Golm Susanne K, Hoffrogge Raimund, Müller Kristian M
Department of Biotechnology, Faculty of Technology, Bielefeld University, 33501 Bielefeld, Germany.
Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, 33501 Bielefeld, Germany.
Int J Mol Sci. 2025 Jun 30;26(13):6315. doi: 10.3390/ijms26136315.
Recombinant adeno-associated virus (rAAV) is the leading vector for gene replacement therapy; however, the roles and regulation of host proteins in rAAV production remain incompletely understood. In this comparative proteomic analysis, we focused on proteins in the nucleus, the epicenter of DNA uptake, transcription, capsid assembly, and packaging. HEK-293 cells were analyzed under the following three conditions: (i) untransfected, (ii) mock-transfected with the ITR and an unrelated plasmid, and (iii) triple-transfected with rAAV2 production plasmids. Cells were harvested at 24 and 72 h post-transfection, and nuclear fractions were processed using filter-aided sample preparation (FASP) followed by nano-scale liquid chromatography-tandem mass spectrometry (nLC-Orbitrap MS/MS). Across all samples, we identified 3384 proteins, revealing significant regulatory changes associated with transfection and rAAV production. Transfection alone accounted for some of the most substantial proteomic shifts, while rAAV production induced diverse regulatory changes linked to cell cycle control, structure, and metabolism. STRING analysis of significantly regulated proteins also identified an enrichment of those associated with the Gene Ontology (GO) term 'response to virus'. Additionally, we examined proteins with reported relation to adenoviral components. Our findings help to unravel the complexity of rAAV production, identify interesting targets for further investigation, and may contribute to improving rAAV yield.
重组腺相关病毒（rAAV）是基因替代疗法的主要载体；然而，宿主蛋白在rAAV生产中的作用和调控仍未完全明确。在这项比较蛋白质组学分析中，我们聚焦于细胞核中的蛋白质，细胞核是DNA摄取、转录、衣壳组装和包装的核心部位。在以下三种条件下对HEK-293细胞进行分析：（i）未转染，（ii）用ITR和无关质粒进行模拟转染，以及（iii）用rAAV2生产质粒进行三重转染。在转染后24小时和72小时收获细胞，使用滤膜辅助样品制备（FASP）处理细胞核组分，随后进行纳升级液相色谱-串联质谱分析（nLC-Orbitrap MS/MS）。在所有样品中，我们鉴定出3384种蛋白质，揭示了与转染和rAAV生产相关的显著调控变化。仅转染就导致了一些最显著的蛋白质组学变化，而rAAV生产则诱导了与细胞周期控制、结构和代谢相关的多种调控变化。对显著调控蛋白的STRING分析还发现，与基因本体论（GO）术语“对病毒的反应”相关的蛋白有所富集。此外，我们还研究了与腺病毒成分有报道关系的蛋白质。我们的研究结果有助于揭示rAAV生产的复杂性，识别出值得进一步研究的有趣靶点，并可能有助于提高rAAV的产量。
