMedGoogle Scholar
  35. 35.
    Hanenberg, H., Hashino, K., Konishi, H., Hock, R.A., Kato, I., Williams, D.A. (1997) Optimization of fibronectin-assisted ret-roviral gene transfer into human CD34+ hematopoietic cells. Hum. Gene Ther. 8, 2193–2206CrossRefPubMedGoogle Scholar
  36. 36.
    Moritz, T., Dutt, P., Xiao, X., Carstanjen, D., Vik, T., Hanenberg, H., et al. (1996) Fibronectin improves transduction of reconstituting hematopoietic stem cells by retro-viral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments. Blood 88, 855–862PubMedGoogle Scholar
  37. 37.
    Moritz, T., Patel, V. P., Williams, D.A. (1994) Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors. J. Clin. Invest. 93, 1451–1457CrossRefPubMedGoogle Scholar
  38. 38.
    Pollok, K.E., Williams, D.A. (1999) Facilitation of retrovirus-mediated gene transfer into hematopoietic stem and progenitor cells and peripheral blood T-lymphocytes utilizing recombinant fibronectin fragments. Curr. Opin. Mol. Ther. 1, 595–604PubMedGoogle Scholar
  39. 39.
    Davis, H.E., Morgan, J.R., Yarmush, M.L. (2002) Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys. Chem. 97, 159–172CrossRefPubMedGoogle Scholar
  40. 40.
    Davis, H.E., Rosinski, M., Morgan, J.R., Yarmush, M.L. (2004) Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation. Biophys. J. 86, 1234–1242CrossRefPubMedGoogle Scholar
  41. 41.
    Kwon, Y.J., Hung, G., Anderson, W. F., Peng, C.A., Yu, H. (2003) Determination of infectious retrovirus concentration from colony-forming assay with quantitative analysis. J. Virol. 77, 5712–5720CrossRefPubMedGoogle Scholar
  42. 42.
    Kwon, Y.J., Peng, C.A. (2002) Transduction rate constant as more reliable index quantifying efficiency of retroviral gene delivery. Biotechnol. Bioeng. 77, 668–677CrossRefPubMedGoogle Scholar
  43. 43.
    Persons, D.A., Mehaffey, M.G., Kaleko, M., Nienhuis, A.W., Vanin, E.F. (1998) An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene. Blood Cells Mol. Dis. 24, 167–182CrossRefPubMedGoogle Scholar
  44. 44.
    Bahnson, A.B., Dunigan, J.T., Baysal, B.E., Mohney, T., Atchison, R.W., Nimgaonkar, M.T., et al. (1995) Centrifugal enhancement of retroviral mediated gene transfer. J. Virol. Methods 54, 131–143CrossRefPubMedGoogle Scholar
  45. 45.
    Campain, J.A., Terrell, K.L., Tomczak, J.A., Shpall, E.J., Hami, L.S., Harrison, G.S. (1997) Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood. Biol. Blood Marrow Transplant. 3, 273–281PubMedGoogle Scholar
  46. 46.
    Kuhlcke, K., Fehse, B., Schilz, A., Loges, S., Lindemann, C., Ayuk, F., et al. (2002) Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels. Mol. Ther. 5, 473–478CrossRefPubMedGoogle Scholar
  47. 47.
    Movassagh, M., Desmyter, C., Baillou, C., Chapel-Fernandes, S., Guigon, M., Klatz-mann, D., et al. (1998) High-level gene transfer to cord blood progenitors using gibbon ape leukemia virus pseudotype retroviral vectors and an improved clinically applicable protocol. Hum. Gene Ther. 9, 225–234CrossRefPubMedGoogle Scholar
  48. 48.
    Sanyal, A., Schuening, F.G. (1999) Increased gene transfer into human cord blood cells by centrifugation-enhanced transduction in fibronectin fragment-coated tubes. Hum. Gene Ther. 10, 2859–2868CrossRefPubMedGoogle Scholar
  49. 49.
