基因替代疗法是治疗Leber先天性黑矇2型（LCA2）的唯一有效手段。但目前应用的低穿透性腺相关病毒AAV2-hRPE65载体依赖于视网膜下腔注射，可造成视细胞损伤且治疗范围局限；也不能阻断视细胞的凋亡进程。针对这两个问题，课题组前期筛选了新型Y-F点突变高穿透性AAV8(Y447F+Y733F)载体；同时证实组蛋白去乙酰酶抑制剂（HDACi）能延缓视细胞凋亡。由此我们得出新的LCA2基因治疗新策略：基于多点突变AAV8载体的基因替代疗法联合抗凋亡基因疗法是中晚期LCA2的优化治疗策略。我们将首先设计和构建新型AAV8载体；特定HDACi治疗后分选视网膜色素上皮细胞和视细胞进行RNA-seq，筛选抗凋亡基因并探索相关机制；评估分别和同时搭载hRPE65基因及抗凋亡基因如Bcl-2-L10的高效载体经玻璃体腔治疗LCA2小鼠的整体效果。课题研究成果将为中晚期视网膜遗传病基因治疗开启新途径。

Leber congenital amaurosis (LCA) is an early onset retinal dystrophy that causes severe visual impairment. It has been reported that gene replacement therapy using AAV vector containing hRPE65 gene following subretinal injection, which usually covers 20-45% retina, can restore the absent RPE65 and visual function in human eyes with LCA2. However, the recent long-term clinical trail data related with middle to late stages of LCA2 patients showed that the retinal detachment caused by subretinal injection can damage cone cells in central fovea of macular, and the apoptotic processes of remaining photoreceptor cells did not completely stop in treated areas; the slow dying of the treated visual cells in middle and late stage LCA2 patients could be due to the gradual invasion of the toxic materials of the dying visual cells from the surrounding untreated retina. Our studies found that histone deacetylase inhibitor (HDACi) can inhibit or delay the death of photoreceptor cells in retinal degeneration mouse models although it could not restore the lost function in these models. We restored visual function by subretinal gene replacement therapy in different ages of rd12 mouse, a naturally occurring animal model of LCA2 with RPE65 mutation. In order to reduce or delay the apoptosis in photoreceptor cells and maintain the long-term visual function restored by gene replacement therapy in older age of rd12 mouse, we explore a new way of LCA2 treatment by using one new efficient AAV8 vector containing hRPE65 gene in combination with the anti-apoptotic gene in the same AAV8 vector, or with another similar AAV8 vector containing the same anti-apoptotic gene such as Bcl-2-L10 through intravitreal injection, which will help us to understand more about the mechanism of apoptosis in inherited retinal diseases. This will not only provide a way to help to fix the problem in the ongoing LCA2 gene replacement therapy, but also open a new strategy to treat other advanced retinal genetic disorders in the future.