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Nucleic Acids Research Advance Access originally published online on April 14, 2008
Nucleic Acids Research 2008 36(9):e49; doi:10.1093/nar/gkn125
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Nucleic Acids Research, 2008, Vol. 36, No. 9 e49
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer

Gary P. Wang1, Alexandrine Garrigue2, Angela Ciuffi1, Keshet Ronen1, Jeremy Leipzig1, Charles Berry3, Chantal Lagresle-Peyrou2,4, Fatine Benjelloun2,4, Salima Hacein-Bey-Abina2,5, Alain Fischer2,4,6, Marina Cavazzana-Calvo2,4,5 and Frederic D. Bushman1,*

1University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA, 2INSERM Unit 768Hôpital Necker Enfants Malades 149 rue de Sèvres, 75015 Paris, France, 3Department of Family/Preventive Medicine, University of California, San Diego School of Medicine, San Diego, CA 92093, USA, 4Faculté de Médecine René Descartes, Université Paris-Descartes, 5Assistance Publique, Département de Biotherapie and 6Assistance Publique, Hôpitaux de Paris (AP/HP), Service d’Immunologie et d’Hématologie Pédiatriques, Hôpital Necker Enfants Malades, Hôpital Necker Enfants Malades 149 rue de Sèvres, 75015 Paris, France

*To whom correspondence should be addressed. Tel: +1 215 573 8732; Fax: +1 215 573 4856; Email: bushman{at}mail.med.upenn.edu

Received January 2, 2008. Revised March 4, 2008. Accepted March 5, 2008.

Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160 232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy—in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.

Present Address: Angela Ciuffi, Institute of Microbiology (IMUL), University Hospital Center and University of Lausanne, Bugnon 48 – CHUV, 1011 Lausanne, Switzerland


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