Human 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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This work was supported by Cancer Research UK and the Medical Research Council, UK. We thank Blair Strang for assistance in the production of the envelope-expressing cell lines and Greg Towers for the Taqman quantitation of MLV vectors. Academic labs may obtain these cells by contacting Mary Collins, and commercial labs by contacting Kyriacos Mitrophanous (K.Mitrophanous@oxfordbiomedica.co.uk).
K.M. is employed by Oxford BioMedica. M.C. and Y.T. are paid consultants for Oxford BioMedica. Oxford BioMedica owns the cell lines described in this publication. These cells may be obtained by academic labs by writing to M.C. Commercial labs may obtain these cell lines by contacting K.M.
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Ikeda, Y., Takeuchi, Y., Martin, F. et al. Continuous high-titer HIV-1 vector production. Nat Biotechnol 21, 569–572 (2003). https://doi.org/10.1038/nbt815
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