    Takiyama, N., Mohney, T., Swaney, W., Bahnson, A.B., Rice, E., Beeler, M., et al. (1998) Comparison of methods for retroviral mediated transfer of glucocerebrosidase gene to CD34+ hematopoietic progenitor cells. Eur. J. Haematol. 61, 1–6CrossRefPubMedGoogle Scholar
  50. 50.
    Zielske, S.P., Gerson, S.L. (2002) Lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro. Mol. Ther. 5, 381–387CrossRefPubMedGoogle Scholar
  51. 51.
    Chono, H., Yoshioka, H., Ueno, M., Kato, I. (2001) Removal of inhibitory substances with recombinant fibronectin-CH-296 plates enhances the retroviral transduction efficiency of CD34(+)CD38(−) bone marrow cells. J. Biochem. 130, 331–334PubMedGoogle Scholar
  52. 52.
    Kustikova, O.S., Wahlers, A., Kuhlcke, K., Stahle, B., Zander, A.R., Baum, C., et al. (2003) Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood 102, 3934–3937CrossRefPubMedGoogle Scholar
  53. 53.
    Suzuki, T., Shen, H., Akagi, K., Morse, H.C., Malley, J.D., Naiman, D.Q., et al. (2002) New genes involved in cancer identified by retroviral tagging. Nat. Genet. 32, 166–174CrossRefPubMedGoogle Scholar
  54. 54.
    Przybylowski, M., Hakakha, A., Stefanski, J., Hodges, J., Sadelain, M., Riviere, I. (2006) Production scale-up and validation of packaging cell clearance of clinical-grade retrovi-ral vector stocks produced in Cell Factories. Gene Ther. 13, 95–100CrossRefPubMedGoogle Scholar
  55. 55.
    Reeves, L., Cornetta, K. (2000) Clinical retroviral vector production: step filtration using clinically approved filters improves titers. Gene Ther. 7, 1993–1998CrossRefPubMedGoogle Scholar
  56. 56.
    Reeves, L., Smucker, P., Cornetta, K. (2000) Packaging cell line characteristics and optimizing retroviral vector titer: the National Gene Vector Laboratory experience. Hum. Gene Ther. 11, 2093–2103CrossRefPubMedGoogle Scholar
  57. 57.
    Miller, A.D., Garcia, J.V., von Suhr, N., Lynch, C.M., Wilson, C., Eiden, M.V. (1991) Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65, 2220–2224PubMedGoogle Scholar
  58. 58.
    Yu, S.S., Kim, J.M., Kim, S. (2000) The 17 nucleotides downstream from the env gene stop codon are important for murine leukemia virus packaging. J. Virol. 74, 8775–8780CrossRefPubMedGoogle Scholar
  59. 59.
    Ory, D.S., Neugeboren, B.A., Mulligan, R.C. (1996) A stable human-derived packaging cell line for production of high titer retrovirus/ vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. U S A 93, 11400–11406CrossRefPubMedGoogle Scholar
  60. 60.
    Riviere, I., Sadelain, M.W.J. (1997) Methods for the construction of retroviral vectors and the generation of high titer producers. In Robbins, P. (ed.) Methods in Molecular Medicine. Totowa : Humana, pp. 59–78Google Scholar
  61. 61.
    Petzer, A.L., Hogge, D.E., Landsdorp, P.M., Reid, D.S., Eaves, C.J. (1996) Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. Proc. Natl. Acad. Sci. U S A 93, 1470–1474CrossRefPubMedGoogle Scholar
  62. 62.
    Schnell, S., Young, J.W., Houghton, A.N., Sadelain, M. (2000) Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells. J. Immunol. 164, 1243–1250PubMedGoogle Scholar
  63. 63.
    Tuschong, L., Soenen, S.L., Blaese, R.M., Candotti, F., Muul, L.M. (2002) Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy. Hum. Gene Ther. 13, 1605–1610CrossRefPubMedGoogle Scholar
  64. 64.
