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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...

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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2011
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.
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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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