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Background: Landmark studies reported on faricimab efficacy and safety predominantly in treatment naïve patients, but outcomes following switch from other anti-VEGF therapies are lacking. We evaluated patients switched to faricimab who had previously shown a partial response to other anti-VEGF injections for neovascular age-related macular degeneration (nAMD) and diabetic macular oedema (DMO)., Methods: Retrospective study at the Oxford Eye Hospital. Patients switched to faricimab from January to April 2023 with six months follow-up were identified via electronic medical records., Results: A total of 116 patients (151 eyes) were included. In 88 patients with nAMD (107 eyes), mean visual acuity remained stable: 62±17 ETDRS letters at baseline; 62±18 at six months (p > 0.05). Central subfield thickness (CST) reduced from 294 ± 73 μm to 270 ± 53 μm (p < 0.05) at six months. Subretinal or intraretinal fluid was present in 102 eyes (95%) at baseline and 75 eyes (70%) at follow-up (p < 0.05). Pigment epithelial detachment height decreased from 233 ± 134 μm to 188 ± 147 μm (p < 0.05). Mean treatment interval increased by 1.7 weeks (p < 0.05) and was extended in 61 eyes (57%) at six months. In 28 patients with DMO (44 eyes), visual acuity remained stable: 69 ± 15 letters at baseline; 70±15 at six months (p > 0.05). CST reduced from 355 ± 87 μm to 317 ± 82 μm (p < 0.05). Mean treatment interval increased by 1.4 weeks (p < 0.05) and was extended in 21 eyes (46%) by six months., Conclusions: Switching to faricimab in treatment resistant eyes led to improved anatomical response and extended treatment interval in a significant proportion of patients. Ongoing review of real-world data will inform longer-term outcomes of safety and effectiveness., (© 2024. The Author(s).)

Background: Danon disease is a rare, multisystemic X-linked dominant disorder caused by variants in the LAMP2 gene. It can be associated with retinal degeneration, but this is not well characterized. Here we describe a late presentation of a mild retinal phenotype, initially diagnosed as choroideremia carrier, associated with a novel variant in the LAMP2 gene., Methods: Retrospective analysis of the case included medical history, ophthalmic examination, multimodal retinal imaging, and microperimetry. Genetic testing was conducted to establish the molecular diagnosis., Results: A 54-year-old female presented with worsening night vision, without any family history. BCVA was 6/6 bilaterally and fundus examination showed light peripheral pigmentary changes bilaterally. FAF demonstrated a widespread speckled pattern and OCT revealed hyper-reflective spots in the outer nuclear layer. Differentials included non-genetic and genetic causes, suspected of being a manifesting choroideremia carrier. However, initial genetic testing by targeted analysis of retinal disorders did not detect a pathogenic variant. Further systems review revealed that the patient had previously been diagnosed with dilated cardiomyopathy, mini-stroke and partial deafness. Subsequent whole mitochondrial genome sequencing analysis did not detect any pathogenic variants too. Finally, whole exome sequencing with targeted analysis of a panel of hypertrophic cardiomyopathy genes identified a novel pathogenic heterozygous variant (c.925del, p.(Ser309fs)) in the LAMP2 gene, confirming the diagnosis of X-linked Danon disease., Conclusion: Recording previous medical history and extraocular symptoms is crucial. The similarity in choroideremia carrier and Danon disease retinal phenotypes suggests a possible common pathway in these two genes where pathogenic variants lead to retinal pigment epithelium degeneration.

Wagner syndrome is a rare autosomal dominant vitreoretinopathy caused by mutations in chondroitin sulphate proteoglycan 2 (CSPG2)/Versican (VCAN). Here, we present a retrospective case series of a family pedigree with genetically confirmed Wagner syndrome (heterozygous VCAN exon 8 deletion), as follows: a 34-year-old mother (P1), 12-year-old daughter (P2), and a 2-year-old son (P3). The phenotype included early-onset cataract (P1), optically empty vitreous with avascular membranes (P1, 2), nasal dragging of optic nerve heads associated with foveal hypoplasia (P1, 2), tractional retinoschisis on optical coherence tomography (P2), and peripheral circumferential vitreo-retinal interface abnormality resembling white-without-pressure (P3) progressing to pigmented chorio-retinal atrophy (P1, 2). P2 developed a macula-off retinal detachment, which was treated initially with encircling band + vitrectomy + gas, followed by vitrectomy + heavy silicone oil tamponade for re-detachment from new inferior breaks. Strong vitreo-retinal adhesion was noted intraoperatively, which prevented the separation of posterior hyaloid beyond the equator. Electroretinograms from P1&2 demonstrated attenuated b-waves, a-waves, and flicker responses in light- and dark-adapted conditions, suggestive of generalised retinal dysfunction. Our patients demonstrated the clinical spectrum of Wagner syndrome, highlighting nasal dragging with foveal disruption as a distinguishing feature from other inherited vitreoretinopathies. Surgical outcomes demonstrate significant challenges in managing vitreo-retinal traction and need for further research into strategies to prevent sight loss.

Background: CFAP410 (Cilia and Flagella Associated Protein 410) encodes a protein that has an important role in the development and function of cilia. In ophthalmology, pathogenic variants in CFAP410 have been described in association with cone rod dystrophy, retinitis pigmentosa, with or without macular staphyloma, or with systemic abnormalities such as skeletal dysplasia and amyotrophic lateral sclerosis. Herein, we report a consanguineous family with a novel homozygous CFAP410 c.335&#95;346del variant with cone only degeneration and no systemic features., Methods: A retrospective analysis of ophthalmic history, examination, retinal imaging, electrophysiology and microperimetry was performed as well as genetic testing with in silico pathogenicity predictions and a literature review., Results: A systemically well 28-year-old female of Pakistani ethnicity with parental consanguinity and no relevant family history, presented with childhood-onset poor central vision and photophobia. Best-corrected visual acuity and colour vision were reduced (0.5 LogMAR, 6/17 Ishihara plates (right) and 0.6 LogMAR, 3/17 Ishihara plates (left). Fundus examination showed no pigmentary retinopathy, no macular staphyloma and autofluorescence was unremarkable. Optical coherence tomography showed subtle signs of intermittent disruption of the ellipsoid zone. Microperimetry demonstrated a reduction in central retinal sensitivity. Electrodiagnostic testing confirmed a reduction in cone-driven responses. Whole-genome sequencing identified an in-frame homozygous deletion of 12 base pairs at c.335&#95;346del in CFAP410 ., Conclusions: The non-syndromic cone dystrophy phenotype reported herein expands the genotypic and phenotypic spectra of CFAP410 -associated ciliopathies and highlights the need for light of potential future genetic therapies.

