The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

抗血管生成和神经源性色素上皮衍生因子（PEDF）在与年龄有关的黄斑变性（AMD）患者中显示了控制脉络膜新血管形成的能力。本研究的目标是开发一种高效安全的技术，将PEDF基因离体整合到视网膜色素上皮（RPE）细胞中，以便以后移植到AMD患者的视网膜下空间，从而允许附近PEDF的连续分泌受影响的黄斑。因为成功的基因治疗方法需要有效的基因传递和稳定的基因表达，所以我们使用了无抗生素的pFAR4微型质粒载体来传递高活性的《睡美人》。转座子系统，介导转基因整合到宿主细胞的基因组中。在最初的研究中，脂质体介导的HeLa细胞与SB100X转座酶基因和pFAR4传递的报道分子标记的共转染显示，与使用pT2载体时相比，转基因细胞的水平高2倍。类似地，使用pFAR4构建体，原代人RPE细胞的电穿孔介导转染导致重组PEDF蛋白分泌高2.4倍，转染后8个月仍保持这种状态。因此，我们的结果表明，pFAR4质粒是将转基因传递和整合到真核细胞中的优良载体。