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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.

The recent approval in the United States of the first adeno-associated viral (AAV) vector for the treatment of an inherited retinal degeneration validates this approach for the treatment of many other diseases. A major limiting factor continues to be the size restriction of the AAV transgene at under 5 kb. Stargardt disease is the most prevalent form of recessively inherited blindness and is caused by mutations in ABCA4, the gene that codes for ATP-binding cassette transporter protein family member 4, which has a coding sequence length of 6.8 kb. Dual vector approaches increase the capacity of AAV gene therapy, but at the cost of substantially reduced levels of target protein, which may be insufficient to achieve a therapeutic effect. Here we show that the efficacy of recombination of dual vectors is dependent on the length of DNA overlap between two transgenes. With optimized recombination, full-length ABCA4 protein is expressed in the photoreceptor outer segments of Abca4-/- mice at levels sufficient to reduce bisretinoid formation and correct the autofluorescent phenotype. These observations support a dual vector approach in future clinical trials using AAV gene therapy to treat Stargardt disease.

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_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_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. [ABSTRACT FROM AUTHOR]

Background: Choroideremia is a monogenic inherited retinal dystrophy that manifests in males with night blindness, progressive loss of peripheral vision, and ultimately profound sight loss, commonly by middle age. It is caused by genetic defects of the CHM gene, which result in a deficiency in Rab-escort protein-1, a key element for intracellular trafficking of vesicles, including those carrying melanin. As choroideremia primarily affects the retinal pigment epithelium, fundus autofluorescence, which focuses on the fluorescent properties of pigments within the retina, is an established imaging modality used for the assessment and monitoring of affected patients. Methods and Results: In this manuscript, we demonstrate the use of both short-wavelength blue and near-infrared autofluorescence and how these imaging modalities reveal distinct disease patterns in choroideremia. In addition, we show how these structural measurements relate to retinal functional measures, namely microperimetry, and discuss the potential role of these retinal imaging modalities in clinical practice and research studies. Moreover, we discuss the mechanisms underlying retinal autofluorescence patterns by imaging with a particular focus on melanin pigment. Conclusions: This could be of particular significance given the current progress in therapeutic options, including gene replacement therapy. [ABSTRACT FROM AUTHOR]

Gene therapies have emerged as promising treatments in clinical development for various retinal disorders, offering hope to patients with inherited degenerative eye conditions. Several gene therapies have already shown remarkable success in clinical trials, with significant improvements observed in visual acuity and the preservation of retinal function. A multitude of gene therapies have now been delivered safely in human clinical trials for a wide range of inherited retinal disorders but there are some gaps in the reported trial data. Some of the most exciting treatment options are not under peer review and information is only available in press release form. Whilst many trials appear to have delivered good outcomes of safety, others have failed to meet primary endpoints and therefore not proceeded to phase III. Despite this, such trials have enabled researchers to learn how best to assess and monitor patient outcomes, which will guide future trials to greater success. In this review, we consider recent and ongoing clinical trials for a variety of potential retinal gene therapy treatments and discuss the positive and negative issues related to these trials. We discuss the treatment potential following clinical trials as well as the potential risks of some treatments under investigation. As these therapies continue to advance through rigorous testing and regulatory approval processes, they hold the potential to revolutionise the landscape of retinal disorder treatments, providing renewed vision and enhancing the quality of life for countless individuals worldwide. [ABSTRACT FROM AUTHOR]

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 lightsensitivity 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 FROM AUTHOR]

