Pastor Marie, Johnen Sandra, Harmening Nina, Quiviger Mickäel, Pailloux Julie, Kropp Martina, Walter Peter, Ivics Zoltán, Izsvák Zsuzsanna, Thumann Gabriele, Scherman Daniel, Marie Corinne
CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258, 75006 Paris, France; Chimie ParisTech, PSL Research University, UTCBS, 75005 Paris, France; Université Paris Descartes, Sorbonne-Paris-Cité, UTCBS, 75006 Paris, France; INSERM, UTCBS U 1022, 75006 Paris, France.
Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany.
Mol Ther Nucleic Acids. 2018 Jun 1;11:57-67. doi: 10.1016/j.omtn.2017.12.017. Epub 2017 Dec 30.
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.
抗血管生成和神经生成的色素上皮衍生因子（PEDF）已证明有能力控制年龄相关性黄斑变性（AMD）患者的脉络膜新生血管形成。本研究的目的是开发一种高效且安全的技术，将PEDF基因在体外整合到视网膜色素上皮（RPE）细胞中，随后移植到AMD患者的视网膜下间隙，以便在受影响的黄斑附近持续分泌PEDF。由于成功的基因治疗方法需要高效的基因传递和稳定的基因表达，我们使用不含抗生素的pFAR4微型质粒载体来递送超活性睡眠美杜莎转座子系统，该系统介导转基因整合到宿主细胞的基因组中。在初步研究中，与使用pT2载体时相比，用SB100X转座酶基因和由pFAR4递送的报告标记物进行脂质体转染介导的HeLa细胞共转染显示转基因修饰细胞水平高出2倍。同样，使用pFAR4构建体时，电穿孔介导的原代人RPE细胞转染导致重组PEDF蛋白分泌高出2.4倍，转染后8个月仍保持该水平。因此，我们的结果表明，pFAR4质粒是将转基因递送至真核细胞并整合到其中的优良载体。
