Limited expression of non-integrating CpG-free plasmid is associated with increased nucleosome enrichment

• Published in: PloS one Vol. 15; no. 12; p. e0244386
• Main Authors: Habib, Omar, Mohd Sakri, Rozita, Ghazalli, Nadiah, Chau, De-Ming, Ling, King-Hwa, Abdullah, Syahril
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.12.2020; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Binding sites; Biocompatibility; Biology and life sciences; Cell Line; Cell lines; CpG Islands; Cytokines; Cytotoxicity; Deoxyribonucleic acid; Depletion; DNA; DNA methylation; Enrichment; Fibroblasts; Gene dosage; Gene Expression; Gene transfer; Green Fluorescent Proteins - genetics; Health sciences; HEK293 Cells; Humans; In vivo methods and tests; Inflammation; Laboratories; Luciferases - genetics; Lungs; MCF-7 Cells; Medical research; Medicine; Medicine and Health Sciences; Mice; mRNA; NIH 3T3 Cells; Nucleosomes; Nucleosomes - genetics; Physical Sciences; Plasmids; Plasmids - genetics; Research and Analysis Methods; Species Specificity; Steady state; Toxicity; Transfection; Transgenes; Malaysia; Selangor Malaysia; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0244386

CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.