Purpose: Clinical trials for X-linked retinitis pigmentosa (RP) often assess retinal structure using optical coherence tomography (OCT) and function using microperimetry to evaluate initial eligibility and endpoints. Therefore, we seek to determine which parameters might be most sensitive in screening new patients for enrollment., Methods: Thirty-one patients (62 eyes) with confirmed retinitis pigmentosa GTPase regulator (RPGR) mutations attending Oxford Eye Hospital were included in this retrospective analysis. Outer retinal structure was investigated by measuring the remaining ellipsoid zone (EZ) and external limiting membrane (ELM) on OCT. Visual function was evaluated by using 10-2 microperimetry mean sensitivity., Results: The median age of patients with RPGR was 31 years (interquartile range [IQR] = 22-39 years). For the right and left eyes, respectively, the median EZ length through the foveal section was 921 µm (IQR = 607-1570) and 865 µm (IQR = 508-1442) and median ELM length was 2056 µm (IQR = 1336-2764) and 1860 µm (IQR = 1152-2680). Similarly, the median microperimetry sensitivity (MS) was 2.0 decibel (dB; IQR = 0.4-5.4) and 1.1 dB (IQR = 0.1-5.4). Linear mixed model regression analysis showed that EZ was significantly positively correlated to ELM (P < 0.001, R² = 0.931). EZ and ELM were significantly correlated with the microperimetry sensitivity with exponential relationship (P < 0.001, R² = 0.71 and 0.72, respectively). Using the exponential equation of regression line, EZ below approximately 600 µm (RE = 637 µm, 95% confidence interval [CI] = 397-877, LE = 586 µm, 95% CI = 356-817) results in microperimetry sensitivity of approximately 0 dB. There was high degree of inter-eye symmetry for progression of EZ, ELM, and microperimetry sensitivity. Age was significantly correlated with the analyzed parameters (P < 0.001), although with low R² for each of them., Discussion: EZ may comprise a surrogate biomarker for prediction of visual function in X-linked RP caused by mutations in RPGR and, in turn, identification of appropriate patients for enrollment in clinical trials. As expected, age plays a role in predicting potential biomarkers and visual function, however, it should not be used to preclude patients for gene therapy due to the poor correlation and heterogeneity of disease onset., Translational Relevance: Biomarkers of visual function in RPGR-associated RP may lead to identification of appropriate patients for enrollment in clinical trials.

Introduction: Retinal focal nodular gliosis (FNG), also known as vasoproliferative tumors (VPTs), are rare, benign vascular tumors associated with exudation with no current consensus on management. Herein, we describe the varied clinical course and management of 3 patients with retinal FNG, one of whom is associated with retinitis pigmentosa., Case Presentations: Case 1 is a 76-year-old female who presented with reduced vision and distortion secondary to a vitreous hemorrhage and epiretinal membrane (ERM) as complications of a known small peripheral retinal FNG. She underwent vitrectomy for the hemorrhage to relieve vascular traction and the ERM peel, and the tumor was kept under observation. Case 2 is a 24-year-old female with genetically uncharacterized retinitis pigmentosa-like phenotype who presented with gradual loss of central vision in one eye due to cystoid macular oedema (CMO). She was found to have two peripheral retinal areas of FNG located inferonasally. Tumors were treated with cryotherapy and adjuvant intraocular steroid implant to control the CMO. Case 3 is a 28-year-old female with retinitis pigmentosa secondary to genetically confirmed variant in CRB1 gene who presented with intractable right eye CMO and localized inferior serous retinal detachment secondary to a large inferotemporal FNG. Her left eye has no light perception vision due to previous extensive serous retinal detachment and anterior segment ischemia. The right eye tumor was managed with multiple rounds of cryotherapy and laser therapy to control the serous detachment. Despite this, the condition progressed and was ultimately treated with plaque brachytherapy. Unfortunately, this resulted in extensive retinal inflammation causing annular tractional retinal detachment which was treated with combined pars plana vitrectomy and scleral buckle., Conclusion: We characterized the retinal phenotype of 3 patients with retinal FNG (VPTs) and found them to have varied clinical courses requiring tailored surgical management. The case associated with retinitis pigmentosa had a known pathogenic variant in Crumbs homolog-1 (CRB1) gene affecting retinal structure and exhibited a more severe clinical course. It is therefore important for patients with retinal dystrophies to undergo thorough peripheral examinations and detect FNG early as they may require prompt, aggressive treatment., Competing Interests: None of the above authors have any conflicts of interests., (© 2024 S. Karger AG, Basel.)

Conventional gene therapy involving supplementation only treats loss-of-function diseases and is limited by viral packaging sizes, precluding therapy of large genes. The discovery of CRISPR/Cas has led to a paradigm shift in the field of genetic therapy, with the promise of precise gene editing, thus broadening the range of diseases that can be treated. The initial uses of CRISPR/Cas have focused mainly on gene editing or silencing of abnormal variants via utilising Cas endonuclease to trigger the target cell endogenous non-homologous end joining. Subsequently, the technology has evolved to modify the Cas enzyme and even its guide RNA, leading to more efficient editing tools in the form of base and prime editing. Further advancements of this CRISPR/Cas technology itself have expanded its functional repertoire from targeted editing to programmable transactivation, shifting the therapeutic focus to precise endogenous gene activation or upregulation with the potential for epigenetic modifications. In vivo experiments using this platform have demonstrated the potential of CRISPR-activators (CRISPRa) to treat various loss-of-function diseases, as well as in regenerative medicine, highlighting their versatility to overcome limitations associated with conventional strategies. This review summarises the molecular mechanisms of CRISPRa platforms, the current applications of this technology in vivo, and discusses potential solutions to translational hurdles for this therapy, with a focus on ophthalmic diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Ltd.)

The use of robotic surgery in ophthalmology has been shown to offer many potential advantages to current surgical techniques. Vitreoretinal surgery requires complex manoeuvres and high precision, and this is an area that exceeds manual human dexterity in certain surgical situations. With the advent of advanced therapeutics such as subretinal gene therapy, precise delivery and minimising trauma is imperative to optimize outcomes. There are multiple robotic systems in place for ophthalmology in pre-clinical and clinical use, and the Preceyes Robotic Surgical System (Preceyes BV) has also gained the CE mark and is commercially available for use. Recent in-vivo and in-human surgeries have been performed successfully with robotics systems. This includes membrane peeling, subretinal injections of therapeutics, and retinal vein cannulation. There is huge potential to integrate robotic surgery into mainstream clinical practice. In this review, we summarize the existing systems, and clinical implementation so far, and highlight the future clinical applications for robotic surgery in vitreo-retina., (© 2024. The Author(s).)

Age-related macular degeneration (AMD) is a growing public health concern given the aging population and it is the leading cause of blindness in developed countries, affecting individuals over the age of 55 years. AMD affects the retinal pigment epithelium (RPE) and Bruch's membrane in the macula, leading to secondary photoreceptor degeneration and eventual loss of central vision. Late AMD is divided into two forms: neovascular AMD and geographic atrophy (GA). GA accounts for around 60% of late AMD and has been the most challenging subtype to treat. Recent advances include approval of new intravitreally administered therapeutics, pegcetacoplan (Syfovre) and avacincaptad pegol (Iveric Bio), which target complement factors C3 and C5, respectively, which slow down the rate of enlargement of the area of atrophy. However, there is currently no treatment to reverse the central vision loss associated with GA. Optogenetics may provide a strategy for rescuing visual function in GA by imparting light-sensitivity to the surviving inner retina (i.e., retinal ganglion cells or bipolar cells). It takes advantage of residual inner retinal architecture to transmit visual stimuli along the visual pathway, while a wide range of photosensitive proteins are available for consideration. Herein, we review the anatomical changes in GA, discuss the suitability of optogenetic therapeutic sensors in different target cells in pre-clinical models, and consider the advantages and disadvantages of different routes of administration of therapeutic vectors., Competing Interests: REM is a co-founder and director of Beacon Therapeutics, a retinal gene therapy company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Borchert, Shamsnajafabadi, Ng, Xue, De Silva, Downes, MacLaren and Cehajic-Kapetanovic.)

