1 Stephen A. Wynn Institute for Vision Research, University of Iowa , Iowa City, Iowa.
2 Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa , Iowa City, Iowa.
Hum Gene Ther. 2018 Apr;29(4):424-436. doi: 10.1089/hum.2017.179. Epub 2018 Feb 23.
Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.
单基因视网膜疾病病因的发现进展，加上向视网膜递送 DNA 的技术，为治疗以前无法治疗的致盲性疾病提供了巨大的机会。然而，为了使基因增强最有效，需要具有正确细胞类型特异性的载体。虽然动物模型非常有用，但它们在视网膜细胞表面受体方面通常与人类视网膜存在差异。本研究评估了使用离体器官型外植体系统来测试七种不同腺相关病毒 2 型 (AAV2) 血清型在人视网膜和视网膜色素上皮脉络膜中的转导效率和趋向性，AAV2/1、AAV2/2、AAV2/4、AAV2/5、AAV2/6、AAV2/8 和 AAV2/9——均在巨细胞病毒启动子的控制下驱动 GFP 的表达。在培养 7 天后，发现 AAV2/4 和 AAV2/5 特别有效地转导光感受器细胞，并且 AAV2/5 对外核层具有高度特异性，而 AAV2/8 对光感受器的转导始终较低。为了验证器官型培养系统的真实性，还在猪模型中比较了相同的一组 AAV 的转导，其中将体内视网膜下注射与离体培养和转导的器官型培养进行了比较。本研究展示了不同 AAV 血清型在人视网膜中的行为，并为进一步研究每种血清型用于基于基因增强的遗传性视网膜变性治疗提供了深入了解。
