Original ResearchVol. 58, Issue 3 • pp. 700-703DOI: 10.3724/abbs.2025104
Authors: Xi Zhang, Rui Liu, Zimeng Han, Zihan Guo, Mengying Ji, Wen Wang, Yanlong Jia, Tianyun Wang, Xiaoyin Wang
Non-viral episomal vectors offer a safe and attractive alternative to viral and integrated vectors by avoiding insertional mutagenesis and position effects, making them ideal expression vectors for gene therapy. The first non-viral episomal vector, pEPI-1, which is based on the full-length scaffold/matrix attachment region (S/MAR), was established by Piechaczek et al. The full-length S/MAR element interacts with the nuclear matrix via the matrix protein, e.g. SAF-A, thereby maintaining mitotic stability and transgene expression. Several strategies, including optimization of the vector backbone and promoter and incorporation of chromatin-modifying elements, have been used to increase expression levels and stability. In our previous work, we constructed the novel vector pEGFP-C1-M on the basis of S/MAR characteristic motifs (only 375 bp). This vector, which is shorter than the prototype episomal vector pEPI-1, resulted in relatively higher transgene expression. Building on the pEGFP-C1-M vector, we further constructed the episomal vector pEMEα with the EF-1α promoter and demonstrated that pEMEα maintained higher transgene expression, stability and copy number. The transgene expression levels of episomal vectors are correlated with gene copy number, that is, the number of plasmid episomes on the host cell chromosome. Previous studies have shown that the episomal maintenance of pEPI-1 vectors is mediated primarily by SAF-A. While the role of SAF-A in maintaining mammalian pEPI-1 episomal vectors has been well established, it remains unknown whether the overexpression of SAF-A promotes transgene expression and stability and whether the 375 bp MAR characteristic sequence retains its interaction with SAF-A. In the present study, we first evaluated whether transgene expression is positively correlated with the expression level of SAF-A. The non-viral episomal vector pEMEα was used as the gene of interest (GOI) vector and was subsequently transfected into CHO-K1 cells using the Lipofectamine 2000 reagent. The cells were cultured in medium containing 800 μg/mL geneticin (G418) 48 h post-transfection, and the G418 concentration was then reduced to 400 μg/mL to obtain monoclonal cell lines using the limiting dilution method. Five monoclonal cell clones were selected, and the eGFP expression levels, measured as the mean fluorescence intensity (MFI), were (6.5 ± 1.0) × 104, (6.8 ± 0.9) × 104, (7.0 ± 1.4) × 104, (23.5 ± 1.2) × 104 and (14.9 ± 0.17) × 104 for Clones 1–5, respectively. qPCR analysis of Clones 1–5 revealed that the relative mRNA levels of SAF-A and eGFP were 0.16 ± 0.13, 0.43 ± 0.11, 0.46 ± 0.23, 2.17 ± 0.41, 1.78 ± 0.15 and 0.51 ± 0.16, 0.71 ± 0.12, 1.05 ± 0.09, 2.47 ± 0.14, and 1.86 ± 0.10, respectively. Our results indicated that eGFP mRNA and protein expression levels are positively correlated with SAF-A mRNA level. To further verify the relationship between SAF-A expression and transgene expression, two shRNA plasmids targeting SAF-A (shRNA1: 5′-GCCACCTGTTGAAGAAGAAGA-3′, and shRNA2: 5′-GCTGGAGGAAGAGCTTCTTAT-3′) which were obtained from Shanghai GenePharma Co., Ltd. were designed and transfected into stable cell pools with the pEMEα vector. qPCR analysis revealed that the relative SAF-A mRNA levels in the shRNA1 and shRNA2 vectors were 0.47 ± 0.01 and 0.19 ± 0.02, respectively, indicating successful downregulation of SAF-A expression. Moreover, flow cytometry and qPCR revealed that, compared with those in the control group, the relative protein and mRNA levels of eGFP were reduced by 0.47- and 0.23-fold, and 0.47- and 0.40-fold in the pools of cells transfected with the shRNA1 and shRNA2 vectors, respectively. On the basis of the above results, the SAF-A overexpression vector pIRES-SAF-A was constructed and transfected into CHO-K1 cells, and the cells were cultured in blasticidin-containing medium 48 h after transfection to obtain stable cell pools. The stable cell pools overexpressing SAF-A were subsequently transfected with the pEMEα vector. Stable cell pools coexpressing SAF-A and GOI were selected, and the relative mRNA levels of SAF-A and eGFP were analyzed. qPCR analysis revealed that the relative mRNA levels of SAF-A and eGFP in the pools of cells overexpressing SAF-A were 2.69-fold and 2.05-fold higher than those in the control group, respectively. Flow cytometry also revealed a 2.07-fold increase in MFI in stable cell pools overexpressing SAF-A compared with the control group. To assess the long-term stability of transgene expression, we measured the MFI in st
