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Introduction: 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., Methods and Materials: 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., Results: 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., Discussion: 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.

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

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

Retinal astrocytes are vital for neuronal homeostasis in the retina. Together with Müller glia, they provide retinal cells with neurotrophic factors, antioxidative support, and defense mechanisms such as the formation of the blood-retinal barrier. Substantial heterogeneity of astrocyte morphology and function represents a challenge for identification of distinct subtypes which may be potential targets for therapeutic purposes. Hence, identification of novel markers of astrocyte subpopulations is highly relevant to better understand the molecular mechanisms involved in retinal development, homeostasis, and pathology. In this study, we observed that the cell cycle regulator, p16 INK4a , is expressed in immature astrocytes in the mouse retina. Immunohistochemical analysis showed p16 INK4a expression in the optic nerve of wild-type mice from 3 days to 3 months of age and in the nerve fiber layer of the adult mouse retina. Colocalization of p16 INK4a expression and glial fibrillary acidic protein (immature/mature astrocyte marker) tends to decrease with age. However, colocalization of p16 INK4a expression and vimentin (immature astrocyte marker) remains high in the optic nerve from the early postnatal period to adulthood. The observations from this study provide a valuable tool for further investigations of ocular astrocytes in the developing retina as well as in degenerative retinopathies.

Pathogenic variants in the Retinitis pigmentosa GTPase regulator (RPGR) gene lead to a clinically severe form of X-linked retinal dystrophy. However, it remains unclear why some variants cause a predominant rod, while others result in a cone-dominated phenotype. Post-translational glutamylation of the photoreceptor-specific RPGR ORF15 isoform by the TTLL5 enzyme is essential for its optimal function in photoreceptors, and loss of TTLL5 leads to retinal dystrophy with a cone phenotype. Here we show that RPGR retinal disease, studied in a single cohort of 116 male patients, leads to a clear progressive shift from rod- to cone-dominating phenotype as the RPGR ORF15 variant location approaches the distal part of the Open Reading Frame 15 (ORF15) region. The rod photoreceptor involvement on the contrary diminishes along the RGPR sequence, and the variants associated with the cone only phenotype are located predominantly in the very distal part, including the C-terminal basic domain. Moreover, these distal truncating RPGR ORF15 variants disrupt the interaction with TTLL5 and lead to a significant impairment of RPGR glutamylation. Thus, consistent with the phenotype of TTLL5 pathogenic variants, our study shows that RPGR ORF15 variants, which disrupt its basic domain and the interaction with TTLL5, also impair RPGR glutamylation and lead to the cone phenotype. This has implications for ongoing gene therapy clinical trials where the application of RPGR with impaired glutamylation may be less effective in treating RGPR dystrophies and may even convert a rod-cone dystrophy into a cone dystrophy phenotype.

Introduction: Mutations in the RPGR gene are responsible for one of the most prevalent and severe types of retinitis pigmentosa. Gene therapy has shown great promise to treat inherited retinal diseases, and currently, four RPGR gene therapy vectors are being evaluated in clinical trials., Areas Covered: This manuscript reviews the gene therapy products that are in development for X-linked retinitis pigmentosa caused by mutations in RPGR , and the challenges that scientists and clinicians have faced., Expert Opinion: The development of a gene therapy product for RPGR -associated retinal degeneration has been a great challenge due to the incomplete understanding of the underlying genetics and mechanism of action of RPGR, and on the other hand, due to the instability of the RPGR gene. Three of the four gene therapy vectors currently in clinical trials include a codon-optimized version of the human RPGR sequence, and the other vector contains a shortened version of the human RPGR . To date, the only Phase I/II results published in a peer-reviewed journal demonstrate a good safety profile and an improvement in the visual field using a codon optimized version of RPGR ORF15 .

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a Rho P23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo., (© 2021. The Author(s).)

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 × 10 9 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., Competing Interests: R.E.M. is a consultant to Biogen, Spark Therapeutics, and Novartis and a scientific advisor to the UK’s National Institute for Health and Care Excellence in relation to retinal gene therapy. The views expressed are those of the authors and do not necessarily represent those of the Wellcome Trust, MRC, or NIHR., (© 2021 The Author(s).)

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.

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

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

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., (© 2019 The Authors.)

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

Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca 2+ -activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents in vitr o electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.

Introduction: X-linked retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator ( RPGR ) gene is the most common form of recessive RP. The phenotype is characterised by its severity and rapid disease progression. Gene therapy using adeno-associated viral vectors is currently the most promising therapeutic approach. However, the construction of a stable vector encoding the full-length RPGR transcript has previously proven to be a limiting step towards gene therapy clinical trials. Recently however, a codon optimised version of RPGR has been shown to increase the stability and fidelity of the sequence, conferring a therapeutic effect in murine and canine animal models., Areas Covered: This manuscript reviews the natural history of X-linked retinitis pigmentosa and the research performed from the discovery of the causative gene, RPGR , to the preclinical testing of potential therapies that have led to the initiation of three clinical trials., Expert Opinion: X-linked retinitis pigmentosa is an amenable disease to be treated by gene therapy. Codon optimisation has overcome the challenge of designing an RPGR vector without mutations, and with a therapeutic effect in different animal models. With the RPGR gene therapy clinical trials still in the early stages, the confirmation of the safety, tolerability and potency of the therapy is still ongoing., Competing Interests: Declaration of Interest RE MacLaren is the academic founder and director of Nightstar Therapeutics, a gene therapy company established by the University of Oxford and originally funded by the Wellcome Trust through Syncona Partners. RE MacLaren and M. Dominik Fischer are names inventors on a patent filed on behalf of the University of Oxford, relating to the expression cassette and codon optimisation of RPGR coding sequence in general. They are also consultants to Nightstar Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

X-linked retinitis pigmentosa (XLRP) is generally a severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGR ORF15 ). Despite successful RPGR ORF15 gene replacement with adeno-associated viral (AAV) vectors being established in a number of animal models of XLRP, progression to human trials has not yet been possible. The inherent sequence instability in the purine-rich region of RPGR ORF15 (which contains highly repetitive nucleotide sequences) leads to unpredictable recombination errors during viral vector cloning. While deleted RPGR may show some efficacy in animal models, which have milder disease, the therapeutic effect of a mutated RPGR variant in patients with XLRP cannot be predicted. Here, we describe an optimized gene replacement therapy for human XLRP disease using an AAV8 vector that reliably and consistently produces the full-length correct RPGR protein. The glutamylation pattern in the RPGR protein derived from the codon-optimized sequence is indistinguishable from the wild-type variant, implying that codon optimization does not significantly alter post-translational modification. The codon-optimized sequence has superior stability and expression levels in vitro. Significantly, when delivered by AAV8 vector and driven by the rhodopsin kinase promoter, the codon-optimized RPGR rescues the disease phenotype in two relevant animal models (Rpgr -/y and C57BL/6J Rd9/Boc ) and shows good safety in C57BL6/J wild-type mice. This work provides the basis for clinical trial development to treat patients with XLRP caused by RPGR mutations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)