Nature Biotechnology
21, 569 - 572 (2003)
Published online: 7 April 2003; | doi:10.1038/nbt815
Continuous high-titer HIV-1 vector productionYasuhiro Ikeda1, Yasuhiro Takeuchi1, Francisco Martin1, Francois-Loic Cosset2, Kyriacos Mitrophanous3
& Mary Collins11
Department of Immunology and Molecular Pathology,
Windeyer Institute, University College London, 46 Cleveland
St., London W1T 4JF, UK. 2
Vectorologie Retrovirale et Therapie Genique,
Ecole Normale Superieure de Lyon, Lyon,
France. 3
Oxford BioMedica Limited, The Oxford Science
Park, Oxford, UK.
Correspondence should be addressed to Mary Collins mary.collins@ucl.ac.ukHuman immunodeficiency virus type 1 (HIV-1)−based vectors are
currently made by transient transfection, or using packaging cell lines in
which expression of HIV-1 Gag and Pol proteins is induced1,
2,
3.
Continuous vector production by cells in which HIV-1 Gag-Pol is stably
expressed would allow rapid and reproducible generation of large vector
batches. However, attempts to make stable HIV-1 packaging cells by transfection
of plasmids encoding HIV-1 Gag-Pol have resulted in cells which secrete only
low levels of p24 antigen (20−80 ng/ml)4,
5,
6, possibly
because of the cytotoxicity of HIV-1 protease7. Infection of
cells with HIV-1 can result in stable virus production8; cell
clones that produce up to 1,000 ng/ml secreted p24 antigen have been
described9. Here we report that expression of HIV-1 Gag-Pol by a
murine leukemia virus (MLV) vector allows constitutive, long-term, high-level
(up to 850 ng/ml p24) expression of HIV-1 Gag. Stable packaging cells were
constructed using codon-optimized HIV-1 Gag-Pol10 and envelope
proteins of gammaretroviruses; these producer cells could make up to
107 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for
at least three months in culture.
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