Abstract

Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification–mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.

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Acknowledgements

We thank I. Kutschera, N. Krenzer, C. Lulay, S. Braun and D. Glagow for technical assistance and O. Danos for fruitful discussions about the model. Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium für Bildung und Forschung (iGene), by the VIth + VIIth Framework Programs of the European Commission (Concerted Safety & Efficiency Evaluation of Retroviral Transgenesis in Gene Therapy of Inherited Diseases (CONSERT), European Network for the Advancement of Clinical Gene Transfer and Therapy (CLINIGENE) and Persisting Transgenesis (PERSIST)) and by the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer.

Author information

Author notes

    • Richard Gabriel
    • , Ralph Eckenberg
    • , Anna Paruzynski
    • , Cynthia C Bartholomae
    • , William Saurin
    •  & Manfred Schmidt

    These authors contributed equally to this work.

Affiliations

  1. Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany.

    • Richard Gabriel
    • , Anna Paruzynski
    • , Cynthia C Bartholomae
    • , Ali Nowrouzi
    • , Anne Arens
    • , Wei Wang
    • , Katrin Faber
    • , Kerstin Schwarzwaelder
    • , Romy Kirsten
    • , Annette Deichmann
    • , Claudia R Ball
    • , Hanno Glimm
    • , Christof von Kalle
    •  & Manfred Schmidt
  2. Genomining, Montrouge, France.

    • Ralph Eckenberg
    •  & William Saurin
  3. Genomics and Proteomics Core Facilities, German Cancer Research Center, Heidelberg, Germany.

    • Anne Arens
  4. Molecular Immunology Unit, Institute of Child Health, University College, London, UK.

    • Steven J Howe
    • , Robin R Ali
    •  & Adrian J Thrasher
  5. Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy.

    • Alessandra Recchia
    •  & Fulvio Mavilio
  6. Cancer Immunotherapy and Gene Therapy Program, Instituto Scientifico H. San Raffaele, Milan, Italy.

    • Claudia Cattoglio
    •  & Fulvio Mavilio
  7. Institute of Ophthalmology, University College London, London, UK.

    • Kamaljit S Balaggan
    •  & Robin R Ali
  8. School of Biological Sciences, Royal Holloway–University of London, Egham, UK.

    • Rafael J Yáñez-Muñoz
  9. Department of Clinical Immunology, Great Ormond Street Hospital NHS Trust, London, UK.

    • H Bobby Gaspar
    •  & Adrian J Thrasher
  10. San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.

    • Luca Biasco
    • , Alessandro Aiuti
    • , Daniela Cesana
    • , Eugenio Montini
    •  & Luigi Naldini
  11. Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy.

    • Alessandro Aiuti
  12. Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquées, Ecole Normale Supérieure de Cachan, Cachan, France.

    • Odile Cohen-Haguenauer
  13. Oncogenetics, Department of Clinical Oncology, Hôpital Saint-Louis, Paris, France.

    • Odile Cohen-Haguenauer
  14. Molecular and Gene Therapy Program, Cincinnati Children's Research Foundation, Cincinnati, Ohio, USA.

    • Christof von Kalle

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Contributions

R.E., C.v.K., W.S. and M.S. conceived of the genome accessibility model and interpreted data. R.E. and W.S. conducted bioinformatics analyses. C.v.K. and M.S., R.G., A.P. and H.G. developed the concept of nrLAM and designed experiments. R.G., A.P., A.N., C.C.B. and C.R.B. performed experiments. W.W. provided lentiviral vectors. D.C., E.M., L.N., L.B., A. Aiuti, O.C.-H., K.S.B., R.J.Y.-M., R.R.A., A.R., C.C. and F.M. provided retroviral integration site data sets; C.C.B. and A. Arens performed LAM-PCR and generated the data on these samples. C.C.B., K.S., A. Arens, K.F. and A.D. generated LAM data on X-SCID samples provided by S.J.H., H.B.G. and A.J.T. R.G., R.E., A.P., C.C.B., R.K., W.S., C.v.K. and M.S. prepared and wrote the manuscript.

Corresponding author

Correspondence to Christof von Kalle.

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DOI

https://doi.org/10.1038/nm.2057

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