Purpose: Surgical innovation in ophthalmology is impeded by the physiological limits of human motion, and robotic assistance may facilitate an expansion of the surgical repertoire. We conducted a systematic review to identify ophthalmic procedures in which robotic systems have been trialled, evaluate their performance, and explore future directions for research and development of robotic techniques., Methods: The Cochrane Library, Embase, MEDLINE, Scopus, and Web of Science were searched. Screening adhered to five criteria: (1) English language; (2) primary research article; (3) human patients; (4) ophthalmological surgery; and (5) robot-assisted surgery. Quality assessment was conducted with Joanna Briggs Institute Tools for Critical Appraisal. The study protocol was registered prospectively (PROSPERO ID CRD42023449793)., Results: Twelve studies were included. In comparative studies, there was no difference in the occurrence of ocular harms in robot-assisted procedures and conventional surgery. However, robotic assistance did not demonstrate consistent benefits over manual surgery in terms of effectiveness or practicality, likely reflecting the learning curve associated with these systems. Single studies indicated the potential of robotic assistance to improve the consistency of subretinal drug infusion and efficiency of instrument manipulation in vitreoretinal surgery., Conclusions: Proof-of-concept studies have demonstrated the potential of robotic assistance to facilitate procedures otherwise infeasible or impractical, and may broaden access to surgery. However, robot-assisted surgery has not yet demonstrated any significant benefits over standard surgical practice. Improving the speed and reducing perioperative requirements of robot-assisted surgery are particular priorities for research and innovation to improve the practicality of these novel techniques., Translational Relevance: This systematic review summarizes the potential and limitations of robotic systems for assisting eye surgery and outlines what is required for these systems to benefit patients and surgeons.

Background: Ciliopathies responsible for retinitis pigmentosa can also cause systemic manifestations. RPGR is a ciliary gene and pathogenic variants in RPGR cause a retinal ciliopathy, the commonest cause of X-linked recessive retinitis pigmentosa. The RPGR protein interacts with numerous other ciliary proteins present in the transition zone of both motile and sensory cilia, and may play an important role in regulating ciliary protein transport. There has been a growing, putative association of RPGR variants with systemic ciliopathies: mainly sino-respiratory infections and primary ciliary dyskinesia., Materials and Methods: Retrospective case series of patients with RPGR-RP presenting to Oxford Eye Hospital with systemic disease., Results: We report three children with RPGR-related rod-cone dystrophy, all of whom have mutations in the N-terminus of RPGR. Two cases co-presented with confirmed diagnoses of primary ciliary dyskinesia and one case with multiple sino-respiratory symptoms strongly suggestive of primary ciliary dyskinesia. These and all previously reported RPGR co-pathologies relate to ciliopathies and have no other systemic associations., Conclusions: The link between RPGR variants and a systemic ciliopathy remains plausible, but currently unproven.

Our study evaluated the morphological and functional outcomes, and the side effects, of voretigene neparvovec (VN) gene therapy for RPE65-mediated inherited retinal dystrophies (IRDs) in 12 eyes (six patients) at the Oxford Eye Hospital with a mean follow-up duration of 8.2 (range 1-12) months. All patients reported a subjective vision improvement 1 month after gene therapy. Best-corrected visual acuity (BCVA) remained stable (baseline: 1.28 (±0.71) vs. last follow-up: 1.46 (±0.60); p = 0.25). Average white Full-Field Stimulus Testing (FST) showed a trend towards improvement (baseline: -4.41 (±10.62) dB vs. last follow-up: -11.98 (±13.83) dB; p = 0.18). No changes in central retinal thickness or macular volume were observed. The side effects included mild intraocular inflammation (two eyes) and cataracts (four eyes). Retinal atrophy occurred in 10 eyes (eight mild, two severe) but did not impact FST measurements during the follow-up period. Increased intraocular pressure (IOP) was noted in three patients (six eyes); four eyes (two patients) required glaucoma surgery. The overall safety and effectiveness of VN treatment in our cohort align with previous VN clinical trials, except for the higher occurrence of retinal atrophy and increased IOP in our cohort. This suggests that raised IOP and retinal atrophy may be more common than previously reported.

Purpose: In patients with choroideremia, it is not known how smooth and mottled patterns on short-wavelength fundus autofluorescence (AF) imaging relate to retinal function., Methods: A retrospective case-note review was undertaken on 190 patients with choroideremia at two specialist centers for retinal genetics. Twenty patients with both smooth and mottled zones on short-wavelength AF imaging and concurrent mesopic microperimetry assessments were included. Mean retinal sensitivities within the smooth and mottled zones were compared between choroideremia patients, and identical points on mesopic microperimetry collected from 12 age-matched controls. Longitudinal analyses were undertaken at 2 and 5 years in a subset of patients., Results: In patients with choroideremia, mean retinal sensitivities at baseline were significantly greater in the smooth zone (26.1 ± 2.0 dB) versus the mottled zone (20.5 ± 4.2 dB) (P < 0.0001). Mean retinal sensitivities at baseline were similar in the smooth zone between choroideremia patients and controls (P = 0.054) but significantly impaired in the mottled zone in choroideremia compared to controls (P < 0.0001). The rate of decline in total sensitivity over 5 years was not significant in either the smooth or mottled zone in a small subset of choroideremia patients (n = 7; P = 0.344)., Conclusions: In choroideremia, retinal sensitivity as determined by microperimetry correlates with patterns on AF imaging: retinal function in the smooth zone, where the retinal pigment epithelium is anatomically preserved, is similar to controls, but retinal sensitivity in the mottled zone is impaired., Translational Relevance: Patterns on AF imaging may represent a novel, objective outcome measure for clinical trials in choroideremia as a surrogate for retinal function.

Age-related macular degeneration (AMD) is the leading cause of vision loss and visual impairment in people over 50 years of age. In the current therapeutic landscape, intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapies have been central to the management of neovascular AMD (also known as wet AMD), whereas treatments for geographic atrophy have lagged behind. Several therapeutic approaches are being developed for geographic atrophy with the goal of either slowing down disease progression or reversing sight loss. Such strategies target the inflammatory pathways, complement cascade, visual cycle or neuroprotective mechanisms to slow down the degeneration. In addition, retinal implants have been tried for vision restoration and stem cell therapies for potentially a dual purpose of slowing down the degeneration and restoring visual function. In particular, therapies focusing on the complement pathway have shown promising results with the FDA approved pegcetacoplan, a complement C3 inhibitor, and avacincaptad pegol, a complement C5 inhibitor. In this review, we discuss the mechanisms of inflammation in AMD and outline the therapeutic landscapes of atrophy AMD. Improved understanding of the various pathway components and their interplay in this complex neuroinflammatory degeneration will guide the development of current and future therapeutic options, such as optogenetic therapy.

Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial genetic disease that causes blindness in young adults. Over 50 inherited mitochondrial DNA (mtDNA) variations are associated with LHON; however, more than 95% of cases are caused by one of three missense variations (m.11778 G > A, m.3460 G > A, and m.14484 T > C) encoding for subunits ND4, ND1, and ND6 of the respiration complex I, respectively. These variants remain silent until further and currently poorly understood genetic and environmental factors precipitate the visual loss. The clinical course that ensues is variable, and a convincing treatment for LHON has yet to emerge. In 2015, an antioxidant idebenone (Raxone) received European marketing authorisation to treat visual impairment in patients with LHON, and since then it was introduced into clinical practice in several European countries. Alternative therapeutic strategies, including gene therapy and gene editing, antioxidant and neurotrophic agents, mitochondrial biogenesis, mitochondrial replacement, and stem cell therapies are being investigated in how effective they might be in altering the course of the disease. Allotopic gene therapies are in the most advanced stage of development (phase III clinical trials) whilst most other agents are in phase I or II trials or at pre-clinical stages. This manuscript discusses the phenotype and genotype of the LHON disease with complexities and peculiarities such as incomplete penetrance and gender bias, which have challenged the therapies in development emphasising the most recent use of gene therapy. Furthermore, we review the latest results of the three clinical trials based on adeno-associated viral (AAV) vector-mediated delivery of NADH dehydrogenase subunit 4 (ND4) with mitochondrial targeting sequence, highlighting the differences in the vector design and the rationale behind their use in the allotopic transfer.

Late-stage retinal degenerative disease involving photoreceptor loss can be treated by optogenetic therapy, cell transplantation and retinal prostheses. These approaches aim to restore light sensitivity to the retina as well as visual perception by integrating neuronal responses for transmission to the cortex. In age-related macular degeneration, some cell-based therapies also aim to restore photoreceptor-supporting tissue to prevent complete photoreceptor loss. In the earlier stages of degeneration, gene-replacement therapy could attenuate retinal-disease progression and reverse loss of function. And gene-editing strategies aim to correct the underlying genetic defects. In this Review, we highlight the most promising gene therapies, cell therapies and retinal prostheses for the treatment of retinal disease, discuss the benefits and drawbacks of each treatment strategy and the factors influencing whether functional tissue is reconstructed and repaired or replaced with an electronic device, and summarize upcoming technologies for enhancing the restoration of vision., (© 2022. Springer Nature Limited.)

Inherited retinal diseases (IRDs) are associated with mutations in over 250 genes and represent a major cause of irreversible blindness worldwide. While gene augmentation or gene editing therapies could address the underlying genetic mutations in a small subset of patients, their utility remains limited by the great genetic heterogeneity of IRDs and the costs of developing individualised therapies. Gene-agnostic therapeutic approaches target common pathogenic pathways that drive retinal degeneration or provide functional rescue of vision independent of the genetic cause, thus offering potential clinical benefits to all IRD patients. Here, we review the key gene-agnostic approaches, including retinal cell reprogramming and replacement, neurotrophic support, immune modulation and optogenetics. The relative benefits and limitations of these strategies and the timing of clinical interventions are discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 John, Quinn, Hu, Cehajic-Kapetanovic and Xue.)

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 RPGR ORF15 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 RPGR ORF15 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 RPGR ORF15 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 RPGR ORF15 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.

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 RPGR ORF15 .

Purpose: Peripheral visual fields have not been as well defined by static automated perimetry as kinetic perimetry in RPGR-related retinitis pigmentosa. This study explores the pattern and sensitivities of peripheral visual fields, which may provide an important end point when assessing interventional clinical trials., Methods: A retrospective observational cross-sectional study of 10 genetically confirmed RPGR subjects was performed. Visual fields were obtained using the Octopus 900 perimeter. Interocular symmetry and repeatability were quantified. Visual fields were subdivided into central and peripheral subfields for analysis., Results: Mean patient age was 32 years old (20 to 49 years old). Average mean sensitivity was 7 dB (SD = 3.67 dB) and 6.8 dB (SD = 3.4 dB) for the right and left eyes, respectively, demonstrating interocular symmetry. Coefficient of repeatability for overall mean sensitivity: <2 dB. Nine out of 10 subjects had a preserved inferotemporal subfield, whose mean sensitivity was highly correlated to the central field (r2 = 0.78, P = 0.002 and r2 = 0.72, P = 0.002 for the right and left eyes, respectively). Within the central field, sensitivities were greater in the temporal than the nasal half (t-test, P = 0.01 and P = 0.03 for the right and left eyes, respectively)., Conclusions: Octopus static-automated perimeter demonstrates good repeatability. Interocular symmetry permits use of the noninterventional eye as an internal control. In this cohort, the inferotemporal and central visual fields are preserved into later disease stages likely mapping to populations of surviving cones., Translational Relevance: A consistently preserved inferotemporal island of vision highly correlated to that of the central visual field may have significance as a possible future therapeutic site.

Purpose: To report the results of a first-in-human study using a robotic device to assist subretinal drug delivery in patients undergoing vitreoretinal surgery for macular hemorrhage., Design: Double-armed, randomized controlled surgical trial (ClinicalTrials.gov identifier: NCT03052881)., Methods: The study was performed at the Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom. In total, 12 participants were recruited-6 in the robot-assisted and 6 in the control manual surgery arm according to the prespecified inclusion and exclusion criteria. All subjects presented with acute loss of vision owing to a subfoveal hemorrhage secondary to neovascular age-related macular degeneration. After standard vitrectomy, intraoperative optical coherence tomography-guided subretinal injection of tissue plasminogen activator (TPA) was performed by either robot-assisted or conventional manual technique under local anesthesia. The robotic part of the procedure involved advancement of a cannula through the retina and stabilizing it during foot-controlled injection of up to 100 µL of TPA solution. We assessed surgical success, duration of surgery, adverse events, and tolerability of surgery under local anesthesia., Results: The procedure was well tolerated by all participants and safely performed in all cases. Total duration of surgery, time taken to complete the injection, and retinal microtrauma were similar between the groups and not clinically significant. Subretinal hemorrhage was successfully displaced at 1 month postintervention, except for 1 control subject, and the median gain in visual acuity was similar in both arms., Conclusions: This first-in-human study demonstrates the feasibility and safety of high-precision robot-assisted subretinal drug delivery as part of the surgical management of submacular hemorrhage, simulating its potential future application in gene or cell therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Mean retinal sensitivity is the main output measure used in microperimetry. It is, however, of limited use in patients with poor vision because averaging is weighted toward zero in those with significant scotomas creating an artificial floor effect. In contrast, volumetric measures avoid these issues and are displayed graphically as a hill of vision., Methods: An open-source program was created to manipulate raw sensitivity threshold data files obtained from MAIA microperimetry. Thin plate spline interpolated heat maps and three-dimensional hill of vision plots with an associated volume were generated. Retrospective analyses of microperimetry volumes were undertaken in patients with a range of retinal diseases to assess the qualitative benefits of three-dimensional visualization and volumetric measures. Simulated pathology was applied to radial grid patterns to investigate the performance of volumetric sensitivity in nonuniform grids., Results: Volumetric analyses from microperimetry in RPGR-related retinitis pigmentosa, choroideremia, Stargardt disease, and age-related macular degeneration were analyzed. In simulated nonuniform testing grids, volumetric sensitivity was able to detect differences in retinal sensitivity where mean sensitivity could not., Conclusions: Volumetric measures do not suffer from averaging issues and demonstrate superior performance in nonuniform testing grids. Additionally, volume measures enable detection of localized retinal sensitivity changes that might otherwise be undetectable in a mean change., Translational Relevance: As microperimetry has become an outcome measure in several gene-therapy clinical trials, three-dimensional visualization and volumetric sensitivity enables a complementary analysis of baseline disease characteristics and subsequent response to treatment, both as a signal of safety and efficacy.

