Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes

Abstract

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.

Expression and subcellular localization of USH1C/harmonin in the human retina provide insights into pathomechanisms and therapy

“…Though harmonin is detected in the ribbon synapses of both PRC types, our immunoelectron microscopy data show that harmonin is more abundant in the synaptic pedicles of cones than in rod presynaptic terminals. This is consistent with the previously reported role of harmonin in synaptic maturation in cone pedicles of zebrafish (Phillips et al, 2011) and with recent findings in our USH1C knock-in pig model indicating that the harmonin deficiency alters the width of cone synaptic pedicles (Grotz et al, 2022). Notably, earlier studies have suggested selective modulation of pre-synaptic L-type calcium currents in cones to broaden the dynamic range of synaptic transfer by controlling the amount of transmitter release from cones (Hosoi et al, 2005).…”

“…Interestingly, harmonin fulfills the criteria of such spacers: as a scaffold protein, harmonin facilitates protein networks, forms homomers, binds to membranes, directly to phospholipids (Bahloul et al, 2010) or by interacting with transmembrane proteins such as the large disc membrane protein rhodopsin, shown herein (Figure 6B,C; Supplemental Figure S1B). Such roles of harmonin in disc morphogenesis and stacking are in line with our recent findings on the altered disc architecture of rod outer segments in the USH1C pig model, revealing vertically oriented membrane discs, disk stacks interrupted by interstitial gaps and vesicle-like structures present at the outer segment base of rods in the absence of harmonin (Grotz et al, 2022).…”

“…The latter either represent group c splice variants of harmonin (Reiners et al, 2003; Scanlan et al, 1999) or protein degradation products of higher molecular mass species. The absence of any harmonin band in the immunoblots of the harmonin deficient tissue of a USH1C pig model (Grotz et al, 2022) or of siRNA-mediated knockdowns of USH1C /harmonin in HEK293T cells further validated the specificity of our H3 antibody (Figure 2D). Immunoblot analysis of fractions enriched for different cell types of the human retina revealed harmonin protein variants in fractions of both MGCs and RNs (Figure 2E).…”

“…Based on the depicted subcellular localization of harmonin isoforms in murine photoreceptor cells (PRCs), others and we have suggested differential scaffold functions of harmonin especially in the outer segment and at the synapses (8, 15, 21, 24, 25). A putative synaptic function has also been indicated in the murine retina (22), confirmed by zebrafish morpholino knock-downs (26) and recently by the USH1C pig model (27).…”

“…Outer retinal atrophy progressed to constriction of the visual field at around 5 degrees and a hyperfluorescent perifoveal ring on the fundus autofluorescence imaging (Figure 7B). Additionally, epiretinal gliosis observed in OCT is a common complication of photoreceptor degeneration and appears to be a hallmark in the retina of the USH1C pig model generated by introducing the human exon 2 bearing the c.91C>T;p.(R31*) nonsense mutation into the porcine USH1C gene (Grotz et al, 2022). The posterior eye pole showed typical bone spicules in the mid periphery and periphery (Fig.…”