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

   


Authors: Brady, T.; Roth, S. L.; Malani, N.; Wang, G. P.; Berry, C. C.; Leboulch, P.; Hacein-Bey-Abina, S.; Cavazzana-Calvo, M.; Papapetrou, E. P.; Sadelain, M.; Savilahti, H.; Bushman, F. D.
Article 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. © 2011 The Author(s).
Keywords: human cell; polymerase chain reaction; gene; in vitro study; nucleotide sequence; gene therapy; retrovirus vector; beta thalassemia; dna sequence; cd3+ t lymphocyte; unindexed sequence; severe combined immunodeficiency; genomic dna; peripheral blood mononuclear cell; pyrosequencing; bacteriophage mu; transposase; dna barcoding; dna integration; dna transposition; hmga2 gene; integration site sequence; restriction mapping
Journal Title: Nucleic Acids Research
Volume: 39
Issue: 11
ISSN: 0305-1048
Publisher: Oxford University Press  
Date Published: 2011-06-01
Start Page: e72
Language: English
DOI: 10.1093/nar/gkr140
PROVIDER: scopus
PUBMED: 21415009
PMCID: PMC3113588
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-79959469934&partnerID=40&md5=071084052ab1cae3edfcb8f0761e5e3c
Notes: --- - "Export Date: 17 August 2011" - "CODEN: NARHA" - "Source: Scopus"
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MSK Authors
  1. Michel W J Sadelain
    583 Sadelain
  2. Eirini P Papapetrou
    24 Papapetrou
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