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Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors

Abstract

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 × 107 transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

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Acknowledgements

We thank L Naldini for providing the basic lentiviral construct, F-L Cosset for the RD114/TR envelope, E Will for providing M57-DAW, and C Klanke and M Id for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (1837/Ba4) and by the Integrated Project CONSERT of the European Union (LSHB-CT-2004-005242).

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Correspondence to J Bohne.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Schambach, A., Mueller, D., Galla, M. et al. Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors. Gene Ther 13, 1524–1533 (2006). https://doi.org/10.1038/sj.gt.3302807

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