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Abstract Uveitis is characterised by breakdown of the blood-retinal barrier (BRB), allowing infiltration of immune cells that mediate intraocular inflammation, which can lead to irreversible damage of the neuroretina and the loss of sight. Treatment of uveitis relies heavily on corticosteroids and systemic immunosuppression due to limited understanding of disease pathogenesis. We performed single-cell RNA-sequencing of retinas, as well as bulk RNA-sequencing of retinal pigment epithelial (RPE) cells from mice with experimental autoimmune uveitis (EAU) versus healthy control. This revealed that the Th1/Th17-driven disease induced strong gene expression changes in response to inflammation in rods, cones, Müller glia and RPE. In particular, Müller glia and RPE cells were found to upregulate expression of chemokines, complement factors, leukocyte adhesion molecules and MHC class II, thus highlighting their contributions to immune cell recruitment and antigen presentation at the inner and outer BRB, respectively. Additionally, ligand-receptor interaction analysis with CellPhoneDB revealed key interactions between Müller glia and T cell / natural killer cell subsets via chemokines, galectin-9 to P4HB/TIM-3, PD-L1 to PD-1, and nectin-2/3 to TIGIT signalling axes. Our findings elucidate mechanisms contributing to breakdown of retinal immune privilege during uveitis and identify novel targets for therapeutic interventions.

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.

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

RNA editing holds great promise for the therapeutic correction of pathogenic, single nucleotide variants (SNV) in the human transcriptome since it does not risk creating permanent off-targets edits in the genome and has the potential for innovative delivery options. Adenine deaminases acting on RNA (ADAR) enzymes catalyse the most widespread form of posttranscriptional RNA editing in humans and their ability to hydrolytically deaminate adenosine to inosine in double stranded RNA (dsRNA) has been harnessed to change pathogenic single nucleotide variants (SNVs) in the human genome on a transcriptional level. Until now, the most promising target editing rates have been achieved by exogenous delivery of the catalytically active ADAR deaminase domain (ADARDD) fused to an RNA binding protein. While it has been shown that endogenous ADARs can be recruited to a defined target site with the sole help of an ADAR-recruiting guide RNA, thus freeing up packaging space, decreasing the chance of an immune response against a foreign protein, and decreasing transcriptome-wide off-target effects, this approach has been limited by a low editing efficiency. Through the recent development of novel circular ADAR-recruiting guide RNAs as well as the optimisation of ADAR-recruiting antisense oligonucleotides, RNA editing with endogenous ADAR is now showing promising target editing efficiency in vitro and in vivo. A target editing efficiency comparable to RNA editing with exogenous ADAR was shown both in wild-type and disease mouse models as well as in wild-type non-human primates (NHP) immediately following and up to 6 weeks after application. With these encouraging results, RNA editing with endogenous ADAR has the potential to present an attractive option for the treatment of inherited retinal diseases (IRDs), a field where gene replacement therapy has been established as safe and efficacious, but where an unmet need still exists for genes that exceed the packaging capacity of an adeno associated virus (AAV) or are expressed in more than one retinal isoform. This review aims to give an overview of the recent developments in the field of RNA editing with endogenous ADAR and assess its applicability for the field of treatment of IRD.

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: 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.

Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45+ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 109 genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation.

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.

Rhodopsin-related dominant retinitis pigmentosa is a degenerative disease of the retina of the eye for which there is no current treatment. In this study, the authors use a novel form of RNA-interference- artificial mirtrons- to slow retinal degeneration in a mouse model of the disease.

