CRISPR/Cas9技术在眼底新生血管性疾病治疗中的应用  

Application of CRISPR/Cas9 technology in the treatment of fundus neovascular diseases

作　　者：刘晋星 周国宏[2] 王文娟[2] 王永瑞 李静[2] 李会琳[3] Liu Jinxing;Zhou Guohong;Wang Wenjuan;Wang Yongrui;Li Jing;Li Huilin(Shanxi Medical University,Taiyuan 030001,China;Eye Hospital of Shanxi Province,Taiyuan 030002,China;Heji Hospital of Changzhi Medical College,Changzhi Shanxi 046001,China)

机构地区：[1]山西医科大学,太原030001 [2]山西省眼科医院,太原030002 [3]长治医学院附属和济医院,山西长治046001

出　　处：《国际眼科纵览》2020年第6期408-413,共6页International Review of Ophthalmology

基　　金：山西省卫生和计划生育委员会科研项目(2017097);山西省留学回国人员科技活动择优资助项目(2017-031)。

摘　　要：成簇的规律间隔的短回文重复序列相关蛋白9(clustered regularly interspaced short palindromic repeats-associated proteins 9,CRISPR/Cas9)技术可用于靶向敲入、替换或敲除真核细胞基因。将血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)组装的特异Cas9核糖核蛋白于小鼠视网膜下注射,可明显减小激光诱导的脉络膜新生血管(choroidal neovascularization,CNV)面积;小分子蛋白CjCas9靶向敲除视网膜色素上皮细胞(retinal pigment epithelium,RPE)中VEGFA或缺氧诱导因子1基因亦可有效减小激光诱导的CNV面积,且其突变后维持时间可超过1年。在视网膜水平,利用CRISPR/Cas9技术将RPE细胞中的VEGFA或人视网膜微血管内皮细胞(human microvascular endothelial cells,HREC)中的VEGFR2基因敲除可明显抑制视网膜新生血管的生成;将内皮细胞诱导的小类泛素蛋白修饰分子(small ubiquit in-like modifier,SUMO)特异性蛋白酶1基因敲低后SUMO化的VEGFR2聚集于高尔基体,亦可有效抑制病理性新生血管。将HREC中的p110δ敲除后,HREC的增生、迁移及成管能力均减弱。以PI3Kδ为靶点的药物idelalisib也有抑制病理性视网膜新生血管的作用。利用CRISPR/Cas9技术对天冬氨酸蛋白磷酸酶1基因敲除可使AKT磷酸化状态改变进而影响新生血管的生成。利用CRISPR/Cas9的协同激活介体系统筛选出AKT的阳性调节子AK023948,将AK023948敲低可抑制AKT的活性,进而抑制新生血管的生成。(国际眼科纵览,2020,44:408-413)Clustered regularly interspaced short palindromic repeats-associated proteins 9(CRISPR/Cas9)technology can be used to knock-in,replacement or knock-out of eukaryotic genes.Injecting the RNP Cas9(VEGFA gene-specific Cas9 ribonucleoproteins)under the mouse retina can significantly reduce the area of laser-induced choroidal neovascularization(CNV).Targeting small molecule protein CjCas9 to knock out VEGFA(vascular endothelial growth factor A)or HIF-1(hypoxia-inducible factor 1)genes of RPE(retinal pigment epithelium)can also effectively reduce the area of laser-induced CNV,and its mutation can maintain exceed 1 year.At the retinal level,knocking out VEGFA in RPE cells or VEGFR2 in human microvascular endothelial cells(HRECs)by CRISPR/Cas9 technology can significantly inhibit the formation of retinal neovascularization.After knocking down the small ubiquit in-like modifier-specific protease 1(SUMO-specific protease 1,SENP1)induced by endothelial cells,the SUMO-turned VEGFR2 in the Golgi can also effectively inhibit pathological neovascularization.After knocking out the p110δin HRECs,the proliferation,migration and tube formation ability of HRECs were all weakened.The drug idelalisib,which targets PI3Kδ,can also inhibit pathological retinal neovascularization.Using CRISPR/Cas9 technology to knock out SCP1(aspartic proteinase 1)can change the phosphorylation state of AKT and affect the formation of new blood vessels.The CRISPR/Cas9 synergistic activation mediator(SAM)system can screen out the positive regulator AK023948 of AKT.Knock down of AK023948 can inhibit the activity of AKT,thereby inhibiting the formation of new blood vessels.(Int Rev Ophthalmol,2020,44:408-413)

关 键 词：成簇的规律间隔的短回文重复序列相关蛋白9 新生血管形成 PI3K/AKT通路 

分 类 号：R73[医药卫生—肿瘤]