Molecular confirmation of pathogenic sequence variants in the CHM gene is required prior to enrolment in retinal gene therapy clinical trials for choroideremia. Individuals with mild choroideremia have been reported. The molecular basis of genotype–phenotype associations is of clinical relevance since it may impact on selection for retinal gene therapy. Genetic testing and RNA analysis were undertaken in a patient with mild choroideremia to confirm the pathogenicity of a novel intronic variant in CHM and to explore the mechanism underlying the mild clinical phenotype. A 42-year-old male presented with visual field loss. Fundoscopy and autofluorescence imaging demonstrated mild choroideremia for his age. Genetic analysis revealed a variant at a splice acceptor site in the CHM gene (c.1350-3C > G). RNA analysis demonstrated two out-of-frame transcripts, suggesting pathogenicity, without any detectable wildtype transcripts. One of the two out-of-frame transcripts is present in very low levels in healthy controls. Mild choroideremia may result from +3 or −3 splice site variants in CHM. It is presumed that the resulting mRNA transcripts may be partly functional, thereby preventing the development of the null phenotype. Choroideremia patients with such variants may present challenges for gene therapy since there may be residual transcript activity which could result in long-lasting visual function which is atypical for this disease. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This review considers research into the treatment of Usher syndrome, a deaf-blindness syndrome inherited in an autosomal recessive manner. Usher syndrome mutations are markedly heterogeneous, involving many different genes, and research grants are limited due to minimal patient populations. Furthermore, gene augmentation therapies are impossible in all but three Usher syndromes as the cDNA sequence exceeds the 4.7 kb AAV packaging limit. It is, therefore, vital to focus research efforts on alternative tools with the broadest applicability. The CRISPR field took off in recent years following the discovery of the DNA editing activity of Cas9 in 2012. New generations of CRISPR tools have succeeded the original CRISPR/Cas9 model to enable more sophisticated genomic amendments such as epigenetic modification and precise sequence alterations. This review will evaluate the most popular CRISPR tools to date: CRISPR/Cas9, base editing, and prime editing. It will consider these tools in terms of applicability (in relation to the ten most prevalent USH2A mutations), safety, efficiency, and in vivo delivery potential with the intention of guiding future research investment. [ABSTRACT FROM AUTHOR]

MERTK (MER proto-oncogene, tyrosine kinase) is a transmembrane protein essential in regulating photoreceptor outer segment phagocytosis. Biallelic mutations in MERTK cause retinal degeneration. Here we present the retinal phenotype of three patients with missense variants in MERTK. All patients underwent a full clinical examination, fundus photography, short-wavelength fundus autofluorescence and optical coherence tomography imaging. Two patients also underwent Goldmann visual field testing and electroretinography was undertaken for the third patient. Molecular genetic testing was undertaken using next generation or whole-exome sequencing with all variants confirmed by Sanger sequencing. The first patient was a 29-year-old female heterozygous for a missense variant (c.1133C>T, p.Thr378 Met) and a nonsense variant (c.1744_1751delinsT, p.Ile582Ter) in MERTK. The second patient was a 26-year-old male homozygous for a c.2163T>A, p.His721Gln variant in MERTK. The third patient was an 11-year-old female heterozygous for a deletion of exons 5–19 and a missense variant (c.1866 G>C, p.Lys622Asn) in MERTK. Reduced night vision was the initial symptom in all patients. Fundoscopy revealed typical signs of retinitis pigmentosa (RP) with early-onset macular atrophy. All three MERTK missense variants affect highly conserved residues within functional domains, have low population frequencies and are predicted to be pathogenic in silico. We report three missense variants in MERTK and present the associated phenotypic data, which are supportive of non-syndromic RP. MERTK is a promising candidate for viral-mediated gene replacement therapy. Moreover, one variant represents a single nucleotide transition, which is theoretically targetable with CRISPR-Cas9 base-editing. [ABSTRACT FROM AUTHOR]

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

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 rodto 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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Voretigene neparvovec (VN) is a gene therapy and the first approved pharmacological treatment for biallelic RPE65-mediated inherited retinal dystrophies (IRD), a rare condition that starts in early life and causes vision to progressively deteriorate towards complete blindness. In a phase III trial, treatment with VN significantly improved functional vision and visual function, and in October 2019 the National Institute for Health and Care Excellence (NICE) Highly Specialised Technologies (HST) process recommended VN for patients in England and Wales. We assessed the cost-effectiveness of VN compared with best supportive care (BSC) in individuals with biallelic RPE65-mediated IRD in the UK.Methods: A Markov model was developed to estimate the incremental cost per quality-adjusted life-year (QALY) gained for VN compared with BSC, from the perspective of the UK National Health Service and Personal Social Services. Phase III trial data were used to inform transition probabilities up to year 1, after which the treatment effect was assumed to be maintained for 40 years, followed by a decline in vision. A bespoke elicitation exercise involving clinical experts, patients and carers was conducted to estimate utility values for each model health state.Results: At list price, VN is associated with incremental costs of £612,404 and incremental QALYs of 6.4, resulting in an incremental cost-effectiveness ratio (ICER) of £95,072 per QALY gained. Voretigene neparvovec is associated with a significant undiscounted QALY gain (20.5) and is therefore eligible for additional QALY weighting under the NICE HST process; an ICER of up to £205,000 per QALY gained could be considered cost-effective under this framework.Conclusion: The results of the model show VN to be a cost-effective use of healthcare resources in the UK at list price. The availability of a commercial discount in the UK (as considered in the NICE appraisal) means that in reality the ICER will be even lower. Plain language summary available for this article. [ABSTRACT FROM AUTHOR]