Purpose: To quantify the effect of cataract on color vision as measured by the low-vision Cambridge Colour Test (lvCCT; Cambridge Research Systems) and to understand whether different types and severities of cataract have different effects on color vision. Design: Cohort study. Participants: Patients aged 18 to 95 undergoing routine cataract surgery at the Oxford Eye Hospital. Methods: The lvCCT was performed to measure color sensitivity in both eyes both before and after surgery. The crystalline lens was examined and graded according to the Lens Opacities Classification System III to determine the type and severity of cataract. Measures of repeatability were performed for the data to explore test–retest bias using Bland–Altman analysis. The Wilcoxon signed-rank test was performed to assess the effect of cataract on color vision by comparing control and surgical test measurements. Three multiple linear regressions were performed to relate cataract grading or severity to color vision measurements. Main Outcome Measures: Color discrimination along each of the protan, deutan, and tritan confusion lines. Results: The Wilcoxon signed-rank test showed a statistically significant difference in both the protan (P = 0.024) and tritan (P = 0.020) axes on comparison of control and surgical test measurements. As severity of cataract increased, color vision sensitivity was affected more greatly, and nuclear sclerotic cataract showed the most profound effect on color vision sensitivity in the lvCCT; however, the linear regression models showed that these observations did not reach statistical significance. Conclusions: Cataract surgery has a statistically significant effect on color vision in both the protan and tritan axes. The effects of specific subtypes of cataract and different severities could not be elucidated because of the high prevalence of patients with mixed cataract. The lvCCT color sensitivity measurements are used regularly as outcome measures in clinical gene therapy trials involving vitreoretinal surgery, and vitrectomy accelerates cataract formation. Therefore, it is important to quantify the effect of cataract on color vision measurements so that it may be taken into account when used as an outcome measure in clinical trials. We were unable to derive a precise correction factor for cataract on color vision measurements.

Recent advances in recombinant adeno-associated virus (rAAV) gene therapy for choroideremia show gene replacement to be a promising approach. It is, however, well known that contact of vector solution with plastic materials in the surgical device may result in non-specific adsorption with resulting loss of physical titer and/or level of protein expression and activity. Here we assessed the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) before and following passage through the injection device over a period of time to mimic the clinical scenario. Three identical devices were screened using two concentrations of vector: high (1E+12 DNase-resistant particles [DRP]/mL) and low (1E+11 DRP/mL), to mimic high- and low-dose administrations of vector product. The low dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF68) or balanced salt solution (BSS). We observed significant losses in the genomic titer of samples diluted with BSS for all time points. The addition of 0.001% PF68 did not, however, affect rAAV physical titer, or REP1 protein expression and biological activity. Hence we observed that neither the genomic titer nor the biological activity of a rAAV2-REP1-containing solution was affected following passage through the surgical device when PF68 was present as a surfactant and this was maintained over a period up to 10 h.

Stargardt macular dystrophy (STGD1) is the most common form of inherited childhood blindness worldwide and for which no current treatments exist. It is an autosomal recessive disease caused by mutations in ABCA4. To date, a variety of gene supplementation approaches have been tested to create a therapy, with some reaching clinical trials. New technologies, such as CRISPR-Cas based editing systems, provide an exciting frontier for addressing genetic disease by allowing targeted DNA or RNA base editing of pathogenic mutations. ABCA4 has ∼1,200 known pathogenic mutations, of which ∼63% are transition mutations amenable to this editing technology. In this report, we screened the known “pathogenic” and “likely pathogenic” mutations in ABCA4 from available data in gnomAD, Leiden Open Variation Database (LOVD), and ClinVar for potential PAM sites of relevant base editors, including Streptococcus pyogenes Cas (SpCas), Staphylococcus aureus Cas (SaCas), and the KKH variant of SaCas (Sa-KKH). Overall, of the mutations screened, 53% (ClinVar), 71% (LOVD), and 71% (gnomAD), were editable, pathogenic transition mutations, of which 35–47% had “ideal” PAM sites. Of these mutations, 16–20% occur within a range of multiple PAM sites, enabling a variety of editing strategies. Further, in relevant patient data looking at three cohorts from Germany, Denmark, and China, we find that 44–76% of patients, depending on the presence of complex alleles, have at least one transition mutation with a nearby SaCas, SpCas, or Sa-KKH PAM site, which would allow for potential DNA base editing as a treatment strategy. Given the complexity of the genetic landscape of Stargardt, these findings provide a clearer understanding of the potential for DNA base editing approaches to be applied as ABCA4 gene therapy strategies.

