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Degenerated pIX-IVa2 adenoviral vector sequences lowers reacquisition of the E1 genes during virus amplification in 293 cells

Gene Therapy volume 8, pages 1713–1720 (2001)Cite this article

Abstract

A critical issue for E1-deleted adenoviral vectors manufactured from 293 cells is the emergence of replication-competent adenovirus (RCA). These contaminants arise through homologous recombination between identical sequences framing the E1 locus displayed by 293 cells, and the vector backbones. Modified recombinogenic sequences (syngen) were thus introduced within the vector backbone, and virus viability and RCA emergence were assessed. Syngen#1 is a synthetic sequence displaying silent point mutations in the pIX and IVa2 coding regions. A side by side comparison of Ad5CMV/p53 (E1-deleted adenovirus expressing the p53 tumor suppressor gene) and AVΔE1#1CMV/p53 (with syngen#1 in place of wild-type sequences) demonstrated a normal productivity for the modified construct. The altered sequences did not impair p53-mediated apoptosis in a model tumor cell line. Most importantly, a statistically significant decrease in terms of RCA occurrence could also be demonstrated. Degenerescence of the recombinogenic sequences could be further accentuated by modifying non-coding pIX region (syngen #2), with no effect on virus productivity and stability. We concluded that these vector modifications constitute a feasible strategy to reduce RCA emergence during amplification in 293 cells. This approach could also be applied to decrease reincorporation of the E1 genes during amplification of ΔE1ΔE4 vectors in 293/E4-transcomplementing cells.

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Acknowledgements

The authors thank Claude Kedinger for anti-pIX and anti-IVa2 antibodies, F Bellot for anti-virion L5 antibodies and T Huet for pIN116 plasmid.

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Authors and Affiliations

  1. Aventis Gencell-CNRS-IGR UMR 1582, Institut Gustave Roussy, Villejuif, France

    J-J Robert, I Gauffeny, C Jullien, E Vigne, M Perricaudet & P Yeh

  2. Aventis Gencell, CRVA, Vitry-sur-Seine Cedex, France

    P Benoit & J Crouzet

  3. INSERM U472, Laboratoire de Recherches en Epidémiologie et Biostatistiques, Villejuif, France

    J Maccario

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  1. J-J Robert
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Robert, JJ., Gauffeny, I., Maccario, J. et al. Degenerated pIX-IVa2 adenoviral vector sequences lowers reacquisition of the E1 genes during virus amplification in 293 cells. Gene Ther 8, 1713–1720 (2001). https://doi.org/10.1038/sj.gt.3301562

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