![]() The first CpG-free pDNA developed was able to significantly reduce inflammation, which includes levels of pro-inflammatory cytokines, and neutrophil infiltration indicative of minimal tissue damage, irrespective of topical lung or systemic delivery. Given these points, removing CpGs from pDNA would be advantageous in an in vivo system. Moreover, studies have shown that the unmethylated CpGs in pDNA were prone to de novo DNA methylation-mediated silencing that resulted in the loss of transgene expression. As a result, CpG-induced inflammation leads to immune-mediated destruction of transgene-carrying cells, repression of transgene expression due to the direct action of pro-inflammatory cytokines, and prolonged inflammation, which could result in the development of autoimmunity. Even the presence of a single CpG in pDNA was enough to elicit profound inflammation in vivo. Toll-like Receptor 9 (TLR9) recognizes non-methylated CpG-containing DNA fragments to be foreign, and will accordingly induce an acute inflammatory response. This distinction is crucial in antigen recognition for immune defense. In contrast, 60–90% of CpGs in vertebrate DNA are methylated. CpGs exist more frequently in bacterial pDNA (1 in 16 dinucleotides compared to 1 in 64 dinucleotides in vertebrates DNA) and are not methylated. One of the more noteworthy improvements is the development of CpG (cytosine-guanine dinucleotides)-depleted pDNA or commonly known as CpG-free pDNA, where CpG dinucleotides are completely depleted from the whole pDNA, including the transgene.ĬpGs in pDNA of bacterial origin differ from CpGs in vertebrate DNA in the frequency and methylation status. Some of the improvements are the incorporation of novel promoters, introns, and chromatin insulators in the pDNA design. As a result, pDNA has undergone various improvements to better facilitate transgene expression. This allows better insight into the biology of a system and consequently offers the ability to control and manipulate such a system for beneficial purposes such as gene therapy. Due to its ability to facilitate transgene expression, it becomes practical to express, silence, augment the levels of a gene(s) of interest, or even introduce foreign gene(s). Plasmid DNA (pDNA) has played an essential role in advancing basic research, biotechnology and medicine. NO - The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.ĬpG-free plasmid backbone containing CpG-free GFP pRGFP,ĬpG-free plasmid backbone containing CpG-rich GFP TLR9,įormaldehyde-Assisted Isolation of Regulatory Elements This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper.įunding: Syahril Abdullah was funded by the Ministry of Higher Education Malaysia, project grant number: 04-01-16-1815FR (FRGS). Received: Accepted: DecemPublished: December 21, 2020Ĭopyright: © 2020 Habib et al. PLoS ONE 15(12):Įditor: Ilayaraja Muthuramu, University of Pennsylvania, UNITED STATES Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.Ĭitation: Habib O, Mohd Sakri R, Ghazalli N, Chau D-M, Ling K-H, Abdullah S (2020) Limited expression of non-integrating CpG-free plasmid is associated with increased nucleosome enrichment. 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. 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. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. 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. 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. 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. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA.
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