Unexpected Amplification of Synergistic Gene Expression to Boom Vascular Flow in Advantageous Dual-Gene Co-expression Plasmid Delivery Systems over Physically Mixed Strategy.

Gene therapy exerts powerful potential in the treatment of various diseases, such as overexpressing pro-angiogenic gene to accelerate angiogenesis and restore vascular flow of ischemic tissue. Tremendous efforts have been invested in developing gene carriers for high transfection efficiency, while little research has been devoted to synergistically expressing functional proteins via optimizing therapeutic genes. Actually, the amplified gene expression is the ultimate goal of gene delivery. Dual-gene co-delivery and coordinate expression become a "breach" of strengthened gene expression. Herein, we explored the synergistic effects on gene expression and pro-angiogenesis by two typical dual-gene delivery strategies to determine which one is more efficient. The physical mixing method used ZNF and VEGF plasmids with a 1/1 weight ratio (p1:1), and the other strategy involved chemically inserting ZNF and VEGF genes into one plasmid as a dual-gene co-expression plasmid (pZNF-VEGF). p1:1 and pZNF-VEGF were loaded by REDV-TAT-NLS-H carrier, a promising peptide carrier, to form corresponding dual-gene delivery systems. Both systems exhibited approximately similar size and zeta potential, guaranteeing almost the same cellular uptake. We comprehensively evaluated two delivery systems through gene expression at mRNA and protein levels and angiogenesis-related activities in vitro and in vivo. Interestingly, the pZNF-VEGF group showed a remarkably amplified synergistic effect in the expression of ZNF and VEGF genes in comparison with the p1:1 group. More importantly, the unexpected amplified synergistic effect of dual-gene co-expression plasmid was further verified for proliferation, migration, and angiogenesis in vitro and in vivo. Accordingly, we believed that the co-delivery of dual genes via constructing co-expression plasmids offers a better option for gene therapy, which can more effectively enhance the synergistic expression of target genes than the physical mixing method.