慢病毒介导的血管内皮生长因子RNA干扰对猴虹膜新生血管化眼的作用

Lentivirus-mediated RNA interference of vascular endothelial growth factor in monkey eyes with iris neovascularization.

作者信息

Yuan Meng-Ke, Tao Yong, Yu Wen-Zhen, Kai Wang, Jiang Yan-Rong

机构信息

Department of Ophthalmology, People's Hospital, Peking University, Beijing, China.

出版信息

Mol Vis. 2010 Aug 25;16:1743-53.

PMID:20806089

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2927369/

Abstract

PURPOSE

To explore the in vivo anti-angiogenesis effects resulting from lentivirus-mediated RNAi of vascular endothelial growth factor (VEGF) in monkeys with iris neovascularization (INV).

METHODS

Five specific recombinant lentiviral vectors for RNA interference, targeting Macaca mulatta VEGFA, were designed and the one with best knock down efficacy (LV-GFP-VEGFi1) in H1299 cells and RF/6A cells was selected by real-time PCR for in vivo use. A laser-induced retinal vein occlusion model was established in one eye of seven cynomolgus monkeys. In monkeys number 1, 3, and 5 (Group 1), the virus (1x10(8) particles) was intravitreally injected into the preretinal space of the animal's eye immediately after laser coagulation; and in monkeys number 2, 4, and 6 (Group 2), the virus (1x10(8) particles) was injected at 10 days after laser coagulation. In monkey number 7, a blank control injection was performed. In monkeys number 1 and 2, virus without RNAi sequence was used; in monkeys number 3 and 4, virus with nonspecific RNAi sequence was used; and in monkeys 5 and 6, LV-GFP-VEGFi1 was used.

RESULTS

In monkey number 5, at 23 days after laser treatment, no obvious INV was observed, while fluorescein angiography of the iris revealed high fluorescence at the margin of pupil and point posterior synechiae. At 50 days after laser treatment, only a slight ectropion uvea was found. However, in the other eyes, obvious INV or hyphema was observed. The densities of new iridic vessels all significantly varied: between monkey number 5 and number 3 (36.01+/-4.49/mm(2) versus 48.68+/-9.30/mm(2), p=0.025), between monkey number 3 and monkey number 7 (48.68+/-9.30/mm(2) versus 74.38+/-9.23/mm(2), p=0.002), and between monkey number 5 and number 7 (36.01+/-4.49/mm(2) versus 74.38+/-9.23/mm(2), p<0.001).

CONCLUSIONS

Lentivirus-mediated RNAi of VEGF may be a new strategy to treat iris neovascularization, while further studies are needed to investigate the long-term effect.

摘要

目的

探讨慢病毒介导的血管内皮生长因子（VEGF）RNA干扰对虹膜新生血管（INV）猴的体内抗血管生成作用。

方法

设计了5种针对猕猴VEGFA的特异性重组RNA干扰慢病毒载体，并通过实时PCR在H1299细胞和RF/6A细胞中筛选出敲低效率最佳的一种（LV-GFP-VEGFi1）用于体内实验。在7只食蟹猴的一只眼睛中建立激光诱导的视网膜静脉阻塞模型。在第1、3和5号猴（第1组）中，激光凝固后立即将病毒（1×10⁸个颗粒）玻璃体内注射到动物眼睛的视网膜前间隙；在第2、4和6号猴（第2组）中，在激光凝固后10天注射病毒（1×10⁸个颗粒）。对第7号猴进行空白对照注射。在第1和2号猴中，使用无RNAi序列的病毒；在第3和4号猴中，使用具有非特异性RNAi序列的病毒；在第5和6号猴中，使用LV-GFP-VEGFi1。

结果

在第5号猴中，激光治疗后23天，未观察到明显的虹膜新生血管，而虹膜荧光血管造影显示瞳孔边缘和后粘连点处有高荧光。激光治疗后50天，仅发现轻微的葡萄膜外翻。然而，在其他眼中，观察到明显的虹膜新生血管或前房积血。新虹膜血管的密度均有显著差异：第5号猴与第3号猴之间（36.01±4.49/mm²对48.68±9.30/mm²，p = 0.025），第3号猴与第7号猴之间（48.68±9.30/mm²对74.38±9.23/mm²，p = 0.002），以及第5号猴与第7号猴之间（36.01±4.49/mm²对74.38±9.23/mm²，p < 0.001）。