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Construction of stable packaging cell lines for clinical lentiviral vector production

Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to...

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Autores principales: Sanber, Khaled S., Knight, Sean B., Stephen, Sam L., Bailey, Ranbir, Escors, David, Minshull, Jeremy, Santilli, Giorgia, Thrasher, Adrian J., Collins, Mary K., Takeuchi, Yasuhiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356972/
https://www.ncbi.nlm.nih.gov/pubmed/25762005
http://dx.doi.org/10.1038/srep09021
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author Sanber, Khaled S.
Knight, Sean B.
Stephen, Sam L.
Bailey, Ranbir
Escors, David
Minshull, Jeremy
Santilli, Giorgia
Thrasher, Adrian J.
Collins, Mary K.
Takeuchi, Yasuhiro
author_facet Sanber, Khaled S.
Knight, Sean B.
Stephen, Sam L.
Bailey, Ranbir
Escors, David
Minshull, Jeremy
Santilli, Giorgia
Thrasher, Adrian J.
Collins, Mary K.
Takeuchi, Yasuhiro
author_sort Sanber, Khaled S.
collection PubMed
description Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10(6) transducing units/ml can be harvested from the final producer clones, which can be increased to 10(8) TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.
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spelling pubmed-43569722015-03-17 Construction of stable packaging cell lines for clinical lentiviral vector production Sanber, Khaled S. Knight, Sean B. Stephen, Sam L. Bailey, Ranbir Escors, David Minshull, Jeremy Santilli, Giorgia Thrasher, Adrian J. Collins, Mary K. Takeuchi, Yasuhiro Sci Rep Article Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10(6) transducing units/ml can be harvested from the final producer clones, which can be increased to 10(8) TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors. Nature Publishing Group 2015-03-12 /pmc/articles/PMC4356972/ /pubmed/25762005 http://dx.doi.org/10.1038/srep09021 Text en Copyright © 2015, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Sanber, Khaled S.
Knight, Sean B.
Stephen, Sam L.
Bailey, Ranbir
Escors, David
Minshull, Jeremy
Santilli, Giorgia
Thrasher, Adrian J.
Collins, Mary K.
Takeuchi, Yasuhiro
Construction of stable packaging cell lines for clinical lentiviral vector production
title Construction of stable packaging cell lines for clinical lentiviral vector production
title_full Construction of stable packaging cell lines for clinical lentiviral vector production
title_fullStr Construction of stable packaging cell lines for clinical lentiviral vector production
title_full_unstemmed Construction of stable packaging cell lines for clinical lentiviral vector production
title_short Construction of stable packaging cell lines for clinical lentiviral vector production
title_sort construction of stable packaging cell lines for clinical lentiviral vector production
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356972/
https://www.ncbi.nlm.nih.gov/pubmed/25762005
http://dx.doi.org/10.1038/srep09021
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