Abstract
Lentiviral vectors have traditionally been produced in human embryonic kidney 293T cells (1), involving transient transfection procedures that were originally established for the production of retroviral vectors based on Moloney murine leukemia (MoMLV) virus (2). Simian virus 40 (SV40)-transformed African green monkey kidney (COS-7) cells (3) and human TE671 rhabdomyosarcoma cells (4) have also been used to generate human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (5–7), but titers obtained in 293T cells are generally higher than those observed in other cell lines. Page et al. (5) were the first to describe an HIV-1-based vector harboring a selectable transgene. The HIV-1 genome was rendered replication defective in this study by replacing the envelope (Env)-encoding gp160 sequence with a guanine-phosphoribosyl transferase (gpt) gene driven by the SV40 early promoter. Transient cotransfection of COS-7 cells with this env-deleted vector and a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. Upon infection of susceptible cells, the gpt drug resistance gene was transmitted and expressed, allowing transduced cells to be selected in mycophenolic acid (5). Landau et al. (8) subsequently demonstrated that expression of heterologous Env proteins, including the MoMLV Env and the human T-cell leukemia virus type I (HTLV-I) Env, in cells transfected with the HIV-gpt vector resulted in the production of vector pseudotypes capable of infecting human as well as murine cells with titers reaching 105 colony-forming units (CFU) per mL.
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References
DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., and Calos, M. P. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell Biol. 7, 379–387.
Soneoka, Y., Cannon, P. M., Ramsdale, E. E., et al. (1995) A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23, 628–633.
Gluzman, Y. (1981) SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175–182.
Stratton, M. R., Reeves, B. R., and Cooper, C. S. (1989) Misidentified cell. Nature 337, 311–312.
Page, K. A., Landau, N. R., and Littman, D. R. (1990) Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J. Virol. 64, 5270–5276.
Reiser, J., Harmison, G., Kluepfel-Stahl, S., Brady, R. O., Karlsson, S., and Schubert, M. (1996) Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. Proc. Natl. Acad. Sci. USA 93, 15,266–15,271.
Chang, L. J., Urlacher, V., Iwakuma, T., Cui, Y., and Zucali, J. (1999) Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther. 6, 715–728.
Landau, N. R., Page, K. A., and Littman, D. R. (1991) Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range. J. Virol. 65, 162–169.
Akkina, R. K., Walton, R. M., Chen, M. L., Li, Q. X., Planelles, V., and Chen, I. S. (1996) High-efficiency gene transfer into CD34+ cells with a human immunode-ficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. J. Virol. 70, 2581–2585.
Naldini, L., Blömer, U., Gallay, P., et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267.
Mochizuki, H., Schwartz, J. P., Tanaka, K., Brady, R. O., and Reiser, J. (1998) High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. J. Virol. 72, 8873–8883.
Dull, T., Zufferey, R., Kelly, M., et al. (1998) A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471.
Hanawa, H., Kelly, P. F., Nathwani, A. C., et al. (2002) Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. Mol. Ther. 5, 242–251.
Carroll, R., Lin, J. T., Dacquel, E. J., Mosca, J. D., Burke, D. S., and St Louis, D. C. (1994) A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector system utilizing stable HIV-1 packaging cell lines. J. Virol. 68, 6047–6051.
Corbeau, P., Kraus, G., and Wong-Staal, F. (1996) Efficient gene transfer by a human immunodeficiency virus type 1 (HIV-1)-derived vector utilizing a stable HIV packaging cell line. Proc. Natl. Acad. Sci. USA 93, 14,070–14,075.
Kaplan, A. H. and Swanstrom, R. (1991) The HIV-1 gag precursor is processed via two pathways: implications for cytotoxicity. Biomed. Biochim. Acta 50, 647–653.
Rogel, M. E., Wu, L. I., and Emerman, M. (1995) The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection. J. Virol. 69, 882–888.
Li, Y., Drone, C., Sat, E., and Ghosh, H. P. (1993) Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains. J. Virol. 67, 4070–4077.
Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551.
No, D., Yao, T. P., and Evans, R. M. (1996) Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, 3346–3351.
Yu, H., Rabson, A. B., Kaul, M., Ron, Y., and Dougherty, J. P. (1996) Inducible human immunodeficiency virus type 1 packaging cell lines. J. Virol. 70, 4530–4537.
Kaul, M., Yu, H., Ron, Y., and Dougherty, J. P. (1998) Regulated lentiviral packaging cell line devoid of most viral cis-acting sequences. Virology 249, 167–174.
Kafri, T., van Praag, H., Ouyang, L., Gage, F. H., and Verma, I. M. (1999) A packaging cell line for lentivirus vectors. J. Virol. 73, 576–584.
Klages, N., Zufferey, R., and Trono, D. (2000) A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol. Ther. 2, 170–176.
Xu, K., Ma, H., McCown, T. J., Verma, I. M., and Kafri, T. (2001) Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors. Mol. Ther. 3, 97–104.
Pacchia, A. L., Adelson, M. E., Kaul, M., Ron, Y., and Dougherty, J. P. (2001) An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins. Virology 282, 77–86.
Farson, D., Witt, R., McGuinness, R., et al. (2001) A new-generation stable inducible packaging cell line for lentiviral vectors. Hum. Gene Ther. 12, 981–997.
Sparacio, S., Pfeiffer, T., Schaal, H., and Bosch, V. (2001) Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors. Mol. Ther. 3, 602–612.
