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

3 型亚瑟综合症新型大型动物模型的生成和表征

• 批准号: 10372342
• 负责人: ASTRA DINCULESCU
• 金额: $ 24.37万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372342
• 关键词: Accounting; Acoustic Nerve; Actins; Adult; Age; Age-Months; Animal Model; Auditory; Biological Assay; Blindness; Bypass; CRISPR/Cas technology; Cause of Death; Cell Adhesion; Cell membrane; Cessation of life; Clarin-1; Clinic; Cochlea; Cochlear implant procedure; Cone; Cytoskeleton; Data; Defect; Development; Diagnosis; Disease; Disease Progression; Effectiveness; Electroretinography; Epitopes; Exons; Eye; Family suidae; Focal Adhesions; Functional disorder; Future; Generations; Genes; Genetically Engineered Mouse; Goals; Guide RNA; Hair Cells; Histologic; Human; In Situ Hybridization; Introns; Knock-in Mouse; Knock-out; Labyrinth; Life; Light; Longevity; Masks; Mediating; Molecular; Morphology; Muller' s cell; Mus; Mutation; Natural History; Night Blindness; Nonsense Codon; Onset of illness; Optical Coherence Tomography; Pathogenicity; Pathologic; Patients; Peripheral; Phenotype; Photoreceptors; Physiologic Intraocular Pressure; Play; Population; Protein Isoforms; Proteins; Publishing; RNA Splicing; Rare Diseases; Reagent; Resources; Retina; Retinal Degeneration; Retinal Photoreceptors; Retinitis Pigmentosa; Rod; Role; Structure; Technology; Testing; Therapeutic; Therapeutic Studies; Tight Junctions; Time; Transcript; Treatment Efficacy; Usher Syndrome Type 3; Variant; Vision; Visual; Visual Fields; Visual impairment; cell type; comparative; deaf; deafness; disease mechanisms study; disease natural history; early onset; effective therapy; efficacy evaluation; experimental study; follower of religion Jewish; fundus imaging; genome editing; hearing impairment; mouse model; mutant; nonhuman primate; novel; offspring; porcine model; prevent; progressive hearing loss; protein expression; recruit; response; single-cell RNA sequencing; tomography; transcriptomics; visual dysfunction

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.

项目总结/摘要 Usher综合征3型（USH 3）是由Clarin-1（CLRN 1）突变引起的常染色体隐性遗传病。 基因这是一种毁灭性的疾病，导致视网膜变性和进行性听力损失， 前庭功能障碍。USH 3被认为是一种孤儿病，约占所有USH的2% 例目前，还没有治疗方法来防止由光感死亡引起的视力丧失。 视网膜光感受器耳蜗植入术通过刺激听神经恢复听力 直接，绕过内耳受损的毛细胞。然而，长期的有效性 方法目前未知。因此，有一个关键的未满足的需要，以制定治疗策略，为这两个 视网膜和耳蜗USH 3表型。在我们最近发表的研究中， 测序方法（scRNAseq），结合原位杂交测定，发现CLRN 1转录物 局限于内层视网膜，并在三个不同的物种，人类，小鼠和非- 人类灵长类动物这种新的观点强调了这种重要的细胞类型在Müller胶质细胞中心疾病中的作用。 未来的治疗研究，以防止USH 3视力丧失。然而，目前可用的基因工程 USH 3（缺乏CLRN 1）小鼠模型在其整个寿命期间保持正常视力（无退化 尽管在1个月大时经历了快速听力损失和深度耳聋。缺乏动物模型 模拟人USH 3眼病的细菌是该领域的主要障碍。它阻止我们理解 疾病机制和评估治疗方法的有效性，以防止USH 3失明。 这项R21提案旨在通过创建USH 3的大型动物模型来满足这一关键需求， 人类表型。为了实现这一目标，我们使用了CRISPR/Cas9基因组编辑技术， 产生缺乏主要CLRN 1同种型的建立者USH 3猪。在具体目标1中，我们将继续描述 在双等位基因建立者中疾病发作和进展的自然史，并创建纯合子单等位基因 USH 3猪我们将采用类似的非侵入性方法描述所有USH 3猪的疾病进展 用于临床诊断和跟踪患者的方法，包括眼底成像、全视野视网膜电图 (ERG)和光谱域光学相干断层扫描（SD-OCT）用于视网膜病变的纵向评估 功能和结构。在具体目标2中，我们将确定CLRN 1的结构和分子影响 通过使用比较组织学和转录组学分析，在USH 3猪的视网膜上缺失。USH3 迫切需要大型动物模型来克服该领域的主要障碍。它具有巨大的潜力， 对预防USH 3患者失明有直接的显着影响，也应该是一种资源， 学习/治疗听力损失。