Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing.
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.
Schmidt
Manfred
M
Department I of Internal Medicine, University of Freiburg, 79106 Freiburg, Germany.
Glimm
Hanno
H
Wissler
Manuela
M
Hoffmann
Gesa
G
Olsson
Karin
K
Sellers
Stephanie
S
Carbonaro
Denise
D
Tisdale
John F
JF
Leurs
Cordula
C
Hanenberg
Helmut
H
Dunbar
Cynthia E
CE
Kiem
Hans-Peter
HP
Karlsson
Stefan
S
Kohn
Donald B
DB
Williams
David
D
Von Kalle
Christof
C
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
D004251:DNA Transposable Elements