    Seppen, J., Kimmel, R.J., Osborne, W.R. (1997) Serum-free production, concentration and purification of recombinant retroviruses. Biotechniques 23, 788–790PubMedGoogle Scholar
  65. 65.
    Kluge, K.A., Bonifacino, A.C., Sellers, S., Agricola, B.A., Donahue, R.E., Dunbar, C.E. (2002) Retroviral transduction and engraftment ability of primate hematopoietic progenitor and stem cells transduced under serum-free versus serum-containing conditions. Mol. Ther. 5, 316–322CrossRefPubMedGoogle Scholar
  66. 66.
    Schilz, A.J., Kuhlcke, K., Fauser, A.A., Eckert, H.G. (2001) Optimization of retro-viral vector generation for clinical application. J. Gene Med. 3, 427–436CrossRefPubMedGoogle Scholar
  67. 67.
    Coroadinha, A.S., Silva, A.C., Pires, E., Coelho, A., Alves, P.M., Carrondo, M.J. (2006) Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability. Biotechnol. Bioeng. 94, 322–329CrossRefPubMedGoogle Scholar
  68. 68.
    Pages, J.C., Loux, N., Farge, D., Briand, P., Weber, A. (1995) Activation of Moloney murine leukemia virus LTR enhances the titer of recombinant retrovirus in psi CRIP packaging cells. Gene Ther. 2, 547–551PubMedGoogle Scholar
  69. 69.
    Forestell, S.P., Bohnlein, E., Rigg, R.J. (1995) Retroviral end-point titer is not predictive of gene transfer efficiency: implications for vector production. Gene Ther. 2, 723–730PubMedGoogle Scholar
  70. 70.
    Kotani, H., Newton, P.B., 3rd, Zhang, S., Chiang, Y.L., Otto, E., Weaver, L., et al. (1994) Improved methods of retroviral vector transduction and production for gene therapy. Hum. Gene Ther. 5, 19–28CrossRefPubMedGoogle Scholar
  71. 71.
    Eckert, H.G., Kuhlcke, K., Schilz, A.J., Lindemann, C., Basara, N., Fauser, A.A., et al. (2000) Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1). Bone Marrow Transplant. 25 (2), S114–117CrossRefPubMedGoogle Scholar
  72. 72.
    Carmo, M., Faria, T.Q., Falk, H., Coroadinha, A.S., Teixeira, M., Merten, O.W., et al. (2006) Relationship between retroviral vector membrane and vector stability. J. Gen. Virol. 87, 1349–1356CrossRefPubMedGoogle Scholar
  73. 73.
    Wikstrom, K., Blomberg, P., Islam, K.B. (2004) Clinical grade vector production: analysis of yield, stability, and storage of gmp-produced retroviral vectors for gene therapy. Biotechnol. Prog. 20, 1198–1203CrossRefPubMedGoogle Scholar
  74. 74.
    Carmo, M., Peixoto, C., Coroadinha, A.S., Alves, P.M., Cruz, P.E., Carrondo, M.J. (2004) Quantitation of MLV-based retroviral vectors using real-time RT-PCR. J. Virol. Methods 119, 115–119CrossRefPubMedGoogle Scholar
  75. 75.
    Sanburn, N., Cornetta, K. (1999) Rapid titer determination using quantitative real-time PCR. Gene Ther. 6, 1340–1345CrossRefPubMedGoogle Scholar
  76. 76.
    Scott-Taylor, T.H., Gallardo, H.F., Gansbacher, B., Sadelain, M. (1998) Adenovirus facilitated infection of human cells with ecotropic retrovirus. Gene Ther. 5, 621–629CrossRefGoogle Scholar
  77. 77.
