DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.

Details

Serval ID
serval:BIB_D47F95402494
Type
Article: article from journal or magazin.
Collection
Publications
Title
DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.
Journal
Nucleic Acids Research
Author(s)
Wang G.P., Garrigue A., Ciuffi A., Ronen K., Leipzig J., Berry C., Lagresle-Peyrou C., Benjelloun F., Hacein-Bey-Abina S., Fischer A., Cavazzana-Calvo M., Bushman F.D.
ISSN
0305-1048
Publication state
Published
Issued date
05/2008
Peer-reviewed
Oui
Volume
36
Number
9
Pages
e49
Language
english
Notes
Publication types: Evaluation Studies ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't Publication Status: ppublish
Abstract
Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160,232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy--in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.
Keywords
Animals, Cell Proliferation, DNA Restriction Enzymes, Disease Models, Animal, Gene Therapy/adverse effects, Gene Transfer Techniques, Genes, Tumor Suppressor, Genetic Vectors, Lentivirus/genetics, Lymphoproliferative Disorders/genetics, Mice, Proto-Oncogenes, Sequence Analysis, DNA/methods
Pubmed
Web of science
Open Access
Yes
Create date
04/02/2009 18:27
Last modification date
20/08/2019 16:54
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