Rescue of cone and rod photoreceptor function in a CDHR1-model of age-related retinal degeneration.

Age-related macular degeneration (AMD) is the most common cause of untreatable blindness in the developed world. Recently, CDHR1 has been identified as the cause of a subset of AMD that has the appearance of the "dry" form, or geographic atrophy. Biallelic variants in CDHR1-a specialized protocadherin highly expressed in cone and rod photoreceptors-result in blindness from shortened photoreceptor outer segments and progressive photoreceptor cell death. Here we demonstrate long-term morphological, ultrastructural, functional, and behavioral rescue following CDHR1 gene therapy in a relevant murine model, sustained to 23-months after injection. This represents the first demonstration of rescue of a monogenic cadherinopathy in vivo. Moreover, the durability of CDHR1 gene therapy seems to be near complete-with morphological findings of the rescued retina not obviously different from wildtype throughout the lifespan of the mouse model. A follow-on clinical trial in patients with CDHR1-associated retinal degeneration is warranted. Hypomorphic CDHR1 variants may mimic advanced dry AMD. Accurate clinical classification is now critical, as their pathogenesis and treatment are distinct.

Impaired glutamylation of RPGRORF15 underlies the cone-dominated phenotype associated with truncating distal ORF15 variants.

Pathogenic 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.

Assessment of Visual Function with Cotoretigene Toliparvovec in X-Linked Retinitis Pigmentosa in the Randomized XIRIUS Phase 2/3 Study.

PURPOSE: Cotoretigene toliparvovec (BIIB112/AAV8-RPGR) is an investigational vector-based gene therapy designed to provide a full-length, codon-optimized retinitis pigmentosa GTPase regulator (RPGR) protein to individuals with RPGR-associated X-linked retinitis pigmentosa (XLRP). We assessed efficacy and tolerability of cotoretigene toliparvovec subretinal gene therapy. DESIGN: Part 2 of the XIRIUS trial (ClinicalTrials.gov identifier, NCT03116113) was a phase 2/3, 12-month, randomized (1:1:1) dose-expansion study. PARTICIPANTS: Male patients ≥10 years of age with RPGR-associated XLRP were included. METHODS: Participants were randomized 1:1:1 to receive low-dose subretinal cotoretigene toliparvovec (5 × 1010 vector genomes/eye), high-dose cotoretigene toliparvovec (2.5 × 1011 vector genomes/eye) or to be an untreated control participant. MAIN OUTCOME MEASURES: The primary end point was the percentage of participants meeting microperimetry responder criteria (≥ 7-dB improvement at ≥ 5 of 16 central loci). Secondary end points included change from baseline in retinal sensitivity at the central 16 loci and the entire 68 loci at 12 months and change from baseline in low-luminance visual acuity (LLVA) at 12 months, as well as the proportion of eyes with a ≥ 15-Early Treatment Diabetic Retinopathy Study ETDRS letter LLVA and ≥ 10-ETDRS letter LLVA change from baseline at month 12. RESULTS: Because of the impact of the COVID-19 pandemic, enrollment ended before reaching the initial target, leaving the trial underpowered. Twenty-nine participants were included (low-dose group, n = 10; high-dose group, n = 10; control group, n = 9). At month 12, the percentage of participants meeting microperimetry responder criteria was not significantly different between either cotoretigene toliparvovec group (low dose, 37.5% [P = 0.3181]; high dose, 25.0% [P = 0.5177]) and the control group (22.2%). However, the mean change from baseline in microperimetry sensitivity improved significantly with the low-dose group versus the control group at month 12 (P = 0.0350). Significant improvement in LLVA occurred in the low-dose group versus the control group at month 12 (33.3% difference [80% confidence interval, 14.7%-55.2%]; P = 0.0498). Three ocular-related serious adverse events (SAEs) occurred in the low-dose group versus 7 SAEs in the high-dose group. CONCLUSIONS: The primary microperimetry end point was not met. Significant improvements in LLVA and mean microperimetry were observed compared with controls and fewer SAEs occured with low-dose compared with high dose cotoretigene toliparvovec. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

