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AAV serotype 2 vectors preferentially integrate into active genes in mice

Nature Genetics volume 34, pages 297–302 (2003)Cite this article

Abstract

Recombinant adeno-associated virus serotype 2 (rAAV2) is a promising vector for gene therapy because it can achieve long-term stable transgene expression in animals and human subjects after direct administration of vectors into various target tissues1. In the liver, although stable transgene expression primarily results from extrachromosomal vector genomes2, a series of experiments has shown that vector genomes integrate into host chromosomes in hepatocytes3,4,5 at a low frequency2. Despite the low integration efficiency, recent reports of retroviral insertional mutagenesis in mice6 and two human subjects7,8 have raised concerns about the potential for rAAV2-mediated insertional mutagenesis. Here we characterize rAAV2-targeted chromosomal integration sites isolated from selected or non-selected hepatocytes in vector-injected mouse livers. We document frequent chromosomal deletions of up to 2 kb at integration sites (14 of 14 integrations, 100%; most of the deletions were <0.3 kb) and preferred integration into genes (21 of 29 integrations, 72%). In addition, all of the targeted genes analyzed (20 of 20 targeted genes, 100%) were expressed in the liver. This is the first report to our knowledge on host chromosomal effects of rAAV2 integration in animals, and it provides insights into the nature of rAAV2 vector integration into chromosomes in quiescent somatic cells in animals and human subjects.

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Figure 1: rAAV2 shuttle vectors, strategy for isolating vector-cellular DNA junctions and structures of rAAV2 proviruses isolated from mouse livers.
Figure 2: Sequences of rAAV2 vector-mouse cellular DNA junctions.
Figure 3: RT–PCR analysis for expression of rAAV2-targeted genes in mouse livers.

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Acknowledgements

We thank J. Park for data analysis. This work was supported by grants from the National Heart, Lung, and Blood Institute of the US National Institutes of Health to M.A.K.

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Author notes

  1. Eugenio Montini

    Present address: Instituto per la cura e la ricerca del Cancro, Candiolo, Torino, 10060, Italy

Authors and Affiliations

  1. Departments of Pediatrics and Genetics, Stanford University School of Medicine, 300 Pasteur Dr. Rm G305A, Stanford, 94305, California, USA

    Hiroyuki Nakai, Sally Fuess, Theresa A Storm & Mark A Kay

  2. Departments of Molecular and Medical Genetics, Oregon Health & Science University, Portland, 97201, Oregon, USA

    Eugenio Montini & Markus Grompe

  3. Department of Pediatrics, Oregon Health & Science University, Portland, 97201, Oregon, USA

    Markus Grompe

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  1. Hiroyuki Nakai
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Correspondence to Mark A Kay.

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M.A.K. is on the Scientific Advisory Board of Avigen Inc., a company involved in AAV gene therapeutics.

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Nakai, H., Montini, E., Fuess, S. et al. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat Genet 34, 297–302 (2003). https://doi.org/10.1038/ng1179

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