Usher Syndrome Research Articles

A Novel Biallelic Variant in CDH23 Gene in a Family with Atypical USH1D Manifestation: A Literature Review and Investigation of Genotype-Phenotype Correlation

Introduction: Usher syndrome (USH) is an autosomal recessive disorder that predominantly affects hearing, vision, and, in some cases, vestibular function. USH, according to the onset age, severity, and progression of symptoms, is categorized into four main types. In addition, there are a significant number of reports that patients’ manifestations deviate from canonical phenotypic criteria of main types of USH, which are named atypical USH. CDH23 is the second most common USH gene in which its defects result in USH1D, non-syndromic autosomal recessive deafness-12 (DFNB12), and in a few cases, atypical USH1D. While some studies have suggested that missense and truncating damaging variants in the CDH23 gene cause DFNB12 and USH1D, respectively, no genotype-phenotype correlation for atypical USH1D has been established. Methods: Using whole-exome sequencing, we studied an Iranian family with two affected siblings who manifested congenital bilateral hearing loss, late-onset nyctalopia, retinitis pigmentosa, and normal vestibular function, indicating that their clinical symptoms are consistent with USH2. Results: Whole-exome data analysis revealed a novel bi-allelic nonsense variant (c.6562G>T; p.Glu2188Ter) in the CDH23 gene, which was confirmed by Sanger sequencing. Surprisingly, CDH23 is a member of the USH1 genes; therefore, our patients suffered from atypical USH1D. Also, by conducting a literature review, we provided a clinical and mutational profile of all reported patients with atypical manifestations or those who refuted the claimed genotype-phenotype correlation. Conclusion: By reporting a novel damaging variant, we expand the mutational spectrum of the CDH23 gene that leads to atypical USH1D. Also, reviewing the literature shows that, contrary to previous claims, different genotypes occur in the CDH23 gene allelic disorders, and there is no clear-cut genotype-phenotype correlation.

Generation of the induced pluripotent stem cell line SFMUi001-A from a patient with usher syndrome type 2 caused by biallelic variants in the USH2A gene

Biallelic variants in the USH2A gene cause Usher syndrome type 2 (USH2), in which patients’ symptoms are progressive night blindness, reduced visual field, decreased central vision and sensorineural hearing impairment. There is currently no effective drug for USH2. In this study, we isolated peripheral blood mononuclear cells from a patient with USH2. The pluripotency of induced cells was verified by the presence of cell surface markers, the expression of pluripotent genes, and the formation of teratomas. The generation of this induced pluripotent stem cell line provides an effective way to study USH2, such as disease modeling and drug screening.Usher syndrome type 2 (USH2) is a genetic disease mainly caused by biallelic variants in the USH2A gene. Patients usually present with progressive night blindness, reduced visual field, and then reduced central vision. Patients with USH2 also have sensorineural hearing impairment. There is currently no effective treatment for USH2, and the pathogenesis is still unclear. Therefore, it is of great significance to study the pathogenic mechanism of USH2A gene variants for the study of therapeutic targets. In this study, we obtained induced pluripotent stem cell (iPSC) line containing USH2A gene variants. We isolated mononuclear cells from the peripheral blood of patient and established iPSCs by reprogramming with nonintegrating vectors. We then confirmed the pluripotency of our generated iPSCs through the detection of multiple cell surface markers, the expression of pluripotency-related genes, and the ability to form teratomas with three germ layer structures in vivo. The generation of this cell line will facilitate research on USH2 disease and will play a role that cannot be underestimated in future organoid generation, drug screening, and research on drug targets as well as mechanisms.

A Review of CRISPR Tools for Treating Usher Syndrome: Applicability, Safety, Efficiency, and In Vivo Delivery.

This review considers research into the treatment of Usher syndrome, a deaf-blindness syndrome inherited in an autosomal recessive manner. Usher syndrome mutations are markedly heterogeneous, involving many different genes, and research grants are limited due to minimal patient populations. Furthermore, gene augmentation therapies are impossible in all but three Usher syndromes as the cDNA sequence exceeds the 4.7 kb AAV packaging limit. It is, therefore, vital to focus research efforts on alternative tools with the broadest applicability. The CRISPR field took off in recent years following the discovery of the DNA editing activity of Cas9 in 2012. New generations of CRISPR tools have succeeded the original CRISPR/Cas9 model to enable more sophisticated genomic amendments such as epigenetic modification and precise sequence alterations. This review will evaluate the most popular CRISPR tools to date: CRISPR/Cas9, base editing, and prime editing. It will consider these tools in terms of applicability (in relation to the ten most prevalent USH2A mutations), safety, efficiency, and in vivo delivery potential with the intention of guiding future research investment.