    Abe, T., Ito, M., Okamoto, Y., Kim, H.J., Takaue, Y., Yasutomo, K., et al. (1997) Trans-duction of retrovirus-mediated NeoR gene into CD34+ cells purified from granulocyte colony-stimulating factor (G-CSF)-mobilized infant and cord blood. Exp. Hematol. 25, 966–971PubMedGoogle Scholar
  78. 78.
    Lu, L., Xiao, M., Clapp, D.W., Li, Z.H., Broxmeyer, H.E. (1993) High efficiency ret-roviral mediated gene transduction into single isolated immature and replatable CD34(3+) hematopoietic stem/progenitor cells from human umbilical cord blood. J. Exp. Med. 178, 2089–2096CrossRefPubMedGoogle Scholar
  79. 79.
    Pollok, K.E., van Der Loo, J.C., Cooper, R.J., Hartwell, J.R., Miles, K.R., Breese, R., et al. (2001) Differential transduction efficiency of SCID-repopulating cells derived from umbilical cord blood and granulocyte colony-stimulating factor-mobilized peripheral blood. Hum. Gene Ther. 12, 2095–2108CrossRefPubMedGoogle Scholar
  80. 80.
    Carow, C.E., Hangoc, G., Broxmeyer, H.E. (1993) Human multipotential progenitor cells (CFU-GEMM) have extensive replating capacity for secondary CFU-GEMM: an effect enhanced by cord blood plasma. Blood 81, 942–949PubMedGoogle Scholar
  81. 81.
    Lu, L., Xiao, M., Grigsby, S., Wang, W.X., Wu, B., Shen, R.N., et al. (1993) Comparative effects of suppressive cytokines on isolated single CD34(3+) stem/progenitor cells from human bone marrow and umbilical cord blood plated with and without serum. Exp. Hematol. 21, 1442–1446PubMedGoogle Scholar
  82. 82.
    Lu, L., Xiao, M., Shen, R.N., Grigsby, S., Broxmeyer, H.E. (1993) Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high prolifer-ative and replating potential. Blood 81, 41–48PubMedGoogle Scholar
  83. 83.
    Noort, W.A., Wilpshaar, J., Hertogh, C.D., Rad, M., Lurvink, E.G., van Luxemburg-Heijs, S.A., et al. (2001) Similar myeloid recovery despite superior overall engraftment in NOD/SCID mice after transplantation of human CD34(+) cells from umbilical cord blood as compared to adult sources. Bone Marrow Transplant. 28, 163–171CrossRefPubMedGoogle Scholar
  84. 84.
    Wilpshaar, J., Bhatia, M., Kanhai, H.H., Breese, R., Heilman, D.K., Johnson, C.S., et al. (2002) Engraftment potential of human fetal hematopoietic cells in NOD/SCID mice is not restricted to mitotically quiescent cells. Blood 100, 120–127CrossRefPubMedGoogle Scholar
  85. 85.
    Wilpshaar, J., Falkenburg, J.H., Tong, X., Noort, W.A., Breese, R., Heilman, D., et al. (2000) Similar repopulating capacity of mitotically active and resting umbilical cord blood CD34(+) cells in NOD/SCID mice. Blood 96, 2100–2107PubMedGoogle Scholar
  86. 86.
    Veena, P., Traycoff, C.M., Williams, D.A., McMahel, J., Rice, S., Cornetta, K., et al. (1998) Delayed targeting of cytokine-non-responsive human bone marrow CD34(+) cells with retrovirus-mediated gene transfer enhances transduction efficiency and long-term expression of transduced genes. Blood 91, 3693–3701PubMedGoogle Scholar
  87. 87.
    Bregni, M., Di Nicola, M., Siena, S., Belli, N., Milanesi, M., Shammah, S., et al. (1998) Mobilized peripheral blood CD34+ cells express more amphotropic retrovirus receptor than bone marrow CD34+ cells. Haematologica 83, 204–208PubMedGoogle Scholar
  88. 88.
