A method to sequence and quantify DNA integration for monitoring outcome in gene therapy

Troy Brady, Shoshannah L. Roth, Nirav Malani, Gary P. Wang, Charles C. Berry, Philippe Leboulch, Salima Hacein-Bey-Abina, Marina Cavazzana-Calvo, Eirini P. Papapetrou, Michel Sadelain, Harri Savilahti, Frederic D. Bushman

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

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.

Original languageEnglish
Pages (from-to)e72
JournalNucleic Acids Research
Volume39
Issue number11
DOIs
StatePublished - Jun 2011
Externally publishedYes

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