LAUSR

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector‐mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long‐term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase‐deficient (Dnmt3a‐deficient) CHO cells were transfected with plasmid‐mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5‐Aza‐2′‐deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in‐situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60‐generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long‐time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a‐deficient cell lines and CHO cells treated with 5‐Aza‐2′‐deoxycytidine. In addition, Dnmt3a‐deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long‐term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.