IZKF Aachen, RWTH Aachen University, Aachen, Germany.
Invest Ophthalmol Vis Sci. 2012 Jul 18;53(8):4787-96. doi: 10.1167/iovs.12-9951.
Subretinal transplantation of retinal (RPE) or iris (IPE) pigment epithelial cells has been advocated as a treatment for retinal degeneration. However, to our knowledge, in patients with age-related macular degeneration no significant beneficial effects on vision have been shown. Since the transplanted cells did not appear to maintain a healthy avascular and neuroprotective environment, we postulate that it will be necessary to transplant cells that express elevated levels of anti-angiogenic and neuroprotective activities. In our study, we provide a protocol for the efficient stable gene transfer and sustained gene expression of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic and neuroprotective factor, using the nonviral Sleeping Beauty transposon system (SB100X).
Pigment epithelial cells were electroporated with a Venus reporter or a PEDF encoding plasmid, controlled by either CMV or CAGGS promoters. Transfection efficiencies and protein expression stability were evaluated by flow cytometry and immunoblotting. Gene expression profiles were analyzed by RT-PCR.
SB100X-based delivery resulted in efficiencies of 100% with the Venus gene and 30% with the PEDF gene. Cell sorting enabled establishment of pure PEDF-transfected ARPE-19 populations. Transfected RPE and IPE cells have been shown to maintain stable PEDF secretion for more than 16 and 6 months, respectively.
Transfection using the nonviral SB100X vector system avoids complications associated with viral gene delivery. SB100X-mediated transfer allows for stable PEDF gene integration into the cell's genome, ensuring continuous expression and secretion of PEDF. Stable expression of the therapeutic gene is critical for the development of cell-based gene addition therapies for retinal degenerative diseases.
视网膜（RPE）或虹膜（IPE）色素上皮细胞的视网膜下移植已被提倡用于治疗视网膜变性。然而，据我们所知，在年龄相关性黄斑变性患者中，视力没有明显的有益效果。由于移植细胞似乎没有保持健康的无血管和神经保护环境，我们推测有必要移植表达高水平抗血管生成和神经保护活性的细胞。在我们的研究中，我们提供了一种使用非病毒 Sleeping Beauty 转座子系统（SB100X）有效稳定基因转移和持续基因表达色素上皮衍生因子（PEDF）的方案，PEDF 是一种有效的抗血管生成和神经保护因子。
用 Venus 报告基因或 PEDF 编码质粒转染色素上皮细胞，由 CMV 或 CAGGS 启动子控制。通过流式细胞术和免疫印迹法评估转染效率和蛋白表达稳定性。通过 RT-PCR 分析基因表达谱。
基于 SB100X 的递送导致 Venus 基因的效率为 100%，PEDF 基因的效率为 30%。细胞分选可建立纯 PEDF 转染 ARPE-19 群体。转染的 RPE 和 IPE 细胞已被证明分别能稳定分泌 PEDF 超过 16 个月和 6 个月。
使用非病毒 SB100X 载体系统进行转染可避免与病毒基因传递相关的并发症。SB100X 介导的转移允许 PEDF 基因稳定整合到细胞基因组中，确保 PEDF 的持续表达和分泌。治疗基因的稳定表达对于开发用于治疗视网膜退行性疾病的基于细胞的基因添加疗法至关重要。