Introduction: There are no currently approved treatments for choroideremia, an X-linked progressive inherited retinal degeneration that leads to blindness by middle age. Several treatment options are being explored, but with major advances in adeno-associated vector (AAV) gene replacement therapy that has reached phase III clinical trials. Areas covered: In this review, we discuss new insights into the clinical phenotyping and genetic testing of choroideremia patients, that aid disease characterization, progression and patient inclusion into clinical trials. Recent advances in in-vitro studies have resulted in the development of functional assays that can be used to confirm the diagnosis in challenging cases and to quantify vector potency for use in clinical trials. We review the progress in current gene therapy trials and some considerations toward gene therapy approval for the treatment of choroideremia. Lastly, we discuss developments in alternative therapies including optogenetics. Expert opinion: AAV gene replacement therapy is the most promising treatment strategy for choroideremia, that has developed exponentially over the last few years with a phase III clinical trial now underway. Optogenetics is a promising alternative strategy that might be applicable in late stages of degeneration. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Gene therapy offers the potential for targeted replacement of single gene defects in inherited retinal degenerations. Areas covered: Choroideremia is an X-linked blinding retinal disease resulting from deficiency of the CHM gene product, REP1. The disease represents an ideal target for retinal gene therapy, as it is readily diagnosed in the clinic, relatively homogenous in phenotype and slow progressing, thereby providing a wide therapeutic window for intervention. Ongoing clinical trials of retinal gene therapy for choroideremia using an adeno-associated viral vector have demonstrated safety and early efficacy. We review the clinical characteristics of the disease with a view to interpreting the findings of gene therapy clinical trials and discuss future directions. Expert commentary: Choroideremia gene therapy has so far demonstrated good safety profile and early functional visual acuity gains in a proportion of trial participants, which appear to be sustained. [ABSTRACT FROM AUTHOR]

Background: Inherited retinal degenerations are a major cause of untreatable blindness in the younger age group. Recent advances in gene therapy using adeno-associated viral (AAV) vectors have raised the possibility of slowing or stopping retinal degenerations with gene replacement in cases of gene deficiency. Materials and methods: In this report, we present a family with autosomal dominant retinitis pigmentosa. A screen for commonADRPgenes was performed with 105 genes targeted. Next generation sequencing was used to identify the mutation which was next confirmed by bidirectional Sanger sequencing. Results: A novel mutation of theTOPORSgene was identified, c.2539C>T p.(Arg847Ter), resulting in a premature termination codon and suggesting haploinsufficiency as the pathological mechanism. Conclusions: Since the cDNA encodingTOPORSis 3,135 nucleotides (within the coding capacity of AAV vectors) and haploinsufficiency is a mechanism relating to inadequate gene expression, gene replacement therapy may be an option for patients with this condition. [ABSTRACT FROM AUTHOR]

The development of a macular hole is relatively common in retinal dystrophies eligible for gene therapy such as choroideremia. However, the subretinal delivery of gene therapy requires an uninterrupted retina to allow dispersion of the viral vector. A macular hole may thus hinder effective gene therapy. Little is known about the outcome of macular hole surgery and its possible beneficial and/or adverse effects on retinal function in patients with choroideremia. We describe a case of a unilateral full-thickness macular hole (FTMH) in a 45year-old choroideremia patient (c.1349_1349+2dup mutation in CHM gene) and its management. Pars plana vitrectomy with internal limiting membrane (ILM) peeling and 20% SF6 gas tamponade was performed, and subsequent FTMH closure was confirmed at 4 weeks, 3 months and 5 months postoperatively. No postoperative adverse events occurred, and fixation stability improved on microperimetry from respectively 11% and 44% of fixation points located within a 1° and 2° radius, preoperatively, to 94% and 100% postoperatively. This case underlines that pars plana vitrectomy with ILM peeling and gas tamponade can successfully close a FTMH in choroideremia patients, with subsequent structural and functional improvement. Macular hole closure may be important for patients to be eligible for future submacular gene therapy. [ABSTRACT FROM AUTHOR]

Choroideremia is a complex and rare disease that is frequently misdiagnosed due to its similar appearance to classic retinitis pigmentosa. Recent advances in genetic testing have identified specific genetic mutations in many retinal dystrophies, and the identification of the mutation of the CHM gene on the X chromosome 25 years ago has paved the way for gene replacement therapy with the first human trials now underway. This article reviews the epidemiological and pathological features of choroideremia and new prospects in imaging to monitor disease progression, as well as potential treatment approaches for choroideremia. [ABSTRACT FROM AUTHOR]