Canivet, M., Hoffman, A. D., Hardy, D., Sernatinger, J., and Levy, J. A. (1990) Replication of HIV-1 in a wide variety of animal cells following phenotypic mixing with murine retroviruses. Virology 178, 543–551.
Lusso, P., di Marzo Veronese, F., Ensoli, B., et al. (1990) Expanded HIV-1 cellular tropism by phenotypic mixing with murine endogenous retroviruses. Science 247, 848–852.
Chesebro, B., Wehrly, K., and Maury, W. (1990) Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses. J. Virol. 64, 4553–4557.
Spector, D. H., Wade, E., Wright, D. A., et al. (1990) Human immunodeficiency virus pseudotypes with expanded cellular and species tropism. J. Virol. 64, 2298–2308.
Zhu, Z. H., Chen, S. S., and Huang, A. S. (1990) Phenotypic mixing between human immunodeficiency virus and vesicular stomatitis virus or herpes simplex virus. J. Acquir. Immune Defic. Syndr. 3, 215–219.
Landau, N. R. and Littman, D. R. (1992) Packaging system for rapid production of murine leukemia virus vectors with variable tropism. J. Virol. 66, 5110–5113.
Sutton, R. E. and Littman, D. R. (1996) Broad host range of human T-cell leukemia virus type 1 demonstrated with an improved pseudotyping system. J. Virol. 70, 7322–7326.
Stitz, J., Buchholz, C. J., Engelstadter, M., et al. (2000) Lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus 10A1. Virology 273, 16–20.
Christodoulopoulos, I. and Cannon, P. M. (2001) Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors. J. Virol. 75, 4129–4138.
Lewis, B. C., Chinnasamy, N., Morgan, R. A., and Varmus, H. E. (2001) Development of an avian leukosis-sarcoma virus subgroup A pseudotyped lentiviral vector. J. Virol. 75, 9339–9344.
Beyer, W. R., Westphal, M., Ostertag, W., and von Laer, D. (2002) Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range. J. Virol. 76, 1488–1495.
Kobinger, G. P., Weiner, D. J., Yu, Q. C., and Wilson, J. M. (2001) Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat. Biotechnol. 19, 225–230.
Chan, S. Y., Speck, R. F., Ma, M. C., and Goldsmith, M. A. (2000) Distinct mechanisms of entry by envelope glycoproteins of Marburg and Ebola (Zaire) viruses. J. Virol. 74, 4933–4937.
Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K. (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, 8033–8037.
Bartz, S. R. and Vodicka, M. A. (1997) Production of high-titer human immuno-deficiency virus type 1 pseudotyped with vesicular stomatitis virus glycoprotein. Methods 12, 337–342.
Reiser, J. (2000) Production and concentration of pseudotyped HIV-1-based gene transfer vectors. Gene Ther. 7, 910–913.
VandenDriessche, T., Naldini, L., Collen, D., and Chuah, M. K. (2002) Oncoretroviral and lentiviral vector-mediated gene therapy. Methods Enzymol. 346, 573–589.
Stein, C. S. and Davidson, B. L. (2002) Gene transfer to the brain using feline immunodeficiency virus-based lentivirus vectors. Methods Enzymol. 346, 433–454.
Nomura, T., Yabe, T., Mochizuki, H., Reiser, J., Becerra, S. P., and Schwartz, J. P. (2001) Survival effects of pigment epithelium-derived factor expressed by a lentiviral vector in rat cerebellar granule cells. Dev. Neurosci. 23, 145–152.
Cui, Y., Golob, J., Kelleher, E., Ye, Z., Pardoll, D., and Cheng, L. (2002) Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells. Blood 99, 399–408.
Pham, L., Ye, H., Cosset, F. L., Russell, S. J., and Peng, K. W. (2001) Concentration of viral vectors by co-precipitation with calcium phosphate. J. Gene Med. 3, 188–194.
Zhang, B., Xia, H. Q., Cleghorn, G., Gobe, G., West, M., and Wei, M. Q. (2001) A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors. Gene Ther. 8, 1745–1751.
Scherr, M., Battmer, K., Blömer, U., et al. (2002) Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells. Blood 99, 709–712.
Cosset, F. L., Takeuchi, Y., Battini, J. L., Weiss, R. A., and Collins, M. K. (1995) High-titer packaging cells producing recombinant retroviruses resistant to human serum. J. Virol. 69, 7430–7436.
Reiser, J., Lai, Z., Zhang, X. Y., and Brady, R. O. (2000) Development of multigene and regulated lentivirus vectors. J. Virol. 74, 10,589–10,599.
Wu, X., Wakefield, J. K., Liu, H., Xiao, H., Kralovics, R., Prchal, J. T., and Kappes, J. C. (2000) Development of a novel trans-lentiviral vector that affords predictable safety. Mol. Ther. 2, 47–55.
Sakoda, T., Kasahara, N., Hamamori, Y., and Kedes, L. (1999) A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. J. Mol. Cell Cardiol. 31, 2037–2047.
Higashikawa, F. and Chang, L. (2001) Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280, 124–131.
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Marino, M.P., Luce, M.J., Reiser, J. (2003). Small- to Large-Scale Production of Lentivirus Vectors. In: Federico, M. (eds) Lentivirus Gene Engineering Protocols. Methods in Molecular Biology™, vol 229. Humana Press. https://doi.org/10.1385/1-59259-393-3:43
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DOI: https://doi.org/10.1385/1-59259-393-3:43
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