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诱导型 EIAV 基于慢病毒载体包装和生产细胞系的开发。

Development of inducible EIAV-based lentiviral vector packaging and producer cell lines.

机构信息

Oxford BioMedica (UK) Ltd, Medawar Centre, Robert Robinson Avenue, The Oxford Science Park, Oxford, UK.

出版信息

Gene Ther. 2009 Jun;16(6):805-14. doi: 10.1038/gt.2009.20. Epub 2009 Mar 5.

Abstract

Large-scale production of gene therapeutics comprising equine infectious anaemia virus (EIAV) -based lentiviral vectors (LVs) would benefit from the development of producer cell lines enabling the generation of larger quantities of vector than achievable by transient systems. Such cell lines would contain three vector components (Gag/Pol, VSV-G envelope and genome expression constructs). As the vesicular stomatitis virus (VSV-G) envelope protein is cytotoxic, its expression must be regulated. It is also desirable to regulate Gag/Pol expression to minimise metabolic burden on the cell. The Tet repressor (TetR) system was selected to regulate expression of VSV-G and Gag/Pol, necessitating the introduction of a fourth construct, encoding TetR, into the cell line. We have generated an inducible packaging cell line that shows tight control of the packaging components, and high-titre vector production on transient transfection of the EIAV genome. The cell line is stable for at least 7 weeks in the absence of selective pressure. To verify that this packaging cell line can support the generation of producer cell lines it was transfected stably with an EIAV genome cassette encoding ProSavin; a gene therapeutic for Parkinson's disease. Producer cell lines were generated, which on induction, yielded ProSavin with titres comparable to the transient system.

摘要

大规模生产包含马传染性贫血病毒(EIAV)的基于慢病毒载体(LV)的基因治疗药物将受益于生产细胞系的发展,这些细胞系能够产生比瞬时系统更多的载体。这些细胞系将包含三个载体组件(Gag/Pol、VSV-G 包膜和基因组表达构建体)。由于水疱性口炎病毒(VSV-G)包膜蛋白具有细胞毒性,因此必须对其表达进行调节。调节 Gag/Pol 表达以最小化细胞的代谢负担也是可取的。Tet 阻遏物(TetR)系统被选择用于调节 VSV-G 和 Gag/Pol 的表达,这需要将第四种构建体,即 TetR,引入细胞系。我们已经生成了一种可诱导的包装细胞系,该细胞系对包装成分具有严格的控制,并且在瞬时转染 EIAV 基因组时能够产生高滴度的载体。在没有选择压力的情况下,该细胞系至少稳定 7 周。为了验证该包装细胞系能够支持生产细胞系的生成,我们将其稳定转染了一个编码 ProSavin 的 EIAV 基因组盒,ProSavin 是一种治疗帕金森病的基因治疗药物。生成了生产细胞系,在诱导后,产生的 ProSavin 的滴度与瞬时系统相当。

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