Myosin VIIa Gene Therapy

描述（由申请人提供）：在Usher综合征中，耳聋患者也会发生视网膜色素变性。Usher综合征1B型与肌球蛋白vla基因（MYO7A）突变有关。在哺乳动物眼中，肌球蛋白VIla蛋白位于感光细胞的纤毛和视网膜色素上皮（RPE）细胞的顶端区域。Shaker1小鼠也携带myosin vla基因突变，目前在这些小鼠的视网膜中已经发现了几种突变表型。本研究旨在确定基因治疗shaker1小鼠肌球蛋白vla缺乏症的疗效。制备高滴度的同时表达人肌球蛋白vla蛋白和绿色荧光蛋白（GFP）的慢病毒，并将其注射到新生小鼠的视网膜下间隙。光感受器和RPE细胞中病毒感染和转基因转导的效率将通过监测肌球蛋白vla和GFP的表达模式来确定。病毒介导的myosin vla表达的挽救作用将通过分析shaker1小鼠视网膜中先前鉴定的突变表型来确定：例如，RPE细胞中色素颗粒的亚细胞分布，连接纤毛的光感受器中的视紫红质分布以及RPE细胞对外节盘的吞噬。拟议的研究将为人类Usher 1b综合征基因治疗的可行性以及慢病毒作为其他遗传性视网膜疾病基因治疗载体的应用提供直接相关的数据。

DESCRIPTION (provided by applicant): In Usher syndrome, deaf patients also develop retinitis pigmentosa. Usher syndrome type 1B has been associated with mutations in the myosin Vlla gene (MYO7A). In the mammalian eye, myosin VIla protein is located in the cilium of photoreceptor cells and in the apical region of the retinal pigmented epithelial (RPE) cells. Shaker1 mice also carry mutations in the myosin Vlla gene, and several mutant phenotypes have now been identified in the retinas of these mice. The proposed research aims to determine the efficacy of gene therapy for myosin Vlla deficiency in the shaker1 mouse. High titer lentiviruses co-expressing the human myosin Vlla protein and the green fluorescent protein (GFP) will be produced and injected into the sub-retinal space of newborn mice. The efficiency of viral infection and transgene transduction in photoreceptors and RPE cells will be determined by monitoring expression patterns of myosin Vlla and GFP. The rescuing effects of the viral mediated myosin Vlla expression will be determined by assaying previously identified mutant phenotypes in the retinas of shaker1 mice: e.g., the subcellular distribution of the pigment granules in the RPE cells, the distribution of rhodopsin in the photoreceptor connecting cilium, and the phagocytosis of the outer segment disks by the RPE cells. The proposed research will provide data directly relevant to the feasibility of gene therapy for the Usher 1 B syndrome in humans and to the application of lentivirus as a gene therapy vehicle for other inherited retinal diseases.