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Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

Gene Therapy volume 17, pages 400–411 (2010)Cite this article

Abstract

Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were ∼10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O6-methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression.

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Acknowledgements

This study was supported by grants of the German Ministry for Research and Education (TreatID, Pidnet), the DAAD (German-Chinese junior research groups), the Deutsche Forschungsgemeinschaft (DFG, that is SFB738 and Excellence Cluster REBIRTH) and the European Union (CONSERT, LSHB-CT-2004-005242; Clinigene, LSHB-CT-2006-018933, Persist), the German National Merit foundation (stipend to TM) and the Else Kröner Fresenius Stiftung (fellowship to AS). We thank Bernhard Gentner and Luigi Naldini for helpful comments and suggestions. We are grateful to Ute Modlich for sharing the qPCR standard, to Don Ganem and Christopher Sullivan for kindly providing the Nodamuravirus B2 expression plasmid, to Dirk Lindemann providing the HFV tas cDNA, to Beau Fenner and Jimmy Kwang for providing the Betanodavirus B2 expression plasmid, to Kajohn Boonrod for providing p19 RNA, and to Roger Y Tsien for providing the mCherry cDNA.

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Authors and Affiliations

  1. Department of Experimental Hematology, Hannover Medical School, Hannover, Germany

    T Maetzig, M Galla, M H Brugman, C Baum & A Schambach

  2. EUFETS AG, Idar-Oberstein, Germany

    R Loew

  3. Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA

    C Baum

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  1. T Maetzig
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  2. M Galla
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  3. M H Brugman
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Correspondence to A Schambach.

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

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Maetzig, T., Galla, M., Brugman, M. et al. Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors. Gene Ther 17, 400–411 (2010). https://doi.org/10.1038/gt.2009.129

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