Gene therapy in simple terms can be defined as a medical treatment that exerts its effects using molecules of DNA or RNA within cells. Most traditional drugs act by mechanisms that include binding to cell surface receptors, inhibiting enzymes in intracellular pathways or by modifying transcription. These approaches rely to some extent on a normal genetic make-up of the cell in the final common pathway, which raises significant challenges in diseases that are caused by specific gene mutations. An alternative gene therapy approach to change the behaviour of cells at the most fundamental level by one single genetic modification is therefore potentially very powerful and wide ranging. This paper presents an overview of retinal gene therapy at the current time and highlights the future therapeutic potential for a number of diseases that are currently incurable. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with this disease. Methods In a multicentre clinical trial, six male patients (aged 35-63 years) with choroideremia were administered AAV.REP1 (0·6−1·0×1010 genome particles, subfoveal injection). Visual function tests included best corrected visual acuity, microperimetry, and retinal sensitivity tests for comparison of baseline values with 6 months after surgery. This study is registered with ClinicalTrials.gov, number NCT01461213. Findings Despite undergoing retinal detachment, which normally reduces vision, two patients with advanced choroideremia who had low baseline best corrected visual acuity gained 21 letters and 11 letters (more than two and four lines of vision). Four other patients with near normal best corrected visual acuity at baseline recovered to within one to three letters. Mean gain in visual acuity overall was 3·8 letters (SE 4·1). Maximal sensitivity measured with dark-adapted microperimetry increased in the treated eyes from 23·0 dB (SE 1·1) at baseline to 25·3 dB (1·3) after treatment (increase 2·3 dB [95% CI 0·8−·8]). In all patients, over the 6 months, the increase in retinal sensitivity in the treated eyes (mean 1·7 [SE 1·0]) was correlated with the vector dose administered per mm2 of surviving retina (r=0·82, p=0·04). By contrast, small non-significant reductions (p>0·05) were noted in the control eyes in both maximal sensitivity (−0·8 dB [1·5]) and mean sensitivity (−1·6 dB [0·9]). One patient in whom the vector was not administered to the fovea re-established variable eccentric fixation that included the ectopic island of surviving retinal pigment epithelium that had been exposed to vector. Interpretation The initial results of this retinal gene therapy trial are consistent with improved rod and cone function that overcome any negative effects of retinal detachment. These findings lend support to further assessment of gene therapy in the treatment of choroideremia and other diseases, such as age-related macular degeneration, for which intervention should ideally be applied before the onset of retinal thinning. [ABSTRACT FROM AUTHOR]

Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4−/− mouse which is a model for Stargardt disease and in the Pde6brd1/rd1 mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4−/− mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments. [ABSTRACT FROM AUTHOR]

Leber Congenital Amaurosis (LCA) and Early Childhood Onset Severe Retinal Dystrophy are clinically and genetically heterogeneous retinal disorders characterised by visual impairment and nystagmus from birth or early infancy. We investigated the prevalence of sequence variants in AIPL1 in a large cohort of such patients (n = 392) and probed the likelihood of disease-causation of the identified variants, subsequently undertaking a detailed assessment of the phenotype of patients with disease-causing mutations. Genomic DNA samples were screened for known variants in the AIPL1 gene using a microarray LCA chip, with 153 of these cases then being directly sequenced. The assessment of disease-causation of identified AIPL1 variants included segregation testing, assessing evolutionary conservation and in silico predictions of pathogenicity. The chip identified AIPL1 variants in 12 patients. Sequencing of AIPL1 in 153 patients and 96 controls found a total of 46 variants, with 29 being novel. In silico analysis suggested that only 6 of these variants are likely to be disease-causing, indicating a previously unrecognized high degree of polymorphism. Seven patients were identified with biallelic changes in AIPL1 likely to be disease-causing. In the youngest subject, electroretinography revealed reduced cone photoreceptor function, but rod responses were within normal limits, with no measurable ERG in other patients. An increasing degree and extent of peripheral retinal pigmentation and degree of maculopathy was noted with increasing age in our series. AIPL1 is significantly polymorphic in both controls and patients, thereby complicating the establishment of disease-causation of identified variants. Despite the associated phenotype being characterised by early-onset severe visual loss in our patient series, there was some evidence of a degree of retinal structural and functional preservation, which was most marked in the youngest patient in our cohort. This data suggests that there are patients who have a reasonable window of opportunity for gene therapy in childhood. [ABSTRACT FROM AUTHOR]

