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

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Author

  • M Schmidt
  • H Glimm
  • M Wissler
  • G Hoffmann
  • Karin Olsson
  • S Sellers
  • D Carbonaro
  • JF Tisdale
  • C Leurs
  • H Hanenberg
  • CE Dunbar
  • HP Kiem
  • Stefan Karlsson
  • DB Kohn
  • D Williams
  • C von Kalle
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Summary, in English

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.

Publishing year

2003

Language

English

Pages

112-121

Publication/Series

Annals of the New York Academy of Sciences (HEMATOPOIETIC STEM CELLS 2002: GENETICS AND FUNCTION: Fourth International Symposium)

Volume

996

Document type

Conference paper

Publisher

New York Academy of Sciences

Topic

  • Hematology

Keywords

  • proviruses
  • therapy
  • insertion sequence elements
  • investigative techniques
  • gene
  • stem cells

Conference name

HEMATOPOIETIC STEM CELLS 2002: GENETICS AND FUNCTION: Fourth International Symposium

Conference date

0001-01-02

Status

Published

ISBN/ISSN/Other

  • ISSN: 0077-8923