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Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy

Gene Therapy volume 10, pages 1703–1711 (2003)Cite this article

Abstract

Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (>3/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1–2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy.

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Acknowledgements

We are grateful to Dr JM Cunningham, Dr F Hanaoka and Dr AD Miller for kindly providing the pJET, pSV2bsr and pLHL plasmids, respectively. We also thank our colleagues Dr A Cuthbert and Dr J Arrand for helpful suggestions and support during this work and Dr B Bigger for statistical analysis. This work was supported by Grants from the Cancer Research UK SP2133/0301) and the European Commission (FIGH-CT1999-00002; QLG1-1999-01341).

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  1. Division of Cell and Molecular Biology, Imperial College, London, UK

    M Themis & C Coutelle

  2. Brunel Institute of Cancer Genetics and Pharmacogenomics, Faculty of Science, Brunel University, Uxbridge, UK

    D May & R F Newbold

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  2. D May
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Themis, M., May, D., Coutelle, C. et al. Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy. Gene Ther 10, 1703–1711 (2003). https://doi.org/10.1038/sj.gt.3302059

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