Yan Junyu, Li Ziqian, Shu Yue, Chen Hui, Wang Tianxingzi, Li Xin, Zhang Yuhang, Li LiLi, Zhang Yuntao
Beijing Institute of Biological Products Company Limited, Beijing, China.
Beijing Institute of Biological Products Company Limited, Beijing, China.
Gene. 2025 Feb 20;938:149122. doi: 10.1016/j.gene.2024.149122. Epub 2024 Nov 23.
The field of gene therapy using Adeno-associated viral (AAV) vector delivery is rapidly advancing in the biotherapeutics industry. Despite its successes, AAV manufacturing remains a challenge due to limited production yields. The triple plasmid transfection of HEK293 cells represents the most extensively utilized system for AAV production. The regulatory factors and mechanisms underlying viral production in HEK293 cells are largely unknown. In this study, we isolated high-titer AAV production clones from a parental HEK293 population using a single limiting dilution step, and subsequently elucidating their underlying molecular mechanisms through whole transcriptome analysis. LncRNA TCONS_00160397 was upregulated in clones and shown to promoted HEK293 cells proliferation and improved the titer of AAV production. Mechanistically, results from proteomics and metabolomics indicated that TCONS_00160397 regulated the ABC transporters pathway. These findings furnish a rich repository of knowledge and actionable targets for the rational optimization of HEK293-based producer lines, thereby paving the way for tangible improvements in AAV vector output and expediting the broad implementation of gene therapies.
使用腺相关病毒（AAV）载体递送的基因治疗领域在生物治疗行业中正在迅速发展。尽管取得了成功，但由于产量有限，AAV的生产仍然是一个挑战。HEK293细胞的三质粒转染是AAV生产中使用最广泛的系统。HEK293细胞中病毒产生的调控因子和机制在很大程度上尚不清楚。在本研究中，我们通过单步有限稀释从亲本HEK293群体中分离出高滴度AAV生产克隆，随后通过全转录组分析阐明其潜在的分子机制。LncRNA TCONS_00160397在克隆中上调，并显示出促进HEK293细胞增殖和提高AAV生产滴度的作用。从机制上讲，蛋白质组学和代谢组学的结果表明，TCONS_00160397调节ABC转运蛋白途径。这些发现为基于HEK293的生产细胞系的合理优化提供了丰富的知识储备和可操作的靶点，从而为切实提高AAV载体产量和加速基因治疗的广泛应用铺平了道路。
