HR9: An Important Cell Penetrating Peptide for Delivery of HCV NS3 DNA into HEK-293T Cells

The delivery of exogenous genes into cells for functional expression is required for development of DNA vaccine and gene therapy in medicine and pharmacology. Cell Penetrating Peptides (CPPs) were considered to mediate gene and drug delivery into living cells. In this study, an attempt was made to evaluate the efficiency of an arginine-rich CPP, HR9, in HCV NS3 gene delivery compared to TurboFect cationic polymer and supercharged +36 GFP into HEK-293T cells. Methods: The recombinant pEGFP-NS3 was constructed and their accuracy was confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into HEK-293T cells by TurboFect, +36 GFP and HR9 gene delivery systems. The expression of NS3 protein was assessed by fluorescent microscopy, flow cytometry and western blotting. Results: Our data indicated that HR9 peptide was able to form stable complexes with plasmid DNA and increased its delivery into HEK-293T cells in a non-covalent manner. Furthermore, treatment of cells with HR9 and HR9/DNA complexes resulted in a viability of 90-95% indicating this CPP was not cytotoxic. The analysis of zeta potential and size showed the importance of interactions between positively-charged HR9/pEGFP-NS3 complexes and negatively-charged plasma membranes. Conclusion: The non-toxic HR9 CPP can be considered an effective carrier for delivering plasmid DNA harboring Hepatitis C virus (HCV) gene in therapeutic vaccine design.