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Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system
The human Adeno-Associated Virus serotype 2 (wtAAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. Although rAAVs are routinely produced in the Baculovirus/Sf9 cell system, wtAAV2 has never been studied in this context. We tried to produce...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033426/ https://www.ncbi.nlm.nih.gov/pubmed/29975713 http://dx.doi.org/10.1371/journal.pone.0199866 |
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author | Savy, Adrien Kaikkonen, Minna U. Léger, Adrien Dickx, Yohann Galibert, Lionel Merten, Otto-Wilhelm |
author_facet | Savy, Adrien Kaikkonen, Minna U. Léger, Adrien Dickx, Yohann Galibert, Lionel Merten, Otto-Wilhelm |
author_sort | Savy, Adrien |
collection | PubMed |
description | The human Adeno-Associated Virus serotype 2 (wtAAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. Although rAAVs are routinely produced in the Baculovirus/Sf9 cell system, wtAAV2 has never been studied in this context. We tried to produce wtAAV2 in the baculovirus/Sf9 cell system hypothesizing that the wtAAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce wtAAV2 in Baculovirus/Sf9, we found that wtAAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system leads to the expression of Rep78 that finally excises wtAAV2 genome from the baculovirus genome during the earliest phases of baculovirus stock production. Via p5 promoter expression kinetics and strand specific RNA-Seq analysis of wtAAV2, rAAV and Rep2/Cap2 cassettes in the baculovirus context we could demonstrate that wtAAV2 native promoters, p5, p19 and p40 are all active in the context of the baculovirus system and lead to the expression of different proteins and peptides. In addition, this study has proven that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle. |
format | Online Article Text |
id | pubmed-6033426 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-60334262018-07-19 Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system Savy, Adrien Kaikkonen, Minna U. Léger, Adrien Dickx, Yohann Galibert, Lionel Merten, Otto-Wilhelm PLoS One Research Article The human Adeno-Associated Virus serotype 2 (wtAAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. Although rAAVs are routinely produced in the Baculovirus/Sf9 cell system, wtAAV2 has never been studied in this context. We tried to produce wtAAV2 in the baculovirus/Sf9 cell system hypothesizing that the wtAAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce wtAAV2 in Baculovirus/Sf9, we found that wtAAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system leads to the expression of Rep78 that finally excises wtAAV2 genome from the baculovirus genome during the earliest phases of baculovirus stock production. Via p5 promoter expression kinetics and strand specific RNA-Seq analysis of wtAAV2, rAAV and Rep2/Cap2 cassettes in the baculovirus context we could demonstrate that wtAAV2 native promoters, p5, p19 and p40 are all active in the context of the baculovirus system and lead to the expression of different proteins and peptides. In addition, this study has proven that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle. Public Library of Science 2018-07-05 /pmc/articles/PMC6033426/ /pubmed/29975713 http://dx.doi.org/10.1371/journal.pone.0199866 Text en © 2018 Savy et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Savy, Adrien Kaikkonen, Minna U. Léger, Adrien Dickx, Yohann Galibert, Lionel Merten, Otto-Wilhelm Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system |
title | Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system |
title_full | Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system |
title_fullStr | Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system |
title_full_unstemmed | Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system |
title_short | Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system |
title_sort | genetics instability of wtaav2 genome and aav promoter activities in the baculovirus/sf9 cells system |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033426/ https://www.ncbi.nlm.nih.gov/pubmed/29975713 http://dx.doi.org/10.1371/journal.pone.0199866 |
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