514 research outputs found
HYPERREFLECTIVE FOCI AS A PATHOGENETIC BIOMARKER IN CHOROIDEREMIA
No full textTo assess hyperreflective foci (HF) number and distribution in choroideremia (CHM) using spectral domain optical coherence tomography
underlies the cone-dominated phenotype associated with truncating distal ORF15 variants
No full textPathogenic variants in the Retinitis pigmentosa GTPase regulator (RPGR) gene lead to a clinically severe form of X-linked retinal dystrophy. However, it remains unclear why some variants cause a predominant rod, while others result in a cone-dominated phenotype. Post-translational glutamylation of the photoreceptor-specific RPGRORF15 isoform by the TTLL5 enzyme is essential for its optimal function in photoreceptors, and loss of TTLL5 leads to retinal dystrophy with a cone phenotype. Here we show that RPGR retinal disease, studied in a single cohort of 116 male patients, leads to a clear progressive shift from rod-to cone-dominating phenotype as the RPGRORF15 variant location approaches the distal part of the Open Reading Frame 15 (ORF15) region. The rod photoreceptor involvement on the contrary diminishes along the RGPR sequence, and the variants associated with the cone only phenotype are located predominantly in the very distal part, including the C-terminal basic domain. Moreover, these distal truncating RPGRORF15 variants disrupt the interaction with TTLL5 and lead to a significant impairment of RPGR glutamylation. Thus, consistent with the phenotype of TTLL5 pathogenic variants, our study shows that RPGRORF15 variants, which disrupt its basic domain and the interaction with TTLL5, also impair RPGR glutamylation and lead to the cone phenotype. This has implications for ongoing gene therapy clinical trials where the application of RPGR with impaired glutamylation may be less effective in treating RGPR dystrophies and may even convert a rod–cone dystrophy into a cone dystrophy phenotype.</p
Optimising CRISPR sgRNA delivery strategies for the treatment of retinal diseases
No full textGene therapy for inherited retinal diseases (IRDs) has traditionally focussed on gene augmentation. However, the advent of CRISPR/Cas9 has changed the research landscape dramatically. Theoretically able to correct almost any mutation, CRISPR/Cas9 is an attractive tool for IRD therapeutics, many untreatable by gene augmentation. However, numerous challenges exist for Cas9 delivery to retina. This thesis explores non-viral, separate delivery of Cas9 sgRNA as a way to overcome these limitations. The ability of unpackaged RNA oligonucleotides to enter retina from intravitreal injection has been shown by chemically modified antisense oligonucleotides (ASOs). Similar modifications applied to Cas9 sgRNA have been used effectively in vitro, and in vivo in liver, but not in the eye. In this thesis I investigate the use of chemically modified Cas9 sgRNA as a proof-of-concept for retinal therapeutics. First, I examined sgRNA modification pattern and number, and showed that variations can enhance or abrogate EGFP and Cas9 knockdown for SpCas9, SaCas9, and a SpCas9 prime editor in vitro in HEKdEGFP cells without toxicity. Next, I examined retinal distribution and survival of unpackaged sgRNA in vivo using qPCR and direct visualisation of fluorescently-labelled chemically modified sgRNA. This revealed that intravitreally-injected sgRNA enters the retinal ganglion cell (RGC) layer and survives for at least 1 month without toxicity, but is rapidly lost from vitreous, while subretinally-injected sgRNA survives in the retina for at least 8 weeks without toxicity. Finally, I examined indel formation in vivo, targeting photoreceptor-level EGFP in the Nrl.EGFP mouse with subretinal unpackaged sgRNA and AAV8-GRK1-Cas9, and RGC-level EGFP using intravitreal unpackaged sgRNA in the Cas9EGFP mouse. This revealed that heavily modified SpCas9 sgRNA successfully effects indel formation in RGCs, but not in photoreceptors, perhaps due to sgRNA trapping in outer segments or within other retinal cells. As part of this thesis, I also characterised retinal Cas9 expression of several transgenic Cas9-expressing mice using Western blot, in vivo imaging, and IHC
Developing gene therapy for inherited retinal degenerations
No full textThe aim of this thesis was to evaluate the efficacy and safety of retinal gene therapy in a pre-clinical model of CDHR1-associated retinal degeneration – a hitherto untreatable, blinding disorder.
Deep phenotyping of the Cdhr1-/- murine model demonstrated severe early deficits in cone and rod photoreceptor function followed by progressive photoreceptor cell death – recapitulating CDHR1 cone-rod dystrophy. Two AAV8 vectors expressing the full-length human CDHR1 coding sequence were designed, manufactured, validated and titrated in vivo.
