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Self-inactivating helper virus for the production of high-capacity adenoviral vectors

Gene Therapy volume 18, pages 1025–1033 (2011)Cite this article

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Abstract

Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which Ψ is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers >1010 infectious units and <0.1% HV contamination. The residual HVs lacked Ψ and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of HC-Ads.

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Acknowledgements

This work was funded in part by Fundacion Ramon Areces; Fundacion MMA; Grants SAF2009-11324, BFU2007-60228; BFU2010-16382 from the Spanish Department of Science; and the UTE project CIMA. MGA was supported by a Torres Quevedo contract from the Spanish Department of Science. The technical support of Marian Fernandez Estevez is acknowledged.

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Authors and Affiliations

  1. Division of Hepatology and Gene Therapy, CIMA, University of Navarra, Foundation for Applied Medical Research, Pamplona, Spain

    M Gonzalez-Aparicio, I Mauleon, P Alzuguren, M Bunuales, G Gonzalez-Aseguinolaza, J Prieto & R Hernandez-Alcoceba

  2. Department of Macromolecular Structure, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain

    C San Martín

  3. CIBERehd, University Clinic of Navarra, Pamplona, Spain

    J Prieto

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  1. M Gonzalez-Aparicio
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Correspondence to R Hernandez-Alcoceba.

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Gonzalez-Aparicio, M., Mauleon, I., Alzuguren, P. et al. Self-inactivating helper virus for the production of high-capacity adenoviral vectors. Gene Ther 18, 1025–1033 (2011). https://doi.org/10.1038/gt.2011.58

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