Nuclear delivery of plasmid DNA determines the efficiency of gene expression

Abstract

As a cationic non‐viral gene delivery vector, poly(agmatine/ N, N′‐cystamine‐bis‐acrylamide) (AGM‐CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM‐CBA/pDNA polyplexes was found to have a non‐linear relationship with AGM‐CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM‐CBA), we used pGL3‐control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM‐CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate‐limiting step for pLUC transfection expression. Further optimization of the non‐viral gene delivery system can be focused on the improvement of gene intracellular availability.

“PFH/AGM-CBA/HSV-TK/LIPOSOME-Affibody”: Novel Targeted Nano Ultrasound Contrast Agents for Ultrasound Imaging and Inhibited the Growth of ErbB2-Overexpressing Gastric Cancer Cells

“…This was due to the intracellular localization effect of guanidine polymers AGM-CBA, whose nuclear localization ability had been reported previously. 9 , 11 , 37 , 38 This greatly assisted the delivery of HSV-TK into the nucleus and led to further successful transfection.…”

“…It has been demonstrated that AGM-CBA is of lower cytotoxicity, higher transfection efficiency, and excellent nuclear localization effect as a gene delivery carrier in various applications. 9–11 …”

COVID-19 Vaccines: Where Did We Stand at the End of 2023?

“…The DNA-based vaccine approach is attractive due to its easy manufacturing and convenient storage temperature and handling [ 151 ]. However, transfection of plasmid DNA is inferior to viral infection and, in contrast to mRNA-based vaccines, translocation of DNA to the cell nucleus is necessary for in vivo RNA transcription [ 152 ]. Although DNA-based vaccine candidates have been subjected to more than 500 clinical trials, only a single DNA vaccine against the H5N1 influenza virus in chicken has been approved by the USDA [ 151 ] prior to granting EUA for DNA-based COVID-19 vaccines.…”

COVID-19 Vaccines: Where Do We Stand at the End of 2023?

“…The DNA-based vaccine approach is attractive due to its easy manufacturing, convenient storage temperature, and handling [131]. However, transfection of plasmid DNA is inferior to viral infection and in contrast to mRNA-based vaccines, translocation of DNA to the cell nucleus is necessary for in vivo RNA transcription [132]. Although DNA-based vaccine candidates have been subjected to more than 500 clinical trials, only a single DNA vaccine against the H5N1 influenza virus in chicken has been approved by the USDA [131] prior to granting EUA for DNA-based COVID-19 vaccines.…”