Wang Xiaoyu, Gao Bin, Zhou Jiaying, Ren Xiang-Kui, Guo Jintang, Xia Shihai, Zhang Wencheng, Feng Yakai
School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin 300350, P. R. China.
Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Weijin Road 92, Tianjin 300072, P. R. China.
ACS Appl Bio Mater. 2020 Oct 19;3(10):7228-7235. doi: 10.1021/acsabm.0c01023. Epub 2020 Sep 21.
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.
基因治疗在多种疾病的治疗中具有强大的潜力，例如过表达促血管生成基因以加速血管生成并恢复缺血组织的血流。人们在开发具有高转染效率的基因载体方面投入了巨大努力，而通过优化治疗基因来协同表达功能蛋白的研究却很少。实际上，基因表达的增强是基因递送的最终目标。双基因共递送和协调表达成为增强基因表达的一个“突破口”。在此，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，以确定哪种策略更有效。物理混合方法使用重量比为1/1的ZNF和VEGF质粒（p1:1），另一种策略是将ZNF和VEGF基因化学插入到一个质粒中作为双基因共表达质粒（pZNF-VEGF）。p1:1和pZNF-VEGF由一种有前景的肽载体REDV-TAT-NLS-H负载，形成相应的双基因递送系统。两种系统表现出大致相似的大小和zeta电位，保证了几乎相同的细胞摄取。我们通过mRNA和蛋白质水平的基因表达以及体外和体内与血管生成相关的活性，对两种递送系统进行了全面评估。有趣的是，与p1:1组相比，pZNF-VEGF组在ZNF和VEGF基因的表达上显示出显著增强的协同效应。更重要的是，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意外的增强协同效应得到了进一步验证。因此，我们认为通过构建共表达质粒进行双基因共递送为基因治疗提供了一个更好的选择，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。
