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.

PROJECT NARRATIVE Usher syndrome type 3 (USH3) is a devastating orphan disease that causes blindness and deafness, resulting from mutations in the Clarin-1 (CLRN1) gene. The lack of animal models that mimic the human USH3 vision loss represents a major barrier to progress in the field, preventing the testing of treatments to prevent blindness. Our proposed studies focus on the development and characterization of novel, large (pig) animal models of USH3, and will allow us to better understand the molecular basis of disease and to develop effective therapies.