Generation and characterization of a human iPSC line (JUFMDOi007-A) from a patient with Usher syndrome due to mutation in USH2A

Usher syndrome type 2A (USH2A) gene mutations have been identified as the most frequent genetic causes of hereditary deafness in Usher syndrome, and an effective treatment has yet to be established. The encoded protein, Usherin, is essential for the ankle link associated with extracellular connections between the stereocilia of inner ear hair cells. We report the generation of a patient-derived USH2A iPSC line with compound mutations c.1907_1912ATGTTT > TCACAG (p.D636V + V637T + C638G) and c.8328_8329delAA (p.L2276fs*12). The iPSC showed the expression of pluripotency markers, the ability to differentiate into three germ layers in vitro, and USH2A mutations with normal karyotype.

Retinitis Pigmentosa Sine Pigmento in a Carrier of Usher Syndrome

We report a carrier of Usher syndrome type I with retinitis pigmentosa sine pigmento. A 71-year-old male was referred for further evaluation of severe, progressive, painless vision loss in both eyes over the course of four years. He had bilateral sensorineural hearing loss. Upon a comprehensive examination, his best-corrected visual acuity was 20/100 in the right eye and 20/40 in the left eye. He had an unremarkable anterior segment examination and normal intraocular pressures in both eyes. Upon fundus examination, the patient had pale discs, optic disc cupping, and multiple scattered drusen in the macula and at the midperiphery of both eyes. Optical coherence tomography showed retinal nerve fiber layer thinning in all quadrants. The visual field was severely constricted in both eyes. A comprehensive workup for infectious and inflammatory causes, as well as a brain MRI, was unremarkable. Sequencing analysis showed that he carried a heterozygous pathogenic mutation, USH1C c.672C>A (p.Cys224*) variant. Usher syndrome is a rare genetic disease characterized by hearing loss and retinitis pigmentosa. Our case suggests that patients and carriers of Usher syndrome may have a phenotype compatible with retinitis pigmentosa sine pigmento.

The adhesion G protein-coupled receptor VLGR1/ADGRV1 controls autophagy.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest known adhesion G protein-coupled receptor. Mutations in VLGR1/ADGRV1 cause Usher syndrome (USH), the most common form of hereditary deaf-blindness, and have been additionally linked to epilepsy. Although VLGR1/ADGRV1 is almost ubiquitously expressed, little is known about the subcellular function and signalling of the VLGR1 protein and thus about mechanisms underlying the development of diseases. Using affinity proteomics, we identified key components of autophagosomes as putative interacting proteins of VLGR1. In addition, whole transcriptome sequencing of the retinae of the Vlgr1/del7TM mouse model revealed altered expression profiles of gene-related autophagy. Monitoring autophagy by immunoblotting and immunocytochemistry of the LC3 and p62 as autophagy marker proteins revealed evoked autophagy in VLGR1-deficient hTERT-RPE1 cells and USH2C patient-derived fibroblasts. Our data demonstrate the molecular and functional interaction of VLGR1 with key components of the autophagy process and point to an essential role of VLGR1 in the regulation of autophagy at internal membranes. The close association of VLGR1 with autophagy helps to explain the pathomechanisms underlying human USH and epilepsy related to VLGR1 defects.

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and invitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Three-Dimensional Structure of Inner Ear Hair Cell Ribbon Synapses in a Zebrafish Model of Usher Syndrome Type 1B.

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa-/- null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa-/- zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa-/- mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa-/- mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa-/- mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.

