Transcriptome analysis in the humanized USH1CR31*pig retina reveals new insights into the pathomechanisms underlying USH1C and novel therapeutic targets

Abstract

Purpose : Human Usher syndrome (USH) is the most common form of hereditary deaf-blindness. To date, there is no therapy for any of the three clinical USH types in the eye, mainly because the pathomechanisms underlying the disease in the retina are still elusive. To get insights into the molecular basis of the disease, we analyzed the differential gene expression in the retina of the humanized USH1C^R31* pig model, which carries a pathological nonsense mutation in the USH1C gene leading to USH1, the most severe form of USH.

Methods : We isolated mRNA from different areas of the retinas of three wild-type (WT) and USH1C^R31* pigs for bulk RNA-Seq analysis. The differentially expressed genes (DEGs) were clustered by GO-Term analysis. To confirm the differential gene expression, RT-qPCR analysis of several genes was performed. Protein expression in the retina was determined by Western blot and immunohistochemistry.

Results : Our results showed 477 DEGs between WT and USH1C^R31* pig retinae. Several DEGs are connected to the cWnt-signaling pathway, which is inter alia regulated by USH1C/harmonin. In addition to dysregulated Müller glia cell (MGC) genes, the USH1C gene is significantly upregulated in the USH1C^R31* pig retinae. Moreover, various clusters such as programmed cell death or miRNA transcription were identified. The differential expression of the genes was confirmed by RT-qPCR and quantitative protein expression analysis.

Conclusions : Our data shows, that the functional loss of USH1C/harmonin affects several processes within the retina. The dysregulation of cWnt-related genes is in line with previous findings in cells and confirms the importance of USH1C/harmonin as a regulator of the cWnt-signaling pathway in vivo. The dysregulated pathways identified are new targets for future therapeutic approaches for USH1C. They also include useful molecular biomarkers for the retinal degeneration in USH1C.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.