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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目概要/摘要 3 型 Usher 综合征 (USH3) 是一种由 Clarin-1 (CLRN1) 突变引起的常染色体隐性遗传疾病这是一种毁灭性的疾病，会导致视网膜变性和渐进性听力损失，其程度各不相同。 USH3 被认为是一种孤儿疾病，约占所有 USH 的 2%。目前，尚无治疗方法可以预防因光感应死亡而导致的视力丧失。视网膜感光器植入通过刺激听觉神经来减轻听力损失。直接，并绕过内耳受损的毛细胞，然而，这种方法的长期有效性。因此，目前迫切需要开发针对这两种疾病的治疗策略。在我们最近发表的研究中，我们采用了单细胞 RNA 来研究视网膜和耳蜗 USH3 表型。测序方法（scRNAseq），结合原位杂交检测，发现CLRN1转录本仅限于视网膜内层，并且在人类、小鼠和非人类这三个不同物种的米勒神经胶质细胞中富集。这种以穆勒胶质细胞为中心的疾病的新观点强调了这种重要细胞类型在人类灵长类动物中的作用。未来预防 USH3 视力丧失的治疗研究然而，目前可用的基因工程。 USH3（缺乏 CLRN1）的小鼠模型在其整个生命周期中保持正常视力（无退化）尽管在 1 个月大时就出现严重耳聋，但听力却迅速丧失缺乏动物模型。模仿人类 USH3 眼部疾病是该领域的一个主要障碍，它阻碍了我们了解这一领域。疾病机制以及评估 USH3 预防失明治疗方法的功效。该 R21 提案旨在通过创建 USH3 的大型动物模型来满足这一迫切需求，该模型概括了为了实现这一目标，我们利用CRISPR/Cas9基因组编辑技术并成功地实现了这一目标。创始人生成的 USH3 猪缺乏主要的 CLRN1 同种型，在具体目标 1 中，我们将继续表征。双等位基因创始人疾病发生和进展的自然史，并创建纯合单等位基因我们将采用类似的非侵入性方法来描述所有 USH3 猪的疾病进展。用于临床诊断和跟踪患者的设备，包括眼底成像、全视野视网膜电图检查 (ERG) 和谱域光学相干断层扫描 (SD-OCT) 用于视网膜纵向评估在具体目标 2 中，我们将确定 CLRN1 的结构和分子影响。通过使用比较组织学和转录组学分析，发现 USH3 猪视网膜上的缺失。迫切需要大型动物模型来克服该领域的一个主要障碍，它具有巨大的潜力。对防止 USH3 患者失明产生直接重大影响，它也应该成为以下资源：研究/治疗听力损失。