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.

中文翻译：

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与物理混合策略相比，在有利的双基因共表达质粒递送系统中意外扩增协同基因表达以促进血管流动

基因治疗在各种疾病的治疗中发挥着强大的潜力，例如过表达促血管生成基因以加速血管生成和恢复缺血组织的血管流动。人们在开发基因载体以提高转染效率方面投入了巨大的努力，而很少有研究致力于通过优化治疗基因来协同表达功能蛋白。实际上，扩增的基因表达是基因传递的最终目标。双基因共传递和协同表达成为加强基因表达的“突破口”。在这里，我们通过两种典型的双基因递送策略探索了对基因表达和促血管生成的协同作用，以确定哪一种更有效。物理混合法使用 ZNF ₅₈₀和 VEGF₁₆₅个重量比为 1/1 (p1:1) 的质粒，另一种策略涉及将 ZNF ₅₈₀和 VEGF ₁₆₅基因化学插入一个质粒作为双基因共表达质粒 (pZNF-VEGF)。p1:1 和 pZNF-VEGF 由 REDV-TAT-NLS-H ₁₂载体（一种有前途的肽载体）加载，形成相应的双基因传递系统。两个系统都表现出大致相似的大小和 zeta 电位，保证了几乎相同的细胞吸收。我们通过 mRNA 和蛋白质水平的基因表达以及体外和体内血管生成相关活动全面评估了两种递送系统。有趣的是，pZNF-VEGF 组在 ZNF ₅₈₀和 VEGF的表达中显示出显着放大的协同效应与 p1:1 组相比，有_{165 个基因。}更重要的是，进一步验证了双基因共表达质粒在体外和体内的增殖、迁移和血管生成方面出人意料的扩增协同效应。因此，我们认为通过构建共表达质粒共递送双基因为基因治疗提供了更好的选择，与物理混合方法相比，它可以更有效地增强靶基因的协同表达。