Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing

Manfred Schmidt, Hanno Glimm, Manuela Wissler, Gesa Hoffmann, Karin Olsson, Stephanie Sellers, Denise Carbonaro, John F. Tisdale, Cordula Leurs, Helmut Hanenberg, Cynthia E. Dunbar, Hans Peter Kiem, Stefan Karlsson, Donald B. Kohn, David Williams, Christof Von Kalle

Research output: Contribution to journalArticle

18 Scopus citations

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 celis 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.

Original languageEnglish (US)
Pages (from-to)112-121
Number of pages10
JournalAnnals of the New York Academy of Sciences
Volume996
DOIs
StatePublished - Jan 1 2003

Keywords

  • Gene therapy
  • Insertion sequence elements
  • Investigative techniques
  • Proviruses
  • Stem cells

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • History and Philosophy of Science

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    Schmidt, M., Glimm, H., Wissler, M., Hoffmann, G., Olsson, K., Sellers, S., Carbonaro, D., Tisdale, J. F., Leurs, C., Hanenberg, H., Dunbar, C. E., Kiem, H. P., Karlsson, S., Kohn, D. B., Williams, D., & Von Kalle, C. (2003). Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing. Annals of the New York Academy of Sciences, 996, 112-121. https://doi.org/10.1111/j.1749-6632.2003.tb03239.x