CRISPR Manipulation of Age-Related Macular Degeneration Haplotypes in the Complement System: Potential Future Therapeutic Applications/Avenues.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss among the elderly in the developed world. Whilst AMD is a multifactorial disease, the involvement of the complement system in its pathology is well documented, with single-nucleotide polymorphisms (SNPs) in different complement genes representing an increased risk factor. With several complement inhibitors explored in clinical trials showing limited success, patients with AMD are still without a reliable treatment option. This indicates that there is still a gap of knowledge in the functional implications and manipulation of the complement system in AMD, hindering the progress towards translational treatments. Since the discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool, the field of molecular biology has been revolutionised. Genetic variants in the complement system have long been associated with an increased risk of AMD, and a variety of haplotypes have been identified to be predisposing/protective, with variation in complement genes believed to be the trigger for dysregulation of the cascade leading to inflammation. AMD-haplotypes (SNPs) alter specific aspects of the activation and regulation of the complement cascade, providing valuable insights into the pathogenic mechanisms of AMD with important diagnostic and therapeutic implications. The effect of targeting these AMD-related SNPs on the regulation of the complement cascade has been poorly explored, and the CRISPR/Cas system provides an ideal tool with which to explore this avenue. Current research concentrates on the association events of specific AMD-related SNPs in complement genes without looking into the effect of targeting these SNPs and therefore influencing the complement system in AMD pathogenesis. This review will explore the current understanding of manipulating the complement system in AMD pathogenesis utilising the genomic manipulation powers of the CRISPR/Cas systems. A number of AMD-related SNPs in different complement factor genes will be explored, with a particular emphasis on factor H (CFH), factor B (CFB), and complement C3 (C3).

A cross-sectional study to assess the clinical utility of modern visual function assessments in patients with inherited retinal disease: a mixed methods observational study protocol.

BACKGROUND: Treatment options for patients with inherited retinal disease are limited, although research into novel therapies is underway. To ensure the success of future clinical trials, appropriate visual function outcome measures that can assess changes resulting from therapeutic interventions are urgently required. Rod-cone degenerations are the most common type of inherited retinal disease. Visual acuity is a standard measure but is typically preserved until late disease stages, frequently making it an unsuitable visual function marker. Alternative measures are required. This study investigates the clinical utility of a range of carefully selected visual function tests and patient reported outcome measures. The aim is to identify suitable outcome measures for future clinical trials that could be considered for regulatory approval. METHODS: This cross-sectional study involves two participant groups, patients with inherited retinal disease (n = 40) and healthy controls (n = 40). The study has been designed to be flexible and run alongside NHS clinics. The study is split into two parts. Part one includes examining standard visual acuity, low luminance visual acuity, the Moorfields acuity chart visual acuity, mesopic microperimetry and three separate patient reported outcome measures. Part two involves 20 min of dark adaptation followed by two-colour scotopic microperimetry. Repeat testing will be undertaken where possible to enable repeatability analyses. A subset of patients with inherited retinal disease will be invited to participate in a semi-structured interview to gain awareness of participants' thoughts and feelings around the study and different study tests. DISCUSSION: The study highlights a need for reliable and sensitive validated visual function measures that can be used in future clinical trials. This work will build on work from other studies and be used to inform an outcome measure framework for rod-cone degenerations. The study is in keeping with the United Kingdom Department of Health and Social Care research initiatives and strategies for increasing research opportunities for NHS patients as part of their NHS care. TRIAL REGISTRATION: ISRCTN registry, ISRCTN24016133, Visual Function in Retinal Degeneration, registered on 18th August 2022.

Harnessing the potential of practice-based clinical optometry research in the United Kingdom.

Research is the core of evidence-based practice across all healthcare, in order to ensure optimum patient care. The College of Optometrists is a national standard setting institution for optometric practice in the United Kingdom. However, the standards are only as good as the available evidence, and currently there is little evidence relating directly to optometric practice. The National Institute of Health and Care Research, the General Medical Council and The College of Optometrists, amongst others, have published research strategies describing ambitious plans to expand the scope of healthcare research. The aim of this article is to raise awareness of these government initiatives and consider how they may relate to optometric practice. To improve optometrist research engagement, we need to address the barriers to research and implement strategies to overcome them. There are many opportunities to support research, with different degrees of involvement, from signposting patients to research studies, supporting recruitment or collecting data for a multicentre clinical trial, as well as undertaking an individual research project. Healthcare research is changing and there is scope for more practice-based research activities in optometry. Research should not be a solo endeavour but a multi-disciplinary effort. Greater collaborations across all stakeholders, including primary care, secondary care, academia, regulators and industry is needed to make this possible.

