C21ORF2相关视网膜纤毛病致病机理与基因治疗研究

Hereditary retinal degeneration causes visual impairment or vision loss for more than 2 millions of people worldwide. To date, effective treatments remain elusive. Genetic defects in cilium formation and function cause a large group of retinal degeneration diseases, so called retinal ciliopathy. Recently, two RNAi-based functional screening for cilium-associated genes have both identified an LRR-containing protein, C21ORF2 with unknown function. The subsequent genetic studies demonstrated that C21ORF2 mutations cause both syndromic and non-syndromic forms of retinal ciliopathy with a variable spectrum of skeletal involvement. The affected patients all share severe cone-rod dystrophy in early childhood. C21ORF2 protein was found to colocalize and to interact with another two ciliopathy-associated proteins NEK1 and SPATA7 at the photoreceptor transition zone (TZ). In photoreceptors, a large amount of phototransduction proteins are transported through TZ into the outer segments (OS) to maintain photoreceptor morphology and function. We hypothesize that C21ORF2 is a component of a functional module that controls protein trafficking through the TZ (the “cilium gate”); dysfunction of C21ORF2 impedes protein targeting to photoreceptor OS and causes early-onset retinal degeneration. Our specific aims of this proposal are: 1) We will identify and map C21ORF2-associated “cilium gate” module components in the photoreceptor transition zone. 2) Next, we will determine whether identified C21ORF2 mutations disrupt its interaction with other module components, ciliogenesis and intraflagellar transport in ciliated cells. 3) Then, we will generate conditional knockout mice by abrogation of C21orf2 in photoreceptors before and after their transition zone formation to determine whether it is essential for photoreceptor ciliogenesis and/or OS protein transport. 4) We will also generate C21ORF2-associated retinal ciliopathy mouse models carrying C21orf2 disease causative mutations via CRISPR/Cas9-mediated genomic editing to study the underlying pathogenesis of their photoreceptor degeneration. 3) By using an AAV gene delivery system, we will develop gene therapy to cure or delay C21ORF2-associated retinal degeneration in the disease mice model by expressing wild-type C21ORF2 in photoreceptors before initiation of retinal degeneration. This study may improve our understanding of ciliary protein trafficking and associated retinal ciliopathy, lead to potential therapy in preserving vision of the patients affected with C21ORF2-associated retinal ciliopathy.

遗传性视网膜变性在全世界导致2百多万儿童和成人视力低下或失明，由于缺乏对其致病机理的清楚认识，目前尚无有效治疗方法。初级纤毛相关基因突变是其中重要致病因素之一，引起一组称作视网膜纤毛病的严重视网膜变性疾病。近期发现，C21ORF2基因突变引起以婴幼儿期锥杆细胞变性为特点的视网膜纤毛病。其编码蛋白位于调节蛋白选择性转运的感光细胞连接纤毛，但其功能和致病机理完全不清楚。因此，我们将通过对C21ORF2和其相互作用的纤毛蛋白在感光细胞连接纤毛的精确定位和相互作用，解析相关“纤毛闸门”的筛选机制；并通过构建和分析感光细胞C21orf2条件敲除和致病突变敲入小鼠，研究其在感光细胞蛋白定向转运中的作用和相关视网膜纤毛病致病机理；最后，利用疾病小鼠模型建立AAV介导的基因治疗方法。该研究必将推动我们对感光细胞结构和功能的清楚认识；揭示视网膜纤毛病发生和发展机理；并最终为这类疾病的治疗提供重要依据和手段。

遗传性视网膜纤毛病是一组由纤毛基因突变引起的视网膜变性疾病，因致病机理不明，目前尚无有效治疗方法。本课题主要对视网膜纤毛病致病基因C21ORF2和SDCCAG8的功能、致病机理及基因治疗进行研究。.为研究C21ORF2的功能，我们构建了2个携带致病突变的质粒GFP-C21ORF2（218G>C)和GFP-C21ORF2 (103delA)，及3个C21orf2特异性siRNA，并将其分别转染IMCD3细胞。与野生型比较，C21orf2基因突变和敲低都不同程度导致培养细胞C21ORF2蛋白表达水平降低，纤毛数量和长度降低，提示C21ORF2在纤毛形成中的重要作用。此外，在我们根据项目计划构建C21orf2基因条件敲除小鼠和携带移码突变103delA的小鼠模型时，因突变基因的先天致死性，导致最终未能按原计划实施项目。携带点突变218G>C的小鼠目前9月龄，尚未发现视网膜或其他器官的形态和功能异常。.为研究SDCCAG8的功能，我们构建了2个携带致病突变的质粒GFP-SDCCAG8(E451GfsX16) 和GFP-SDCCAG8(Y236X)，并将其转染到IMCD3细胞。与野生型相比，突变基因致SDCCAG8表达蛋白截短和表达水平降低。同时，我们构建携带致病点突变708C>G的基因敲入小鼠。与野生型细胞相比，突变小鼠胚胎成纤维细胞纤毛形成的数量和长度严重降低。突变小鼠呈现早期视网膜退行性变，以感光细胞层外节段和外核层进行性缩短为特点，同时小鼠视网膜感光功能降低，外节段特异性蛋白转运严重受阻。此外，该突变小鼠模型呈现多个器官和组织的纤毛病综合征，包括多趾畸形、早发肾囊肿和纤维化、肾小管上皮细胞纤毛缺失等。因此，我们首次建立携带SDCCAG8致病突变，导致多个器官和组织表型的纤毛病综合征小鼠模型，揭示该基因在多种细胞纤毛形成和维持中的重要作用，为研究此类疾病致病机理和治疗策略提供重要的依据。