Multicentric Longitudinal Prospective Study in a European Cohort of MYO7A Patients: Disease Course and Implications for Gene Therapy.

Purpose: We investigated the natural history of retinal dystrophy owing to variants in the MYO7A gene. Methods: Fifty-three patients (mean age, 33.6 ± 16.7 years) with Usher syndrome owing to biallelic, mostly pathogenic, variants in MYO7A underwent baseline and two annual follow-up visits. Best-corrected visual acuity (BCVA), semiautomatic kinetic visual field, full-field electroretinogram, color fundus imaging, microperimetry, spectral-domain optical coherence tomography, and fundus autofluorescence were assessed. Results: At baseline, all patients presented with decreased BCVA (66.4 ± 17.9 Early Treatment Diabetic Retinopathy score and 59.5 ± 21.7 Early Treatment Diabetic Retinopathy score, in the better- and worse-seeing eyes, respectively), restricted semiautomatic kinetic visual field (III4e area, 3365.8 ± 4142.1°2; 4176.4 ± 4400.3°2) and decreased macular sensitivity (9.7 ± 9.9 dB; 9.0 ± 10.2 dB). Spectral-domain optical coherence tomography revealed reduced central macular thickness (259.6 ± 63.0 µm; 250.7 ± 63.3 µm) and narrowed ellipsoid zone band width (2807.5 ± 2374.6 µm; 2615.5 ± 2370.4 µm). Longitudinal analyses (50 patients) showed a significant decrease of BCVA in better-seeing eyes, whereas no changes were observed in worse-seeing eyes for any parameter. BCVA, semiautomatic kinetic visual field (III4e and V4e) and macular sensitivity were related significantly to age at baseline. Hyperautofluorescent foveal patch (16 eyes [31.4%]) and abnormal central hypoautofluorescence (9 eyes [17.6%]) were significantly associated with worse morphological and functional read-outs compared with the hyperautofluorescent ring pattern (22 eyes [43.1%]). Conclusions: Our European multicentric study offers the first prospective longitudinal analysis in one of the largest cohorts of MYO7A patients described to date, confirming the slow disease progression. More important, this study emphasizes the key role of fundus autofluorescence patterns in retinal impairment staging and advocates its adoption as an objective biomarker in patient selection for future gene therapy clinical trials.

Extended time frame for restoring inner ear function through gene therapy in Usher1G preclinical model.

Neonatal gene therapy has been shown to prevent inner ear dysfunction in mouse models of Usher syndrome type I (USH1), the most common genetic cause of combined deafness-blindness and vestibular dysfunction. However, hearing onset occurs after birth in mice and in utero in humans, making it questionable how to transpose murine gene therapy outcomes to clinical settings. Here, we sought to extend the therapeutic time window in a mouse model for USH1G to periods corresponding to human neonatal stages, more suitable for intervention in patients. Mice with deletion of Ush1g (Ush1g-/-) were subjected to gene therapy after the hearing onset. The rescue of inner ear hair cell structure was evaluated by confocal imaging and electron microscopy. Hearing and vestibular function were assessed by recordings of the auditory brain stem response and vestibulo-ocular reflex and by locomotor tests. Up to P21, gene therapy significantly restored both the hearing and balance deficits in Ush1g-/- mice. However, beyond this age and up to P30, vestibular function was restored but not hearing. Our data show that effective gene therapy is possible in Ush1g-/- mice well beyond neonatal stages, implying that the therapeutic window for USH1G may be wide enough to be transposable to newborn humans.

[Advances on gene therapy for USH2A exon 13 related inherited retinal dystrophy].

Biallelic pathogenic variants in the USH2A gene result in Usher syndrome type Ⅱ and non-syndromic retinitis pigmentosa, both of which entail the progressive loss of photoreceptors leading to blindness. The cDNA of the USH2A gene is extensive, consisting of 15 606 base pairs, rendering it impractical for delivery via adeno-associated virus vectors for gene replacement therapy. Notably, exon 13 has emerged as a focal point for therapeutic intervention, given its predilection for harboring the most pathogenic variants within USH2A. Recent intervention studies targeting USH2A exon 13 through the utilization of antisense oligonucleotides, genome editing, and RNA editing approaches have exhibited promising therapeutic potential. This paper provides a comprehensive overview of the molecular mechanisms, outcome data, and the challenges associated with the application of these interventions in this domain.

