Gene augmentation therapy attenuates retinal degeneration in a knockout mouse model of Fam161a retinitis pigmentosa.

Photoreceptor cell degeneration and death is the major hallmark of a wide group of human blinding diseases including age-related macular degeneration and inherited retinal diseases such as retinitis pigmentosa. In recent years, inherited retinal diseases have become the "testing ground" for novel therapeutic modalities, including gene and cell-based therapies. Currently there is no available treatment for retinitis pigmentosa caused by FAM161A biallelic pathogenic variants. In this study, we injected an adeno-associated virus encoding for the longer transcript of mFam161a into the subretinal space of P24-P29 Fam161a knockout mice to characterize the safety and efficacy of gene augmentation therapy. Serial in vivo assessment of retinal function and structure at 3, 6, and 8 months of age using the optomotor response test, full-field electroretinography, fundus autofluorescence, and optical coherence tomography imaging as well as ex vivo quantitative histology and immunohistochemical studies revealed a significant structural and functional rescue effect in treated eyes accompanied by expression of the FAM161A protein in photoreceptors. The results of this study may serve as an important step toward future application of gene augmentation therapy in FAM161A-deficient patients by identifying a promising isoform to rescue photoreceptors and their function.

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

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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. Translational Relevance: 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.

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 in vitro. 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.

ABCA4 c.859-25A>G, a Frequent Palestinian Founder Mutation Affecting the Intron 7 Branchpoint, Is Associated With Early-Onset Stargardt Disease.

Purpose: The effect of noncoding variants is often unknown in the absence of functional assays. Here, we characterized an ABCA4 intron 7 variant, c.859-25A>G, identified in Palestinian probands with Stargardt disease (STGD) or cone-rod dystrophy (CRD). We investigated the effect of this variant on the ABCA4 mRNA and retinal phenotype, and its prevalence in Palestine. Methods: The ABCA4 gene was sequenced completely or partially in 1998 cases with STGD or CRD. The effect of c.859-25A>G on splicing was investigated in silico using SpliceAI and in vitro using splice assays. Homozygosity mapping was performed for 16 affected individuals homozygous for c.859-25A>G. The clinical phenotype was assessed using functional and structural analyses including visual acuity, full-field electroretinography, and multimodal imaging. Results: The smMIPs-based ABCA4 sequencing revealed c.859-25A>G in 10 Palestinian probands from Hebron and Jerusalem. SpliceAI predicted a significant effect of this putative branchpoint-inactivating variant on the nearby intron 7 splice acceptor site. Splice assays revealed exon 8 skipping and two partial inclusions of intron 7, each having a deleterious effect. Additional genotyping revealed another 46 affected homozygous or compound heterozygous individuals carrying variant c.859-25A>G. Homozygotes shared a genomic segment of 59.6 to 87.9 kb and showed severe retinal defects on ophthalmoscopic evaluation. Conclusions: The ABCA4 variant c.859-25A>G disrupts a predicted branchpoint, resulting in protein truncation because of different splice defects, and is associated with early-onset STGD1 when present in homozygosity. This variant was found in 25/525 Palestinian inherited retinal dystrophy probands, representing one of the most frequent inherited retinal disease-causing variants in West-Bank Palestine.

A deep intronic substitution in CNGB3 is one of the major causes of achromatopsia among Jewish patients.

Purpose: Although most (or even all) genes that can cause achromatopsia (ACHM) when mutated are known, some patients are still negative for mutations even after screening the coding sequence of all known genes. Our aim was to characterize the genetic and clinical aspects of a deep intronic (c.1663-1205G>A, IVS14-1205G>A) CNGB3 variant. Methods: Clinical evaluation included visual acuity testing, refractive error, a full clinical eye exam, full-field electroretinography (ffERG), color vision testing, and retinal imaging. Genetic analysis of CNGB3 exons, as well as part of intron 14, was performed by Sanger sequencing of PCR products. Results: Screening for the CNGB3 c.1663-1205G>A variant revealed 17 patients belonging to 12 unrelated families who were either homozygous for this variant (7 cases, 5 families) or heterozygous in combination with another heterozygous known CNGB3 mutation (10 cases, 7 families). All patients were diagnosed with cone-dominated disease, mainly complete ACHM. In all cases, the disease had an early, congenital onset. Visual acuity was markedly impaired, ranging between 0.07 and 0.32 on the Early Treatment Diabetic Retinopathy Study (ETDRS) scale (logarithm of the minimum angle of resolution [LogMAR] +1.18 to +0.50), with a mean visual acuity of 0.15 ETDRS (LogMAR +0.80). Additional typical signs of ACHM, including impaired color vision, light aversion, and nystagmus, were also noted in all patients. As is common in ACHM, fundus exams were largely unremarkable in most patients, with mild foveal RPE changes seen in some cases at older ages. ERG was available for 14 out of 17 patients, and in all of them-including infants from the age of 6 months-cone responses were nondetectable. In a few cases, rod involvement was also evident, with a mild reduction of amplitudes. Optical coherence tomography (OCT) imaging showed irregularity of the ellipsoid zone in the foveal area in some patients. Conclusions: CNGB3 is the most common cause of ACHM in patients of European descent; this is mainly due to a panethnic founder mutation, c.1148del. Here, we report on an intronic CNGB3 variant that is more frequent than the c.1148del mutation in our cohort of Jewish patients. Among our ACHM cohort, 63.7% of patients had biallelic CNGA3 mutations and 26.4% had biallelic CNGB3 mutations. The phenotype of patients harboring the intronic mutation falls largely within the spectrum commonly seen in ACHM. Since gene therapy for CNGB3 is currently under investigation, these patients might benefit from this promising therapy. Given that this variant is not detectable by current commonly used genetic testing platforms, these patients could easily be missed.