Developmental Expression of the Cell Cycle Regulator p16INK4a in Retinal Glial Cells: A Novel Marker for Immature Ocular Astrocytes?

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, p16INK4a, is expressed in immature astrocytes in the mouse retina. Immunohistochemical analysis showed p16INK4a 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 p16INK4a expression and glial fibrillary acidic protein (immature/mature astrocyte marker) tends to decrease with age. However, colocalization of p16INK4a 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.

A Novel Postoperative Drop Regimen Reduces Risk of Ocular Hypertension Following Pars Plana Vitrectomy.

INTRODUCTION: Postoperative steroid/antibiotic drop regimens are known to effectively suppress inflammation and infection following pars plana vitrectomy (PPV), but the steroid frequently induces ocular hypertension (OHT). The aim of this contemporaneous cohort-control study was to assess safety and efficacy of a novel post-PPV drop regimen conceived to address this problem. METHODS: Electronic case notes of consecutive eyes undergoing PPV between December 2020 and April 2021 at St. Thomas' Hospital, London, UK, were reviewed retrospectively. Postoperative drops in the intervention cohort consisted of 1-week g. dexamethasone 0.1%/antibiotic QDS and 1-month g. ketorolac TDS. Standard care controls received 1-month g. dexamethasone 0.1%/antibiotic QDS. RESULTS: Fifty-eight patients were in the intervention cohort, and 151 received standard care. The primary outcome measure was IOP ≥30 mm Hg 2 weeks postoperatively. This occurred in none of the intervention group but in 14% of controls (p = 0.01). Secondary outcomes of rates of anterior uveitis and cystoid macular edema did not differ significantly between the groups, but those in the intervention cohort had fewer hospital visits (p = 0.0004). CONCLUSION: A post-PPV drop regimen of 1-week dexamethasone 0.1%/antibiotic and 1-month ketorolac may be as effective as an anti-inflammatory but safer in terms of OHT incidence than standard care 1-month dexamethasone 0.1%.

Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa.

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 RhoP23H 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.

Effect of AAV-Mediated Rhodopsin Gene Augmentation on Retinal Degeneration Caused by the Dominant P23H Rhodopsin Mutation in a Knock-In Murine Model.

Mutations in the rhodopsin gene may cause photoreceptor degeneration in autosomal dominant retinitis pigmentosa (ADRP) by dominant negative or toxic gain-of-function mechanisms. Controversy exists as to the mechanism by which the widely studied P23H mutation induces rod cell dysfunction and death. Inherited disease caused by dominant negative mutations may be amenable to treatment using wild-type gene augmentation. Indeed, prior studies in the RHOP23H, Rho+/- transgenic mouse model of ADRP have suggested that a therapeutic benefit may be achieved when wild-type rhodopsin is overexpressed following subretinal delivery of a recombinant adeno-associated viral (AAV) vector. In this study, we investigated the effect of wild-type rhodopsin supplementation on the rate of retinal degeneration in the more clinically relevant RhoP23H/+ knock-in mouse model of ADRP. Four AAVs carrying the human rhodopsin coding sequence were first designed and compared for efficacy in the rhodopsin knockout mouse. All four vectors were capable of driving expression of the human transgene in the knockout retina with the protein being appropriately trafficked to de novo rod outer segments. The most efficient of these vectors was injected at one of two doses into the subretinal space of RhoP23H/+ mice and the effect on retinal structure and function determined longitudinally by spectral-domain optical coherence tomography and electroretinography, respectively, over a 3-month period. Although significant overexpression of rhodopsin protein was achieved in this model, no beneficial effect on retinal structure or function was observed at either dose. Lack of therapeutic efficacy in this model may be attributable to the relative rapidity of degeneration in the RhoP23H/+ mouse relative to the human disease, over- or under dosing at the level of individual photoreceptors, late timing of the intervention, or a possible predominant toxic gain-of-function mechanism of degeneration.

Dynamic in vivo quantification of rod photoreceptor degeneration using fluorescent reporter mouse models of retinitis pigmentosa.

Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP). Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho-/-) or heterozygous for knock-in of P23H mutant rhodopsin (Nrl-EGFP, RhoP23H/+) were used in this study. These novel strains have green fluorescent rods which undergo a progressive degeneration. Fundus imaging was performed at three-weekly intervals by near infrared reflectance (NIR) and blue light autofluorescence (BAF) confocal scanning laser ophthalmoscopy (cSLO). Mean grey values (mGV), which quantify fluorescence levels within such images, were compared for degenerate and age-matched non-degenerate (Nrl-EGFP, Rho+/+) controls. Mean grey value significantly decreased over time in the Rho-/- and RhoP23H/+ groups but was maintained in Rho+/+ mice (P < 0.001, two-way ANOVA). This corresponded to outer nuclear layer (ONL) thinning as observed by histology. The mGV of superior retina was significantly greater than that of inferior retina in RhoP23H/+ (P = 0.0024) but not in age-matched Rho+/+ (P = 0.45) or Rho-/- (P = 0.65) mice reflecting histological findings. Focal loss of rods could be visualised and mapped in vivo with this technique following a toxic insult, with thinning of the ONL being confirmed in hypofluorescent regions by spectral domain ocular coherence tomography (OCT). Fluorescence labelling of rods permits in vivo characterisation of models of RP and may provide new insights into patterns of degeneration, or rescue effect after treatment. mGV can be used in such cases as a semiquantitative metric of ONL degeneration, and can be used to identify regional variations in photoreceptor loss.

Filtration of Short-Wavelength Light Provides Therapeutic Benefit in Retinitis Pigmentosa Caused by a Common Rhodopsin Mutation.

Purpose: The role of light exposure in accelerating retinitis pigmentosa (RP) remains controversial. Faster degeneration has however been observed in the inferior than superior retina in several forms ("sector" RP), including those caused by the rhodopsin P23H mutation, suggesting a modifying role of incident light exposure in such cases. Rearing of equivalent animal models in complete darkness has been shown to slow the degeneration. Here we investigate the use of red filters as a potential treatment strategy, with the hypothesis that minimizing retinal exposure to light <600 nm to which rods are maximally sensitive may provide therapeutic benefit. Methods: Knockin mice heterozygous for the P23H dominant rhodopsin mutation (RhoP23H/+) housed in red-tinted plastic cages were divided at weaning into either untinted or red-tinted cages. Subsequently, photoreceptor layer (PRL) thickness was measured by spectral-domain ocular coherence tomography, retinal function quantified by ERG, and cone morphology determined by immunohistochemical analysis (IHC) of retinal flatmounts. Results: Mice remaining in red-tinted cages had a significantly greater PRL thickness than those housed in untinted cages at all time points. Red housing also led to a highly significant rescue of retinal function as determined by both dark- and light-adapted ERG responses. IHC further revealed a dramatic benefit on cone morphology and number in the red- as compared with the clear-housed group. Conclusions: Limitation of short-wavelength light exposure significantly slows degeneration in the RhoP23H/+ mouse model. Red filters may represent a cost-effective and low-risk treatment for patients with rod-cone dystrophy in whom a sectoral phenotype is noted.

A Novel Achromatopsia Mouse Model Resulting From a Naturally Occurring Missense Change in Cngb3.

Purpose: A local colony of inbred mice (129S6/SvEvTac origin), in isolation for over a decade, were found to have absent light-adapted electroretinogram (ERG) responses. We investigated the inheritance and genetic basis of this phenotype of cone photoreceptor function loss. Methods: An affected 129S6/SvEvTac colony animal was outcrossed to a C57BL/6J mouse and intercrossed to investigate inheritance in the F2 generation. We performed ERG testing and targeted resequencing on genes of interest (Gnat2, Cnga3, Cngb3, Pde6c, Hcn1, Syne2). The eyes of a subset of animals underwent histologic immunostaining. Results: All 129S6/SvEvTac colony animals tested lacked cone pathway function by ERG testing (n = 12), although rod pathway-based ERG responses remained unaffected. Outcross-intercross breeding showed a recessive inheritance pattern. A novel missense mutation was identified in the Cngb3 gene, which causes an amino acid substitution at a conserved residue (NM_013927)c.692G>A; p.(R231H). The recessive phenotype only affected homozygotes (χ2 = 39, P = 3.2e-10). Cones had normal morphology at postnatal day (PND) 70, but cone cell counts declined from PND 30 to PND 335 (P = 0.038), indicating progressive cone photoreceptor death. Conclusions: We identified the spontaneous occurrence of a 10th model of cone photoreceptor function loss (cpfl10) in an isolated line of inbred mice. Our results indicate that this is caused by a novel missense mutation in the Cngb3 gene, with a fully recessive inheritance pattern. This mouse may provide a more appropriate background against which to assess CNGB3 achromatopsia gene therapy for missense mutations.

Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia.

Retinal gene therapy is increasingly recognized as a novel molecular intervention that has huge potential in treating common causes of blindness, the majority of which have a genetic aetiology1-5. Choroideremia is a chronic X-linked retinal degeneration that was first described in 18726. It leads to progressive blindness due to deficiency of Rab-escort protein 1 (REP1). We designed an adeno-associated viral vector to express REP1 and assessed it in a gene therapy clinical trial by subretinal injection in 14 patients with choroideremia. The primary endpoint was vision change in treated eyes 2 years after surgery compared to unoperated fellow eyes. Despite complications in two patients, visual acuity improved in the 14 treated eyes over controls (median 4.5 letter gain, versus 1.5 letter loss, P = 0.04), with 6 treated eyes gaining more than one line of vision (>5 letters). The results suggest that retinal gene therapy can sustain and improve visual acuity in a cohort of predominantly late-stage choroideremia patients in whom rapid visual acuity loss would ordinarily be predicted.

Human Retinal Explant Culture for Ex Vivo Validation of AAV Gene Therapy.

Recombinant adeno-associated viral (AAV) vectors have been successfully employed as the mode of gene delivery in several clinical trials for the treatment of inherited retinal diseases to date. The design of such vectors is critical in determining cellular tropism and level of subsequent gene expression that may be achieved following viral delivery. Here we describe a system for living retinal tissue extraction, ex vivo culture, viral transduction and assessment of transgene expression that may be used to assess viral constructs for gene therapy in the human retina at a preclinical stage.

Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis.

PURPOSE: Treatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown. METHODS: The effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by optical coherence tomography, autofluorescence, dark-and light-adapted full-field electroretinography. RESULTS: DB and MB were not toxic at any concentration tested, SF only when undiluted. The presence of dyes did not adversely affect the genomic titer. DB even increased the values, due to presence of surfactant in the formulation. AAV2-GFP transduction efficiency was not reduced by the dyes. No structural and functional toxic effects were observed following subretinal delivery of DB. CONCLUSIONS: Only undiluted SF affected cell viability. No effects on qPCR titer and transduction efficiency were observed. DB does not appear toxic when delivered subretinally and improves titer accuracy. DB may therefore be a safe and helpful adjunct during gene therapy surgery. TRANSLATIONAL RELEVANCE: This paper might be of interest to the retinal gene therapy community: it is a "bench to bedside" research paper about the potential use of dyes as a surgical adjunct during the gene therapy surgery. We have tested the potential toxicity and impact on transduction efficiency in an in vitro and in vivo model.

Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina.

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.

Dysthyroid Orbitopathy Presenting with Gaze-Evoked Amaurosis: Case Report and Review of the Literature.

BACKGROUND: Gaze-evoked amaurosis (GEA) describes visual loss associated with eccentric gaze that recovers when the eye is returned to primary position. Here we describe an unusual case of bilateral GEA as the presenting feature of dysthyroid orbitopathy. This is only the third such case to be reported in the literature and the first to feature bilateral GEA in all positions of gaze without accompanying proptosis or ophthalmoplegia. CASE PRESENTATION: A 50-year-old man who had recently commenced treatment for thyrotoxicosis presented with a 3-week history of typical GEA in both eyes in all positions of gaze. He subsequently developed a bilateral compressive optic neuropathy which was only partially responsive to high dose steroid therapy. CONCLUSION: Although an uncommon presenting feature of dysthyroid orbitopathy, GEA is an ominous symptom that may precede sight-threatening optic nerve compromise. When present, early immunosuppressive and/or decompressive treatment should be considered.

Anti-VEGF Therapy and the Retina: An Update.

Ocular angiogenesis and macular oedema are major causes of sight loss across the world. Aberrant neovascularisation, which may arise secondary to numerous disease processes, can result in reduced vision as a result of oedema, haemorrhage, and scarring. The development of antivascular endothelial growth factor (anti-VEGF) agents has revolutionised the treatment of retinal vasogenic conditions. These drugs are now commonly employed for the treatment of a plethora of ocular pathologies including choroidal neovascularisation, diabetic macular oedema, and retinal vein occlusion to name a few. In this paper, we will explore the current use of anti-VEGF in a variety of retinal diseases and the impact that these medications have had on visual outcome for patients.