High-throughput monitoring of integration site clonality in preclinical and clinical gene therapy studies

• Verfasst von: Giordano, Frank Anton [VerfasserIn]
• Verfasst von: Appelt, Jens-Uwe [VerfasserIn]
• Verfasst von: Link, Barbara [VerfasserIn]
• Verfasst von: Gerdes, Sebastian [VerfasserIn]
• Verfasst von: Scholz, Simone [VerfasserIn]
• Verfasst von: Paruzynski, Anna [VerfasserIn]
• Verfasst von: Röder, Ingo [VerfasserIn]
• Verfasst von: Wenz, Frederik [VerfasserIn]
• Verfasst von: Glimm, Hanno [VerfasserIn]
• Verfasst von: Kalle, Christof von [VerfasserIn]
• Verfasst von: Grez, Manuel [VerfasserIn]
• Verfasst von: Schmidt, Manfred [VerfasserIn]
• Verfasst von: Maier-Laufs, Stephanie [VerfasserIn]
• Titel: High-throughput monitoring of integration site clonality in preclinical and clinical gene therapy studies
• Verf.angabe: Frank A. Giordano, Jens-Uwe Appelt, Barbara Link, Sebastian Gerdes, Christina Lehrer, Simone Scholz, Anna Paruzynski, Ingo Roeder, Frederik Wenz, Hanno Glimm, Christof von Kalle, Manuel Grez, Manfred Schmidt and Stephanie Laufs
• Jahr: 2015
• Umfang: 8 S.
• Fussnoten: Elektronische Reproduktion der Druckausgabe ; Gesehen am 11.08.2021
• Titel Quelle: Enthalten in: Molecular therapy. Methods & clinical development
• Ort Quelle: New York, NY : Nature Publishing Group, 2014
• Jahr Quelle: 2015
• Band/Heft Quelle: 2(2015), Artikel-ID 14061, Seite 1-8
• ISSN Quelle: 2329-0501
• DOI: doi:10.1038/mtm.2014.61
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1766107656

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.