A Packaging Cell Line for Lentivirus Vectors

ABSTRACT Lentivirus vectors can transduce dividing and nondividing cells. Using three-plasmid transient transfections, high-titer (>109 IU/ml) recombinant lentivirus vectors pseudotyped with vesicular stomatitis virus G (VSV-G) protein can be generated (T. Kafri et al., Nat. Genet. 17:314–317, 1997; H. Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319–10323, 1997; L. Naldini et al., Science 272:263–267, 1996). The recombinant lentiviruses can efficiently infect brain, liver, muscle, and retinal tissue in vivo. Furthermore, the transduced tissues demonstrated long-term expression of reporter genes in immunocompetent rodents. We now report the generation of a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 106 IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated to titers of 109 IU/ml and can efficiently transduce nondividing cells in vitro and in vivo. The availability of a lentivirus packaging cell line will significantly facilitate the production of high-titer lentivirus vectors for gene therapy and study of human immunodeficiency virus biology.

[1]  A. Lever,et al.  Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region , 1993, Journal of virology.

[2]  F. Gage,et al.  In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector , 1996, Science.

[3]  D. Peterson,et al.  Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors , 1997, Nature Genetics.

[4]  R. Mulligan,et al.  A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[5]  B. Turner,et al.  Histone acetylation and control of gene expression. , 1991, Journal of cell science.

[6]  T. Masuda,et al.  Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr , 1995, Journal of virology.

[7]  Y. Ron,et al.  Inducible human immunodeficiency virus type 1 packaging cell lines , 1996, Journal of virology.

[8]  D. Burke,et al.  A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector system utilizing stable HIV-1 packaging cell lines , 1994, Journal of virology.

[9]  H. Okayama,et al.  Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. , 1988, BioTechniques.

[10]  A. Panganiban,et al.  Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles , 1993, Journal of virology.

[11]  A. Kaplan,et al.  The HIV-1 gag precursor is processed via two pathways: implications for cytotoxicity. , 1991, Biomedica biochimica acta.

[12]  K. Arai,et al.  SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat , 1988, Molecular and cellular biology.

[13]  R. Schlegel,et al.  pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles. , 1984, The Journal of biological chemistry.

[14]  M. Emerman,et al.  The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection , 1995, Journal of virology.

[15]  A. Panganiban,et al.  Human immunodeficiency virus vectors for inducible expression of foreign genes , 1992, Journal of virology.

[16]  H. Buc,et al.  HIV-1 reverse transcription. A termination step at the center of the genome. , 1994, Journal of molecular biology.

[17]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[18]  F. Wong-Staal,et al.  Efficient gene transfer by a human immunodeficiency virus type 1 (HIV-1)-derived vector utilizing a stable HIV packaging cell line. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[19]  F. Gage,et al.  Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Gossen,et al.  Transcriptional activation by tetracyclines in mammalian cells. , 1995, Science.

[21]  A. Wolffe,et al.  Histone acetylation: chromatin in action. , 1997, Trends in biochemical sciences.

[22]  F. Gage,et al.  Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  I. Verma,et al.  Gene therapy - promises, problems and prospects , 1997, Nature.

[24]  Fred H. Gage,et al.  Development of a Self-Inactivating Lentivirus Vector , 1998, Journal of Virology.

[25]  T Friedmann,et al.  Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.