Third-generation lentiviral gene therapy rescues function in a mouse model of Usher 1B.

Usher syndrome 1B (USH1B) is a devastating genetic disorder with congenital deafness, loss of balance, and blindness caused by mutations in the myosin-VIIa (MYO7A) gene, for which there is currently no cure. We developed a gene therapy approach addressing the vestibulo-cochlear deficits of USH1B using a third-generation, high-capacity lentiviral vector system capable of delivering the large 6,645-bp MYO7A cDNA. Lentivirally delivered MYO7A and co-encoded dTomato were successfully expressed in the cochlear cell line HEI-OC1. In normal-hearing mice, both cochlea and the vestibular organ were efficiently transduced, and ectopic MYO7A overexpression did not show any adverse effects. In Shaker-1 mice, an USH1B disease model based on Myo7a mutation, cochlear and vestibular hair cells, the main inner ear cell types affected in USH1B, were successfully transduced. In homozygous mutant mice, delivery of MYO7A at postnatal day 16 resulted in a trend for partial recovery of auditory function and in strongly reduced balance deficits. Heterozygous mutant mice were found to develop severe hearing loss at 6 months of age without balance deficits, and lentiviral MYO7A gene therapy completely rescued hearing to wild-type hearing thresholds. In summary, this study demonstrates improved hearing and balance function through lentiviral gene therapy in the inner ear.

Dual AAV-based PCDH15 gene therapy achieves sustained rescue of visual function in a mouse model of Usher syndrome 1F.

Mutations in the PCDH15 gene, encoding protocadherin-15, are among the leading causes of Usher syndrome type 1 (USH1F), and account for up to 12% USH1 cases worldwide. A founder truncating variant of PCDH15 has a ∼2% carrier frequency in Ashkenazi Jews accounting for nearly 60% of their USH1 cases. Although cochlear implants can restore hearing perception in USH1 patients, presently there are no effective treatments for the vision loss due to retinitis pigmentosa. We established a founder allele-specific Pcdh15 knockin mouse model as a platform to ascertain therapeutic strategies. Using a dual-vector approach to circumvent the size limitation of adeno-associated virus, we observed robust expression of exogenous PCDH15 in the retinae of Pcdh15KI mice, sustained recovery of electroretinogram amplitudes and key retinoid oxime, substantially improved light-dependent translocation of phototransduction proteins, and enhanced levels of retinal pigment epithelium-derived enzymes. Thus, our data raise hope and pave the way for future gene therapy trials in USH1F subjects.

Expression of two major isoforms of MYO7A in the retina: Considerations for gene therapy of Usher syndrome type 1B.

Usher syndrome type 1B (USH1B) is a deaf-blindness disorder, caused by mutations in the MYO7A gene, which encodes the heavy chain of an unconventional actin-based motor protein. Here, we examined the two retinal isoforms of MYO7A, IF1 and IF2. We compared 3D models of the two isoforms and noted that the 38-amino acid region that is present in IF1 but absent from IF2 affects the C lobe of the FERM1 domain and the opening of a cleft in this potentially important protein binding domain. Expression of each of the two isoforms of human MYO7A and pig and mouse Myo7a was detected in the RPE and neural retina. Quantification by qPCR showed that the expression of IF2 was typically âˆ¼ 7-fold greater than that of IF1. We discuss the implications of these findings for any USH1B gene therapy strategy. Given the current incomplete knowledge of the functions of each isoform, both isoforms should be considered for targeting both the RPE and the neural retina in gene augmentation therapies.

Optimization of Capillary-Based Western Blotting for MYO7A.