Monitoring paxillin in astrocytes reveals the significance of the adhesion G protein coupled receptor VLGR1/ADGRV1 for focal adhesion assembly.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest adhesion G protein-coupled receptor (aGPCR). Mutations in VLGR1/ADGRV1 are associated with human Usher syndrome, the most common form of deaf-blindness, and also with epilepsy in humans and mice. VLGR1 is expressed almost ubiquitously but is mainly found in the CNS and in the sensory cells of the eye and inner ear. Little is known about the pathogenesis of the diseases related to VLGR1. We previously identified VLGR1 as a vital component of focal adhesions (FAs) serving as a metabotropic mechanoreceptor controls cell spreading and migration. FAs are highly dynamic and turnover in response to internal and external signals. Here, we aimed to elucidate how VLGR1 participates in FA turnover. Nocodazole washouts and live cell imaging of paxillin-DsRed2 consistently showed that FA disassembly was not altered, but de novo assembly of FA was significantly delayed in Vlgr1-deficient astrocytes, indicating that VLGR1 is enrolled in FA assembly. In FRAP experiments, recovery rates were significantly reduced in Vlgr1-deficient FAs, indicating reduced turnover kinetics in VLGR1-deficient FAs. We showed that VLGR1 regulates cell migration by controlling the FA turnover during their assembly and expect novel insights into pathomechanisms related to pathogenic dysfunctions of VLGR1.

Choroidal structure investigated by choroidal vascularity index in patients with inherited retinal diseases

PurposeTo evaluate the choroidal structure in patients with inherited retinal diseases (IRDs) by investigating the choroidal vascularity index (CVI).MethodsThe present study was conducted on 113 IRD patients and 113 sex- and age-matched healthy individuals. Patients’ data was extracted from the Iranian National Registry for IRDs (IRDReg®). Total choroidal area (TCA) was determined between retinal pigment epithelium and choroid-scleral junction,1500 microns on either side of the fovea. Luminal area (LA) was considered as the black area corresponding to the choroidal vascular spaces, following Niblack binarization. CVI was calculated as the ratio of the LA to the TCA. CVI and other parameters were compared among different types of IRD and the control group.ResultsThe IRD diagnosis included retinitis pigmentosa (n = 69), cone-rod dystrophy (n = 15), Usher syndrome (n = 15), Leber congenital amaurosis (n = 9), and Stargardt disease (n = 5). Sixty-one (54.0%) individuals of each of the study and control groups were male. The average CVI was 0.65 ± 0.06 in the IRD patients and 0.70 ± 0.06 in the control group (P < 0.001). Accordingly, the average of TCA and LA were 2.32 ± 0.63 and 1.52 ± 0.44 mm [1] in patients with IRDs, respectively. The measurements for the TCA and the LA were significantly lower in all subtypes of IRD (P-values < 0.05).ConclusionCVI is significantly lower in patients with IRD than in healthy age-matched individuals. Choroidal changes in IRDs may be related to the changes in the lumen of the choroidal vessels rather than the stromal changes.

Change in Cone Structure Over 24 Months in USH2A-Related Retinal Degeneration

To describe cone structure changes using adaptive optics scanning laser ophthalmoscopy (AOSLO) in the Rate of Progression of USH2A-related Retinal Degeneration (RUSH2A) study. Multicenter, longitudinal natural history study. AOSLO images were acquired at 4 centers, twice at baseline and annually for 24 months in this natural history study. For each eye, at least 10 regions of interest (ROIs) with ≥50 contiguous cones were analyzed by masked, independent graders. Cone spacing Z-scores, standard deviations from the normal mean at the measured location, were compared between graders and tests at baseline. The association of cone spacing with clinical characteristics was assessed using linear mixed effects regression models weighted by image quality score. Annual rates of change were calculated based on differences between visits. Fourteen eyes of 14 participants were imaged, with 192 ROIs selected at baseline. There was variability among graders, which was greater in images with lower image quality score (P < .001). Cone spacing was significantly correlated with eccentricity, quality score, and disease duration (P < .02). On average, the cone spacing Z-score increased 0.14 annually (about 9%, P < .001). We observed no significant differences in rate of change between disease type (Usher syndrome or retinitis pigmentosa), imaging site, or grader. Using current methods, the analysis of quantitative measures of cone structure showed some challenges, yet showed promise that AOSLO images can be used to characterize progressive change over 24 months. Additional multicenter studies using AOSLO are needed to advance cone mosaic metrics as sensitive outcome measures for clinical trials. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Natural Disease Course in Usher Syndrome Patients Harboring USH2A Variant p.Cys870* in Exon 13, Amenable to Exon Skipping Therapy.

