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

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

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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.

项目摘要/摘要 USHER综合征3（USH3）是由Clarin-1突变引起的常染色体隐性疾病（CLRN1）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，有变化前庭功能障碍。 USH3被认为是孤儿病，约占所有USH的2％案例。目前，没有治疗方法可以防止因光感应死亡而导致视力丧失视网膜感受器。人工耳蜗植入通过刺激听觉神经来减轻听力损失直接绕过内耳内部受损的毛细胞。但是，这是长期有效性方法目前未知。那是为了制定理论策略的重要需求视网膜和人工耳蜗USH3表型。在最近发表的研究中，我们采用了单细胞RNA 测序方法（SCRNASEQ）与原位杂交分析结合在一起，发现CLRN1转录本局限于内部视网膜，并在三种不同的物种中富含穆勒胶质人类隐私。这种以穆勒胶质性疾病为中心的新型观点突出了这种重要细胞类型在未来的治疗研究，以防止USH3的视力丧失。但是，目前可用的一般设计 USH3的鼠标模型（缺乏CLRN1）在整个生命周期中保持正常视力（无变性）尽管经历了迅速的听力损失和1个月大的死亡。缺乏动物模型模仿人类USH3眼病是该领域的主要障碍。它使我们无法理解疾病机制以及评估治疗方法的有效性，以防止USH3失明。该R21提案旨在通过创建一个大型的USH3动物模型来满足这种关键需求人类表型。为了实现这一目标，我们使用了CRISPR/CAS9基因组编辑技术并成功产生的创始人USH3猪缺乏主要的CLRN1同工型。在特定目标1中，我们将继续描述疾病发作和疾病的自然历史，并创建纯合单相关的疾病。 USH3猪。我们将以非侵入性方法相似的所有USH3猪的疾病进展表征用于诊断和跟踪诊所患者的人，包括眼底成像，全场电视图（ERG）和光谱域光学相干断层扫描（SD-OCT）用于纵向评估功能和结构。在特定的目标2中，我们将确定CLRN1的结构和分子影响使用比较的组织学和转录组分析，在USH3猪中的视网膜缺失。一个USH3 迫切需要大型动物模型克服该领域的主要障碍。它具有巨大的潜力立即对防止USH3患者失明产生重大影响，这也应该是研究/治疗听力损失。