Interdisciplinary Center for Clinical Research Aachen, Rheinisch-Westfälische Technische Hochschule Aachen University, Germany.
Invest Ophthalmol Vis Sci. 2011 Jul 23;52(8):5400-7. doi: 10.1167/iovs.10-6178.
Genetic modification of cells before transplantation may allow the delivery of neuroprotective and other functional molecules to patients with neurodegenerative diseases. To avoid complications associated with virally transfected cells, we have explored the use of nonviral methods to insert genetic material into RPE cells.
After transfection with plasmids encoding different pigment epithelium-derived factor (PEDF) fusion proteins, transfected cells were established and passaged up to 100 times. Gene expression of PEDF, ZnT3, ZIP2, CRALBP, CATD, and ZO-1 was determined by RT-PCR. Secretion dynamics were analyzed using ELISA and a spheroid-based assay was used to confirm the anti-angiogenic activity of the recombinant PEDF.
Transfection efficiency reached up to 98.7% with a plasmid encoding PEDF and enhanced green fluorescent protein (EGFP) separately and 87.2% with a plasmid encoding an EGFP-PEDF fusion. Immunoblotting revealed that transfected RPE cells express the appropriate PEDF or EGFP-PEDF. Expression of recombinant PEDF is stable, as shown by its secretion for the 2 years and the 100 passages the cells have been followed. PEDF expression was overexpressed and the transfected cells exhibited increased proliferation, up-regulation of ZnT3 and ZIP2, and inhibited sprouting in human umbilical vein endothelial cell spheroids.
Genetic in vitro modification of pigment epithelial cells using nonviral transfection protocols should improve the potential therapeutic treatment of neurodegenerative diseases by transplantation of genetically modified cells without the disadvantages of virally mediated transfection. Here we have shown that genetically modified RPE cells overexpress a functional human recombinant PEDF, as evidenced by the autogenic regulation of proliferation, up-regulation of two distinct zinc transporters, and in vitro inhibition of endothelial cell sprouting.
细胞在移植前进行基因修饰可能允许将神经保护和其他功能分子递送至神经退行性疾病患者。为避免与病毒转染细胞相关的并发症，我们探索了使用非病毒方法将遗传物质插入 RPE 细胞。
用编码不同色素上皮衍生因子（PEDF）融合蛋白的质粒转染后，建立转染细胞并传代 100 次以上。通过 RT-PCR 测定 PEDF、ZnT3、ZIP2、CRALBP、CATD 和 ZO-1 的基因表达。使用 ELISA 分析分泌动力学，并使用球体测定法证实重组 PEDF 的抗血管生成活性。
用编码 PEDF 和增强型绿色荧光蛋白（EGFP）的质粒转染效率分别高达 98.7%和 87.2%，用编码 EGFP-PEDF 融合蛋白的质粒转染效率为 87.2%。免疫印迹显示转染的 RPE 细胞表达适当的 PEDF 或 EGFP-PEDF。重组 PEDF 的表达是稳定的，从细胞已经传代 2 年和 100 次可以看出。PEDF 表达过表达，转染细胞表现出增殖增加、ZnT3 和 ZIP2 的上调以及人脐静脉内皮细胞球体发芽的抑制。
使用非病毒转染方案对色素上皮细胞进行体外基因修饰，应通过移植遗传修饰的细胞改善神经退行性疾病的潜在治疗方法，而没有病毒介导转染的缺点。在这里，我们已经表明，遗传修饰的 RPE 细胞过表达功能性人重组 PEDF，这可以通过增殖的自体调节、两种不同的锌转运体的上调以及内皮细胞发芽的体外抑制来证明。