The aim of the study was to determine the rate of retinal degeneration in patients with c.2610C>A (p.Cys870*) in USH2A exon 13, amenable to exon skipping therapy. There were nine patients from seven families, three of whom were male (two were homozygous). Seven patients had follow-up data (median of 11 years). Analysis included best corrected visual acuity (BCVA, decimal Snellen), visual field (Goldmann perimetry target II/4), fundus autofluorescence (FAF), optical coherence tomography (OCT), and microperimetry (MP). The median age at the onset of nyctalopia was 20 years (range, 8-35 years of age). At the first exam, at a median age of 42 years, the median BCVA was 0.5 (0.2-1.0), and the median visual field diameter was 23° (5°-114°). Imaging showed a hyperautofluorescent ring delineating preserved foveal photoreceptors in 78% (7/9) of patients, while 22% (2/9) had a hyperautofluorescent patch or atrophy, reflecting advanced disease. Survival analysis predicted that 50% of patients reach legal blindness based on a visual field diameter < 20° at the age of 52 (95% CI, 45-59) and legal blindness based on a BCVA ≤ 0. 1 (20/200) at the age of 55 (95% CI, 46-66). Visual field constriction occurred at the median rate of radial 1.5 deg/year, and hyperautofluorescent ring constriction occurred at the median rate of 34 μm/year. A non-null second allele was found in two patients: p.Thr4315Pro and p.Arg303His; the patient with p.Arg303His had a milder disease. The rates of progression will be useful in the design and execution of clinical trials.

Homozygous Knockout of Cep250 Leads to a Relatively Late-Onset Retinal Degeneration and Sensorineural Hearing Loss in Mice.

Usher syndrome (USH) is the most common syndromic inherited retinal disease, causing retinitis pigmentosa and sensorineural hearing loss. We reported previously that a nonsense mutation in the centrosome-associated protein CEP250 gene (encoding C-Nap1) causes atypical USH in patients of Iranian Jewish origin. To better characterize CEP250, we aimed to generate and study a knockout (KO) mouse model for Cep250. Mice heterozygous for a "knockout-first" Cep250 construct were generated and bred with Cre recombinase mice to generate the null allele and produce homozygous Cep250 KO mice. Retinal function was evaluated by full-field electroretinography (ffERG) at variable ages, and retinal structure changes were examined using histological analysis. Hearing thresholds were detected using auditory brainstem response (ABR) at the age of 20 months. The Cep250 KO mouse model was generated by activating a construct harboring a deletion of exons 6 and 7. At 6 months, the ffERG was normal, but it decreased gradually with age. For both photopic and scotopic ffERG responses, very low amplitudes were evident at 20 months. Histological analysis confirmed late-onset retinal degeneration. ABR tests illustrated that hearing threshold significantly increased at the age of 20 months. Although most USH animal models have normal retinal function and structure, the Cep250 KO mouse model shows both retinal degeneration and hearing loss with a relatively late age of onset. This model may shed more light on CEP250-associated retinal and hearing deficits and represents an efficient platform for the development of treatment modalities for USH. Our study demonstrates better understanding of Cep250-associated retinal and hearing disease in a mouse model and may help in developing more efficient gene therapy modalities.

Analysis of specific risk factors of neurodevelopmental disorder in hearing-impaired infants under ten months of age: "EnTNDre" an opening research stemming from a transdisciplinary partnership.

Approximately 20% of children born with severe to profound hearing loss (HL) have an associated disorder that poses a neurodevelopmental risk [1]. The objective of this study is to identify the criteria and profiles of deaf infants at risk of neurodevelopmental disorders (NDD) to provide early intervention. Twenty-two infants aged three to ten months with bilateral congenital deafness were included. Each child attended a consultation with a psychiatrist specializing in the development of hearing-impaired infants as part of their ENT follow-up. The quality of their early development was analyzed using the Olliac grid and well-known postural and sensorimotor criteria. The children were then classified into three groups: normal examination (Group 1), evident NDD (Group 2), and intermediate examination (Group 3). Early medical history, the etiology of deafness, cerebral imaging, and vestibular test results were collected and compared in the different groups. The average age of the children at the time of observation was seven months (3-10 months). All had sensorineural HL, with identified causes in 13 out of 22 cases: five cases of connexin 26 gene mutation, three cases of CHARGE syndrome, two CMV infections, one Usher syndrome, one GATA3 mutation, and one LHPL5 mutation. The average score on the Olliac grid was nine (0-15), and abnormal postural and sensorimotor behaviors were found in 15 cases out of 22 (68%). 27% of the children were classified in Group 1, 45% in Group 2, and 27% in Group 3. Children with non-isolated HL, abnormal brain MRI (8/22), malformations outside the auditory system (10/22), vestibular impairments (9/22), and/or CMV infections (2/22) were distributed as follows: 2/6 in Group 1, 9/10 in Group 2, and 3/6 in Group 3. 8/22 children had suffered perinatal complications (0/6 in Group 1, 6/10 in Group 2, and 2/5 in Group 3). Only one child had a first-degree relative with NDD. He belonged to Group 2. To our knowledge, this paper is the first to describe the development of infants with congenital deafness. It is based on an observation time that had been included in the procedure of multidisciplinary evaluations prior to cochlear implantation (CI), thanks to the partnership between a psychiatric center for deaf children and an ENT-pediatric implantology service. This consultation was aimed at assessing the quality of neurodevelopment and identifying NDD without a specific referral, with good acceptability for families. Using the Olliac grid and postural and sensorimotor criteria developed to be assessed in the routine care consultation, we identified evident early signs of NDD in 45% of infants. This group (Group 2) requires early, targeted, developmental support. Some children in an intermediate zone (Group 3) require further observation and support. The very early identification of NDD seems to be relevant in the care of hearing-impaired infants. The use of the Olliac grid and developmental scales seems relevant to identifying infants at risk for NDD.

