CpG-free plasmid DNA exhibits limited in vitro reporter gene expression due to transcriptional incongruity

The use of efficient vector is essential in gene therapy, which has the promising potential to treat not only genetic diseases but also acquired diseases. Although plasmid DNAs (pDNA) have been shown to be a much safer alternative compared to viral vectors, limitations, like detrimental inflammatory response and transient transgene expression, hinder its clinical application. This is mainly due to the presence of unmethylated cytosine guanine (CpG) motifs on pDNA. As a solution, CpG-free pDNA (p0CpG) was developed, where CpG motifs are completely depleted from the pDNA backbone and transgene. In addition, the safety and efficacy of the p0CpG were further reinforced with significant therapeutic effect observed in a cystic fibrosis clinical trial. Given these points, extending the application of clinically relevant p0CpG towards ex vivo gene therapy would be a novel approach to treat many disorders. However, it was recently reported that the depletion of CpG motifs from transgene could affect the in vitro gene expression negatively. Similar results may also extend to p0CpG, as the p0CpG is also devoid of CpG motifs. Thus, the main objective of this study is to evaluate the performance of p0CpG in vitro, specifically the effect of CpG depletion from the transgene. First, expression of luciferase reporter gene was compared between p0CpG and a CpG-containing pDNA in multiple human cell lines. Surprisingly, pCpG-free exhibited poor expression in vitro that contradicts published in vivo reports. To determine if this was due to the CpG depletion from transgene, two novel pDNAs were constructed where each has CpG-free backbone with Green Fluorescent Protein (GFP) gene that is either CpG-free (pZGFP) or contains 60 CpGs (pRGFP). pZGFP showed significantly inferior transgene expression when compared to pRGFP, despite using different gene transfer agents (lipid or polymer-based) or cell types (human, mouse cell lines & primary mouse cells). Besides that, there was no significant difference in pDNA copy number and toxicity between the pDNAs. Therefore, the low expression of pZGFP is attributed to CpG depletion from transgene. Upon further investigation, the lower expression of pZGFP was not due to mRNA export but was due to lower transcriptional rate as observed in mRNA distribution studies. In conclusion, complete depletion of CpG motifs from transgene in p0CpG resulted in reduced transcription rate. Data obtained from this study could be used to improve and optimize p0CpG for ex vivo gene therapy approach in the future.

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