Generation and Characterization of Novel Large Animal Models of Usher Syndrome Type 3

项目摘要/摘要 Usher综合征3型(USH3)是一种由Clarin-1(CLRN1)基因突变引起的常染色体隐性遗传病 吉恩。它是一种毁灭性的疾病，会导致视网膜变性和进行性听力损失，其变量 前庭功能障碍。USH3被认为是一种孤儿疾病，约占所有USH的2% 案子。目前，还没有治疗方法来防止由于光感知的死亡而导致的视力丧失。 视网膜光感受器。人工耳蜗术通过刺激听神经减轻听力损失 直接，绕过内耳受损的毛细胞。然而，这一举措的长期有效性 目前尚不清楚这一方法。因此，迫切需要为这两种疾病制定治疗策略，但尚未得到满足。 视网膜和耳蜗型USH3表型。在我们最近发表的研究中，我们用单细胞RNA 测序方法(ScRNAseq)结合原位杂交分析，发现CLRN1转录本 仅限于视网膜内部，并在三个不同的物种中富含Müler胶质细胞，人、小鼠和非 人类灵长类动物。这一关于以胶质细胞为中心的疾病的新观点强调了这种重要的细胞类型在 未来USH3预防视力丧失的治疗研究。然而，目前可用的基因工程 缺乏CLRN1的USH3小鼠模型在其整个生命周期中保持正常视力(不退化) 尽管在1个月大的时候经历了迅速的听力损失和严重的耳聋。缺乏动物模型 模拟人类USH3眼病是该领域的一大障碍。它阻碍了我们理解 来自对USH3中预防失明的治疗方法的有效性的评估。 这项R21提案旨在通过创建一个大型动物模型USH3来满足这一关键需求，该模型概括了 人类的表型。为此，我们使用了CRISPR/Cas9基因组编辑技术，并成功地 产生了缺乏主要CLRN1亚型的创始人USH3猪。在具体目标1中，我们将继续描述 双等位基因创建者疾病发生和发展的自然历史，并创造纯合单等位基因 USH3头猪。我们将用类似的非侵入性方法描述所有USH3猪的疾病进展 到临床上用于诊断和跟踪患者的设备，包括眼底成像、全视野视网膜电图仪 (ERG)和光谱域光学相干断层扫描(SD-OCT)用于视网膜纵向评估 功能和结构。在具体目标2中，我们将确定CLRN1的结构和分子影响 USH3猪视网膜缺失的比较组织学和转录学分析。USH3 迫切需要大型动物模型来克服该领域的一大障碍。它具有巨大的潜力 对USH3患者预防失明的即时显著影响，它也应该是 研究/治疗听力损失。

Project Summary/Abstract Usher syndrome type 3 (USH3) is an autosomal recessive disorder caused by mutations in the Clarin-1 (CLRN1) gene. It is a devastating disease, leading to retinal degeneration and progressive hearing loss, with variable vestibular dysfunction. USH3 is considered an orphan disease, accounting for approximately 2% of all USH cases. Currently, there are no therapeutic approaches to prevent vision loss caused by the death of light-sensing retinal photoreceptors. Cochlear implantation alleviates the hearing loss by stimulating the auditory nerve directly, and bypassing the damaged hair cells in the inner ear. However, the long-term effectiveness of this approach is currently unknown. Thus, there is a critical unmet need to develop therapeutic strategies for both the retinal and cochlear USH3 phenotypes. In our recently published study, we took a single-cell RNA sequencing approach (scRNAseq), combined with in-situ hybridization assays, and found that CLRN1 transcripts are confined to the inner retina, and are enriched in Müller glia in three distinct species, human, mouse and non- human primates. This novel view of Müller glia-centered disease highlights the role of this important cell-type in future therapeutic studies to prevent vision loss in USH3. However, currently available genetically engineered mouse models of USH3 (lacking CLRN1) maintain normal vision (no degeneration) throughout their lifespan despite undergoing rapid hearing loss and profound deafness by 1 month of age. The lack of an animal model that mimics the human USH3 ocular disease is a major barrier in the field. It prevents us from understanding the disease mechanisms and from evaluating the efficacy of therapeutic approaches to prevent blindness in USH3. This R21 proposal aims to fill this critical need by creating a large animal model of USH3 that recapitulates the human phenotype. Toward this goal, we used CRISPR/Cas9 genome editing technology and successfully generated founder USH3 pigs lacking the main CLRN1 isoform. In Specific Aim 1, we will continue to characterize the natural history of disease onset and progression in biallelic founders, and create homozygous monoallelic USH3 pigs. We will characterize the disease progression in all USH3 pigs with noninvasive approaches similar to those used to diagnose and track patients in the clinic, including fundus imaging, full-field electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT) for longitudinal assessment of retinal function and structure. In Specific Aim 2, we will determine the structural and molecular impact of CLRN1 absence on the retina in USH3 pigs by using comparative histological and transcriptomic analyses. An USH3 large animal model is urgently needed to overcome a major barrier in the field. It has a tremendous potential for immediate significant impact on preventing blindness in USH3 patients and it should also be a resource for studying/treating hearing loss.