Hernandez Maria, Recalde Sergio, Garcia-Garcia Laura, Bezunartea Jaione, Miskey Csaba, Johnen Sandra, Diarra Sabine, Sebe Attila, Rodriguez-Madoz Juan Roberto, Pouillot Severine, Marie Corinne, Izsvák Zsuzsanna, Scherman Daniel, Kropp Martina, Prosper Felipe, Thumann Gabriele, Ivics Zoltán, Garcia-Layana Alfredo, Fernandez-Robredo Patricia
Experimental Ophthalmology Laboratory, Ophthalmology, Clínica Universidad de Navarra, IdiSNA, Pamplona, Spain.
Red Temática de Investigación Cooperativa Sanitaria en Enfermedades Oculares, Oftared, ISCIII, Madrid, Spain.
Mol Ther Methods Clin Dev. 2019 Nov 9;15:403-417. doi: 10.1016/j.omtm.2019.10.013. eCollection 2019 Dec 13.
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 cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral () 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--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 non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.
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失衡。本研究的目的是评估使用不含抗生素抗性标记的微型质粒（pFAR4）递送的非病毒（）转座子系统进行基于细胞的基因治疗对稳定表达人色素上皮衍生因子（PEDF）释放的影响。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（ITRs）CMV-PEDF-BGH和pFAR4-CMV--SV40质粒共转染。在大鼠中进行激光诱导的脉络膜新生血管形成（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细胞在第7天和第14天显著减少渗漏面积，在第7天显著减少CNV面积。tRPE细胞使PEDF和PEDF/VEGF比值显著增加，MMP-2活性降低。我们的数据表明，非病毒基因治疗可减少CNV，可能是治疗血管生成性视网膜疾病的一种有效且安全的治疗选择。
