Construction of a CRISPR/Cas9-mediated VEGFA gene knockout vector and its effects on ARPE-19 cells
10.3980/j.issn.1672-5123.2026.7.04
- VernacularTitle:基于CRISPR/Cas9技术敲除VEGFA基因药物的制备及其在ARPE-19细胞中的作用
- Author:
Wenhua ZHANG
1
;
Jingxuan XU
1
;
Keying CAO
1
;
Chunmei WANG
1
;
Zongming SONG
1
;
Xiaoli LI
1
Author Information
1. Henan Provincial People's Hospital;Henan Eye Hospital;People's Hospital of Zhengzhou University;School of Clinical Medicine Henan University, Zhengzhou 450003, Henan Province, China
- Publication Type:Journal Article
- Keywords:
VEGFA;
CRISPR/Cas9;
retinal neovascularization;
gene knockout;
retinal pigment epithelium(RPE)cells
- From:
International Eye Science
2026;26(7):1127-1132
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To develop a novel gene-delivery therapeutic based on CRISPR/Cas9 genome editing technology capable of specifically targeting and knocking out the VEGFA gene, thereby achieving sustained suppression of VEGFA expression in retinal pigment epithelial(RPE)cells and providing a new strategy for gene therapy in retinal neovascular diseases.METHODS:Single guide RNAs targeting the human VEGFA gene for knockout were designed, and corresponding recombinant plasmids were constructed. A novel polymer(PTEE)was used to encapsulate the plasmids to prepare a PTEE-loaded anti-VEGFA plasmid(PLAP)gene delivery system. PTEE materials at concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 μg/μL were co-incubated with ARPE-19 cells, and the biocompatibility of PTEE was evaluated using the cell counting kit-8(CCK-8)assay. Recombinant plasmids expressing green fluorescent protein(GFP)were constructed. Lipofectamine 3000 and jetOPTIMUS®DNA transfection reagents were used as control groups, and PTEE nanomaterials were used as the experimental group to encapsulate the plasmids. When the cell confluence reached 80%, the formulations were transfected into ARPE-19 and 293T cells. GFP expression was observed under light microscopy, and the transfection efficiencies of each group were compared. ARPE-19 cells were induced under hypoxia, and PLAP was transfected into the cells. The expression level of VEGFA was detected by enzyme-linked immunosorbent assay(ELISA)to evaluate the efficacy of this novel gene delivery system.RESULTS: After co-incubation of ARPE-19 cells with different concentrations of PTEE for 24 h and 48 h, no significant effect on cell viability was observed in any group. The transfection efficiency of PLAP in ARPE-19 cells was higher than that in the Lipo3000 and jetOPTIMUS groups, with statistically significant differences(P<0.01). Hypoxia for 6 h significantly induced the upregulation of VEGFA mRNA expression in ARPE-19 cells, and under hypoxic conditions, the PTEE group exhibited a significant inhibitory effect on VEGFA expression(P<0.01).CONCLUSION:PLAP exhibits favorable biocompatibility and prominent VEGFA inhibitory effects in vitro, making it a potential candidate drug for gene therapy of retinal neovascular diseases.
