Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

Abstract

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.

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

“…At 48 h post-transfection, eGFP transient expression was observed by fluorescence microscopy (Nikon ECLIPSE, Nikon, Japan) and mean fluorescence intensity (MFI) was detected by flow cytometry as previously described ( Wang et al, 2019 ). Briefly, cells were collected after pancreatic enzyme digestion, a total of 100,000 fluorescent events were acquired using a 530/15 bandpass filter for the green fluorescent signal, which was acquired with an emission wave length of 530 nm.…”

Stabilizing and Anti-Repressor Elements Effectively Increases Transgene Expression in Transfected CHO Cells

“…At 48-h post transfection, the transfection efficiency of each expression vector was observed under a fluorescence microscope (ECLIPSE Ti; Nikon, Tokyo, Japan), and mean fluorescence intensity (MFI) was detected by flow cytometry as previously described ( Wang et al, 2019 ; Wang et al, 2022 ). Briefly, a total of 100,000 fluorescent events were obtained using a 530/15 bandpass filter for the green fluorescent signal, which was acquired with an emission wave length of 530 nm.…”

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells

“…( Mohan et al, 2008 ; Li et al, 2018 ). In addition, by constructing DNA methyltransferase-deficient CHO cells, the stability of expression for the recombinant protein can be enhanced by inactivating DNA methylation ( Jia et al, 2018 ; Wang et al, 2019 ).…”