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NGS library preparation may generate artifactual integration sites of AAV vectors

Nature Medicine volume 20, pages 577–578 (2014)Cite this article

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To the Editor:

The treatment of monogenic diseases using recombinant adeno-associated virus (rAAV) vectors has recently gained maturity with the approval of Glybera (AAV1-LPLS447X) by the European Medicines Agency1 for the treatment of lipoprotein lipase (LPL) deficiency. This milestone in gene therapy paves the way for future development of other rAAV products against a wide range of human diseases. rAAV vectors are generally considered safe and efficient, but some studies have raised concerns about their potential genotoxicity and highlighted the need for further investigation regarding their possible role in cancer development2,3. In this context, Kaeppel et al.4 recently published an analysis of rAAV integration sites (ISs) in human DNA following Glybera administration. They first applied linear amplification–mediated (LAM) PCR on human biopsies and mouse samples following intramuscular and intravenous injections of AAV1-LPLS447X. As previously described in preclinical samples5, they found random integration into genomic DNA5, but they also identified probable ISs in mitochondrial DNA (mtDNA). In the second part of the article, the authors developed a new strategy to specifically assess the ability of rAAV to integrate into mtDNA. The principle of this method substantially differed from that of LAM-PCR and was based on mitochondria enrichment from fresh tissue followed by next-generation sequencing (NGS). We have performed a closely related protocol and have gathered strong evidence that this approach generates artifactual junctions between rAAV and cellular genomes that are indistinguishable from true insertion events (IEs). We think that the lack of appropriate controls led Kaeppel et al.4 to misinterpret the data from NGS regarding integration of the rAAV genome into mitochondrial DNA and to formulate erroneous conclusions with major scientific and clinical implications.

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Figure 1: Distribution of junction breakpoints along rAAV genomes.

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Acknowledgements

This study has been supported by funds from the Association Française contre les Myopathies and the French Regional Council of Pays-de-la-Loire. We thank the genomics and bioinformatics core facilities of Nantes (Biogenouest) for their expert services. We are also grateful to M. Haskins for his critical reading.

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Authors and Affiliations

  1. INSERM, UMR 1089, Nantes, France

    Benjamin Cogné, Richard Snyder, Jean-Baptiste Dupont, Philippe Moullier & Adrien Leger

  2. University of Nantes, Nantes, France

    Benjamin Cogné, Pierre Lindenbaum, Jean-Baptiste Dupont, Richard Redon, Philippe Moullier & Adrien Leger

  3. Nantes University Hospital, Nantes, France

    Benjamin Cogné, Pierre Lindenbaum, Jean-Baptiste Dupont, Richard Redon, Philippe Moullier & Adrien Leger

  4. Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, USA

    Richard Snyder & Philippe Moullier

  5. Center of Excellence for Regenerative Health Biotechnology, University of Florida, Alachua, Florida, USA

    Richard Snyder

  6. INSERM, UMR 1087, L'Institut du Thorax, Nantes, France

    Pierre Lindenbaum & Richard Redon

  7. CNRS, UMR 6291, Nantes, France

    Pierre Lindenbaum & Richard Redon

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  1. Benjamin Cogné
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Correspondence to Adrien Leger.

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Competing interests

R.S. is an inventor on patents related to recombinant AAV and owns equity in a gene therapy company, AGCT, that is commercializing AAV for gene therapy applications.

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Supplementary Methods, Supplementary Table 1 and Supplementary Figures 1–3 (PDF 342 kb)

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Cogné, B., Snyder, R., Lindenbaum, P. et al. NGS library preparation may generate artifactual integration sites of AAV vectors. Nat Med 20, 577–578 (2014). https://doi.org/10.1038/nm.3578

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