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.
Keywords: 293 cell line; CR2; E1A; Replication-Competent Adenovirus.