Maximilian Klimpel, Monica Terrao, Melina Bräuer, Herbert Dersch, Martina Biserni, Larissa Melo Do Nascimento, Sarah Schwingal, Jessica E. Vogel, Cathrin Ferlemann, Tobias Brandt, Nikki Indresh Lal, Krystal Bridgeman, Alex Petzke, Eva McDwyer, Jo Leen Lim, Seungyoul Oh, Gabriela Brumatti, Albert Garcia Minambres, Ellen Otte, Thomas Noll, Vicky Pirzas, Holger Laux
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引用次数: 0
摘要
用水泡性口炎病毒包膜糖蛋白(VSV-G)伪型慢病毒载体(LV)的生产受到了包膜相关细胞毒性和生产方法(如瞬时转染粘附细胞系)的限制。在这项研究中,我们建立了稳定的悬浮生产细胞系,用于规模化和无血清生产 LV,这些细胞系来自两个稳定的诱导包装细胞系,分别命名为 GPRG 和 GPRTG。已建立的多克隆生产者细胞系可在摇瓶中以半灌流方式生产携带 WAS-T2A-GFP 构建体的自失活(SIN)LV,平均感染滴度高达 4.64 × 107 TU mL-1,且可在两个月内生成。衍生的单克隆细胞系在连续培养过程中功能稳定,在半灌注摇瓶过程中产生的平均感染滴度高达 9.38 × 107 TU mL-1。在非优化的 5 L 搅拌罐生物反应器灌注工艺中,生产克隆能够连续 29 天保持 >1 × 107 TU mL-1 的生产率,这是 LV 生产领域的一个重要里程碑。由于生产细胞系基于可诱导的 Tet-off 表达系统,所建立的工艺可在没有抗生素等诱导剂的情况下生产 LV。纯化的 LV 能有效转导人类 CD34+ 细胞,减少基因和细胞疗法应用所需的 LV 数量。
Generation of stable suspension producer cell lines for serum-free lentivirus production
The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL−1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL−1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL−1 day−1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.
Biotechnology JournalBiochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍:
Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances.
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