Outcomes and Adverse Effects of Voretigene Neparvovec Treatment for Biallelic RPE65-Mediated Inherited Retinal Dystrophies in a Cohort of Patients from a Single Center.

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.

Correlation Between Fundus Autofluorescence Pattern and Retinal Function on Microperimetry in Choroideremia.

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.

Current and Future Landscape in Genetic Therapies for Leber Hereditary Optic Neuropathy.

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.

The Role of Inflammation in Age-Related Macular Degeneration-Therapeutic Landscapes in Geographic Atrophy.

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.

Is RPGR-related retinal dystrophy associated with systemic disease? A case series.

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.

Bioengineering strategies for restoring vision.

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.

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.

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.

Compound dominant-null heterozygosity in a family with RP1-related retinal dystrophy.

Purpose: To report on the presence of autosomal dominant and compound dominant-null RP1-related retinitis pigmentosa in the same non-consanguineous family. Observation: The father was minimally symptomatic and referred by his optometrist aged 38. He was diagnosed with rod-cone dystrophy, confirmed to be caused by the previously reported RP1 c.2613dupA mutation. He was reassured that his 11-year-old daughter had a 50% chance of inheriting the same mutation and that the condition, if she had it, would most likely be similar. Clinical phenotyping of his daughter however revealed an early onset cone-rod dystrophy. The mother was entirely asymptomatic and clinically normal. Sanger sequencing of the RP1 gene in the daughter confirmed the presence of biallelic mutations - the dominant c.2613dupA variant from her father and a c.3843dupT truncating variant inherited from her mother, both located in exon 4 of the RP1 gene. The maternal c.3843dupT has previously been reported. Conclusions and importance: Pathogenic variants in exon 4 of RP1 are known to cause differential dominant and recessive disease. The presence of both phenotypes in a single family has not yet been reported. The father, being minimally symptomatic, is affected by a known dominant variant which truncates the RP1 protein more proximally. However, inheritance of both variants in a compound heterozygous state in the daughter resulted in a much more severe, early onset cone-rod phenotype in a pattern akin to recessive disease. This raises challenges for genetic counselling and development of gene-based therapies for RP1 mutations.

Characterizing Visual Fields in RPGR Related Retinitis Pigmentosa Using Octopus Static-Automated Perimetry.

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.

First-in-Human Robot-Assisted Subretinal Drug Delivery Under Local Anesthesia.

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.

Gene-agnostic therapeutic approaches for inherited retinal degenerations.

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.

CRISPR genome engineering for retinal diseases.

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.

Microperimetry Hill of Vision and Volumetric Measures of Retinal Sensitivity.

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.

Clinical applications of microperimetry in RPGR-related retinitis pigmentosa: a review.

Microperimetry, or fundus-tracked perimetry, is a precise static-automated perimetric technique to assess central retinal function. As visual acuity only deteriorates at a late disease stage in RPGR-related retinitis pigmentosa (RP), alternative markers for disease progression are of great utility. Microperimetry assessment has been of critical value as an outcome measure in a recently reported phase I/II gene therapy trial for RPGR-related RP, both in terms of detecting safety and efficacy signals. Here, we performed a review of the literature. We describe the principles of microperimetry before outlining specific parameters that may be useful as outcome measures in clinical trial settings. The current state of structure-function correlations between short-wavelength autofluorescence, optical coherence tomography and adaptive optics in RPGR-related retinitis pigmentosa are also summarized.

A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting.

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.

AAV Induced Expression of Human Rod and Cone Opsin in Bipolar Cells of a Mouse Model of Retinal Degeneration.

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.

Early Cone Photoreceptor Outer Segment Length Shortening in RPGR X-Linked Retinitis Pigmentosa.

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.

Genome-Editing Strategies for Treating Human Retinal Degenerations.

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.