对 HEK293T 细胞中 rAAV 生产过程中分子和细胞反应的时空洞察

IF 4.6 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
Alok Tanala Patra, Evan Tan, Yee Jiun Kok, Say Kong Ng, Xuezhi Bi
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引用次数: 0

摘要

基因治疗领域正在寻求经济高效地大规模生产重组腺相关病毒(rAAV)载体,用于大剂量治疗。尽管悬浮细胞培养和转染优化等策略取得了一定的成功,但大规模应用仍面临挑战。为了揭示影响 rAAV 生产的分子和细胞机制,我们对使用标准、次优和最优条件转染的 HEK293T 细胞进行了 SWATH-MS 蛋白组学分析。基因本体和通路分析表明,在最佳转染条件下,蛋白质表达发生了显著变化,尤其是在与细胞稳态、代谢调节、囊泡转运、核糖体生物发生和细胞增殖相关的过程中。这使得 rAAV 滴度比标准方案提高了 50%。此外,我们还发现了宿主细胞蛋白中对 AAV mRNA 稳定性和基因翻译至关重要的修饰,特别是在最佳转染条件下 AAV 荚膜转录本的修饰。我们的研究发现了 124 种与 AAV 复制和组装相关的宿主蛋白,每种蛋白在最佳转染条件下的整个 rAAV 生产阶段都表现出不同的表达模式。这项研究揭示了在 HEK293T 细胞中生产 rAAV 所涉及的细胞机制,并提出了在生产过程中进一步提高 rAAV 滴度的可行途径。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells
The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.
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来源期刊
Molecular Therapy-Methods & Clinical Development
Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
9.90
自引率
4.30%
发文量
163
审稿时长
12 weeks
期刊介绍: The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.
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