Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.
BACKGROUND
The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed.


METHODS
Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated.


RESULTS
The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors.


CONCLUSIONS
The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.
Department of Molecular and Medical Virology, Ruhr-University Bochum, D-44780 Bochum, Germany.
Seraphin
S
Kuate
Daniela
D
Stefanou
Dennis
D
Hoffmann
Oliver
O
Wildner
Klaus
K
Uberla
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D015685:Fusion Proteins, gag-pol / Q000235:genetics; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D015302:Simian Immunodeficiency Virus / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D014161:Transduction, Genetic
D015685:Fusion Proteins, gag-pol; D013752:Tetracycline