CLARIN 1 RETINAL FUNCTION AND THERAPEUTIC IMPLICATIONS FOR USH3

摘要 USH综合征(USH)是一种遗传性疾病，导致一半以上的混合性耳聋和 人类的失明。它同时影响光感受器和耳蜗毛细胞，即 分别是视网膜和内耳。Usher综合征III型(USH3)由Clarin-1基因突变引起 (CLRN1)基因，导致视力和正常听力的逐渐和永久性丧失。耳蜗骨 植入在一定程度上缓解了听力障碍，但没有治疗方法可以防止 导致失明的进行性感光细胞死亡。我们的目标是加深对 CLRN1在视网膜中的功能，从而为USH3患者开发一种合理的基因治疗方法。 缺乏USH3动物阻碍了寻找有效的视力丧失治疗策略的努力 模仿人类视网膜疾病的模型，以及关于CLRN1‘S内源性细胞的相互矛盾的报告 定位，只有一项研究表明它存在于光感受器中。所有当前的USH3鼠标型号 表现为听力快速丧失，但视网膜功能和形态正常。作为合作努力的一部分，我们 研究表明，使用腺相关病毒载体(AAVs)的CLRN1基因替代疗法可以预防 USH3小鼠模型人工耳蜗植入后听力损失至少1年，首次成功 这种障碍的概念验证实验。我们还表明，视网膜功能在手术后得以保留 在一种新的小鼠模型USH3中，采用类似的基因治疗方法治疗视网膜，这种模型表现出缓慢的， 随着年龄的增长，视网膜功能逐渐丧失。这些结果表明，出生后CLRN1的分娩可以 防止感觉细胞功能的进一步丧失，对于更晚期的患者来说是一个特别相关的问题 疾病的各个阶段。我们的中心假设是CLRN1在光感受器细胞中扮演着重要的角色， 通过与其他USH蛋白相互作用在结构上稳定脆弱的纤毛区 细胞骨架元素。我们对这一假说有三个具体的目的：1)识别CLRN1的S 小鼠视网膜的内源性视网膜定位和与CLRN1相互作用的蛋白质；2)确定 突变型CLRN1蛋白在体内的视网膜表达模式及致病作用；3)检测多个基因 在USH3小鼠模型中，针对整个视网膜或仅针对光感受器的治疗选择。 这些目标的完成将坚定地确定CLRN1的S的一般职能，并至关重要的是，有助于准确地 需要哪些细胞作为USH3基因替代治疗的靶点以防止失明 病人。

ABSTRACT Usher syndrome (USH) is an inherited disorder responsible for more than half of combined deafness and blindness in humans. It affects both photoreceptors and cochlear hair cells, the sensory neurons of the retina and inner ear, respectively. Usher syndrome type III (USH3) is caused by mutations in clarin-1 (CLRN1) gene, leading to the gradual and permanent loss of both vision and normal hearing. Cochlear implantation alleviates to some degree the hearing impairment, but there is no treatment that prevents the progressive photoreceptor cell death that leads to blindness. Our goal is to develop an understanding of CLRN1 function in the retina so that a rational gene therapy approach for USH3 patients can be developed. Efforts to identify efficient therapeutic strategies for vision loss are hindered by the lack of USH3 animal models that mimic the human retinal disorder, and by conflicting reports on CLRN1’s endogenous cellular localization, with only one study showing its presence in photoreceptors. All current USH3 mouse models display a rapid hearing loss but normal retinal function and morphology. As part of a collaborative effort, we showed that CLRN1 gene replacement therapy using adeno-associated viral vectors (AAVs) can prevent hearing loss for at least 1 year following cochlear delivery in a mouse model of USH3, the first successful proof of concept experiment for this disorder. We also showed that retinal function is preserved following a similar gene therapy approach to treat the retina in a new mouse model of USH3, which displays a slow, progressive loss of retinal function with aging. These results suggest that postnatal CLRN1 delivery can prevent further loss of function in sensory cells, a particularly relevant issue for patients with more advanced stages of disease. Our central hypothesis is that CLRN1 plays an essential role in photoreceptor cells, structurally stabilizing the fragile connecting cilium region by interacting with other USH proteins and cytoskeletal elements. We address this hypothesis with three specific aims: 1) identify CLRN1’s endogenous retinal localization and the proteins that interact with CLRN1 in the mouse retina; 2) determine the retinal expression pattern and pathogenic effects of mutant CLRN1 proteins in-vivo; 3) test several gene therapy options in USH3 mouse models, targeting either the entire retina, or the photoreceptors alone. Completion of these goals would firmly identify CLRN1’s general function and, critically, serve to pinpoint which cells would need to be targeted for USH3 gene replacement therapy to prevent the loss of vision in patients.