Rolling F, Shen W Y, Tabarias H, Constable I, Kanagasingam Y, Barry C J, Rakoczy P E
Lions Eye Institute, Perth, Western Australia, Australia.
Hum Gene Ther. 1999 Mar 1;10(4):641-8. doi: 10.1089/10430349950018715.
The purpose of this study was to evaluate recombinant adeno-associated virus (AAV) as an in vivo gene transfer vector for the retina and to explore the possibility of monitoring the expression of green fluorescent protein (GFP) using a noninvasive method. Rats were injected subretinally with rAAV-gfp or rAAV-lacZ. Strong expression of the reporter gene in a circular area surrounding the injection site was observed in retinal whole mounts and tissue sections. Higher magnification revealed that cells demonstrating high levels of green fluorescence were hexagonal in shape, indicating they were retinal pigment epithelium (RPE) cells. Histological observation of retinal sections demonstrated that recombinant AAV specifically transduced RPE cells. Ten animals were injected with rAAV-gfp for longitudinal studies and the fluorescence was monitored by retinal fluorescence photography. The GFP signal was detected in 100% of the animals as early as 2 weeks postinjection and remained present throughout the experimental period of 4 months. After 2 weeks, a gradual increase in the number of transduced cells occurred before reaching maximal levels of GFP expression at 8 weeks. This was followed by a small decrease over 4 weeks before reaching stable expression at 16 weeks. Our results demonstrated that rAAV efficiently transduces rat RPE cells and that retinal fluorescence photography is suitable for monitoring GFP expression. By using this noninvasive technique, we demonstrated that repetitive measurements of GFP expression in vivo in the rAAV-gfp-transduced retina are possible. This study demonstrated that retinal fluorescence photography is a potent tool for studying AAV-mediated gene delivery in the retina.
本研究的目的是评估重组腺相关病毒（AAV）作为视网膜体内基因转移载体，并探索使用非侵入性方法监测绿色荧光蛋白（GFP）表达的可能性。将大鼠视网膜下注射rAAV-gfp或rAAV-lacZ。在视网膜全层铺片和组织切片中观察到报告基因在注射部位周围的圆形区域强烈表达。更高倍放大显示，呈现高水平绿色荧光的细胞呈六边形，表明它们是视网膜色素上皮（RPE）细胞。视网膜切片的组织学观察表明，重组AAV特异性转导RPE细胞。十只动物注射rAAV-gfp进行纵向研究，并通过视网膜荧光摄影监测荧光。早在注射后2周，100%的动物检测到GFP信号，并且在整个4个月的实验期内一直存在。2周后，转导细胞数量逐渐增加，在8周时达到GFP表达的最大水平。随后在4周内略有下降，然后在16周时达到稳定表达。我们的结果表明，rAAV有效地转导大鼠RPE细胞，并且视网膜荧光摄影适用于监测GFP表达。通过使用这种非侵入性技术，我们证明了在rAAV-gfp转导的视网膜中对GFP表达进行体内重复测量是可行的。这项研究表明，视网膜荧光摄影是研究AAV介导的视网膜基因递送的有力工具。
