In vitro transcriptional aberration of CpG-free plasmid DNA.

• ¹ Universiti Putra Malaysia, Genetics & Regenerative Medicine Research Centre, Faculty of Medicine & Health Sciences, Malaysia
• ² Universiti Putra Malaysia, Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences , Malaysia
• ³ Abubakar Tafawa Balewa University Bauchi, Department of Human Anatomy, College of Medicine & Health Sciences, Nigeria
• ⁴ Universiti Putra Malaysia, Institute of Bioscience, Malaysia

Plasmid DNA (pDNA) with complete depletion of CpG motifs (CpG-free pDNA) was proven to facilitate prolonged transgene expression while avoiding detrimental inflammatory response in mouse lungs. In addition, CpG-free pDNA has been reported to provide a significant outcome in a recent cystic fibrosis gene therapy clinical trial. However, we observed limited transgene expression in cell lines when extending this promising pDNA towards a potential ex vivo approach. Based on a recent report, we postulate that this is due to the CpG depletion. Hence, our main objective is to determine the basis for the poor performance of the CpG-depleted pDNA expression in vitro. In this study, two novel pDNAs of identical CpG-free pDNA backbone with Green Fluorescent Protein (GFP) reporter gene of variable CpG content were constructed. These pDNAs were transfected separately into human embryonic kidney cell line (HEK-293FT) using lipid-based gene transfer agent and GFP proteins expression were compared up to 14 days using flow cytometry (n=3). Following this, evaluation of gene dosage by plasmid copy number was performed at Day 1 post-transfection by extracting total DNA (n=3) and performing absolute qPCR. Next, to determine if the difference in gene expression was at mRNA export level, nuclear and cytoplasmic RNA were extracted from the transfected cells (n=3) at Day 1 post-transfection and the rate of mRNA export was measured using absolute qPCR. The results showed that the constructed CpG-free pDNAs were designed to encode identical GFP protein to avoid translational discrepancies. However, at the DNA sequence level, one pDNA carries GFP devoid of CpG (pZGFP) while the other pDNA has GFP with 60 CpGs (pRGFP). Upon transfection into HEK-293FT, pZGFP exhibited significantly lower GFP expressing (≈ 20% less, p<0.05) at Day 1, and the lowest expression was detected at Day 7 where pZGFP gave ≈ 40% (p<0.05) lower GFP expression when compared to pRGFP. In addition, expression of pZGFP remained significantly low until Day 14. Furthermore, the Mean Fluorescence Intensity (MFI) of pZGFP, which indicates average of single cell expression, was also significantly lower than pRGFP. This difference was not due to disparity in gene dosage as there is no significant difference in plasmid copy number at Day 1 post-transfection between the two pDNAs. Interestingly, the low pZGFP expression was also not due to a discrepancy at the mRNA export level as both pZGFP and pRGFP mRNAs have similar export rate. Instead, the rate of transcription in pZGFP was significantly lower than pRGFP (p<0.05). In conclusion, CpG motif plays a role in improving in vitro transgene transcription where complete depletion of CpG resulted in lower expression. Our data highly suggest that the CpG-depeleted transgene in CpG-free pDNA resulted in reduced rate of transcription.