Genome Editing of Pik3cd Impedes Abnormal Retinal Angiogenesis
Genome Editing of Pik3cd Impedes Abnormal Retinal Angiogenesis
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Pik3cd 的基因组编辑阻碍异常视网膜血管生成
DOI：
10.1089/hum.2022.079
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发表时间：
2022
影响因子：
4.2
通讯作者：
Hetian Lei
中科院分区：
文献类型：
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作者：
Wenyi Wu;Gaoen Ma;Hui Qi;Lijun Dong;fang chen;Yun Wang;Xingxing Mao;Xiaoqing Guo;Jing Cui;Joanne Matsubara;Bart Vanhaesebroeck;Xiaohe Yan;Guoming Zhao;Shaochong Zhang;Hetian Lei
异常血管生成与无数人类疾病相关，包括增殖性糖尿病视网膜病变（PDR）。磷脂酰肌醇3-激酶（PI 3 Ks）信号转导在血管生成中起着关键作用。在此，我们发现p110δ，PI 3 K δ的催化亚基，在氧诱导视网膜病变（OIR）小鼠模型的病理性视网膜血管内皮细胞（EC）和PDR患者的纤维血管膜中高度表达。为了探索PI 3 K δ表达的新干预，我们开发了用于递送CRISPR/Cas9的重组双腺相关病毒（rAAV）系统，其中化脓链球菌（Sp）Cas9表达由细胞间粘附分子2（pICAM 2）的内皮特异性启动子驱动以编辑基因组Pik 3cd（编码p110δ的基因）。然后，我们证明了用靶向genomicPik 3cd的rAAV 1-pICAM 2-SpCas 9和rAAV 1-SpGuide的双重rAAV 1感染培养的小鼠血管EC导致genomicPik 3cd位点中80%的DNA插入/缺失和70%的p110δ表达缺失。此外，我们发现，在OIR编辑视网膜Pik 3cd的小鼠模型中，双rAAV 1不仅导致p110δ表达和Akt活化的显著降低，而且还导致病理性视网膜血管生成的显著减少。这些发现表明Pik 3cdetiting是治疗异常视网膜血管生成的新方法。
Abnormal angiogenesis is associated with myriad human diseases, including proliferative diabetic retinopathy (PDR). Signaling transduction through phosphoinositide 3-kinases (PI3Ks) plays a critical role in angiogenesis. Herein, we showed that p110δ, the catalytic subunit of PI3Kδ, was highly expressed in pathological retinal vascular endothelial cells (ECs) in a mouse model of oxygen-induced retinopathy (OIR) and in fibrovascular membranes from patients with PDR. To explore novel intervention with PI3Kδ expression, we developed a recombinant dual adeno-associated viral (rAAV) system for delivering CRISPR/Cas9 in whichStreptococcus pyogenes(Sp) Cas9 expression was driven by an endothelial specific promoter of the intercellular adhesion molecule 2 (pICAM2) to edit genomicPik3cd, the gene encoding p110δ. We then demonstrated that infection of cultured mouse vascular ECs with the dual rAAV1s of rAAV1-pICAM2-SpCas9 and rAAV1-SpGuide targeting genomicPik3cdresulted in 80% DNA insertion/deletion in the locus of genomicPik3cdand 70% depletion of p110δ expression. Furthermore, we showed that in the mouse model of OIR editing retinalPik3cdwith the dual rAAV1s resulted in not only a significant decrease in p110δ expression, and Akt activation, but also a dramatic reduction in pathological retinal angiogenesis. These findings reveal thatPik3cdediting is a novel approach to treating abnormal retinal angiogenesis.