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A method to sequence and quantify DNA integration for monitoring outcome in gene therapy
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 anal...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113588/ https://www.ncbi.nlm.nih.gov/pubmed/21415009 http://dx.doi.org/10.1093/nar/gkr140 |
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author | Brady, Troy Roth, Shoshannah L. Malani, Nirav Wang, Gary P. Berry, Charles C. Leboulch, Philippe Hacein-Bey-Abina, Salima Cavazzana-Calvo, Marina Papapetrou, Eirini P. Sadelain, Michel Savilahti, Harri Bushman, Frederic D. |
author_facet | Brady, Troy Roth, Shoshannah L. Malani, Nirav Wang, Gary P. Berry, Charles C. Leboulch, Philippe Hacein-Bey-Abina, Salima Cavazzana-Calvo, Marina Papapetrou, Eirini P. Sadelain, Michel Savilahti, Harri Bushman, Frederic D. |
author_sort | Brady, Troy |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3113588 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-31135882011-06-14 A method to sequence and quantify DNA integration for monitoring outcome in gene therapy Brady, Troy Roth, Shoshannah L. Malani, Nirav Wang, Gary P. Berry, Charles C. Leboulch, Philippe Hacein-Bey-Abina, Salima Cavazzana-Calvo, Marina Papapetrou, Eirini P. Sadelain, Michel Savilahti, Harri Bushman, Frederic D. Nucleic Acids Res Methods Online 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. Oxford University Press 2011-06 2011-03-16 /pmc/articles/PMC3113588/ /pubmed/21415009 http://dx.doi.org/10.1093/nar/gkr140 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Brady, Troy Roth, Shoshannah L. Malani, Nirav Wang, Gary P. Berry, Charles C. Leboulch, Philippe Hacein-Bey-Abina, Salima Cavazzana-Calvo, Marina Papapetrou, Eirini P. Sadelain, Michel Savilahti, Harri Bushman, Frederic D. A method to sequence and quantify DNA integration for monitoring outcome in gene therapy |
title | A method to sequence and quantify DNA integration for monitoring outcome in gene therapy |
title_full | A method to sequence and quantify DNA integration for monitoring outcome in gene therapy |
title_fullStr | A method to sequence and quantify DNA integration for monitoring outcome in gene therapy |
title_full_unstemmed | A method to sequence and quantify DNA integration for monitoring outcome in gene therapy |
title_short | A method to sequence and quantify DNA integration for monitoring outcome in gene therapy |
title_sort | method to sequence and quantify dna integration for monitoring outcome in gene therapy |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113588/ https://www.ncbi.nlm.nih.gov/pubmed/21415009 http://dx.doi.org/10.1093/nar/gkr140 |
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