TY - JOUR
T1 - Continuous high-titer HIV-1 vector production
AU - Ikeda, Yasuhiro
AU - Takeuchi, Yasuhiro
AU - Martin, Francisco
AU - Cosset, Francois Loic
AU - Mitrophanous, Kyriacos
AU - Collins, Mary
N1 - Funding Information:
Acknowledgments 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).
PY - 2003/5/1
Y1 - 2003/5/1
N2 - 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 induced. 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), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. 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-Pol and envelope proteins of gammaretroviruses; these producer cells could make up to 10 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.
AB - 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 induced. 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), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. 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-Pol and envelope proteins of gammaretroviruses; these producer cells could make up to 10 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.
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U2 - 10.1038/nbt815
DO - 10.1038/nbt815
M3 - Article
C2 - 12679787
AN - SCOPUS:0038333587
SN - 1087-0156
VL - 21
SP - 569
EP - 572
JO - Nature biotechnology
JF - Nature biotechnology
IS - 5
ER -