通过质粒转染 HEK293 细胞生产重组腺相关病毒的多组学动力学分析。

IF 2.5 3区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Min Lu, Zion Lee, Wei-Shou Hu
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引用次数: 0

摘要

重组腺相关病毒(rAAV)是基因治疗最常用的载体之一。它通常通过转染 HEK293 细胞生产,每个细胞含有三个质粒,每个质粒都含有载体基因组,包括感兴趣基因(GOI)、辅助功能以及用于基因组复制和形成囊盖的 rep 和 cap 基因。为了满足潜在的临床需求,需要提高生产系统的生产率。对生产系统进行更好的过程表征将进一步促进我们对如何提高生产率的深入了解。在这里,我们采用了转录组分析来量化病毒转录本不同同工酶的动态变化,评估细胞生理学的转变,并部署了靶向蛋白质组分析来绝对量化病毒蛋白质,以及串联质量标记(TMT)来评估蛋白质水平上的细胞反应。转录组和蛋白质组水平的功能分析确定了防御和免疫反应、未折叠蛋白反应、p53 信号转导的丰富程度。基于功能分析的小分子添加剂干预研究显示了这种以组学为指导的生产率提高的潜力。多组学分析共同推进了对 rAAV 生产的理解,并为通过质粒转染提高 rAAV 产量提供了见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Multi-omics kinetic analysis of recombinant adeno-associated virus production by plasmid transfection of HEK293 cells

Multi-omics kinetic analysis of recombinant adeno-associated virus production by plasmid transfection of HEK293 cells

Recombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics-guided productivity enhancement. Together, multi-omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection.

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来源期刊
Biotechnology Progress
Biotechnology Progress 工程技术-生物工程与应用微生物
CiteScore
6.50
自引率
3.40%
发文量
83
审稿时长
4 months
期刊介绍: Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries. Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.
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