Vascular endothelial growth factor A (VEGFA) is involved in the pathogenesis of vasoproliferative retinal diseases, such as exudative age-related macular degeneration (AMD). The objective of this study was to investigate whether dual-acting therapy based on the simultaneous expression of anti-VEGFA microRNAs (miRNAs) and the secreted, antiangiogenic protein pigment endothelial-derived factor (PEDF) delivered by adeno-associated virus (AAV) vectors provides improved protection against choroidal neovascularization (CNV). To investigate this, a multigenic AAV vector allowing retina pigment epithelium (RPE)-specific expression of anti-VEGFA miRNAs and PEDF was engineered. Robust expression of PEDF, driven by the RPE-specific vitelliform macular dystrophy 2 promoter, was observed in human cells and in mouse retina. A significant reduction in CNV was observed in a laser-induced CNV mouse model 57 days post-injection of the AAV5 particles conveying either anti-VEGFA miRNA and PEDF dual therapy or anti-VEGFA miRNA monotherapy. Overall, CNV reduction was most prominent in animals receiving dual-acting therapy. In both cases, the reduction in CNV was accompanied by a significant attenuation of VEGFA. In conclusion, the presented data reveal that gene therapy targeting VEGFA via multigenic AAV vectors displays combined efficacy, suggesting that dual-acting therapy is an important tool in future eye gene therapy for the treatment of neovascular ocular diseases, including AMD.

血管内皮生长因子A（VEGFA）参与血管增生性视网膜疾病的发病机制，例如渗出性年龄相关性黄斑变性（AMD）。这项研究的目的是研究是否基于同时表达抗VEGFA microRNA（miRNA）和腺相关病毒（AAV）载体传递的分泌性抗血管生成蛋白色素内皮细胞因子（PEDF）的双重作用疗法提供了针对脉络膜新血管形成（CNV）的增强保护。为了对此进行研究，设计了一种多基因AAV载体，该载体允许视网膜色素上皮（RPE）特异性表达抗VEGFA miRNA和PEDF。在人细胞和小鼠视网膜中观察到由RPE特异性玻璃体黄斑营养不良2启动子驱动的PEDF的稳健表达。在注射诱导抗VEGFA miRNA和PEDF双重疗法或抗VEGFA miRNA单一疗法的AAV5颗粒注射后57天，在激光诱导的CNV小鼠模型中观察到CNV显着降低。总体而言，在接受双重作用治疗的动物中，CNV降低最为明显。在这两种情况下，CNV的降低都伴随着VEGFA的显着减弱。总之，目前的数据表明，通过多基因AAV载体靶向VEGFA的基因疗法显示出综合的功效，这表明双重作用疗法是未来眼基因疗法中用于治疗包括AMD在内的新生血管眼疾病的重要工具。在接受双重作用治疗的动物中，CNV降低最为明显。在这两种情况下，CNV的降低都伴随着VEGFA的显着减弱。总之，目前的数据表明，通过多基因AAV载体靶向VEGFA的基因疗法显示出综合的功效，这表明双重作用疗法是未来眼基因疗法中用于治疗包括AMD在内的新生血管眼疾病的重要工具。在接受双重作用治疗的动物中，CNV降低最为明显。在这两种情况下，CNV的降低都伴随着VEGFA的显着减弱。总之，目前的数据表明，通过多基因AAV载体靶向VEGFA的基因疗法显示出综合的功效，这表明双重作用疗法是未来眼基因疗法中用于治疗包括AMD在内的新生血管眼疾病的重要工具。