Purpose: To report the use of flying baby spectral domain optical coherence tomography (SD-OCT) on infants with advanced retinopathy of prematurity (ROP), where clinical findings alone failed to differentiate between retinoschisis and retinal detachment., Methods: Prospective, non-interventional case-series study of three premature infants with advanced ROP of clinically uncertain stage, after examination by indirect ophthalmoscopy. To confirm the diagnosis, table-mounted SD-OCT retinal imaging was performed with the infant held in the flying baby position under topical ocular anaesthesia only. Spectral domain optical coherence tomography (SD-OCT) findings were correlated with clinical examination and ultra-widefield scanning laser ophthalmoscopy to determine disease stage and appropriate management., Results: The flying baby position was well tolerated, and SD-OCT images of central and peripheral retina were successfully obtained in all three cases. Additional information provided by the SD-OCT changed the ROP staging from 3 to 4 in one case, which subsequently required surgical treatment. In two other cases, clinical suspicion of stage 4 ROP was overruled as SD-OCT revealed tractional retinoschisis rather than full-thickness retinal detachment, thereby avoiding the need for immediate surgical intervention., Conclusions: In this case-series study, flying baby SD-OCT provided a rapid and widely accessible imaging approach that overruled clinical findings and altered classification and management of infants with advanced ROP. The methodology was suitable for outpatient settings with no risks associated with systemic anaesthesia. The increased use of OCT imaging will make apparent how structural information is useful in management of ROP and may influence future classification of the disease., (© 2020 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Inherited retinal degenerations (IRDs) are a leading cause of blindness. Although gene-supplementation therapies have been developed, they are only available for a small proportion of recessive IRD mutations. In contrast, genome editing using clustered-regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated (Cas) systems could provide alternative therapeutic avenues for treating a wide range of genetic retinal diseases through targeted knockdown or correction of mutant alleles. Progress in this rapidly evolving field has been highlighted by recent Food and Drug Administration clinical trial approval for EDIT-101 (Editas Medicine, Inc., Cambridge, MA), which has demonstrated efficacious genome editing in a mouse model of CEP290 -associated Leber congenital amaurosis and safety in nonhuman primates. Nonetheless, there remains a significant number of challenges to developing clinically viable retinal genome-editing therapies. In particular, IRD-causing mutations occur in more than 200 known genes, with considerable heterogeneity in mutation type and position within each gene. Additionally, there are remaining safety concerns over long-term expression of Cas9 in vivo . This review highlights (i) the technological advances in gene-editing technology, (ii) major safety concerns associated with retinal genome editing, and (iii) potential strategies for overcoming these challenges to develop clinical therapies.

Vision loss caused by inherited retinal degeneration affects millions of people worldwide, and clinical trials involving gene supplementation strategies are ongoing for select forms of the disease. When early therapeutic intervention is not possible and patients suffer complete loss of their photoreceptor cells, there is an opportunity for vision restoration techniques, including optogenetic therapy. This therapy provides expression of light-sensitive molecules to surviving cell types of the retina, enabling light perception through residual neuronal pathways. To this end, the bipolar cells make an obvious optogenetic target to enable upstream processing of visual signal in the retina. However, while AAV transduction of the bipolar cells has been described, the expression of human opsins in these cell types within a model of retinal degeneration ( rd1 ) has been less successful. In this study, we have expanded the optogenetic toolkit and shown successful expression of human rhodopsin driven by an ON-bipolar cell promoter ( Grm6 ) in the rd1 mouse model using modified AAV capsids (AAV2.4YF, AAV8.BP2, and AAV2.7m8) delivered via intraocular injection. We also show the first presentation of ectopic expression of human cone opsin in the bipolar cells of rd1 mice. These data provide evidence of an expansion of the optogenetic toolkit with the potential to restore useful visual function, setting the stage for future trials in human patients., Competing Interests: Conflicts of Interest The authors declare that there is no conflict of interest.

Inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensing photoreceptors. The most promising therapeutic approach for IRDs is gene supplementation therapy using viral vectors, which requires the presence of viable photoreceptors at the time of intervention. At later disease stages, photoreceptors are lost and can no longer be rescued with this approach. For these patients, conferring light-sensing abilities to the remaining interneurons of the ON circuit (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment strategy. Such treatments, however, are hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on the effective isolation of these cells to facilitate tool development. Herein, we describe a method to selectively isolate ON bipolar cells via fluorescence-activated cell sorting (FACS), based on the expression of two intracellular markers. We show that the method is compatible with highly sensitive downstream analyses and suitable for the isolation of ON bipolar cells from healthy as well as degenerated mouse retinas. Moreover, we demonstrate that this approach works effectively using non-human primate (NHP) retinal tissue, thereby offering a reliable pipeline for universal screening strategies that do not require inter-species adaptations or transgenic animals., Competing Interests: J.C.-K. is a scientific advisor to Kubota Vision, Inc., S.M. is a co-founder and shareholder of ViGeneron GmbH, and D.G. is a co-founder and shareholder of AaviGen GmbH. The remaining authors declare no competing interests., (© 2021 The Authors.)

