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Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring γ-retrovirus-encoded hsvtk suicide genes

Gene Therapy volume 15, pages 1454–1459 (2008)Cite this article

Abstract

The in vivo regulation of T lymphocyte activity by the activation of a suicide mechanism is an essential paradigm for the safety of adoptive cell therapies. In light of reports showing that γ-retroviral vector-encoded herpes simplex virus thymidine kinase (hsvtk) undergoes recombination, we undertook a thorough investigation of the genomic stability of SFG-based vectors using two variants of the wild-type hsvtk gene. In a large panel of independent clones, we demonstrate that both hsvtk genes undergo recombination with molecular signatures indicative of template switching in GC-rich regions displaying homology at the deletion junctions or RNA splicing. In the absence of ganciclovir selection, the frequency of recombination is 3% per retroviral replication cycle. Our results underscore the importance of the five nucleotide difference between the two hsvtk genes that account for the presence of recombinogenic hot spots in one variant and not the other, indicating that the probability of RNA splicing is influenced by minute nucleotide changes in sequences adjacent to the splice donor and acceptor sites. Furthermore, our mutational analysis in an unbiased panel of human lymphoid cells (that is, without immune or ganciclovir-mediated selective pressure) provides a robust in vitro assay to predict and quantify clinically relevant mutations in hsvtk suicide genes, which can be applied to studying and improving the stability of any transgene expressed in γ-retroviral or lentiviral vectors.

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Acknowledgements

Genomic DNA from GMC/G1TK cells and PA317-G1TK was kindly provided by C Ferrand, Laboratoire de Thérapeutique Immuno-Moléculaire, Besancon, France. We thank Michael Newman for editorial assistance. This work is supported by P30 CA-008748, PO1 CA-059350, PO1 CA-023766, P50 CA086438 and by Mr William H Goodwin and Mrs Alice Goodwin of the Commonwealth Cancer Foundation for Research and the Experimental Therapeutics Center of MSKCC.

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Authors and Affiliations

  1. Gene Transfer and Somatic Cell Engineering Facility, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    X Wang, M Olszewska, V Capacio, J Stefanski, M Przybylowski, M Sadelain & I Rivière

  2. Gene Transfer and Gene Expression Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    S Samakoglu, A H Chang & M Sadelain

  3. Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    M Sadelain & I Rivière

  4. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    M Sadelain & I Rivière

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  1. X Wang
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  2. M Olszewska
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  9. I Rivière
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Correspondence to I Rivière.

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The authors declare no competing financial interests.

Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Wang, X., Olszewska, M., Capacio, V. et al. Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring γ-retrovirus-encoded hsvtk suicide genes. Gene Ther 15, 1454–1459 (2008). https://doi.org/10.1038/gt.2008.103

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