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Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination

Nature Biotechnology volume 19, pages 582–585 (2001)Cite this article

Abstract

Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression1,2,3,4,5,6,7,8,9,10,11,12. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal1,3,4,5,6,7,8,9,10,11,12. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus13. By using a very efficient recombinase, in vitro–evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone1,7,8,10,11,12, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography–based virus purification13.

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Figure 1: An E1-, E3-deleted helper adenovirus (FL helper) with a packaging signal sensitive to FLPe-mediated excision.
Figure 2: Rescue and amplification of a β-galactosidase–expressing HD virus using 293-FLPe cells and FL helper virus.
Figure 3: In vivo β-galactosidase expression in rat brain infected with either GS46-FLPe HD vector or first-generation adenovirus (RAd35).

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References

  1. Maione, D. et al. Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector. Hum. Gene Ther. 11, 859–868 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Mitani, K., Graham, F.L., Caskey, C.T. & Kochanek, S. Rescue, propagation, and partial purification of a helper virus–dependent adenovirus vector. Proc. Natl. Acad. Sci. USA 92, 3854–3858 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kochanek, S. et al. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc. Natl. Acad. Sci. USA 93, 5731–5736 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Parks, R.J. et al. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. USA 93, 13565–13570 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y. & Philips, M.L. Construction of adenovirus vectors through Cre-lox recombination. J. Virol. 71, 1842–1849 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen, H.H. et al. Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc. Natl. Acad. Sci. USA 94, 1645–1650 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schiedner, G. et al. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat. Genet. 18, 180–183 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Morsy, M.A. et al. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc. Natl. Acad. Sci. USA 95, 7866–7871 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Morral, N. et al. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc. Natl. Acad. Sci. USA 96, 12816–12821 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Thomas, C.E., Schiedner, G., Kochanek, S., Castro, M.G. & Lowenstein, P.R. Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases. Proc. Natl. Acad. Sci. USA 97, 7482–7487 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sandig, V. et al. Optimization of the helper-dependent adenovirus system for production and potency in vivo. Proc. Natl. Acad. Sci. USA 97, 1002–1007 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Thomas, C.E. et al. Pre-existing anti-adenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors. Hum. Gene Ther., in press (2001).

  13. Blanche, F. et al. An improved anion-exchange HPLC method for the detection and purification of adenoviral particles. Gene Ther. 7, 1055–1062 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Buchholz, F., Angrand, P.O. & Stewart, A.F. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat. Biotechnol. 16, 657–662 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Ringrose, L. et al. Comparative kinetic analysis of FLP and Cre recombinases: mathematical models for DNA binding and recombination. J. Mol. Biol. 284, 363–384 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Grable, M. & Hearing, P. cis and trans requirements for the selective packaging of adenovirus type 5 DNA. J. Virol. 66, 723–731 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Bett, A.J., Haddara, W., Prevec, L. & Graham, F.L. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc. Natl. Acad. Sci. USA 91, 8802–8806 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Umaña, P., Jean-Mairet, J. & Bailey, J.E. Tetracycline-regulated over-expression of glycosyltransferases in Chinese hamster ovary cells. Biotechnol. Bioeng. 65, 542–549 (1999).

    Article  PubMed  Google Scholar 

  19. Southgate, T.D., Kingston, P.A. & Castro, M.G. Adenoviral vectors for gene transfer into neural cells in primary culture. In Current protocols in neuroscience. (eds Sibley, D.R. et al.) 4.23.1–4.23.40 (John Wiley and Sons, New York, NY; 2000).

    Google Scholar 

  20. Gerdes, C.A., Castro, M.G. & Lowenstein, P.R. Strong promoters are the key to highly efficient noninflammatory and noncytotoxic adenoviral-mediated trangene delivery into the brain in vivo. Mol. Ther. 2, 330–338 (2000).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants from the Medical Research Council (MRC, UK), Biotechnology and Biological Sciences Research Council (BBSRC, UK), The Parkinson's Disease Society, The Royal Society, and European Union-Biomed program grants Contract No. BMH4-CT98-3277 and BMH4-CT98-0297. P.R.L. was a research fellow of The Lister Institute of Preventive Medicine, and D.S. was funded through a BBSRC studentship. We kindly thank A. Francis Stewart for providing us with the pCAGGSFLPe-IRESpuro plasmid, and Stefan Kochanek and Gudrun Schiedner for providing us with the pGS46 plasmid.

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  1. Pablo Umaña

    Present address: GlycArt Biotechnology AG, Einsteinstrasse, 8093, Zurich, Switzerland

Authors and Affiliations

  1. Molecular Medicine and Gene Therapy Unit, Room 1.302 Stopford Building, School of Medicine, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom

    Pablo Umaña, Christian A. Gerdes, Daniel Stone, Maria G. Castro & Pedro R. Lowenstein

  2. Endocrine Sciences Research Group, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, United Kingdom

    Julian R.E. Davis

  3. ARC Epidemiology Unit, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, United Kingdom

    Daniel Ward

  4. Address from 1 July 1 2001: Board of Governors Gene Therapeutics Research Institute, 5th Floor Room R-5089, Research Pavilion, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, 90048-1860, CA, USA

    Maria G. Castro & Pedro R. Lowenstein

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Correspondence to Pedro R. Lowenstein.

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Umaña, P., Gerdes, C., Stone, D. et al. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nat Biotechnol 19, 582–585 (2001). https://doi.org/10.1038/89349

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