Factors affecting rAAV titers during triple-plasmid transient transfection in HEK-293 cells

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2024-09-11 DOI:10.1007/s10529-024-03520-0

Martina Pistek, Peter Andorfer, Reingard Grabherr, Barbara Kraus, Juan A. Hernandez Bort

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Abstract

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficiency utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhance fully packaged rAAV particle yield, we discovered cell-line-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.

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影响 HEK-293 细胞中三重质粒瞬时转染过程中 rAAV 滴度的因素

通过研究两种不同的 HEK-293 细胞系，分析了三重质粒转染在重组腺相关病毒（rAAV）生产中的效率。根据它们在相同条件下不同的生产水平，将它们分为高生产者（HP）和低生产者（LP）。对病毒基因 RNA 表达水平的分析表明，两种细胞系的质粒衍生基因表达存在差异。利用标记质粒对转染效率进行的进一步评估显示，LP 细胞系的质粒吸收率较低，核转运效率较低。此外，我们还观察到 LP 细胞系的翻译活性较低，这也是其整体生产率较低的原因之一。在尝试优化质粒比例以提高全包装 rAAV 粒子产量的过程中，我们发现了细胞系特有的优化潜力。这些发现凸显了转染的复杂性，促使我们根据每个细胞系的特点，为提高 rAAV 的产量制定量身定制的策略，从而加深了对 rAAV 生产效率的理解，并为进一步优化 rAAV 生产效率提供了指导。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.