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Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors

Gene Therapy volume 18, pages 674–681 (2011)Cite this article

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Abstract

Cutaneous gene therapy can be envisioned through the use of keratinocyte stem cell clones in which retroviral genotoxic risks can be pre-assessed. While transactivation of cellular genes by the retroviral long terminal repeat enhancer has been proven in experimental and clinical settings, the formation of chimeric viral–cellular transcripts originated by the inefficient termination (read-through) of retroviral transcripts remains to be studied in depth. We now demonstrate the widespread presence of viral–cellular fusion transcripts derived from integrated proviruses in keratinocytes transduced with self-inactivating (SIN) retroviral vectors. We have detected high molecular weight RNAs in northern blot analysis of retroviral vector expression in individual cell clones. Characterization of some of these transcripts revealed that they originate from genes located at the proviral integration sites. One class of transcripts corresponds to fusions of the viral vectors with intronic sequences, terminating at cryptic polyadenylation sites located in introns. A second class comprises fusion transcripts with coding sequences of genes at the integration sites. These are generated through splicing from a cryptic, not previously described donor site in the lentiviral vectors to exons of cellular genes, and have the potential to encode unintended open reading frames, although they are downregulated by cellular mechanisms. Our data contribute to a better understanding of the impact of SIN lentiviral vector integration on cellular gene transcription, and will be helpful in improving the design of this type of vectors.

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Acknowledgements

We thank Mrs Blanca Duarte for excellent technical help. This work was supported in part by grants, SAF2007-61019 from the Spanish Ministry of Science and Innovation; PI081054 from FIS and P-BIO-0306-2006 from Comunidad de Madrid. The CIBER for rare diseases is an initiative of the Spanish ISCIII.

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Authors and Affiliations

  1. Epithelial Biomedicine Division, Cutaneous Disease Modelling Unit, CIEMAT-CIBERER (Centre for Biomedical Research on Rare Diseases) U714, Madrid, Spain

    D Almarza, M Navarro, F Larcher & R Murillas

  2. Department of Biomedical Sciences, Centre for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy

    G Bussadori & F Mavilio

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  1. D Almarza
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  2. G Bussadori
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  3. M Navarro
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  4. F Mavilio
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  5. F Larcher
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  6. R Murillas
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Correspondence to F Larcher or R Murillas.

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Almarza, D., Bussadori, G., Navarro, M. et al. Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors. Gene Ther 18, 674–681 (2011). https://doi.org/10.1038/gt.2011.12

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