Zhao Guozhi, Zhao Piao, Wang Yonghui, Zhang Hui, Zhu Yi, Zhong Jiamin, You Wulin, Shen Guowei, Luo Changqi, Mei Ou, Wu Xingye, Li Jingjing, Shu Yi, Wang Hongwei, Wagstaff William, Luu Hue H, Bi Yang, Shi Lewis L, Reid Russell R, He Tong-Chuan, Jiang Li, Tang Wei, Fan Jiaming, Tang Ziwei
Departments of Urology, Endocrinology, Orthopedic Surgery, and Gastroenterological Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA.
Genes Dis. 2024 May 31;11(6):101344. doi: 10.1016/j.gendis.2024.101344. eCollection 2024 Nov.
Recombinant adenovirus (rAdV) is a commonly used vector system for gene transfer. Efficient initial packaging and subsequent production of rAdV remains time-consuming and labor-intensive, possibly attributable to rAdV infection-associated oxidative stress and reactive oxygen species (ROS) production. Here, we show that exogenous GAPDH expression mitigates adenovirus-induced ROS-associated apoptosis in HEK293 cells, and expedites adenovirus production. By stably overexpressing GAPDH in HEK293 (293G) and 293pTP (293GP) cells, respectively, we demonstrated that rAdV-induced ROS production and cell apoptosis were significantly suppressed in 293G and 293GP cells. Transfection of 293G cells with adenoviral plasmid pAd-G2Luc yielded much higher titers of Ad-G2Luc at day 7 than that in HEK293 cells. Similarly, Ad-G2Luc was amplified more efficiently in 293G than in HEK293 cells. We further showed that transfection of 293GP cells with pAd-G2Luc produced much higher titers of Ad-G2Luc at day 5 than that of 293pTP cells. 293GP cells amplified the Ad-G2Luc much more efficiently than 293pTP cells, indicating that exogenous GAPDH can further augment pTP-enhanced adenovirus production. These results demonstrate that exogenous GAPDH can effectively suppress adenovirus-induced ROS and thus accelerate adenovirus production. Therefore, the engineered 293GP cells represent a superfast rAdV production system for adenovirus-based gene transfer and gene therapy.
重组腺病毒（rAdV）是一种常用的基因转移载体系统。rAdV的高效初始包装及后续生产仍然耗时且费力，这可能归因于rAdV感染相关的氧化应激和活性氧（ROS）生成。在此，我们表明外源性甘油醛-3-磷酸脱氢酶（GAPDH）表达可减轻腺病毒诱导的HEK293细胞中与ROS相关的细胞凋亡，并加快腺病毒生产。通过分别在HEK293（293G）和293pTP（293GP）细胞中稳定过表达GAPDH，我们证明在293G和293GP细胞中rAdV诱导的ROS生成和细胞凋亡被显著抑制。用腺病毒质粒pAd-G2Luc转染293G细胞，在第7天产生的Ad-G2Luc滴度比在HEK293细胞中高得多。同样，Ad-G2Luc在293G细胞中的扩增效率比在HEK293细胞中更高。我们进一步表明，用pAd-G2Luc转染293GP细胞在第5天产生的Ad-G2Luc滴度比293pTP细胞高得多。293GP细胞扩增Ad-G2Luc的效率比293pTP细胞高得多，表明外源性GAPDH可进一步增强pTP增强的腺病毒生产。这些结果表明，外源性GAPDH可有效抑制腺病毒诱导的ROS，从而加速腺病毒生产。因此，工程化的293GP细胞代表了一种用于基于腺病毒的基因转移和基因治疗的超快速rAdV生产系统。