The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification.

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye.

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.

Choroideremia: The Endpoint Endgame.

Choroideremia is an X-linked retinal degeneration resulting from the progressive, centripetal loss of photoreceptors and choriocapillaris, secondary to the degeneration of the retinal pigment epithelium. Affected individuals present in late childhood or early teenage years with nyctalopia and progressive peripheral visual loss. Typically, by the fourth decade, the macula and fovea also degenerate, resulting in advanced sight loss. Currently, there are no approved treatments for this condition. Gene therapy offers the most promising therapeutic modality for halting or regressing functional loss. The aims of the current review are to highlight the lessons learnt from clinical trials in choroideremia, review endpoints, and propose a future strategy for clinical trials.

Emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa.

INTRODUCTION: Mutations in the RPGR gene are responsible for one of the most prevalent and severe types of retinitis pigmentosa. Gene therapy has shown great promise to treat inherited retinal diseases, and currently, four RPGR gene therapy vectors are being evaluated in clinical trials. AREAS COVERED: This manuscript reviews the gene therapy products that are in development for X-linked retinitis pigmentosa caused by mutations in RPGR, and the challenges that scientists and clinicians have faced. EXPERT OPINION: The development of a gene therapy product for RPGR-associated retinal degeneration has been a great challenge due to the incomplete understanding of the underlying genetics and mechanism of action of RPGR, and on the other hand, due to the instability of the RPGR gene. Three of the four gene therapy vectors currently in clinical trials include a codon-optimized version of the human RPGR sequence, and the other vector contains a shortened version of the human RPGR. To date, the only Phase I/II results published in a peer-reviewed journal demonstrate a good safety profile and an improvement in the visual field using a codon optimized version of RPGRORF15.

EFFICACY OF HALF-DOSE PHOTODYNAMIC THERAPY VERSUS HIGH-DENSITY SUBTHRESHOLD MICROPULSE LASER FOR TREATING PIGMENT EPITHELIAL DETACHMENTS IN CHRONIC CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: Comparing the effect of half-dose photodynamic therapy and high-density subthreshold micropulse laser treatment on retinal pigment epithelial detachments (PEDs) in chronic central serous chorioretinopathy. METHODS: This study included data from the PLACE trial, a prospective randomized controlled trial comparing half-dose photodynamic therapy and high-density subthreshold micropulse laser treatment in chronic central serous chorioretinopathy. Main outcome measurements were changes in both the foveal PED and the highest PED within the macula at baseline compared with first and final evaluation visit. RESULTS: At baseline, a macular PED was detected in 76.9% of patients (123/160), and a PED within 1,500 µm from the foveal center in 37.5% of patients (60/160). In the half-dose photodynamic therapy arm (61 patients), there was a significantly larger decrease in the highest macular PED compared with the high-density subthreshold micropulse laser treatment arm (62 patients) at both first and final evaluation visits (P < 0.001 and P = 0.012, respectively). The decrease of highest foveal PED was significant at first visit (P = 0.025). CONCLUSION: Half-dose photodynamic therapy is superior to high-density subthreshold micropulse laser treatment with regard to a statistically significant reduction in the height of macular PEDs in active chronic central serous chorioretinopathy. These findings may also have implications for other diseases within the pachychoroid disease spectrum that can present with PEDs.

Gene therapy for inherited retinal disease: long-term durability of effect.

