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VIIa基因（MYO7A）突变相关。在哺乳动物眼中，肌球蛋白VIla蛋白位于感光细胞的纤毛中和视网膜色素上皮（RPE）细胞的顶端区域中。Shaker1小鼠也携带肌球蛋白VIIa基因突变，现在已经在这些小鼠的视网膜中鉴定出几种突变表型。 这项研究旨在确定基因治疗对shaker1小鼠肌球蛋白VIIa缺乏症的疗效。将产生共表达人肌球蛋白VIIa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中。通过监测肌球蛋白VIIa和GFP的表达模式来确定光感受器和RPE细胞中病毒感染和转基因转导的效率。病毒介导的肌球蛋白VIIa表达的拯救作用将通过测定先前鉴定的shaker 1小鼠视网膜中的突变体表型来确定：例如，色素颗粒在RPE细胞中的亚细胞分布、视紫红质在感光细胞连接纤毛中的分布以及RPE细胞对外节盘的吞噬作用。拟议的研究将提供数据直接相关的基因治疗的可行性Usher 1 B综合征在人类和应用慢病毒作为基因治疗车辆的其他遗传性视网膜疾病。

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.