Establishment of iPS cell line (KLRMMEi003-A) from a patient with Usher syndrome due to USH2A mutation

We generated an induced pluripotent stem (iPS) cell line by reprogramming peripheral blood mononuclear cells of a patient with Usher syndrome type II carrying USH2A gene mutation (c.8559-2A > G). The iPS cell line with confirmed patient-specific point mutation exhibited typical iPS cell characteristics and maintained a normal karyotype. It can be used as 2D and 3D models to investigate the underlying pathogenic mechanism and lay a solid foundation for future personalized therapy.

The Enigmatic Genetic Landscape of Hereditary Hearing Loss: A Multistep Diagnostic Strategy in the Italian Population.

Hearing loss is the most frequent sensorineural disorder, affecting approximately 1:1000 newborns. Hereditary forms (HHL) represent 50-60% of cases, highlighting the relevance of genetic testing in deaf patients. HHL is classified as non-syndromic (NSHL-70% of cases) or syndromic (SHL-30% of cases). In this study, a multistep and integrative approach aimed at identifying the molecular cause of HHL in 102 patients, whose GJB2 analysis already showed a negative result, is described. In NSHL patients, multiplex ligation probe amplification and long-range PCR analyses of the STRC gene solved 13 cases, while whole exome sequencing (WES) identified the genetic diagnosis in 26 additional ones, with a total detection rate of 47.6%. Concerning SHL, WES detected the molecular cause in 55% of cases. Peculiar findings are represented by the identification of four subjects displaying a dual molecular diagnosis and eight affected by non-syndromic mimics, five of them presenting Usher syndrome type 2. Overall, this study provides a detailed characterisation of the genetic causes of HHL in the Italian population. Furthermore, we highlighted the frequency of Usher syndrome type 2 carriers in the Italian population to pave the way for a more effective implementation of diagnostic and follow-up strategies for this disease.

The usherin mutation c.2299delG leads to its mislocalization and disrupts interactions with whirlin and VLGR1

Usher syndrome (USH) is the leading cause of combined deafness-blindness with type 2 A (USH2A) being the most common form. Knockout models of USH proteins, like the Ush2a-/- model that develops a late-onset retinal phenotype, failed to mimic the retinal phenotype observed in patients. Since patient’s mutations result in the expression of a mutant protein and to determine the mechanism of USH2A, we generated and evaluated an usherin (USH2A) knock-in mouse expressing the common human disease-mutation, c.2299delG. This mouse exhibits retinal degeneration and expresses a truncated, glycosylated protein which is mislocalized to the photoreceptor inner segment. The degeneration is associated with a decline in retinal function, structural abnormalities in connecting cilium and outer segment and mislocaliztion of the usherin interactors very long G-protein receptor 1 and whirlin. The onset of symptoms is significantly earlier compared to Ush2a-/-, proving expression of mutated protein is required to recapitulate the patients’ retinal phenotype.

USH2A Gene Mutations in Rabbits Lead to Progressive Retinal Degeneration and Hearing Loss.

