用于非病毒基因治疗的哺乳动物原代色素上皮细胞的分离、培养和遗传工程。

Experimental Ophthalmology, University of Geneva; Department of Ophthalmology, University Hospitals of Geneva;

Experimental Ophthalmology, University of Geneva; Department of Ophthalmology, University Hospitals of Geneva.

J Vis Exp. 2021 Feb 26(168). doi: 10.3791/62145.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

年龄相关性黄斑变性（AMD）是 60 岁以上患者失明的最常见原因，影响全球约 3000 万人。AMD 是一种多因素疾病，受环境和遗传因素影响，导致视网膜色素上皮（RPE）细胞退化，随后光感受器降解，从而导致视网膜功能障碍。理想的治疗方法包括移植分泌神经营养因子的健康 RPE 细胞，以防止 RPE 细胞死亡和光感受器退化。由于功能和遗传上的相似性以及进行较少侵入性活检的可能性，提出了移植虹膜色素上皮（IPE）细胞作为退化 RPE 的替代品。通过 Sleeping Beauty（SB100X）转座子介导的基因转染，可以用少量视网膜下移植细胞分泌神经营养因子，基因编码色素上皮衍生因子（PEDF）和/或粒细胞巨噬细胞集落刺激因子（GM-CSF）。我们从包括啮齿动物、猪和牛在内的各种物种中建立了 RPE 和 IPE 细胞的分离、培养和 SB100X 介导的转染。眼球被取出，角膜和晶状体被去除以接触虹膜和视网膜。使用特制的刮刀，从分离的虹膜中去除 IPE 细胞。为了收获 RPE 细胞，可能需要根据物种进行胰蛋白酶孵育。然后，使用 RPE 定制刮刀，将细胞悬浮在培养基中。接种后，每周监测细胞两次，达到汇合后，通过电穿孔进行转染。通过 qPCR、WB、ELISA、免疫荧光和功能测定证实了基因整合、表达、蛋白分泌和功能。根据物种的不同，每只眼可分离 3 万至 500 万（RPE）和 1 万至 150 万（IPE）个细胞。基因修饰细胞表现出显著的 PEDF/GM-CSF 过表达，具有减轻氧化应激的能力，并提供了一个灵活的体外分析和体内研究系统，可转移到人类，以开发眼部基因治疗方法。