The recent approval of voretigene neparvovec (Luxturna®) for patients with biallelic RPE65 mutation-associated inherited retinal dystrophy with viable retinal cells represents an important step in the development of ocular gene therapies. Herein, we review studies investigating the episomal persistence of different recombinant adeno-associated virus (rAAV) vector genomes and the pre-clinical and clinical evidence of long-term effects of different RPE65 gene replacement therapies. A targeted review of articles published between 1974 and January 2021 in Medline®, Embase®, and other databases, was conducted, followed by a descriptive longitudinal analysis of the clinical trial outcomes of voretigene neparvovec. Following an initial screening, 14 publications examining the episomal persistence of different rAAV genomes and 71 publications evaluating gene therapies in animal models were included. Viral genomes were found to persist for at least 22 months (longest study follow-up) as transcriptionally active episomes. Treatment effects lasting almost a decade were reported in canine disease models, with more pronounced effects the earlier the intervention. The clinical trial outcomes of voretigene neparvovec are consistent with pre-clinical findings and reveal sustained results for up to 7.5 years for the full-field light sensitivity threshold test and 5 years for the multi-luminance mobility test in the Phase I and Phase III trials, respectively. In conclusion, the therapeutic effect of voretigene neparvovec lasts for at least a decade in animal models and 7.5 years in human subjects. Since retinal cells can retain functionality over their lifetime after transduction, these effects may be expected to last even longer in patients with a sufficient number of outer retinal cells at the time of intervention.

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.

Minicircle Delivery to the Neural Retina as a Gene Therapy Approach.

Non-viral gene therapy has the potential to overcome several shortcomings in viral vector-based therapeutics. Methods of in vivo plasmid delivery have developed over recent years to increase the efficiency of non-viral gene transfer, yet further improvements still need to be made to improve their translational capacity. Gene therapy advances for inherited retinal disease have been particularly prominent over the recent decade but overcoming physical and physiological barriers present in the eye remains a key obstacle in the field of non-viral ocular drug delivery. Minicircles are circular double-stranded DNA vectors that contain expression cassettes devoid of bacterial DNA, thereby limiting the risks of innate immune responses induced by such elements. To date, they have not been extensively used in pre-clinical studies yet remain a viable vector option for the treatment of inherited retinal disease. Here, we explore the potential of minicircle DNA delivery to the neural retina as a gene therapy approach. We consider the advantages of minicircles as gene therapy vectors as well as review the challenges involved in optimising their delivery to the neural retina.

Prevalence and phenotype associations of complement factor I mutations in geographic atrophy.

Rare variants in the complement factor I (CFI) gene, associated with low serum factor I (FI) levels, are strong risk factors for developing the advanced stages of age-related macular degeneration (AMD). No studies have been undertaken on the prevalence of disease-causing CFI mutations in patients with geographic atrophy (GA) secondary to AMD. A multicenter, cross-sectional, noninterventional study was undertaken to identify the prevalence of pathogenic rare CFI gene variants in an unselected cohort of patients with GA and low FI levels. A genotype-phenotype study was performed. Four hundred and sixty-eight patients with GA secondary to AMD were recruited to the study, and 19.4% (n = 91) demonstrated a low serum FI concentration (below 15.6 µg/ml). CFI gene sequencing on these patients resulted in the detection of rare CFI variants in 4.7% (n = 22) of recruited patients. The prevalence of CFI variants in patients with low serum FI levels and GA was 25%. Of the total patients recruited, 3.2% (n = 15) expressed a CFI variant classified as pathogenic or likely pathogenic. The presence of reticular pseudodrusen was detected in all patients with pathogenic CFI gene variants. Patients with pathogenic CFI gene variants and low serum FI levels might be suitable for FI supplementation in therapeutic trials.

Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.

Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).

An analysis of the Kozak consensus in retinal genes and its relevance to gene therapy.

