Efficient Characterization of Retro-, Lenti-, and Foamyvector-Transduced Cell Populations by High-Accuracy Insertion Site Sequencing

Authors

  • MANFRED SCHMIDT,

    1. Department I of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany
    2. Institute for Molecular Medicine and Cell Research, University of Freiburg, 79106 Freiburg, Germany
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  • HANNO GLIMM,

    1. Department I of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany
    2. Institute for Molecular Medicine and Cell Research, University of Freiburg, 79106 Freiburg, Germany
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  • MANUELA WISSLER,

    1. Department I of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany
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  • GESA HOFFMANN,

    1. Department I of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany
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  • KARIN OLSSON,

    1. Department of Molecular Medicine and Gene Therapy, Lund University, BMC A12, 22184 Lund, Sweden
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  • STEPHANIE SELLERS,

    1. Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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  • DENISE CARBONARO,

    1. Division of Research Immunology/B.M.T., Childrens Hospital Los Angeles, and the Departments of Pediatrics and Microbiology, University of Southern California Keck School of Medicine, Los Angeles, California 90029, USA
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  • JOHN F. TISDALE,

    1. Molecular and Clinical Hematology Branch, National Institute for Digestive, Diabetes and Kidney, National Institutes of Health, Bethesda, MD 20892, USA
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  • CORDULA LEURS,

    1. Division of Pediatric Hematology and Oncology, Childrens Health Center, University of Düsseldorf, 40225 Düsseldorf, Germany
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  • HELMUT HANENBERG,

    1. Division of Pediatric Hematology and Oncology, Childrens Health Center, University of Düsseldorf, 40225 Düsseldorf, Germany
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  • CYNTHIA E. DUNBAR,

    1. Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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  • HANS-PETER KIEM,

    1. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
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  • STEFAN KARLSSON,

    1. Department of Molecular Medicine and Gene Therapy, Lund University, BMC A12, 22184 Lund, Sweden
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  • DONALD B. KOHN,

    1. Division of Research Immunology/B.M.T., Childrens Hospital Los Angeles, and the Departments of Pediatrics and Microbiology, University of Southern California Keck School of Medicine, Los Angeles, California 90029, USA
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  • DAVID WILLIAMS,

    1. Division of Experimental Hematology, Cincinnati Children's Research Foundation, Cincinnati Ohio 45229, USA
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  • CHRISTOF VON KALLE

    Corresponding author
    1. Department I of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany
    2. Institute for Molecular Medicine and Cell Research, University of Freiburg, 79106 Freiburg, Germany
    3. Division of Experimental Hematology, Cincinnati Children's Research Foundation, Cincinnati Ohio 45229, USA
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Address for correspondence: Christof von Kalle, Hugstetter Str. 55, Institute for Molecular Medicine and Cell Research, University of Freiburg, 79106 Freiburg, Germany. Voice: +49 761-270-7181; fax: +49 761-270-7177. Kalle@ukl.uni.-freiburg.de)

Abstract

Abstract: The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro-, lenti- and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification-mediated PCR (LAM-PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid-phase. Using LAM-PCR, we show the previously unfeasible in vivo retro-, lenti- and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID-X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.

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