Clinical and preclinical therapeutic outcome metrics for USH2A-related disease

Abstract

USH2A variants are the most common cause of Usher syndrome type 2, characterized by congenital sensorineural hearing loss and retinitis pigmentosa (RP), and also contribute to autosomal recessive non-syndromic RP. Several treatment strategies are under development; however, sensitive clinical trial endpoint metrics to determine therapeutic efficacy have not been identified. In the present study, we have performed longitudinal retrospective examination of the retinal and auditory symptoms in (i) 56 biallelic molecularly confirmed USH2A patients and (ii) ush2a mutant zebrafish to identify metrics for the evaluation of future clinical trials and rapid preclinical screening studies. The patient cohort showed a statistically significant correlation between age and both rate of constriction for the ellipsoid zone length and hyperautofluorescent outer retinal ring area. Visual acuity and pure tone audiograms are not suitable outcome measures. Retinal examination of the novel ush2au507 zebrafish mutant revealed a slowly progressive degeneration of predominantly rods, accompanied by rhodopsin and blue cone opsin mislocalization from 6 to 12 months of age with lysosome-like structures observed in the photoreceptors. This was further evaluated in the ush2armc zebrafish model, which revealed similar changes in photopigment mislocalization with elevated autophagy levels at 6 days post fertilization, indicating a more severe genotype-phenotype correlation and providing evidence of new insights into the pathophysiology underlying USH2A-retinal disease.

Involvement of Oxidative and Endoplasmic Reticulum Stress in RDH12-Related Retinopathies

“…These mice also had a higher level of 11-cis, all-trans, and 13-cis retinal [32]. In a zebrafish model of USH2A-related RP, the ush2a rmc1 mutant, disruption in rhodopsin localisation and autophagy was also noted, with increased autophagosomes and increased gene expression of atg5 and atg12 and LC3 protein expression [33]. Similar to RDH12-related RP, the retinal degeneration in USH2A patients is later onset, developing from adolescence, compared to those with LCA.…”

“…CRISPR guides were designed using Benchlings CRISPR Guide Design Software, targeting exon 1 of rdh12, and the following guide was used: 5 -TGGCGTTCGCGGCGGGTTTAGGG. Mutagenesis was carried out as previously described [33]. Mutations were analysed by Sanger sequencing using the forward primer 5 -GGAGGCTGCTGAACACATTC and reverse primer 5 -CGATTTCTGGAGCAGATCATGTC.…”

“…Enucleated eyes from 12 mpf zebrafish were fixed and embedded as previously described [33]. Blocks were sectioned using a UC7 ultramicrotome (Leica Microsystems) and 70 nm sections were picked up on Formvar-coated 2 mm slot copper grids (Gilder Grids Ltd., Grantham, UK) and post-stained with Reynolds lead citrate for 5 min.…”

Usher syndrome: clinical features, molecular genetics and advancing therapeutics

“…A recent investigation into an Usher 2 patient population carrying a range of USH2A variants used retrospective longitudinal data to identify suitable clinical outcome metrics. 151 Both EZ line and hyperautofluorescent outer retinal ring area showed significant reductions within the follow-up period (2–5 years); however, there was considerable variability in the population. Visual acuity was not found to be a suitable measurement due to its slow decline, consistent with previous work.…”

“…This is especially important when the treatments are administered systemically, such as orally, and the untreated eye cannot be used as a control. As the hearing loss is congenital and relatively stable throughout the lifetime of Usher patients (aside from Usher 3), a number of natural history studies have focussed on the progress of the retinal disease in Usher patients; these have included longitudinal assessment of patients with MYO7A 9 , 10 , 84 , 149 and USH2A 9 , 98 , 150 – 152 mutations using various clinical functional and structural measures, including visual acuity, perimetry, ERG, fundus autofluorescence and OCT-derived measurements.…”