文章：

新型HEK293细胞株：E1A基因CR2区域缺失有效预防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

原文发布日期：5 December 2023

DOI: 10.3390/cancers15245713

类型: Article

开放获取: 是

英文摘要：

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.

摘要翻译： 

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污染。方法：首先，我们采用Q-PCR技术检测了不同来源293细胞的E1A拷贝数，筛选出E1A拷贝数较低且腺病毒生产能力较强的JH293细胞系（购自ATCC）亚克隆JH293-C21。随后，利用CRISPR-Cas9系统敲除该亚克隆E1A基因的保守区2，获得E1A表达降低的稳定细胞克隆JH293-C21-C14，但其复制型腺病毒形成未见显著减少。继而再次应用CRISPR-Cas9系统对JH293-C21-C14细胞进行CR2区段敲除，获得命名为JH293-C21-C14-C28的细胞株。最后，系统检测该细胞株在重组腺病毒生产过程中的细胞增殖能力、腺病毒产量及复制型腺病毒形成情况。结果：JH293-C21-C14-C28细胞在细胞增殖能力和人腺病毒产量方面与JH293-C21细胞相当。最重要的是，该细胞株的复制型腺病毒产生量显著低于JH293-C21细胞。结论：CR2区段敲除的人腺病毒生产细胞克隆JH293-C21-C14-C28能有效抑制复制型溶瘤腺病毒的产生，这为基因治疗的实用性与安全性提供了重要保障。

原文链接：

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