Mutations in USH2A gene are responsible for the greatest proportion of the Usher Syndrome (USH) population, among which more than 30% are frameshift mutations on exon 13. A clinically relevant animal model has been absent for USH2A-related vision loss. Here we sought to establish a rabbit model carrying USH2A frameshift mutation on exon 12 (human exon 13 equivalent). CRISPR/Cas9 reagents targeting the rabbit USH2A exon 12 were delivered into rabbit embryos to produce an USH2A mutant rabbit line. The USH2A knockout animals were subjected to a series of functional and morphological analyses, including acoustic auditory brainstem responses, electroretinography, optical coherence tomography, fundus photography, fundus autofluorescence, histology, and immunohistochemistry. The USH2A mutant rabbits exhibit hyper-autofluorescent signals on fundus autofluorescence and hyper-reflective signals on optical coherence tomography images as early as 4 months of age, which indicate retinal pigment epithelium damage. Auditory brainstem response measurement in these rabbits showed moderate to severe hearing loss. Electroretinography signals of both rod and cone function were decreased in the USH2A mutant rabbits starting from 7 months of age and further decreased at 15 to 22 months of age, indicating progressive photoreceptor degeneration, which is confirmed by histopathological examination. Disruption of USH2A gene in rabbits is sufficient to induce hearing loss and progressive photoreceptor degeneration, mimicking the USH2A clinical disease. To our knowledge, this study presents the first mammalian model of USH2 showing the phenotype of retinitis pigmentosa. This study supports the use of rabbits as a clinically relevant large animal model to understand the pathogenesis and to develop novel therapeutics for Usher syndrome.

Efficacy, pharmacokinetics, and safety in the mouse and primate retina of dual AAV vectors for Usher syndrome type 1B

Gene therapy of Usher syndrome type 1B (USH1B) due to mutations in the large Myosin VIIA (MYO7A) gene is limited by the packaging capacity of adeno-associated viral (AAV) vectors. To overcome this, we have previously developed dual AAV8 vectors which encode human MYO7A (dual AAV8.MYO7A). Here we show that subretinal administration of 1.37E+9 to 1.37E+10 genome copies of a good-manufacturing-practice-like lot of dual AAV8.MYO7A improves the retinal defects of a mouse model of USH1B. The same lot was used in non-humanprimates at doses 1.6× and 4.3× the highest dose proposedfor the clinical trial which was based on mouse efficacy data. Long-lasting alterations in retinal function and morphology were observed following subretinal administration of dual AAV8.MYO7A at the high dose. These findings were modest and improved over time in the low-dose group, as also observed in other studies involving the use of AAV8 in non-human primates and humans. Biodistribution andshedding studies confirmed the presence of vector DNA mainly in the visual pathway. Accordingly, we detected human MYO7A mRNA expression predominantly in the retina. Overall, these studies pave the way for the clinical translation of subretinal administration of dual AAV vectors in USH1B subjects.

Static Perimetry in the Rate of Progression in USH2A-related Retinal Degeneration (RUSH2A) Study: Assessment Through 2 Years

To evaluate disease progression using static perimetry (SP) in patients with USH2A-related retinal degeneration, including Usher syndrome type 2 (USH2) and nonsyndromic autosomal recessive retinitis pigmentosa. Prospective, observational cohort study. A total of 102 patients with biallelic disease-causing sequence variants in USH2A with baseline best-corrected visual acuity (BCVA) letter score ≥54 were recruited from 16 clinical sites in Europe and North America. SP, BCVA, full-field stimulus thresholds, spectral domain optical coherence tomography macular scans, and fundus-guided mesopic microperimetry were performed at baseline and annually. The main outcome measures were total hill of vision (VTOT), hill of vision in the central 30° (V30), VTOT minus V30 (VPERIPH), and mean sensitivity. The average decline (95% CI) was 2.05 (1.40, 2.70) decibel-steradian (dB-sr)/y for VTOT, 0.48 (0.32, 0.65) dB-sr/y for V30, 1.53 (0.97, 2.08) dB-sr/y for VPERIPH, and 0.55 (0.40, 0.71) dB/y for mean sensitivity. Average percentage decline per year was 8.3 (5.5, 11.1) for VTOT, 5.2 (3.0, 7.4) for V30, 16.0 (9.5, 22.0) for VPERIPH, and 5.1 (3.5, 6.7) for mean sensitivity. Changes from baseline to year 2 in all SP measures were highly correlated (r's ranging from 0.52 [V30 vs VPERIPH] to 0.98 [VTOT vs VPERIPH]). Quantitative measures of SP declined significantly over 2 years in USH2A-related retinal degeneration. The annual percentage rate of change was greatest for VTOT and VPERIPH, whereas V30 and mean sensitivity changed least, reflecting earlier and more severe peripheral degeneration compared with central loss.