一种缺失E1A基因CR2区域的新型HEK293细胞可防止复制型腺病毒的出现。

A New HEK293 Cell with CR2 Region of E1A Gene Deletion Prevents the Emergence of Replication-Competent Adenovirus.

作者信息

Lian Xueqi, Zhao Xiaoyan, Zhong Jingjing, Zhang Chenglin, Chu Yongchao, Wang Yaohe, Lu Shuangshuang, Wang Zhimin

机构信息

National Center for International Research in Cell and Gene Therapy, Sino-British Research Centre for Molecular Oncology, School of Basic Medical Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450052, China.

Centre for Biomarkers & Biotherapeutics, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK.

出版信息

Cancers (Basel). 2023 Dec 5;15(24):5713. doi: 10.3390/cancers15245713.

DOI:10.3390/cancers15245713

PMID:38136259

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10742158/

Abstract

PURPOSE

To eliminate the contaminants of Replication-Competent Adenovirus (RCA) during high titer recombinant oncolytic adenovirus production.

METHODS

At first, we detected E1A copy numbers of different sources of 293 cells using Q-PCR, and we screened a subclone JH293-C21 of the JH293 cell line (purchased from ATCC) with lower early region 1A (E1A) copy numbers and higher adenovirus production ability. Then, we deleted the conserved region (CR)2 of the E1A gene in this subclone using the CRISPR-Cas9 system and obtained a stable cell clone JH293-C21-C14 with lower E1A expression, but the RCA formation had no significant reduction. Then, we further deleted the CR2 of JH293-C21-C14 cells with the CRISPR-Cas9 system and obtained a strain of cells named JH293-C21-C14-C28. Finally, we detected the capacity for cell proliferation, adenovirus production, and RCA formation in the production of recombinant adenovirus.

RESULTS

The JH293-C21-C14-C28 cells had a similar cell proliferation ability and human adenovirus production as JH293-C21 cells. Most importantly, RCA production in JH293-C21-C14-C28 cells was lower than in JH293-C21 cells.

CONCLUSION

Human adenovirus producer cell clone JH293-C21-C14-C28 with CR2 deletion can effectively prevent the RCA production of replication-competent oncolytic adenovirus; this will provide significant advantages in utility and safety in gene therapy.

摘要

目的

在高滴度重组溶瘤腺病毒生产过程中消除复制型腺病毒（RCA）污染物。

方法

首先，我们使用Q-PCR检测不同来源的293细胞的E1A拷贝数，并筛选出JH293细胞系（购自ATCC）的一个亚克隆JH293-C21，其早期区域1A（E1A）拷贝数较低且腺病毒生产能力较高。然后，我们使用CRISPR-Cas9系统删除该亚克隆中E1A基因的保守区域（CR）2，获得了一个E1A表达较低的稳定细胞克隆JH293-C21-C14，但RCA形成没有显著减少。接着，我们用CRISPR-Cas9系统进一步删除JH293-C21-C14细胞的CR2，获得了一株名为JH293-C21-C14-C28的细胞。最后，我们检测了重组腺病毒生产中细胞增殖、腺病毒产生和RCA形成的能力。