Sub-retinal injection of AAV8.GRK1.CDHR1.pA (1.5 x 108 vg) in Cdhr1-/- mice at 3-4 weeks of age resulted in functional rescue of cone and rod photoreceptors with improved response amplitudes and decreased implicit times to 12-months post-injection; a slowing of photoreceptor cell death with regeneration of full-length photoreceptor outer segments confirmed on ultrastructural analysis to 21-months post-injection; and a behavioural rescue effect on photopic and scotopic optomotor testing, sustained to 21-months post-injection. AAV8.GRK1.CDHR1.pA at 1.5 x 108 vg appeared safe in C57BL/6J mice by the same outcome measures to 22-months post-injection.
Genetic prevalence estimates presented herein suggest more than 200,000 affected individuals worldwide. We characterised 146 individuals with biallelic variants in CDHR1, describing the retinal phenotype, natural history, genotype-phenotype associations, with 25 novel pathological sequence variants.
This is the first therapy shown to improve retinal structure and function in a pre-clinical model of CDHR1-associated retinal degeneration. A patent to protect the vectors described herein has been filed on behalf of the University of Oxford, with commercial agreements obtained in preparation for an onward phase 1 clinical trial
Current Management of Inherited Retinal Degeneration Patients in Europe: Results of a 2-Year Follow-Up Multinational Survey by the European Vision Institute Clinical Research Network - EVICR.net
No full textIntroduction: An increasing number of gene-specific therapies are being developed for IRDs. Identification of well characterized patients is an emerging need. We conducted the second multinational survey among the EVICR.net and ERN-EYE members to understand the management and treatment of IRDs in Europe and compared it to the 2019 survey.Methods: An electronic survey questionnaire was developed and sent to 124 clinical centers (25 countries) by June/July 2021. Statistical analysis was performed with Excel and R.Results: The overall response rate was 44% but varied among countries. Only 9% of responding centers do not see IRD patients (2019 survey 14%); 42% follow at least 200 patients per year, 18% follow 500-999 and 2% more than 1000. Databases exist in 86% of the centers (local 86%; national web-based 12%). IRD patients are referred to EVICR.net and ERN-EYE centers mainly by general ophthalmologists, patient self-referral, or medical retina specialists. Most IRD patients are first seen as adults. Signs and symptoms depend on age of onset: in infancy nystagmus, at older age night blindness and reduced visual field; reduced visual acuity (VA) is described at any age. Comprehensive ophthalmic examination always includes VA, and almost always visual fields multimodal retinal imaging, electrophysiology, color vision testing, and refraction. Identification of genotypes is successful in 72% of centers in 40-80% of cases (2019 survey 69% of centers). The time for confirmation of the genetic diagnosis varies from 2-4 weeks to 24 months (2019 survey > 4 weeks <= 10 years). Genetic testing is covered by public health service in 83%, private health insurance in 29%, research funds in 24%; 5% do not have access to genetic testing (2019 survey 15%). The most striking result is the high increase in the involvement of centers in natural history and gene therapy trials that for the latter more than doubled. Discussion/Conclusion: This second multinational survey on management of IRDs in Europe highlights persistent important differences in the number of IRD patients managed per center, comparable diagnostic work-up, and increasing genotyping in diagnostic laboratories. The important increase of involvement of centers in natural history and gene therapy trials reflects the rapidly evolving field of gene therapy development. The survey provides important follow-up data for researchers, clinicians, care givers, patient advocate groups, pharmaceutical companies and investors
Current management of patients with RPE65 mutation-associated Inherited Retinal Degenerations (RPE65-IRD) in Europe. Results of a 2 years follow-up multinational survey
No full textIntroduction: To evaluate the current management of RPE65-biallelic mutation-associated inherited retinal degeneration (RPE65-IRD) in Europe since market authorization of Voretigene Neparvovec (VN, LuxturnaTM) in 2018. By July 2022, over 200 patients have been treated outside the USA, of whom about 90% in Europe. We conducted among all centers of the European Vision Institute Clinical Research Network (EVICR.net) and health care providers (HCPs) of the European Reference Network dedicated to Rare Eye Diseases (ERN-Eye) the second multinational survey on management of IRDs in Europe elaborated by EVICR.net with a special focus on RPE65-IRD.
Methods: An electronic survey questionnaire with 48 questions specifically addressing RPE65-IRD (2019 survey 35) was developed and sent by June 2021 to 95 EVICR.net centers and 40 ERN-EYE HCPs and affiliated members. Of note, 11 centers are members of both networks. Statistical analysis was performed with Excel and R.
