title: A method to sequence and quantify DNA integration for monitoring outcome in gene therapy.
abstract: Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.
authors.forename: Troy
authors.initials: T
authors.lastname: Brady
authors.forename: Shoshannah L
authors.initials: SL
authors.lastname: Roth
authors.forename: Nirav
authors.initials: N
authors.lastname: Malani
authors.forename: Gary P
authors.initials: GP
authors.lastname: Wang
authors.forename: Charles C
authors.initials: CC
authors.lastname: Berry
authors.forename: Philippe
authors.initials: P
authors.lastname: Leboulch
authors.forename: Salima
authors.initials: S
authors.lastname: Hacein-Bey-Abina
authors.forename: Marina
authors.initials: M
authors.lastname: Cavazzana-Calvo
authors.forename: Eirini P
authors.initials: EP
authors.lastname: Papapetrou
authors.forename: Michel
authors.initials: M
authors.lastname: Sadelain
authors.forename: Harri
authors.initials: H
authors.lastname: Savilahti
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
mesh_terms: D010583:Bacteriophage mu / Q000235:genetics; D002460:Cell Line; D018390:Gene Targeting; D015316:Genetic Therapy; D006801:Humans; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*