Myosin VIIA (MYO7A)-associated Usher syndrome type 1B (USH1B) is a severe disorder that impacts the auditory, vestibular, and visual systems of affected patients. Due to the large size (~7.5 kb) of the MYO7A coding sequence, we have designed a dual adeno-associated virus (AAV) vector-based approach for the treatment of USH1B-related vision loss. Due to the added complexity of dual-AAV gene therapy, careful attention must be paid to the protein products expressed following vector recombination. In order to improve the sensitivity and quantifiability of our immunoassays, we adapted our traditional western blot protocol for use with the Jess™ Simple Western System. Following several rounds of testing, we optimized our protocol for the detection of MYO7A in two of our most frequently used sample types, mouse eyes, and infected HEK293 cell lysates.

Rescue of hearing by adenine base editing in a humanized mouse model of Usher syndrome type 1F.

Usher syndrome type 1F (USH1F), characterized by congenital lack of hearing and balance and progressive loss of vision, is caused by mutations in the PCDH15 gene. In the Ashkenazi population, a recessive truncation mutation accounts for a large proportion of USH1F cases. The truncation is caused by a single C→T mutation, which converts an arginine codon to a stop (R245X). To test the potential for base editors to revert this mutation, we developed a humanized Pcdh15R245X mouse model for USH1F. Mice homozygous for the R245X mutation were deaf and exhibited profound balance deficits, while heterozygous mice were unaffected. Here we show that an adenine base editor (ABE) is capable of reversing the R245X mutation to restore the PCDH15 sequence and function. We packaged a split-intein ABE into dual adeno-associated virus (AAV) vectors and delivered them into cochleas of neonatal USH1F mice. Hearing was not restored in a Pcdh15 constitutive null mouse despite base editing, perhaps because of early disorganization of cochlear hair cells. However, injection of vectors encoding the split ABE into a late-deletion conditional Pcdh15 knockout rescued hearing. This study demonstrates the ability of an ABE to correct the PCDH15 R245X mutation in the cochlea and restore hearing.

Successful large gene augmentation of USH2A with non-viral episomal vectors.

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.

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.

Mini-PCDH15 gene therapy rescues hearing in a mouse model of Usher syndrome type 1F.

Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3-5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.

Identifying and Overcoming Challenges in Developing Effective Treatments for Usher 1B: A Workshop Report.

Purpose: To identify challenges and opportunities for the development of treatments for Usher syndrome (USH) type 1B. Methods: In September 2021, the Foundation Fighting Blindness hosted a virtual workshop of clinicians, academic and industry researchers, advocates, and affected individuals and their families to discuss the challenges and opportunities for USH1B treatment development. Results: The workshop began with insights from individuals affected by USH1B. Presentation topics included myosin VIIA protein function in the ear and eye and its role in disease pathology; challenges with the USH1B mouse model most used in disease research to date; new investigations into alternative disease models that may provide closer analogues to USH1B in the human retina, including retinal organoids and large animal models; and learnings from and limitations of available disease natural history data. Participants discussed the need for an open dialogue between researchers and regulators to design USH1B clinical trials with appropriate outcome measures of vision improvement, along with multimodal imaging of the retina and other testing approaches that can help inform trial designs. The workshop concluded with presentations and a roundtable reviewing emerging treatments, including USH1B-targeted genetic augmentation therapy and gene-agnostic approaches. Conclusions: Initiatives like this workshop are important to foster all stakeholders in support of achieving the shared goal of treating and curing USH1B. Translational Relevance: Presentations and discussions focused on overcoming disease modeling and clinical trial design challenges to facilitate development, testing, and implementation of effective USH1B treatments.

Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

New CRISPR Tools to Correct Pathogenic Mutations in Usher Syndrome.

Inherited retinal degenerations are a leading cause of blindness in the UK. Significant advances have been made to tackle this issue in recent years, with a pioneering FDA approved gene therapy treatment (Luxturna®), which targets a loss of function mutation in the RPE65 gene. However, there remain notable shortcomings to this form of gene replacement therapy. In particular, the lack of viability for gene sequences exceeding the 4.7 kb adeno-associated virus (AAV) packaging limit or for toxic gain of function mutations. The USH2A gene at ~15.7 kb for instance is too large for AAV delivery: a safe and effective vehicle capable of transducing photoreceptor cells for gene replacement therapy. Usher Syndrome is a clinically and genetically heterogenous deaf-blindness syndrome with autosomal recessive inheritance. The USH2A gene encodes the protein usherin, which localises to the photoreceptor cilium and cochlear hair cells. Mutations in the USH2A gene cause Usher Syndrome type II (USH2), which is the most common subtype of Usher Syndrome and the focus of this review. To date, researchers have been unable to create an efficient, safe editing tool that is small enough to fit inside a single AAV vector for delivery into human cells. This article reviews the potential of CRISPR technology, derived from bacterial defence mechanisms, to overcome these challenges; delivering tools to precisely edit and correct small insertions, deletions and base transitions in USH2A without the need to deliver the full-length gene. Such an ultra-compact therapy could make strides in combating a significant cause of blindness in young people.

