Department of Ophthalmology, University of Florida, Gainesville, Florida, United States.
Department of Ophthalmology, John A. Moran Eye Center, University of Utah Health Science Center, Salt Lake City, Utah, United States.
Invest Ophthalmol Vis Sci. 2019 Aug 1;60(10):3644-3651. doi: 10.1167/iovs.19-27079.
Previously we showed that AAV5-mediated expression of either human M- or L-opsin promoted regrowth of cone outer segments and rescued M-cone function in the treated M-opsin knockout (Opn1mw-/-) dorsal retina. In this study, we determined cone viability and window of treatability in aged Opn1mw-/- mice.
Cone viability was assessed with antibody against cone arrestin and peanut agglutinin (PNA) staining. The rate of cone degeneration in Opn1mw-/- mice was quantified by PNA staining. AAV5 vector expressing human L-opsin was injected subretinally into one eye of Opn1mw-/- mice at 1, 7, and 15 months old, while the contralateral eyes served as controls. M-cone-mediated retinal function was analyzed 2 and 13 months postinjection by full-field ERG. L-opsin transgene expression and cone outer segment structure were examined by immunohistochemistry.
We showed that dorsal M-opsin dominant cones exhibit outer segment degeneration at an early age in Opn1mw-/- mice, whereas ventral S-opsin dominant cones were normal. The remaining M-opsin dominant cones remained viable for at least 15 months, albeit having shortened or no outer segments. We also showed that AAV5-mediated expression of human L-opsin was still able to rescue function and outer segment structure in the remaining M-opsin dominant cones when treatment was initiated at 15 months of age.
Our results showing that the remaining M-opsin dominant cones in aged Opn1mw-/- mice can still be rescued by gene therapy is helpful for establishing the window of treatability in future blue cone monochromacy clinical trials.
我们之前曾表明，AAV5 介导的人 M-或 L-视蛋白的表达促进了锥体外节的再生，并挽救了治疗的 M-视蛋白敲除（Opn1mw-/-）背侧视网膜中的 M-锥体功能。在这项研究中，我们确定了老年 Opn1mw-/-小鼠中的锥体存活率和治疗窗。
通过针对锥体 arrestin 和花生凝集素（PNA）染色的抗体评估锥体存活率。通过 PNA 染色量化 Opn1mw-/-小鼠中锥体变性的速率。在 1、7 和 15 月龄时，将表达人 L-视蛋白的 AAV5 载体通过视网膜下注射到 Opn1mw-/-小鼠的一只眼，而对侧眼作为对照。注射后 2 和 13 个月，通过全视野 ERG 分析 M-锥体介导的视网膜功能。通过免疫组织化学检查 L-视蛋白转基因表达和锥体外节结构。
我们表明，Opn1mw-/- 小鼠中背侧 M-视蛋白优势锥体在早期就表现出外节变性，而腹侧 S-视蛋白优势锥体正常。至少 15 个月，剩余的 M-视蛋白优势锥体仍然存活，尽管外节缩短或不存在。我们还表明，当在 15 月龄时开始治疗时，AAV5 介导的人 L-视蛋白的表达仍然能够挽救剩余的 M-视蛋白优势锥体的功能和外节结构。
我们的研究结果表明，在老年 Opn1mw-/- 小鼠中，剩余的 M-视蛋白优势锥体仍可通过基因治疗挽救，这有助于确定未来蓝锥单色临床研究中的治疗窗。