Inherited retinal diseases (IRD) are a clinically and genetically heterogenous group of diseases and a leading cause of blindness in the working-age population. Even though gene augmentation therapies have shown promising results, they are only feasible to treat a small number of autosomal recessive IRDs, because the size of the gene is limited by the vector used. DNA editing however could potentially correct errors regardless of the overall size of the gene and might also be used to correct dominant mutations. Prime editing is a novel CRISPR/Cas9 based gene editing tool that enables precise correction of point mutations, insertions, and deletions without causing double strand DNA breaks. Due to its versatility and precision this technology may be a potential treatment option for virtually all genetic causes of IRD. Since its initial description, the prime editing technology has been further improved, resulting in higher efficacy and a larger target scope. Additionally, progress has been achieved concerning the size-related delivery issue of the prime editor components. This review aims to give an overview of these recent advancements and discusses prime editing as a potential treatment for IRDs.

Color vision is considered a marker of cone function and its assessment in patients with retinal pathology is complementary to the assessments of spatial vision [best-corrected visual acuity (BCVA)] and contrast detection (perimetry). Rod-cone and chorioretinal dystrophies—such as choroideremia—typically cause alterations to color vision, making its assessment a potential outcome measure in clinical trials. However, clinical evaluation of color vision may be compromised by pathological changes to spatial vision and the visual field. The low vision Cambridge Color Test (lvCCT) was developed specifically to address these latter issues. We used the trivector version of the lvCCT to quantify color discrimination in a cohort of 53 patients with choroideremia. This test enables rapid and precise characterization of color discrimination along protan, deutan, and tritan axes more reliably than the historically preferred test for clinical trials, namely the Farnsworth Munsell 100 Hue test. The lvCCT demonstrates that color vision defects—particularly along the tritan axis—are seen early in choroideremia, and that this occurs independent of changes in visual acuity, pattern electroretinography and ellipsoid zone area on optical coherence tomography (OCT). We argue that the selective loss of tritan color discrimination can be explained by our current understanding of the machinery of color vision and the pathophysiology of choroideremia.

The discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool have revolutionized the field of molecular biology and generated excitement for its potential to treat a wide range of human diseases. As a gene therapy target, the retina offers many advantages over other tissues because of its surgical accessibility and relative immunity privilege due to its blood–retinal barrier. These features explain the large advances made in ocular gene therapy over the past decade, including the first in vivo clinical trial using CRISPR gene-editing reagents. Although viral vector-mediated therapeutic approaches have been successful, they have several shortcomings, including packaging constraints, pre-existing anti-capsid immunity and vector-induced immunogenicity, therapeutic potency and persistence, and potential genotoxicity. The use of nanomaterials in the delivery of therapeutic agents has revolutionized the way genetic materials are delivered to cells, tissues, and organs, and presents an appealing alternative to bypass the limitations of viral delivery systems. In this review, we explore the potential use of non-viral vectors as tools for gene therapy, exploring the latest advancements in nanotechnology in medicine and focusing on the nanoparticle-mediated delivery of CRIPSR genetic cargo to the retina.

While anti-angiogenic therapies for wet age-related macular degeneration (AMD) are effective for many patients, they require multiple injections and are expensive and prone to complications. Gene therapy could be an elegant solution for this problem by providing a long-term source of anti-angiogenic proteins after a single administration. Another potential issue with current therapeutic proteins containing a fragment crystallizable (Fc) domain (such as whole antibodies like bevacizumab) is the induction of an unwanted immune response. In wet AMD, a low level of inflammation is already present, so to avoid exacerbation of disease by the therapeutic protein, we propose single-chain fragment variable (scFv) antibodies, which lack the Fc domain, as a safer alternative. To investigate the feasibility of this, anti-vascular endothelial growth factor (VEGF)-blocking antibodies in two formats were produced and tested in vitro and in vivo. The scFv transgene was then cloned into an adeno-associated virus (AAV) vector. A therapeutic effect in a mouse model of choroidal neovascularization (CNV) was demonstrated with antibodies in both scFv and immunoglobulin G1 (IgG1) formats (p < 0.04). Importantly, the scFv anti-VEGF antibody expressed from an AAV vector also had a significant beneficial effect (p = 0.02), providing valuable preclinical data for future translation to the clinic. Keywords: AAV, vector, gene therapy, AMD, CNV mouse model, anti-VEGF scFv antibodies, single-chain, angiogenesis

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.