Pathogenesis and Treatment of Usher Syndrome Type IIA.

Usher syndrome (USH) is the most common form of deaf-blindness, with an estimated prevalence of 4.4 to 16.6 per 100,000 people worldwide. The most common form of USH is type IIA (USH2A), which is caused by homozygous or compound heterozygous mutations in the USH2A gene and accounts for around half of all USH cases. USH2A patients show moderate to severe hearing loss from birth, with diagnosis of retinitis pigmentosa in the second decade of life and variable vestibular involvement. Although hearing aids or cochlear implants can provide some mitigation of hearing deficits, there are currently no treatments aimed at preventing or restoring vision loss in USH2A patients. In this review, we first provide an overview of the molecular biology of the USH2A gene and its protein isoforms, which include a transmembrane protein (TM usherin) and an extracellular protein (EC usherin). The role of these proteins in the inner ear and retina and their impact on the pathogenesis of USH2A is discussed. We review animal cell-derived and patient cell-derived models currently used in USH2A research and conclude with an overview of potential treatment strategies currently in preclinical development and clinical trials.

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

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.

Usher syndrome IIIA: a review of the disorder and preclinical research advances in therapeutic approaches.

Usher syndrome (USH) is an autosomal recessive disorder characterized by sensorineural hearing loss, progressive pigmentary retinopathy, and vestibular dysfunction. The degree and onset of hearing loss vary among subtypes I, II, and III, while blindness often occurs in the second to fourth decades of life. Usher type III (USH3), characterized by postlingual progressive sensorineural hearing loss, varying levels of vestibular dysfunction, and varying degrees of visual impairment, typically manifests in the first to second decades of life. While USH3 is rare, it is highly prevalent in certain populations. RP61, USH3, and USH3A symbolize the same disorder, with the latter symbol used more frequently in recent literature. This review focuses on the clinical features, epidemiology, molecular genetics, treatment, and research advances for sensory deficits in USH3A.

Genetics, pathogenesis and therapeutic developments for Usher syndrome type 2.

Usher syndrome (USH) is a rare, autosomal recessively inherited disorder resulting in a combination of sensorineural hearing loss and a progressive loss of vision resulting from retinitis pigmentosa (RP), occasionally accompanied by an altered vestibular function. More and more evidence is building up indicating that also sleep deprivation, olfactory dysfunction, deficits in tactile perception and reduced sperm motility are part of the disease etiology. USH can be clinically classified into three different types, of which Usher syndrome type 2 (USH2) is the most prevalent. In this review, we, therefore, assess the genetic and clinical aspects, available models and therapeutic developments for USH2. Mutations in USH2A, ADGRV1 and WHRN have been described to be responsible for USH2, with USH2A being the most frequently mutated USH-associated gene, explaining 50% of all cases. The proteins encoded by the USH2 genes together function in a dynamic protein complex that, among others, is found at the photoreceptor periciliary membrane and at the base of the hair bundles of inner ear hair cells. To unravel the pathogenic mechanisms underlying USH2, patient-derived cellular models and animal models including mouse, zebrafish and drosophila, have been generated that all in part mimic the USH phenotype. Multiple cellular and genetic therapeutic approaches are currently under development for USH2, mainly focused on preserving or partially restoring the visual function of which one is already in the clinical phase. These developments are opening a new gate towards a possible treatment for USH2 patients.

Proposed therapy, developed in a Pcdh15-deficient mouse, for progressive loss of vision in human Usher syndrome.

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9-cis retinal improved ERG amplitudes in Pcdh15R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients.