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Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus
A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturi...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Mary Ann Liebert, Inc., publishers
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590824/ https://www.ncbi.nlm.nih.gov/pubmed/33117614 http://dx.doi.org/10.1089/biores.2020.0031 |
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author | Tang, Qiushi Keeler, Allison M. Zhang, Songbo Su, Qin Lyu, Zhuoyao Cheng, Yangfan Gao, Guangping Flotte, Terence R. |
author_facet | Tang, Qiushi Keeler, Allison M. Zhang, Songbo Su, Qin Lyu, Zhuoyao Cheng, Yangfan Gao, Guangping Flotte, Terence R. |
author_sort | Tang, Qiushi |
collection | PubMed |
description | A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids. |
format | Online Article Text |
id | pubmed-7590824 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Mary Ann Liebert, Inc., publishers |
record_format | MEDLINE/PubMed |
spelling | pubmed-75908242020-10-27 Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus Tang, Qiushi Keeler, Allison M. Zhang, Songbo Su, Qin Lyu, Zhuoyao Cheng, Yangfan Gao, Guangping Flotte, Terence R. Biores Open Access Original Research Article A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids. Mary Ann Liebert, Inc., publishers 2020-10-16 /pmc/articles/PMC7590824/ /pubmed/33117614 http://dx.doi.org/10.1089/biores.2020.0031 Text en © Qiushi Tang et al., 2020; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Research Article Tang, Qiushi Keeler, Allison M. Zhang, Songbo Su, Qin Lyu, Zhuoyao Cheng, Yangfan Gao, Guangping Flotte, Terence R. Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus |
title | Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus |
title_full | Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus |
title_fullStr | Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus |
title_full_unstemmed | Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus |
title_short | Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus |
title_sort | two-plasmid packaging system for recombinant adeno-associated virus |
topic | Original Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590824/ https://www.ncbi.nlm.nih.gov/pubmed/33117614 http://dx.doi.org/10.1089/biores.2020.0031 |
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