Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo
The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector...
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Format: | Article |
Language: | English |
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Elsevier
2021-09-01
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Series: | Molecular Therapy: Methods & Clinical Development |
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Online Access: | http://www.sciencedirect.com/science/article/pii/S232905012100111X |
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doaj-3a3bb4272fd348898a6be51f1a71a71e |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Qingyun Zheng Tianyi Wang Xiangying Zhu Xiao Tian Chen Zhong Guolin Chang Gai Ran Yilin Xie Bing Zhao Liqing Zhu Chen Ling |
spellingShingle |
Qingyun Zheng Tianyi Wang Xiangying Zhu Xiao Tian Chen Zhong Guolin Chang Gai Ran Yilin Xie Bing Zhao Liqing Zhu Chen Ling Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo Molecular Therapy: Methods & Clinical Development rAAV vector production lipopolysaccharide NF-κB signal pathway ClearColi technology |
author_facet |
Qingyun Zheng Tianyi Wang Xiangying Zhu Xiao Tian Chen Zhong Guolin Chang Gai Ran Yilin Xie Bing Zhao Liqing Zhu Chen Ling |
author_sort |
Qingyun Zheng |
title |
Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo |
title_short |
Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo |
title_full |
Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo |
title_fullStr |
Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo |
title_full_unstemmed |
Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo |
title_sort |
low endotoxin e. coli strain-derived plasmids reduce raav vector-mediated immune responses both in vitro and in vivo |
publisher |
Elsevier |
series |
Molecular Therapy: Methods & Clinical Development |
issn |
2329-0501 |
publishDate |
2021-09-01 |
description |
The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development. |
topic |
rAAV vector production lipopolysaccharide NF-κB signal pathway ClearColi technology |
url |
http://www.sciencedirect.com/science/article/pii/S232905012100111X |
work_keys_str_mv |
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doaj-3a3bb4272fd348898a6be51f1a71a71e2021-09-15T04:22:20ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012021-09-0122293303Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivoQingyun Zheng0Tianyi Wang1Xiangying Zhu2Xiao Tian3Chen Zhong4Guolin Chang5Gai Ran6Yilin Xie7Bing Zhao8Liqing Zhu9Chen Ling10State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaZhejiang Hengyu Biological Technology Co., Ltd., Jiaxing, Zhejiang Province 314113, ChinaState Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaDepartment of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province 325000, ChinaState Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaState Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, ChinaDepartment of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province 325000, ChinaState Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China; Corresponding author: Chen Ling, PhD, State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China.The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.http://www.sciencedirect.com/science/article/pii/S232905012100111XrAAV vector productionlipopolysaccharideNF-κB signal pathwayClearColi technology |