Purpose: The classic Kozak consensus is a critical genetic element included in gene therapy transgenes to encourage the translation of the therapeutic coding sequence. Despite optimizations of other transgene elements, the Kozak consensus has not yet been considered for potential tissue-specific sequence refinement. We screened the -9 to -1 region relative to the AUG start codon of retina-specific genes to identify whether a Kozak consensus that is different from the classic sequence may be more appropriate for inclusion in gene therapy transgenes that treat inherited retinal disease. Methods: Sequences for 135 genes known to cause nonsyndromic inherited retinal disease were extracted from the NCBI database, and the -9 to -1 nucleotides were compared. This panel was then refined to 75 genes with specific retinal functions, for which the -9 to -1 nucleotides were placed in front of a GFP transcript sequence and RNAfold predictions performed. These were compared with a GFP sequence with the classic Kozak consensus (GCCGCCACC), and sequences from retinal genes with minimum free energy (MFE) predictions greater than the reference sequence were selected to generate an optimized Kozak consensus sequence. The original Kozak consensus and the refined retina Kozak consensus were placed upstream of the Renilla luciferase coding sequence, which were used to transfect retinoblastoma cell lines Y-79 and WERI-RB-1 and HEK 293T/17 cells. Results: The nucleotide frequencies of the original panel of genes were determined to be comparable to the classic Kozak consensus. RNAfold analysis of a GFP transcript with the classic Kozak sequence in the 5' untranslated region (UTR) generated an MFE prediction of -503.3 kcal/mol. RNAfold analysis was then performed with a GFP transcript containing each -9 to -1 Kozak sequence of 75 retinal genes. Thirty-eight of the 75 genes provided a greater MFE value than -503.3 kcal/mol and exhibited an absence of stable secondary structures before the AUG codon. The -9 to -1 nucleotide frequencies of these genes identified a Kozak consensus of ACCGAGACC, differing from the classic Kozak consensus at positions -9, -5, and -4. Applying this sequence to the GFP transcript increased the MFE prediction to -500.1 kcal/mol. The newly identified retina Kozak sequence was also applied to Renilla luciferase plus the REP1 and RPGR transcripts used in current clinical trials. In all examples, the predicted transcript MFE score increased when compared with the current transcript sequences containing classic Kozak consensus sequences. In vitro transfections identified a 7%-9% increase in Renilla activity when incorporating the optimized Kozak sequence. Conclusions: The Kozak consensus is a critical element of eukaryotic genes; therefore, it is a required feature of gene therapy transgenes. To date, the classic sequence of GCCRCC (-6 to -1) has typically been incorporated in gene therapy transgenes, but the analysis described here suggests that, for vectors targeting the retina, using a Kozak consensus derived from retinal genes can provide increased expression of the target product.

Deep phenotyping of the Cdhr1-/- mouse validates its use in pre-clinical studies for human CDHR1-associated retinal degeneration.

PURPOSE: To validate the Cdhr1-/- mouse as a model for human CDHR1-associated retinal degeneration, which may present as cone-rod dystrophy or geographic atrophy. METHODS: Deep phenotyping of Cdhr1-/-(n = 56) and C57BL6J wildtype control mice (n = 45) was undertaken using in vivo multimodal retinal imaging and dark- and light-adapted electroretinography (ERG) over 15 months to evaluate rod- and cone-photoreceptor responses and retinal morphology. RESULTS: Cdhr1-/- retinas exhibited outer retinal thinning on optical coherence tomography (OCT) at 1-month versus C57BL6J (mean 14.6% reduction; P < 0.0001), with progressive degeneration to 15 months. The OCT layer representing photoreceptor outer segments was more significantly shortened in Cdhr1-/- eyes at 1 month (mean 33.7% reduction; P < 0.0001), remained stable to 3 months and was not identifiable at later timepoints. Outer retinal thinning was more pronounced at inferior versus superior retinal locations in Cdhr1-/- eyes (P < 0.002 at 3-9 months). Dark-adapted ERG identified severe functional deficits in Cdhr1-/- mice at 1 month of age versus C57BL6J (mean 62% reduction) that continued to decline to 15 months (P < 0.0001). Light-adapted flicker identified severe deficits in cone function at 1 month (mean 70% reduction), with improved function to 3 months followed by progressive decline (P < 0.0001). CONCLUSIONS: The Cdhr1-/- mouse exhibits structural and functional evidence of progressive outer retinal degeneration at a slow rate. Early functional deficits affecting both rod and cone photoreceptors in the context of relatively mild structural changes reflect the human phenotype. This study validates the use of the Cdhr1-/- mouse for the pre-clinical evaluation of therapeutics for human CDHR1-associated retinal degeneration.

ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.

PURPOSE: Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. METHODS: Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. RESULTS: A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. CONCLUSION: Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies.

Chimeric human opsins as optogenetic light sensitisers.

Human opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild-type LWS opsin and LWS opsin/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry (ICC) demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies.