Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
Grousbeck Gene Therapy Center, Schepens Eye Research Institute and Massachusetts Eye and Ear, Boston, MA, USA.
Sci Rep. 2017 Mar 31;7:45329. doi: 10.1038/srep45329.
Widespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye.
广泛的基因转移到视网膜是具有挑战性的，因为它需要载体系统来克服物理和生化障碍，进入并扩散到整个视网膜组织。我们研究了外泌体相关的腺相关病毒（exo-AAV）是否能够在玻璃体内（IVT）注射后实现广泛的视网膜靶向，因为已经证明外泌体可以穿透生物屏障，并在全身注射后介导广泛的分布。我们将编码绿色荧光蛋白（GFP）的 AAV 基因组包装到常规 AAV2 和 exo-AAV2 载体中。载体通过 IVT 注射到成年小鼠的眼睛中。通过眼底成像非侵入性地监测 GFP 表达，并在注射后 4 周通过 qRT-PCR 和组织学分析分析视网膜表达。基于眼底图像分析和 qRT-PCR，exo-AAV2 在 GFP 表达方面优于常规 AAV2。Exo-AAV2 在视网膜中的穿透更深，有效地到达内核层和外丛状层，在较小程度上到达外核层。细胞靶标是神经节细胞、双极细胞、Müller 细胞和光感受器。Exo-AAV2 是一种用于小鼠视网膜的强大基因传递工具，其生产和分离的简单性应使其广泛适用于眼部基础研究。