Results: The overall response rate was 44% (55/124); 26 centers follow RPE65 biallelic mutation-associated IRD patients. By June 2021, 8/26 centers have treated 57 RPE65-IRD cases (1 - 19/center, median 6), and 43 planned for treatment (range 0 - 10/center, median 6). The overall age range was 3 - 52 years, and on average 22% of the patients did not (yet) qualify for treatment (range 2 - 60%/center, median 15%). Main reasons were too advanced (range 0-100, median 75%) or mild disease (range 0-100, median 0). Eighty-three percent of centers (10/12) that follow RPE65 mutation-associated IRD patients treated with VN participate in the PERCEIVE registry (EUPAS31153, http://www.encepp.eu/encepp/viewResource.htm?id=37005). Quality of life and full field stimulus test (FST) improvements had the highest scores of the survey-reported outcome parameters in VN treatment follow-up.
Discussion/conclusion: This second multinational survey on management of RPE65-IRD by EVICR.net centers and ERN-Eye HCPs in Europe indicates that RPE65-IRD might be diagnosed more reliably in 2021 compared to 2019. By June 2021, 8/26 centers reported detailed results including VN treatment. Main reasons for non-treatment were too advanced or mild disease, followed by absence of two class 4 or 5 mutations on both alleles, or because of a too young age. Patient satisfaction with treatment was estimated to be high by 50% of the centers
The Abca4-/- mouse model of Stargardt disease – phenotype and therapeutic strategies
No full textStargardt disease is caused by mutations in the ABCA4 gene and is probably the commonest inherited cause for retinal degeneration in youth with progressive visual deterioration. The Abca4-/- mouse is an animal model mimicking certain aspects of the human disease, including an accumulation of autofluorescent lipofuscin in the retinal pigment epithelium (RPE). The model is therefore ideally suited for preclinical investigation of novel treatment approaches for Stargardt disease. Imaging of lipofuscin- and melanin-related fundus autofluorescence (AF) was optimized in mice, which subsequently allowed investigating the mouse ocular phenotype in vivo. The Abca4-/- mouse showed an age-related increase in lipofuscin- and melanin-related AF intensity, correlating to an increase of ex vivo assessed bis-retinoid-fluorophores and formation of melanolipofuscin granules, respectively. Retinal function remained largely unaffected by those changes within the RPE. Abca4-/- mice were fed with C20-deuterized vitamin A (C20dVitA) which had been shown to inhibit lipofuscin-formation in the RPE. The diet markedly reduced lipofuscin- and melanin-related AF intensity to levels measured in wild type animals on a normal diet. This treatment did not affect retinal function. The possibility of performing similar fundus AF measurements in humans may allow fast translation of this therapy into clinical trials. The only causative treatment approach for Stargardt disease will be gene replacement therapy. Investigation of various mutant adeno-associated viruses (AAVs) as vector for delivering ABCA4 revealed that photoreceptors in Abca4-/- mice were more difficult to transduce than photoreceptors in wild type mice. This indicates an influence of the diseased retina on gene delivery. Thus, very efficient viruses might be needed to achieve relevant ABCA4 expression in the retina of patients with Stargardt disease. In summary, application of a clinically relevant imaging method allows to assess the ocular phenotype of the mouse model for Stargardt disease and to investigate novel treatment strategies
IMPG2-related maculopathy
No full textPurpose: to investigate the phenotype, variability, and penetrance of IMPG2-related maculopathy.Design: retrospective observational case series.Methods: clinical evaluation, multimodal retinal imaging, genetic testing, and molecular modeling.Results: a total of 25 individuals with a mono-allelic IMPG2 variant were included, 5 of whom were relatives of patients with IMPG2-associated retinitis pigmentosa. A distinct maculopathy was present in 17 individuals (median age, 52 years; range, 20-72 years), and included foveal elevation with or without subretinal vitelliform material or focal atrophy of the retinal pigment epithelium. Best-corrected visual acuity (BCVA) was ≥20/50 in the better eye (n = 15), and 5 patients were asymptomatic. Longitudinal observation (n = 8, up to 19 years) demonstrated stable maculopathy (n = 3), partial/complete resorption (n = 4) or increase (n = 1) of the subretinal material, with overall stable vision (n = 6). No manifest maculopathy was observed in 8 individuals (median age, 58 years; range, 43-83 years; BCVA ≥20/25), all were identified through segregation analysis. All 8 individuals were asymptomatic, with minimal foveal changes observed on optical coherence tomography in 3 cases. A total of 18 different variants were detected, 11 of them truncating. Molecular modeling of 5 missense variants [c.727G>C, c.1124C>A, c.2816T>A, c.3047T>C, and c.3193G>A] supported the hypothesis that these have a loss-of-function effect.Conclusions: mono-allelic IMPG2 variants may result in haploinsufficiency manifesting as a maculopathy with variable penetrance and expressivity. Family members of patients with IMPG2-related retinitis pigmentosa may present with vitelliform lesions. The maculopathy often remains limited to the fovea and is usually associated with moderate visual impairment.</p
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