title: Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing.
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.
authors.affiliation: Department I of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.forename: Manuela
authors.initials: M
authors.lastname: Wissler
authors.forename: Gesa
authors.initials: G
authors.lastname: Hoffmann
authors.forename: Karin
authors.initials: K
authors.lastname: Olsson
authors.forename: Stephanie
authors.initials: S
authors.lastname: Sellers
authors.forename: Denise
authors.initials: D
authors.lastname: Carbonaro
authors.forename: John F
authors.initials: JF
authors.lastname: Tisdale
authors.forename: Cordula
authors.initials: C
authors.lastname: Leurs
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Kiem
authors.forename: Stefan
authors.initials: S
authors.lastname: Karlsson
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
authors.forename: David
authors.initials: D
authors.lastname: Williams
authors.forename: Christof
authors.initials: C
authors.lastname: Von Kalle
mesh_terms: D000818:Animals; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells / Q000145:classification* / Q000166:cytology*; D006801:Humans; D008253:Macaca mulatta; D010215:Papio; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: D004251:DNA Transposable Elements