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Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors
Plasmid transfection of mammalian cells is the dominant platform used to produce adeno‐associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9314941/ https://www.ncbi.nlm.nih.gov/pubmed/35182435 http://dx.doi.org/10.1002/bit.28068 |
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author | Dash, Shantoshini Sharon, David M. Mullick, Alaka Kamen, Amine A. |
author_facet | Dash, Shantoshini Sharon, David M. Mullick, Alaka Kamen, Amine A. |
author_sort | Dash, Shantoshini |
collection | PubMed |
description | Plasmid transfection of mammalian cells is the dominant platform used to produce adeno‐associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection‐based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation‐specific antibody staining and flow cytometry, we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5%–10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection‐based AAV vector manufacturing platforms. |
format | Online Article Text |
id | pubmed-9314941 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-93149412022-07-30 Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors Dash, Shantoshini Sharon, David M. Mullick, Alaka Kamen, Amine A. Biotechnol Bioeng COMMUNICATION Plasmid transfection of mammalian cells is the dominant platform used to produce adeno‐associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection‐based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation‐specific antibody staining and flow cytometry, we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5%–10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection‐based AAV vector manufacturing platforms. John Wiley and Sons Inc. 2022-02-28 2022-06 /pmc/articles/PMC9314941/ /pubmed/35182435 http://dx.doi.org/10.1002/bit.28068 Text en © 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | COMMUNICATION Dash, Shantoshini Sharon, David M. Mullick, Alaka Kamen, Amine A. Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors |
title | Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors |
title_full | Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors |
title_fullStr | Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors |
title_full_unstemmed | Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors |
title_short | Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors |
title_sort | only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors |
topic | COMMUNICATION |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9314941/ https://www.ncbi.nlm.nih.gov/pubmed/35182435 http://dx.doi.org/10.1002/bit.28068 |
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