Clinical Relevance: Choroideremia is a progressive X-linked inherited rod-cone dystrophy. Patients present with nyctalopia and progressive visual field loss, but visual acuity remains well preserved early on. This study showed that low-luminance visual acuity may be a useful clinical outcome measure during earlier disease stages., Background: Choroideremia is a progressive X-linked inherited rod-cone dystrophy. Patients present with nyctalopia and progressive visual field loss. However, visual acuity remains well preserved until late in the disease process, limiting its usefulness as a clinical trial endpoint across the disease spectrum. Visual acuity measurements under low-luminance and low-contrast conditions may be affected sooner and have been suggested as early markers in other ocular diseases. This study assesses whether low-luminance visual acuity and low-contrast visual acuity provide useful endpoints in choroideremia clinical trials., Method: Standard high-contrast and low-luminance visual acuity was obtained on 29 choroideremia subjects and 16 healthy controls, using a logMAR chart, set at four metres. Low-luminance visual acuity was tested using a 2.0-log unit neutral density filter, with the same chart set-up, without formal dark adaptation. This was followed by low-contrast visual acuity measured using 1.25 per cent and 2.5 per cent low-contrast logMAR charts placed also at four metres. Data from the right eyes only were analysed using non-parametric statistics. High-contrast visual acuity minus low-luminance and low-contrast visual acuity provided the low-luminance and low-contrast difference scores., Results: A higher number of choroideremia subjects were able to complete the low-luminance test than the low-contrast visual acuity tests. Choroideremia subjects had significantly higher low luminance, 2.5 per cent low-contrast and 1.25 per cent low-contrast difference scores compared with controls (p < 0.01, Mann-Whitney U-test); 1.25 per cent low-contrast visual acuity revealed the poorest performance. A strong positive correlation was found between low-luminance and high-contrast visual acuities (ρ = 0.818, p < 0.001) and 2.5 per cent low-contrast and high-contrast visual acuities (ρ = 0.671, p < 0.001)., Conclusion: The low-luminance visual acuity test may be a useful additional clinical trial outcome measure for early-to-moderate disease, when high-contrast visual acuity is preserved.

Novel gene therapy treatments for inherited retinal diseases have been at the forefront of translational medicine over the past couple of decades. Since the discovery of CRISPR mechanisms and their potential application for the treatment of inherited human conditions, it seemed inevitable that advances would soon be made using retinal models of disease. The development of CRISPR technology for gene therapy and its increasing potential to selectively target disease-causing nucleotide changes has been rapid. In this chapter, we discuss the currently available CRISPR toolkit and how it has been and can be applied in the future for the treatment of inherited retinal diseases. These blinding conditions have until now had limited opportunity for successful therapeutic intervention, but the discovery of CRISPR has created new hope of achieving such, as we discuss within this chapter., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: Introduction of retinal gene therapy requires established outcome measures along with thorough understanding of the pathophysiology. Evidence of early, thinned outer segments in RPGR X-linked retinitis pigmentosa could help understand how the level of cone photoreceptor involvement translates to visual potential., Objective: Analysis of foveal photoreceptor outer segment length in a young cohort of RPGR patients to help clarify the reason for absent maximal visual acuity seen., Methods: Case-control study of RPGR patients. Quantitative measurement of photoreceptor outer segment by OCT., Results: Eighteen male RPGR patients and 30 normal subjects were included. Outer segment thickness differed significantly between the RPGR and normal eyes (p < 0.0005). Mean outer segment values were 35.6 ± 2.3 µm and 35.4 ± 2.6 µm for RPGR right and left eyes, respectively. In normal eyes, the mean outer segment thickness was 61.4 ± 0.7 µm for right eyes and 62.4 ± 0.7 µm for left eyes., Conclusions: Patients with RPGR X-linked retinitis pigmentosa show thinning of the foveal photoreceptor outer segment thickness early in the disease course, which could be an explanation for the lower maximum visual acuity seen. These findings must be taken into consideration when assessing efficacy outcome measures in retinal gene therapy trials., (© 2020 S. Karger AG, Basel.)

The degeneration of light-detecting rod and cone photoreceptors in the human retina leads to severe visual impairment and ultimately legal blindness in millions of people worldwide. Multiple therapeutic options at different stages of degeneration are being explored but the majority of ongoing clinical trials involve adeno-associated viral (AAV) vector-based gene supplementation strategies for select forms of inherited retinal disease. Over 300 genes are associated with inherited retinal degenerations and only a small proportion of these will be suitable for gene replacement therapy. However, while the origins of disease may vary, there are considerable similarities in the physiological changes that occur in the retina. When early therapeutic intervention is not possible and patients suffer loss of photoreceptor cells but maintain remaining layers of cells in the neural retina, there is an opportunity for a universal gene therapy approach that can be applied regardless of the genetic origin of disease. Optogenetic therapy offers such a strategy by aiming to restore vision though the provision of light-sensitive molecules to surviving cell types of the retina that enable light perception through the residual neurons. Here we review the recent progress in attempts to restore visual function to the degenerate retina using optogenetic therapy. We focus on multiple pre-clinical models used in optogenetic strategies, discuss their strengths and limitations, and highlight considerations including vector and transgene designs that have advanced the field into two ongoing clinical trials., (Copyright © 2020 McClements, Staurenghi, MacLaren and Cehajic-Kapetanovic.)

Purpose: To report the highest attained visual acuity with an electronic retinal implant for the treatment of advanced retinal degeneration following a novel intensive period of visual training., Methods: A case study as part of the prospective, international, multi-centre, interventional clinical trial (ClinicalTrials.gov NCT02720640 and NCT01024803) of patients with the Retina Implant Alpha AMS (Retina Implant AG, Reutlingen, Germany) for advanced retinal degeneration. A patient with subretinal device implanted into worse-seeing eye with no useful perception of light vision secondary to USH2A retinal degeneration underwent intensive period of visual training., Results: The device remains functional with no safety concerns at 3 years postsurgical implantation, and following visual training, the patient achieved the highest visual acuity so far with an electronic retinal device, with real, digitally unenhanced, reading vision of 0.04 decimal (equivalent to 1.39 LogMAR and 20/500 or 6/150 Snellen). In addition, perception as well as partial identification of obstacles and evaluation of distances was possible in both daylight and night-time settings., Conclusions: Retinal implants are currently the only available therapy option for advanced retinal degeneration. Visual rehabilitation postimplantation has potential to maximize visual percepts. The novel concept of intensive visual training presented herein shows what is achievable with electronic retinal implants and has implications for other therapeutic options, such as optogenetics, that aim to stimulate remaining inner retinal cells in advanced retinal degeneration., (© 2020 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Importance: Pathogenic variants in retinitis pigmentosa GTPase regulator (RPGR) gene typically lead to a severe form of X-linked retinitis pigmentosa, which is associated with early severe vision loss., Objective: To investigate an X-linked retinal degeneration family with atypical preservation of visual acuity in the presence of a novel deep intronic splice site RPGR c.779-5T>G variant., Design, Setting, and Participants: In this case series, 3 members of an X-linked retinal degeneration family were studied by in-depth phenotyping and genetic screening at a single center. Data were collected and analyzed from November 2018 to March 2020., Main Outcomes and Measures: Data were collected on full ophthalmic history, examination, and retinal imaging. A full retinitis pigmentosa gene panel was analyzed by next-generation sequencing. The pathogenicity of the RPGR c.779-5T>G variant was assessed by in silico splice prediction tools and by purpose-designed in vitro splicing assay., Results: An 84-year-old man was referred with clinical diagnosis of choroideremia and possible inclusion into a gene therapy trial. He presented with late-stage retinal degeneration and unusually preserved visual acuity (78 and 68 ETRDS letters) that clinically resembled choroideremia. His 23-year-old grandson was still in early stages of degeneration but showed a very different clinical picture, typical of retinitis pigmentosa. Next-generation sequencing identified a sole RPGR c.779-5T>G variant of undetermined pathogenicity in both cases. The daughter of the proband showed an RPGR carrier phenotype and was confirmed to carry the same variant. The molecular analysis confirmed that the RPGR c.779-5T>G variation reduced the efficiency of intron splicing compared with wild type, leading to a population of mutant and normal transcripts. The predicted consequences of the pathogenic variant are potential use of an alternative splice acceptor site or complete skipping of exon 8, resulting in truncated forms of the RPGR protein with different levels of glutamylation., Conclusions and Relevance: These results support the importance of careful interpretation of inconsistent clinical phenotypes between family members. Using a molecular splicing assay, a new pathogenic variant in a noncoding region of RPGR was associated with a proportion of normal and hypomorphic RPGR, where cones are likely to survive longer than expected, potentially accounting for the preserved visual acuity observed in this family.

