Burkhart Madina, Langenbach Katrin, Holzmann Karlheinz, Hornung Nadine, Baiz Jamie-Ann, Otte Kerstin
Ulm University, Helmholtzstr. 16, Ulm, Germany.
Institute for Applied Biotechnology, University of Applied Sciences Biberach, Biberach, Germany.
Biotechnol J. 2025 Aug;20(8):e70092. doi: 10.1002/biot.70092.
Recombinant adeno-associated viruses (rAAVs) play a pivotal role in gene therapy, yet the molecular interactions underlying rAAV production in host cells remain incompletely understood. Non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs), are increasingly recognized as key regulators of viral and cellular processes. This study investigates the dynamic expression profiles of miRNAs and snoRNAs during rAAV plasmid transfection and vector production in HEK293F cells. A total of 142 miRNAs were differentially expressed during the peak phase of rAAV production, with 128 associated with the Gene Ontology term "viral process", indicating broad involvement in host-virus interactions. Target gene analysis linked these miRNAs to biological pathways such as nucleocytoplasmic transport, innate immunity, apoptosis, and transcriptional regulation, highlighting potential roles of miRNAs in shaping the cellular environment during viral vector assembly. In contrast, snoRNAs exhibited more modest changes in expression, yet five were significantly differentially expressed during active production, suggesting a possible, underexplored involvement in viral replication. These findings illuminate the underexplored contributions of ncRNAs to the host response during rAAV biogenesis and provide a valuable resource for understanding how cellular regulatory networks are engaged throughout vector production.
重组腺相关病毒（rAAVs）在基因治疗中发挥着关键作用，但宿主细胞中rAAV产生背后的分子相互作用仍未完全了解。非编码RNA（ncRNAs），特别是微小RNA（miRNAs）和小核仁RNA（snoRNAs），越来越被认为是病毒和细胞过程的关键调节因子。本研究调查了HEK293F细胞中rAAV质粒转染和载体生产过程中miRNAs和snoRNAs的动态表达谱。在rAAV产生的高峰期，共有142种miRNAs差异表达，其中128种与基因本体术语“病毒过程”相关，表明其广泛参与宿主-病毒相互作用。靶基因分析将这些miRNAs与核质运输、先天免疫、细胞凋亡和转录调控等生物学途径联系起来，突出了miRNAs在病毒载体组装过程中塑造细胞环境的潜在作用。相比之下，snoRNAs的表达变化较为适度，但在活跃生产期间有5种显著差异表达，表明其可能在病毒复制中发挥未被充分探索的作用。这些发现揭示了ncRNAs在rAAV生物发生过程中对宿主反应的未被充分探索的贡献，并为理解整个载体生产过程中细胞调控网络的参与情况提供了宝贵资源。