A bstract In the developed world, diseases of the retina are common causes of untreatable blindness. In many cases, a genetic component to the aetiology has been identified, making the development of gene-based treatments a logical long-term goal. The clinical strategy for retinal gene therapy broadly encompasses two distinct advantages over systemic drug delivery. First is that gene delivery can limit expression of a therapeutic protein to a specific target cell, which is rarely possible even with local drug delivery methods. Second, by delivering DNA that remains stable and non-degraded, gene expression and hence protein production could in theory be indefinite, obviating the need for repeated tablets or injections. Viruses have evolved distinct mechanisms, such as receptor mediated uptake and genomic integration, which efficiently encompass these two properties. For non-viral gene therapy approaches, however, nuclear localization and stable long-term transgene expression remain significant hurdles that need to be overcome. The challenge of non-viral gene therapy is therefore to harness current laboratory and molecular-based techniques to develop a man-made system that can approach the efficiency of a natural biological process. In the unique environment of the retina, this goal may not be insurmountable and would overcome the major limiting factor of adeno-associated viral vectors, which is the size of gene that can be delivered. [ABSTRACT FROM AUTHOR]

Abstract: Ciliary neurotrophic factor (CNTF) has been shown to protect ganglion cells in a variety of acute ischaemia models. Here we assess the efficacy of local CNTF gene transfer in protecting retinal ganglion cells when there is focal ischaemia combined with interruption of axoplasmic flow. This dual injury may be more representative of the pathological mechanisms operating in acute retinal diseases, such as vascular events acting at the optic nerve head. Fourteen rats received an intravitreal injection of an adeno-associated viral (AAV) vector expressing a secretable form of CNTF into the right eye and a control vector into the left eye. Three weeks later, each rat underwent a symmetrical small vertical 2mm standardised retinal crush injury approximately 2mm temporal to the optic disc. The injury also occluded the temporal retinal arteriole so that the axon crush was combined with an acute retinal infarction visible on fundoscopy. Changes in the damaged sector were compared histologically four weeks after injury and ganglion cell survival was estimated by comparing cell counts on retinal flat-mounts immunostained with RT-97 antibody. This mode of injury led to a profound loss of both the inner nuclear and ganglion cell layers, but was limited to the lesioned sector. In AAV.CNTF-treated eyes approximately 12% of ganglion cells survived compared with approximately 2% in control eyes (p =0.01). The scotopic electroretinogram (ERG), however, was reduced to about 50% in AAV.CNTF-treated eyes, both before and after injury. We therefore show that CNTF gene transfer confers neuroprotection to ganglion cells undergoing an acute ischaemic injury combined with interruption of axoplasmic flow. This approach may be relevant to optic nerve trauma and a variety of retinal vascular diseases that lead to swelling of the optic nerve head, provided CNTF can be delivered in a way that does not significantly suppress retinal function. [Copyright &y& Elsevier]

Despite being the most prevalent cause of inherited blindness in children, Stargardt disease is yet to achieve the same clinical trial success as has been achieved for other inherited retinal diseases. With an early age of onset and continual progression of disease over the life course of an individual, Stargardt disease appears to lend itself to therapeutic intervention. However, the aetiology provides issues not encountered with the likes of choroideremia and X-linked retinitis pigmentosa and this has led to a spectrum of treatment strategies that approach the problem from different aspects. These include therapeutics ranging from small molecules and anti-sense oligonucleotides to viral gene supplementation and cell replacement. The advancing development of CRISPR-based molecular tools is also likely to contribute to future therapies by way of genome editing. In this we review, we consider the most recent pre-clinical and clinical trial data relating to the different strategies being applied to the problem of generating a treatment for the large cohort of Stargardt disease patients worldwide. [ABSTRACT FROM AUTHOR]

The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario. [ABSTRACT FROM AUTHOR]

Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the Rho−/− mouse model. To account for gene expression changes attributed to loss of cone photoreceptor cells, we normalized PCR in the remaining number of cones to a cone cell reporter (OPN1-GFP). Gene expression profiles of key components involved in the cone phototransduction cascade were correlated with tests of retinal cone function prior to cell loss. A significant downregulation of the photoreceptor transcription factor Crx was observed, which preceded a significant downregulation in cone opsin transcripts that coincided with declining cone function. Our data add to the growing understanding of molecular changes that occur prior to cone dysfunction in a model of rod-cone dystrophy. It is of interest that gene supplementation of CRX by adeno-associated viral vector delivery prior to cone cell loss did not prevent cone photoreceptor degeneration in this mouse model. [ABSTRACT FROM AUTHOR]