Purpose: Choroideremia is an X-linked retinal disease characterized by early retinal pigment epithelium (RPE) loss and subsequent retinal degeneration. The RPE adopts either a smooth or mottled appearance on fundus autofluorescence (FAF). It is not known how RPE changes predict the health of the overlying ellipsoid zone (EZ)., Methods: A retrospective review of FAF and optical coherence tomography (OCT) images from 20 patients with choroideremia was performed. The percentage of intact EZ in each smooth and mottled FAF region was determined using one horizontal trans-foveal OCT section., Results: Fourteen out of 20 patients had distinct smooth and mottled areas in both eyes and were included in the sub-analysis. On average, 62.5 ± 10.1% of the EZ in each smooth region of the right eyes was intact compared to 10.0 ± 4.3% in the mottled areas. The same trend was observed in left eyes: 76.5 ± 7.2% of the EZ was intact in the smooth regions versus 9.8 ± 3.9% in the mottled areas (two-way anova, p < 0.0001). Thus, the mottled FAF regions were associated with EZ disruption more so than the smooth areas., Conclusion: Retinal pigment epithelium (RPE) changes correlate with the health of the overlying EZ in choroideremia. The smooth FAF region likely represents early stages of the disease, with most of the area containing preserved EZ, whereas the mottled zone indicates more advanced stages and has mostly disrupted EZ. Because of the clear relationship between FAF findings and EZ integrity, FAF imaging can be used to monitor disease progression and identify areas of preserved EZ that could be rescued by gene therapy., (© 2019 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Retinal gene therapy has shown great promise in treating retinitis pigmentosa (RP), a primary photoreceptor degeneration that leads to severe sight loss in young people. In the present study, we report the first-in-human phase 1/2, dose-escalation clinical trial for X-linked RP caused by mutations in the RP GTPase regulator (RPGR) gene in 18 patients over up to 6 months of follow-up (https://clinicaltrials.gov/: NCT03116113). The primary outcome of the study was safety, and secondary outcomes included visual acuity, microperimetry and central retinal thickness. Apart from steroid-responsive subretinal inflammation in patients at the higher doses, there were no notable safety concerns after subretinal delivery of an adeno-associated viral vector encoding codon-optimized human RPGR (AAV8-coRPGR), meeting the pre-specified primary endpoint. Visual field improvements beginning at 1 month and maintained to the last point of follow-up were observed in six patients.

Purpose: To report combined viewpoints on ocular gene therapy from a select group of clinician scientists and a patient advocacy group., Methods: With the support of Randy Wheelock and Dr. Chris Moen from the Choroideremia Research Foundation (CRF), a special interest group at the 2019 Annual meeting of the Association for Research in Vision and Ophthalmology in Vancouver, Canada, shared their knowledge, experience, concepts, and ideas and provided a forum to discuss therapeutic strategies for the treatment of inherited retinal disorders, using experience in choroideremia (CHM) as a model., Results: A member of the CRF presented the patient perspective and role in clinical trials. Five clinician scientists presented reasons for limited long-term visual improvement in many gene therapy trials, including challenges with dose, incomplete understanding of photoreceptor metabolism, vector delivery, inflammation, and identification of patients likely to benefit from treatment., Conclusions: The shared experience of the five clinician scientists indicates that the results of ocular gene therapy for choroideremia have been less successful than for RPE65 -related Leber congenital amaurosis. Improvement in vector delivery and developing a better understanding of gene expression in target tissues, treatment dose and side effects, and inflammation, as well as identifying patients who are most likely to benefit without suffering excessive risk, are necessary to advance the development of effective therapies for inherited retinal degenerations., Translational Relevance: Additional long-term data are required to determine if ocular gene therapy will be sufficient to alter natural progression in choroideremia. Combination therapies may have to be considered, as well as alternative vectors that minimize risk., Competing Interests: Disclosure: I.M. MacDonald, None; C. Moen, None; J.L. Duncan, Spark Therapeutics (C), AGTC (C, F), ProQR Therapeutics (C), 4D Therapeutics (C), Biogen (C), Horama (C), ProQR (C), SparingVision (C), Acucela (F), Allergan (F), Biogen (F), Neurotech USA (F), Second Sight (F); S.H. Tsang, None; J. Cehajic-Kapetanovic, None; T.S. Aleman, None, (Copyright 2020 The Authors.)

Introduction: Retinitis pigmentosa (RP) is the most common form of inherited retinal degenerations with an estimated prevalence of 1 in 4,000 and more than 1 million individuals affected worldwide. With the introduction of the first retinal gene therapy in 2017 the importance of understanding the mechanisms of retinal degeneration and its natural progression has shifted from being of academic interest to being of pivotal for the development of new therapies., Areas Covered: This review covers standard and innovative diagnostic techniques and complementary examinations needed for the evaluation and treatment of RP. It includes chapters on the assessment of visual function, retinal morphology, and genotyping., Expert Opinion: Monitoring the progression of RP can best be achieved by combining assessments of both visual function and morphology. Visual acuity testing using ETDRS charts should be complemented by low-luminance visual acuity and colour vision tests. Assessment of the visual field can also be useful in less advanced cases. In those with central RP involvement measuring retinal sensitivity using microperimetry is recommended. Retinal morphology is best assessed by OCT and autofluorescence. Genetic testing is pivotal as it contributes to the pathophysiological understanding and can guide clinical management as well as identify individuals that could benefit from retinal gene therapy.

Introduction : Currently, there are three Phase I/II clinical trials based on gene therapy ongoing to test different AAV.RPGR or deleted RPGR vectors on patients affected by X-linked retinitis pigmentosa. These three vectors differ in the adeno-associated viral (AAV) vector capsid used, and the coding sequences: two contain codon optimized versions of RPGR which give the full-length protein, whilst the third uses a wild-type sequence that contains a large deletion encoding part of the functional domain of the RPGR protein. Areas covered : This review approaches the different studies that have led to the initiation of three different clinical trials for RPGR related X-linked retinitis pigmentosa. Expert opinion : The development of a gene therapy vector to deliver a normal copy of the RPGR gene into the photoreceptors has presented a challenge for the scientific community. The instability of its sequence and the fact that its function is not well understood can lead to the production of a nonfunctional or deleterious protein for the human retina. Since the RPGR protein undergoes post-translational glutamylation in the protein domain that may be particularly affected by gene instability, a functional assay of glutamylation is essential to verify the correct coding sequence.

