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Comprehensive genomic access to vector integration in clinical gene therapy

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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Figure 1: Modeling the genomic accessibility of viral integration sites.
Figure 2: Limitations in effective co-amplification of long LAM–PCR amplicons.
Figure 3: Limitations in integration site retrieval for a leukemic human subject sample from an X-SCID gene therapy trial.
Figure 4: Comparison between standard and nrLAM–PCR.
Figure 5: nrLAM-PCR on mono-, oligo- and polyclonal clinical and preclinical samples.

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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.

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Authors and Affiliations

Authors

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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Supplementary Figures 1–4, Supplementary Tables 1–9 and Supplementary Methods (PDF 1044 kb)

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Gabriel, R., Eckenberg, R., Paruzynski, A. et al. Comprehensive genomic access to vector integration in clinical gene therapy. Nat Med 15, 1431–1436 (2009). https://doi.org/10.1038/nm.2057

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