    Dunbar, C.E., Seidel, N.E., Doren, S., Sellers, S., Cline, A.P., Metzger, M.E., et al. (1996) Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor. Proc. Natl. Acad. Sci. U S A 93, 11871–11876CrossRefPubMedGoogle Scholar
  89. 89.
    Hematti, P., Sellers, S.E., Agricola, B.A., Metzger, M.E., Donahue, R.E., Dunbar, C.E. (2003) Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates. Blood 101, 2199–2205CrossRefPubMedGoogle Scholar
  90. 90.
    Hematti, P., Tuchman, S., Larochelle, A., Metzger, M.E., Donahue, R.E., Tisdale, J.F. (2004) Comparison of retroviral transduction efficiency in CD34+ cells derived from bone marrow versus G-CSF-mobilized or G-CSF plus stem cell factor-mobilized peripheral blood in nonhuman primates. Stem Cells 22, 1062–1069CrossRefPubMedGoogle Scholar
  91. 91.
    Thomasson, B., Peterson, L., Thompson, J., Goerner, M., Kiem, H.P. (2003) Direct comparison of steady-state marrow, primed marrow, and mobilized peripheral blood for transduction of hematopoietic stem cells in dogs. Hum. Gene Ther. 14, 1683–1686CrossRefPubMedGoogle Scholar
  92. 92.
    Dunbar, C.E., Takatoku, M., Donahue, R.E. (2001) The impact of ex vivo cytokine stimulation on engraftment of primitive hemat-opoietic cells in a non-human primate model. Ann. N. Y. Acad. Sci. 938, 236–244CrossRefPubMedGoogle Scholar
  93. 94.
    Sellers, S.E., Tisdale, J.F., Agricola, B.A., Donahue, R.E., Dunbar, C.E. (2004) The presence of the carboxy-terminal fragment of fibronectin allows maintenance of non-human primate long-term hematopoietic repopulating cells during extended ex vivo culture and transduction. Exp. Hematol. 32, 163–170CrossRefPubMedGoogle Scholar
  94. 95.
    Koizumi, K., Nishio, M., Endo, T., Takashima, H., Haseyama, Y., Fujimoto, K., et al. (2000) Large scale purification of human blood CD34+ cells from cryopreserved peripheral blood stem cells, using a nylon-fiber syringe system and immunomagnetic microspheres. Bone Marrow Transplant. 26, 787–793CrossRefPubMedGoogle Scholar
  95. 96.
    McNiece, I.K., Stoney, G.B., Kern, B.P., Briddell, R.A. (1998) CD34+ cell selection from frozen cord blood products using the Isolex 300i and CliniMACS CD34 selection devices. J. Hematother. 7, 457–461CrossRefPubMedGoogle Scholar
  96. 97.
    Rubinstein, P., Dobrila, L., Rosenfield, R.E., Adamson, J.W., Migliaccio, G., Migliaccio, A.R., et al. (1995) Processing and cryopreser-vation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc. Natl. Acad. Sci. U S A 92, 10119–10122CrossRefPubMedGoogle Scholar
  97. 98.
    Perotti, C.G., Del Fante, C., Viarengo, G., Papa, P., Rocchi, L., Bergamaschi, P., et al. (2004) A new automated cell washer device for thawed cord blood units. Transfusion 44, 900–906CrossRefPubMedGoogle Scholar
  98. 99.
    Martinson, J.A., Loudovaris, M., Smith, S.L., Bender, J.G., Vachula, M., van Epps, D.E., et al. (1997) Ex vivo expansion of frozen/ thawed CD34+ cells isolated from frozen human apheresis products. J. Hematother. 6, 69–75CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Tulin Budak-Alpdogan
    • 1
  • Isabelle Rivière
    • 2
  1. 1.Department of Medicine, The Cancer Institute of New Jersey, Robert Wood Johnson Medical SchoolUniversity of Medicine and Dentistry of New JerseyNew BrunswickUSA
  2. 2.The Gene Transfer and Somatic Cell Engineering Facility, Department of Medicine and Immunology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA

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