Background : Gene editing has shown huge potential in correcting aberrant splicing and Cas13 has been identified as being particularly suitable for targeting RNA. It has therefore become increasingly important to highlight new splice site mutations that may be correctable, particularly in genes that are too large to be encoded by AAV vectors. About 20% of Usher Type 1 cases are caused by mutations in CDH23 . Purpose : To report a novel splice site mutation of CDH23 associated with Usher Type 1D. Materials and Methods : Case report. Results : A 35-year-old Caucasian female who is congenitally deaf with vestibular dysfunction presented with visual acuity of 6/12 in both eyes. Fundus examination revealed findings typical of retinitis pigmentosa with foveal preservation of photoreceptor layer. Next generation sequencing analysis revealed a novel homozygous variant, c.9319 + 1G>T in CDH23 consistent with the diagnosis of Usher Syndrome Type 1D. The c.9319 + 1G>T variant is predicted to affect splicing at the exon 65/intron 65 boundary, which highly likely leads to complete skipping of exon 65. Conclusions : We describe a case of a typical Usher Syndrome Type 1D caused by a novel splice site variant in CDH23 . Currently there are no treatments for CDH23 related retinal degeneration, partly because the cDNA size of 10kb is too large for AAV vector gene augmentation therapy. Alternative strategies include CRISPR-Cas9 adenine base editors and RNA editing with CRISPR-Cas13. Single-nucleotide editing represents a promising approach for targeting this variant in CDH23 to restore the wildtype splice donor site at this position.

Advances in molecular research have culminated in the development of novel gene-based therapies for inherited retinal diseases. We have recently witnessed several groundbreaking clinical studies that ultimately led to approval of Luxturna, the first gene therapy for an inherited retinal disease. In parallel, international research community has been engaged in conducting gene therapy trials for another more common inherited retinal disease known as choroideremia and with phase III clinical trials now underway, approval of this therapy is poised to follow suit. This chapter discusses new insights into clinical phenotyping and molecular genetic testing in choroideremia with review of molecular mechanisms implicated in its pathogenesis. We provide an update on current gene therapy trials and discuss potential inclusion of female carries in future clinical studies. Alternative molecular therapies are discussed including suitability of CRISPR gene editing, small molecule nonsense suppression therapy and vision restoration strategies in late stage choroideremia.

Mutations affecting the Retinitis Pigmentosa GTPase Regulator ( RPGR ) gene are the commonest cause of X-linked and recessive retinitis pigmentosa (RP), accounting for 10%-20% of all cases of RP. The phenotype is one of the most severe amongst all causes of RP, characteristic for its early onset and rapid progression to blindness in young people. At present there is no cure for RPGR -related retinal disease. Recently, however, there have been important advances in RPGR research from bench to bedside that increased our understanding of RPGR function and led to the development of potential therapies, including the progress of adeno-associated viral (AAV)-mediated gene replacement therapy into clinical trials. This manuscript discusses the advances in molecular research, which have connected the RPGR protein with an important post-translational modification, known as glutamylation, that is essential for its optimal function as a key regulator of photoreceptor ciliary transport. In addition, we review key pre-clinical research that addressed challenges encountered during development of therapeutic vectors caused by high infidelity of the RPGR genomic sequence. Finally, we discuss the structure of three current phase I/II clinical trials based on three AAV vectors and RPGR sequences and link the rationale behind the use of the different vectors back to the bench research that led to their development., Competing Interests: REM receives grant funding from Nightstar Therapeutics (now Biogen Inc.). REM is a consultant to Nightstar Therapeutics and Spark Therapeutics. These companies did not have any input into the work presented. No other authors have a conflict of interest.

Importance: The PROM1 gene, commonly associated with cone-rod dystrophies, may have dominant or recessive phenotypes that influence disease onset and severity., Objective: To characterize the clinical phenotype and molecular genetic variations in patients with PROM1 variants., Design, Setting, and Participants: This case-series study was conducted at 2 specialist retinal genetics clinics and examined 19 consecutively enrolled patients with PROM1-related retinal degeneration. Data were collected and analyzed from May 2018 to December 2018., Main Outcomes and Measures: Results of ophthalmic examination, retinal imaging, and molecular genetic analysis by next-generation sequencing., Results: Of 19 patients, 13 (68%) were women, and age ranged from 11 to 70 years. All patients presented with central visual loss, with or without photophobia. Individuals with recessive variants commonly had severe loss of visual acuity by their 20s, whereas the dominant variant was associated with a milder phenotype, with most patients retaining good vision into late adulthood. The recessive cases were associated with a panretinal dystrophy of cone-rod phenotype with early macular involvement, whereas the dominant variants were associated with a cone-rod phenotype that was restricted to the macula with predominantly cone dysfunction. Next-generation sequencing identified 3 novel and 9 previously reported variants in PROM1. Recessive mutations included 6 truncating variants (3 nonsense and 3 frameshift), 4 splice site variants, and 1 missense variant. All 6 dominant variants were associated with a c.1117C>T missense variant. The variants were distributed throughout the PROM1 genomic sequence with no specific clustering on protein domains., Conclusions and Relevance: In this case-series study, PROM1 recessive variants were associated with early-onset, severe panretinal degeneration. The similar phenotypes observed in patients with homozygous missense variants and splice site variants compared with similarly aged patients with truncating variants suggests that all recessive variants have a null (or loss of function close to null) outcome on PROM1 function. In contrast, the dominant missense cases were associated with a milder, cone-driven phenotype, suggesting that the dominant disease is preferentially associated with cones. This has implications for the development of treatments for this severely blinding disease, and adeno-associated viral vector-based gene therapy and optogenetics could become successful treatment options.

Background: Nuclear hormone receptor gene, NR2E3, plays a critical role in retinogenesis and determination of the rod photoreceptor phenotype. Mutations in NR2E3 typically lead to recessive enhanced S-cone syndrome (ESCS), where affected individuals show higher sensitivity to short wavelength light and early onset rod dysfunction. Patients with ESCS present in early childhood with nyctalopia, enhanced sensitivity to blue light and display a very heterogeneic retinal phenotype with varying degrees of clumped pigmentation and occasional retinoschisis., Purpose: To confirm the pathogenicity of a novel mutation in NR2E3 using electrophysiological studies., Materials and Methods: Patient underwent detailed clinical evaluation and ophthalmic imaging followed by next generation sequencing analysis and electrophysiological studies., Results: We describe a case of a young man of Greek descent with a family history of retinal degeneration. His fundal features at presentation were atypical of ESCS, with striking macular involvement in both eyes, including fibrotic subretinal material overlying the pigment epithelial detachment in one eye and schisis in the other. Genetic testing revealed a novel homozygous variant in NR2E3 gene of uncertain pathogenicity. Instead of performing further genetic analyses, electrophysiological studies showed pathognomonic changes in the S-cone response., Conclusions: With the recent clinical endorsement of a gene therapy for RPE65 related-inherited retinal degeneration it is of paramount importance to correctly identify the pathogenic genetic mutation. In this particular syndrome, we highlight the value of electrophysiology to confirm the pathogenicity of a novel mutation in NR2E3 and aid the diagnosis of ESCS, with potential for gene therapy in the future.