Huang Xionggao, Wu Wenyi, Qi Hui, Yan Xiaohe, Dong Lijun, Yang Yanhui, Zhang Qing, Ma Gaoen, Zhang Guoming, Lei Hetian
Department of Ophthalmology, The First Affiliated Hospital of Hainan Medical University, Haikou, China.
Department of Ophthalmology, Hunan Key Laboratory of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.
J Adv Res. 2025 Jun;72:121-133. doi: 10.1016/j.jare.2024.07.006. Epub 2024 Jul 10.
Aberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders.
The goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy.
An advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis.
NILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology.
DN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.
异常血管生成在包括增殖性糖尿病视网膜病变在内的多种人类疾病发展中起重要作用，目前仍有众多患者面临治疗挑战。碱基编辑（PE）是一种通用的基因编辑方法，为基因校正具有挑战性的疾病提供了新机会。
本研究的目标是通过使用先进的碱基编辑系统编辑基因组DNA，构建一种显性负性（DN）血管内皮生长因子受体（VEGFR）2，以阻断氧诱导视网膜病变小鼠模型中的异常视网膜血管生成。
在两个慢病毒载体中建立了一种先进的碱基编辑系统（称为PE6x），一个载体携带增强的碱基编辑引导RNA和与优化逆转录酶融合的经典Cas9切口酶，另一个载体携带切口引导RNA和DN-MLH1以提高碱基编辑效率。然后使用由两个慢病毒PE6x载体产生的双非整合慢病毒（NILV），通过在体外和体内编辑小家鼠VEGFR2基因座，产生VEGFR2 T17967A突变，导致产生提前终止密码子（TAG，K796stop）以产生DN-VEGFR2，从而干扰血管生成所必需的野生型VEGFR2。
通过下一代测序分析，导入培养的小鼠血管内皮细胞中的靶向VEGFR2的NILV导致基因组中51.06%的VEGFR2 T17967A并产生DN-VEGFR2，蛋白质免疫印迹分析表明，DN-VEGFR2可抑制VEGF诱导的VEGFR2磷酸化。在氧诱导视网膜病变模型中，将双NILV玻璃体内注射到出生后第12天的小鼠中，导致出生后第17天的小鼠产生视网膜DN-VEGFR2，其阻断视网膜VEGFR2表达和激活以及异常视网膜血管生成，而不干扰视网膜结构和功能，这通过视网膜电图、光学相干断层扫描、眼底荧光血管造影和组织学评估。
利用PE6x系统编辑基因组VEGFR2产生的DN-VEGFR2可用于治疗眼内病理性血管生成。
