目的：基于CRISPR/Cas9基因编辑技术，研发一种能够靶向敲除VEGFA基因的新型载基因药物，实现对人视网膜色素上皮(RPE)细胞系VEGFA表达的长效抑制，为视网膜新生血管性疾病的基因治疗提供新策略。

方法：设计靶向敲除人VEGFA基因的向导RNA并构建出重组质粒，采用一种新型高分子聚合物(PTEE)对其进行包裹，制备PTEE loading anti-VEGFA plasmid(PLAP)载基因药物。将浓度为0.1、0.2、0.4、0.8、1.6 μg/μL的PTEE材料与ARPE-19细胞共孵育，通过CCK-8法评估PTEE的生物相容性。构建含有GFP蛋白的重组质粒，将脂质体Lipofectamine 3000和jetOPTIMUS^®；DNA体外转染试剂作为对照组，PTEE纳米材料作为实验组，对质粒进行包裹。待细胞融合度达80%时，将药物转染至ARPE-19细胞和293T细胞中，通过光学显微镜观察GFP蛋白的表达情况并比较各组的转染效率。低氧诱导ARPE-19细胞，将PLAP转染至细胞内，利用ELISA检测VEGFA表达水平，评价该新型基因药物的有效性。

结果：不同浓度梯度的PTEE材料与ARPE-19细胞共孵育24 h和48 h，各组细胞活力均未受到明显影响。PLAP在ARPE-19细胞中的转染效率高于lipo3000组与jetOPTIMUS组，差异具有统计学意义(P<0.01)。低氧6 h能显著诱导ARPE-19细胞中VEGFA mRNA的表达升高，且低氧状态下，PTEE组对VEGFA的表达有明显的抑制作用(P<0.01)。

结论：PLAP在体外实验中显示出良好的生物相容性和显著的VEGFA抑制效果，有潜力成为视网膜新生血管性疾病基因治疗的候选药物。

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.