Center for Complex Systems and Brain Sciences, Charles E. Schmidt College of Science, Florida Atlantic University, Boca Raton, FL 33431, USA.
Invest Ophthalmol Vis Sci. 2011 Nov 1;52(12):8562-70. doi: 10.1167/iovs.10-6835.
Retinal Müller cells span the retina and secrete several trophic factors and represent the functional link between blood vessels and neurons, making them attractive targets for gene therapy. Therefore, a hypoxia-regulated, retinal glial cell-specific vector was constructed and tested for its response to hypoxia.
A hybrid promoter containing domains of human glial fibrillary acidic protein (GFAP) and several hypoxia-responsive and aerobically silenced elements (HRSE) was incorporated separately into plasmid vectors for generation of self-complementary adeno-associated virus. Müller cells trasfected with plasmids or virus were compared with other cell lines using standard
The mouse model of oxygen-induced retinopathy (OIR) was used to analyze retinas from mice exposed to high oxygen or room air to evaluate the induction of the regulated promoter.
The regulated promoter was silenced under aerobic conditions in comparison with unregulated promoter in Müller cells. Hypoxia induced a 12-fold and 16-fold increase in promoter activity in primary Müller cells and human Müller cell lines, respectively. In the OIR model, intravitreal injection of the regulated promoter at postnatal day 7 (P7) resulted in high levels of green fluorescent protein expression only in retinal Müller cells at P17. GFP expression was absent in retinas of mice only exposed to room air. In vivo studies confirm normoxia silencing, hypoxic induction, and cell specificity of the regulated promoter in the mouse retina.
This hypoxia-regulated, retinal glial cell-specific AAV vector provides a platform for gene therapy within regions of retinal hypoxia which are found in diabetic retinopathy and age-related macular degeneration.
视网膜 Müller 细胞横跨视网膜并分泌多种营养因子，是血管和神经元之间的功能连接，使其成为基因治疗的有吸引力的靶点。因此，构建了一种缺氧调节的、视网膜神经胶质细胞特异性的载体，并对其对缺氧的反应进行了测试。
包含人胶质纤维酸性蛋白 (GFAP) 和几个缺氧反应和需氧沉默元件 (HRSE) 的杂交启动子分别被整合到质粒载体中，用于生成自我互补的腺相关病毒。用质粒或病毒转染的 Müller 细胞与其他细胞系进行了比较。
与未调节的启动子相比，Müller 细胞在有氧条件下调节的启动子被沉默。缺氧诱导原代 Müller 细胞和人 Müller 细胞系的启动子活性分别增加了 12 倍和 16 倍。在氧诱导的视网膜病变 (OIR) 模型中，在出生后第 7 天 (P7) 玻璃体腔内注射调节启动子，仅在 P17 时在视网膜 Müller 细胞中引起高水平的绿色荧光蛋白表达。仅在暴露于室内空气的小鼠的视网膜中未观察到 GFP 表达。体内研究证实了该调节启动子在小鼠视网膜中的正常氧抑制、缺氧诱导和细胞特异性。
这种缺氧调节的、视网膜神经胶质细胞特异性的 AAV 载体为糖尿病性视网膜病变和年龄相关性黄斑变性中发现的视网膜缺氧区域的基因治疗提供了一个平台。
