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

Abstract

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 ZNF580 and VEGF165 plasmids with a 1/1 weight ratio (p1:1), and the other strategy involved chemically inserting ZNF580 and VEGF165 genes into one plasmid as a dual-gene co-expression plasmid (pZNF–VEGF). p1:1 and pZNF–VEGF were loaded by REDV–TAT–NLS–H12 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 ZNF580 and VEGF165 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.

CAG peptide functionalized graphene quantum dots-cationic polymer composite gene carriers

“…ZNF580 has been shown to have pro-angiogenic effects and high endothelialization efficiency. 38 The effect of NPs on the in vitro angiogenic capacity of HUVECs was evaluated by an in vitro angiogenesis assay, with Lip2000/pZNF580 as a positive control group. The results of the in vitro angiogenesis assay showed (Fig.…”

Chitosan Hydrogel as Tissue Engineering Scaffolds for Vascular Regeneration Applications

“…Our group reviewed recently developed strategies for improving bioavailability of therapeutic drugs through systemic administration [238]. Additionally, it was confirmed that dual-gene co-delivery and coordinate expression became a "breach" of strengthened gene expression [239]. Double genes coordinate interactions have a significant impact on new blood vessels [240].…”

“…Drugs, gaseous signal molecules, genes, and microRNAs are commonly employed to modulate cell behaviors for the regeneration and tissue engineering, such as cell adhesion, growth, proliferation, and migration. Pharmaceutical molecule and gene therapies offer a promising strategy for promoting angiogenesis and accelerating endothelialization in tissue engineering [225][226][227][228][229][230][231][232][233][234][235][236][237][238][239][240][241][242][243].…”

Recent advances in inhibiting atherosclerosis and restenosis: from pathogenic factors, therapeutic molecules to nano-delivery strategies

“…Inspired by the promotion of the ZNF580 protein on VEGF transcription, we explored the synergistic effect of ZNF580 gene and VEGF gene on EC behaviors via co-delivering these two genes at different weight ratios, as well as constructing a co-expression plasmid of these two genes (Table 1). 31,32 A tremendously increased mRNA and protein expression of ZNF580 and VEGF was found in co-delivery and co-expression groups in comparison with single-gene groups. More importantly, the co-delivery strategy synergistically promoted the migration, proliferation, and tube formation of ECs.…”