Summary: Increased calpain activity is linked to neuroinflammation including a heritable retinal disease caused by hyper-activating mutations in the calcium-activated calpain-5 (CAPN5) protease. Although structures for classical calpains are known, the structure of CAPN5, a non-classical calpain, remains undetermined. Here we report the 2.8 Å crystal structure of the human CAPN5 protease core (CAPN5-PC). Compared to classical calpains, CAPN5-PC requires high calcium concentrations for maximal activity. Structure-based phylogenetic analysis and multiple sequence alignment reveal that CAPN5-PC contains three elongated flexible loops compared to its classical counterparts. The presence of a disease-causing mutation (c.799G>A, p.Gly267Ser) on the unique PC2L2 loop reveals a function in this region for regulating enzymatic activity. This mechanism could be transferred to distant calpains, using synthetic calpain hybrids, suggesting an evolutionary mechanism for fine-tuning calpain function by modifying flexible loops. Further, the open (inactive) conformation of CAPN5-PC provides structural insight into CAPN5-specific residues that can guide inhibitor design. : Velez et al. report the crystal structure of the calpain-5 protease core (CAPN5-PC). Sequence- and structure-based phylogenetic analysis reveals that CAPN5-PC contains three elongated loops compared to classical calpains. One loop contains a hyperactivating mutation that causes neovascular inflammatory vitreoretinopathy, revealing a function in this region for regulating proteolytic activity. Keywords: calpain, CAPN5, NIV, protease, uveitis, disease-causing mutations, structural phylogenetics, structural phylogenetics, crystal structure, synthetic biology, calcium-binding protein, enzyme kinetics

Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene. CHM is ubiquitously expressed in human cells and encodes Rab escort protein 1 (REP1). REP1 plays a key role in intracellular trafficking through the prenylation of Rab GTPases, a reaction that can be reproduced in vitro. With recent advances in adeno-associated virus (AAV) gene therapy for CHM showing gene replacement to be a promising approach, an assay to assess the biological activity of the vectors is of the uttermost importance. Here we sought to compare the response of two Rab proteins, RAB27A and RAB6A, to the incorporation of a biotinylated lipid donor in a prenylation reaction in vitro. First, we found the expression of REP1 to be proportional to the amount of recombinant AAV (rAAV)2/2-REP1 used to transduce the cells. Second, prenylation of RAB6A appeared to be more sensitive to REP1 protein expression than prenylation of RAB27A. Moreover, the method was reproducible in other cell lines. These results support the further development of a prenylation reaction using a biotinylated lipid donor and RAB6A to assess the biological activity of AAV vectors for CHM gene therapy. Keywords: AAV gene therapy, choroideremia, prenylation, potency assay

Abstract Background With increasing availability of toric intraocular lenses (IOL) for cataract surgery, real-world refractive outcome data is needed to aid the counselling of patients regarding lens choice. We aim to assess the outcomes of toric intraocular lens use in the non-specialist environment of a typical United Kingdom NHS cataract service. Methods A retrospective cohort study conducted at the Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, UK. All patients who received a toric IOL implant over a 10 months period. Patients underwent pre-operative corneal marking, phacoemulsification and toric IOL implantation. Biometry was obtained using a Zeiss IOLMaster 500 and the toric IOLs were selected using the manufacturers’ online calculators. Post-operative refractions were obtained from optometrist’s manifest refraction or by autorefraction. The outcome measures were post-operative unaided visual acuity (UVA), spherical equivalent refraction, cylindrical correction and all complications. Results Thirty-two eyes of 24 patients aged 21–86 years (mean 66.4, SD 14.5) were included. UVA was superior to pre-operative best-corrected visual acuity (BCVA) in 81% of eyes, same in 16% and inferior in 3%, resulting in a median improvement of 0.20 LogMAR (IQR 0.10 to 0.30). 56%, 81%, 94% and 100% of eyes were within ±0.5, ±1.0, ±1.5 and ±2.0 D of predicted spherical equivalent, respectively. Three (9%) eyes required further surgery to rectify significant IOL rotation. Conclusions Reduced cylindrical correction and improved UVA could be expected in the majority of patients undergoing toric IOL implantation. Patients should be counselled about the risk of lens rotation.

