title: High-throughput monitoring of integration site clonality in preclinical and clinical gene therapy studies.
abstract: Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR) has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies), it is not useful for large-scale analyses. To explore whether vector integration site (IS) recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings. We subjected all mixes to either linear amplification-mediated PCR (LAM-PCR) or nonrestrictive LAM-PCR (nrLAM-PCR), both combined with 454 sequencing. We showed that nrLAM-PCR/454-detected clonality allows estimating qPCR-detected clonality in vitro. We then followed the kinetics of two clones detected in a patient enrolled in a clinical gene therapy trial using both, nrLAM-PCR/454 and qPCR and also saw nrLAM-PCR/454 to correlate to qPCR-measured clonal contributions. The method presented here displays a feasible high-throughput strategy to monitor clonality in clinical gene therapy trials is at hand.
authors.affiliation: Department of Radiation Oncology, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University , Mannheim, Germany.
authors.forename: Frank A
authors.initials: FA
authors.lastname: Giordano
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany ; CLC bio , Aarhus, Denmark.
authors.forename: Jens-Uwe
authors.initials: JU
authors.lastname: Appelt
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Link
authors.affiliation: Institute for Medical Informatics and Biometry, Faculty of Medicine Carl Gustav Carus, Technische Universität , Dresden, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Gerdes
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Lehrer
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Scholz
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Paruzynski
authors.affiliation: Institute for Medical Informatics and Biometry, Faculty of Medicine Carl Gustav Carus, Technische Universität , Dresden, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Roeder
authors.affiliation: Department of Radiation Oncology, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University , Mannheim, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Wenz
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.affiliation: Institute for Biomedical Research, Georg-Speyer-Haus , Frankfurt, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Grez
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ) , Heidelberg, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Laufs