Gene Therapy of Myosin VIIa Null Mice

描述(申请人提供)：在亚瑟综合征中，失聪患者也会发展为视网膜色素变性。Usher综合征1B型与肌球蛋白Vlla基因(MYO7A)突变有关。在哺乳动物眼睛中，肌球蛋白Vila蛋白定位于光感受器细胞的纤毛和视网膜色素上皮(RPE)细胞的顶端。Shaker1小鼠也携带肌球蛋白Vlla基因突变，现在已经在这些小鼠的视网膜中发现了几种突变表型。这项拟议的研究旨在确定基因治疗Shaker1小鼠肌球蛋白Vlla缺乏症的疗效。将产生共表达人肌球蛋白V11a蛋白和绿色荧光蛋白(GFP)的高滴度慢病毒，并将其注射到新生小鼠的视网膜下空间。病毒感染和转基因在光感受器和RPE细胞中的效率将通过监测肌球蛋白V11a和GFP的表达模式来确定。病毒介导的肌球蛋白V11a表达的救援效果将通过分析先前在Shaker1小鼠视网膜中发现的突变表型来确定：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视紫红质的分布，以及RPE细胞对外节圆盘的吞噬作用。这项拟议的研究将提供与人类Usher 1B综合征基因治疗的可行性直接相关的数据，以及将慢病毒作为基因治疗载体应用于其他遗传性视网膜疾病的数据。

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.