Müller glia, the major glial cell types in the retina, maintain retinal homeostasis and provide structural support to retinal photoreceptors. They also possess regenerative potential that might be used for retinal repair in response to injury or disease. In teleost fish (such as zebrafish), the Müller glia response to injury involves reprogramming events that result in a population of proliferative neural progenitors that can regenerate the injured retina. Recent studies have revealed several important mechanisms for the regenerative capacity of Müller glia in fish, which may shed more light on the mechanisms of Müller glia reprogramming and regeneration in mammals. Mammalian Müller glia can adopt stem cell characteristics, and in response to special conditions, be persuaded to proliferate and regenerate, although their native regeneration potential is limited. In this review, we consider the work to date revealing the regenerative potential of the mammalian Müller glia and discuss whether they are a potential source for cell regeneration therapy in humans.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector. Keywords: AAV2, WPRE, gene therapy, mouse retina, human retina, choroideremia

Multiple gene therapy trials are occurring for a variety of ophthalmic diseases around the world. The safety of gene therapy in the eye has been established, and the next step is to reliably assess efficacy. This is primarily done through the use of imaging techniques and visual function measures. Standardized visual function assessments, however, were originally developed for a clinical setting and may not be suitable for detecting and quantifying therapeutic changes. This scoping review takes a comprehensive look at current practice in terms of the outcome measures defined at trial registration. These were compared to the outcome measures reported in the literature. All published trials reported the pre-registered primary outcome measure. A range of additional secondary outcomes were reported that were not originally planned. Gaps in gene therapy assessment exist and further discussion are required to find a way forward, particularly as more conditions progress to phase 2 and 3 trials. Several factors impacting on trial design and outcome measure choice are discussed.

Previous studies have used fluorescently labelled cells to demonstrate the incorporation of transplanted photoreceptor precursors into the mouse retina. Here, the authors show that fluorescent proteins are passed between the host and transplanted cells rather than migration of donor cells into the retina.

Random mutagenesis can uncover novel genes involved in phenotypic traits. Here the authors perform a large-scale phenotypic screen on over 100 mouse strains generated by ENU mutagenesis to identify mice with age-related diseases, which they attribute to specific mutations revealed by whole-genome sequencing.

Purpose: We assessed the safety and efficacy of a technically advanced subretinal electronic implant, RETINA IMPLANT Alpha AMS, in end stage retinal degeneration in an interim analysis of two ongoing prospective clinical trials. The purpose of this article is to describe the interim functional results (efficacy).Methods: The subretinal visual prosthesis RETINA IMPLANT Alpha AMS (Retina Implant AG, Reutlingen, Germany) was implanted in 15 blind patients with hereditary retinal degenerations at four study sites with a follow-up period of 12 months (www.clinicaltrials.gov NCT01024803 and NCT02720640). Functional outcome measures included (1) screen-based standardized 2- or 4-alternative forced-choice (AFC) tests of light perception, light localization, grating detection (basic grating acuity (BaGA) test), and Landolt C-rings; (2) gray level discrimination; (3) performance during activities of daily living (ADL-table tasks).Results: Implant-mediated light perception was observed in 13/15 patients. During the observation period implant mediated localization of visual targets was possible in 13/15 patients. Correct grating detection was achieved for spatial frequencies of 0.1 cpd (cycles per degree) in 4/15; 0.33 cpd in 3/15; 0.66 cpd in 2/15; 1.0 cpd in 2/15 and 3.3 cpd in 1/15 patients. In two patients visual acuity (VA) assessed with Landolt C- rings was 20/546 and 20/1111. Of 6 possible gray levels on average 4.6 ± 0.8 (mean ± SD, n = 10) were discerned. Improvements (power ON vs. OFF) of ADL table tasks were measured in 13/15 patients. Overall, results were stable during the observation period. Serious adverse events (SAEs) were reported in 4 patients: 2 movements of the implant, readjusted in a second surgery; 4 conjunctival erosion/dehiscence, successfully treated; 1 pain event around the coil, successfully treated; 1 partial reduction of silicone oil tamponade leading to distorted vision (silicon oil successfully refilled). The majority of adverse events (AEs) were transient and mostly of mild to moderate intensity.Conclusions: Psychophysical and subjective data show that RETINA IMPLANT Alpha AMS is reliable, well tolerated and can restore limited visual functions in blind patients with degenerations of the outer retina. Compared with the previous implant Alpha IMS, longevity of the new implant Alpha AMS has been considerably improved. Alpha AMS has meanwhile been certified as a commercially available medical device, reimbursed in Germany by the public health system.