Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.

中文翻译：

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使用睡美人转座子系统进行脉络膜新生血管的细胞基因疗法的临床前评估。


年龄相关性黄斑变性（AMD）是一种进行性视网膜疾病，其特征是促血管生成信号和抗血管生成信号不平衡。本研究的目的是评估基于体外细胞的基因治疗的效果，使用由不含抗生素的小质粒传递的非病毒睡美人 (SB100X) 转座子系统稳定表达人类色素上皮衍生因子 (PEDF) 释放抗性标记（pFAR4）。视网膜色素上皮 (RPE) 细胞和虹膜色素上皮 (IPE) 细胞用 pFAR4 反向末端重复序列 (ITR) CMV-PEDF-BGH 和 pFAR4-CMV-SB100X-SV40 质粒共转染。在大鼠中进行激光诱导脉络膜新生血管（CNV），并将转染的原代细胞（转染的RPE [tRPE]和转染的IPE [tIPE]细胞）注射到视网膜下间隙。测量渗漏和 CNV 面积、血管内皮生长因子 (VEGF)、PEDF 蛋白表达、金属蛋白酶 2 和 9 (MMP-2/9) 以及小胶质细胞/巨噬细胞标记物。注射 tRPE/IPE 细胞显着减少了第 7 天和第 14 天的渗漏面积以及第 7 天的 CNV 面积。 tRPE 细胞的 PEDF 和 PEDF/VEGF 比率显着增加，而 MMP-2 活性降低。我们的数据表明，离体非病毒基因疗法可减少 CNV，并且可能是血管生成性视网膜疾病的有效且安全的治疗选择。