Wu Wenyi, Duan Yajian, Ma Gaoen, Zhou Guohong, Park-Windhol Cindy, D'Amore Patricia A, Lei Hetian
Schepens Eye Research Institute of Massachusetts Eye and Ear; Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
Department of Ophthalmology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
Invest Ophthalmol Vis Sci. 2017 Dec 1;58(14):6082-6090. doi: 10.1167/iovs.17-21902.
Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events.
The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined.
AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs.
AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.
病理性血管生成是许多疾病的一个组成部分，包括新生血管性年龄相关性黄斑变性、增殖性糖尿病视网膜病变以及肿瘤生长和转移。本项目的目的是研究腺相关病毒（AAV）介导的成簇规律间隔短回文重复序列（CRISPR）相关核酸酶（Cas）9系统是否可用于在体外耗尽人血管内皮细胞中血管内皮生长因子受体2（VEGFR2）的表达，从而抑制其下游信号事件。
将化脓性链球菌的CRISPR/Cas9双AAV系统（AAV-SpGuide和-SpCas9）用于编辑原代人视网膜微血管内皮细胞（HREC）中的基因组VEGFR2。在该系统中，将细胞间黏附分子2（ICAM2）的内皮特异性启动子克隆到SpGuide和SpCas9的双AAV载体中，分别用于驱动绿色荧光蛋白（GFP）和SpCas9的表达。将这两种AAV载体用于生产重组AAV血清型5（rAAV5），用于感染HREC以耗尽VEGFR2。通过蛋白质印迹法测定蛋白质表达；并检测细胞增殖、迁移以及管形成。
AAV5有效感染血管内皮细胞（EC）和视网膜色素上皮（RPE）细胞；ICAM2启动子在HREC中驱动GFP和SpCas9的表达，但在RPE细胞中不驱动。结果表明，rAAV5-CRISPR/Cas9使VEGFR2减少80%，并完全阻断VEGF诱导的Akt激活以及HREC的增殖、迁移和管形成。
AAV-CRISRP/Cas9介导的VEGFR2耗尽是病理性血管生成的一种潜在治疗策略。
Zotero 插件
