Inherited retinal dystrophies (IRDs) are a major cause of early-onset blindness. Biallelic RPE65-mediated IRD, the most severe form of IRD, occurs when there are mutations in both alleles of the RPE65 gene in retinal pigment epithelium (RPE) cells. Voretigene neparvovec, developed by Spark Therapeutics, Inc., Pennsylvania, US, is a gene therapy designed to deliver a normal copy of the RPE65 gene to the RPE cells that are lacking a normally functioning RPE65 gene. This is the first gene therapy that has completed a phase III clinical trial — a randomized, open-label, controlled trial assessing the safety and efficacy of voretigene neparvovec for the treatment of biallelic RPE65-mediated IRD. In the phase III trial, patients treated with voretigene neparvovec showed significant improvement in navigational ability in dimly light conditions, compared with the control group, at one year. This treatment was associated with mild to moderate ocular adverse events; one patient experienced a loss of visual acuity in the first assigned eye. Improvement in visual function appears to remain durable for up to three years based on current data. Longer-term safety and efficacy data for voretigene neparvovec are needed to confirm its duration of effect, its impact on retinal degeneration, and the impact on the quality of life of participants treated with this therapy. The US FDA approved voretigene neparvovec (voretigene neparvovec-rzyl as per the FDA label) on December 19, 2017 under the trade name Luxturna. The drug is also currently under review by the European Medicines Agency, with a decision expected in the latter half of 2018. In the US, the price of voretigene neparvovev-rzyl has been set to US$425,000 per eye (US$850,000 for bilateral disease); this is a one-time treatment.

RPE65-associated Leber congenital amaurosis (RPE65-LCA) is a progressive severe retinal dystrophy with early profound dysfunction of rod photoreceptors followed by progressive cone photoreceptor degeneration. We aim to provide detailed information about how cone dysfunction affects color discrimination.

Seven adults (aged 16-21) with RPE65-LCA underwent monocular color discrimination assessment using the Trivector and Ellipse versions of three computerized tests: Cambridge Colour Test (CCT), low vision version of the Cambridge Colour Test (lvvCCT), and the Universal Colour Discrimination Test (UCDT). For comparison, subjects were also tested using the American Optical Hardy Rand Rittler (AO-HRR) plates. Each assessment was repeated three times.

The Trivector version of the tests demonstrated that color discrimination along the tritan axis was undetectable in four subjects, and severely reduced in three subjects. These findings were confirmed by the Ellipse version of the tests. Color discrimination along the protan and deutan axes was evident but reduced in six of seven subjects. Four of seven subjects were unable to read any of the HRR plates.

The computerized color vision tests adopted in this study provide detailed information about color discrimination in adult RPE65-LCA patients. The condition is associated with severe impairment of color discrimination, particularly along the tritan axis indicating possible early involvement of S-cones, with additional protan and deutan loss to a lesser extent. This psychophysical assessment strategy is likely to be valuable in measuring the impact of therapeutic intervention on cone function.

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward β-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce β-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.

Keywords: RPE65; apocarotenoid; nonheme iron; regioselectivity; β-apo-14′-carotenal.

Recent work suggested that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is increased in the retinal pigment epithelium (RPE) of age-related macular degeneration (ARMD) patients and therefore could be an attractive therapeutic target. Notably, ERK1/2 pathway inhibitors are used in cancer therapy, with severe and noncharacterized ocular side effects. To decipher the role of ERK1/2 in RPE cells, we conditionally disrupted the Erk1 and Erk2 genes in mouse RPE. The loss of ERK1/2 activity resulted in a significant decrease in the level of RPE65 expression, a decrease in ocular retinoid levels concomitant with low visual function, and a rapid disorganization of RPE cells, ultimately leading to retinal degeneration. Our results identify the ERK1/2 pathway as a direct regulator of the visual cycle and a critical component of the viability of RPE and photoreceptor cells. Moreover, our results caution about the need for a very fine adjustment of kinase inhibition in cancer or ARMD treatment in order to avoid ocular side effects.

The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC50 of 6 μM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.

The aim of this review is to provide a comprehensive summary of current gene therapy clinical trials for monogenic and optic nerve disorders.The number of genes for which gene-based therapies are being developed is growing. At the time of writing this review gene-based clinical trials have been registered for Leber congenital amaurosis 2 (LCA2), retinitis pigmentosa 38, Usher syndrome 1B, Stargardt disease, choroideremia, achromatopsia, Leber hereditary optic neuropathy (LHON) and X-linked retinoschisis. Apart from RPE65 gene therapy for LCA2 and MT-ND4 for LHON which has reached phase III, all other trials are in investigation phase I and II, i.e. testing the efficacy and safety.Because of the relatively easy accessibility of the retina and its ease of visualization which allows monitoring of efficacy, gene-based therapies for inherited retinal disorders represent a very promising treatment option. With the development of novel therapeutic approaches, the importance of establishing not only clinical but also molecular genetic diagnosis is obvious.Key words: gene therapy, monogenic retinal diseases, optic nerve atrophy, mitochondrial disease.

Purpose: The c.1430A > G (Asp477Gly) variant in RPE65 has been reported in Irish and Scottish families with either an autosomal dominant retinal dystrophy (adRD) that resembles choroideremia, a vitelliform macular dystrophy or an isolated macular atrophy. We report novel features on multimodal imaging and the natural history of a family harbouring this variant in combination with the BEST1 c.37C > T (Arg13Cys) variant.

Methods: Members of a family with an adRD were examined clinically to ascertain phenotype and underwent genetic testing. Multimodal imaging included widefield colour fundus photography, quantitative autofluorescence (qAF) and spectral domain optical coherence tomography. Electrophysiology and microperimetry were also performed.

Results: Vision loss was attributed to foveal atrophy in the proband and choroidal neovascularisation and a vitello-eruptive lesion in one affected son. Peripheral retinal white dots corresponding to subretinal deposits were seen in three patients. The median qAF₈ values in the proband (I:1) were low (40 and 101 in OD and OS) at age 79. Similarly, the qAF₈ values for the middle son (II:2) were also low (100 and 87 in ODS and OS) at age 60. Electrophysiology showed disproportionate reduction in Arden ratio prior to the gradual loss of full-field responses. Microperimetry demonstrated an enlarging scotoma in the proband.

Conclusions: The coexistence of the pathogenic BEST1 c.37C > T variant may modify clinical features observed in RPE65 adRD. This study expands our understanding of RPE65 adRD as a retinoid cycle disorder supported by the reduced qAF, fine white retinal dots and corresponding subretinal deposits on OCT in affected members.

Keywords: Best disease; Choroidal neovascularisation; Inherited retinal disease; Microperimetry; Natural history study; Quantitative fundus autofluorescence; Retinitis pigmentosa; Vitelliform macular dystrophy.

Purpose: Leber congenital amaurosis type 2 (LCA2) and early-onset severe retinal dystrophy (EOSRD) are linked to visual impairment with nyctalopia and visual acuity reduction in early childhood. In 2017, the first gene therapy voretigene neparvovec (Luxturna™) for patients with LCA and EOSRD cause by bi-allelic mutations in the RPE65 gene has been approved. Here we report on an example of short-term change in the foveal morphology after functionally successful gene therapy with voretigene neparvovec in a 15-year old patient.

Methods: The clinical examinations included best corrected visual acuity (BCVA), spectral domain optical coherence tomography (OCT) and adaptive optics retinal imaging.

Results: During follow-up over a period of 3 months after the treatment, an improvement of the central foveal morphology could be observed in OCT, with a clear demarcation of the external limiting membrane and changes in the photoreceptor mosaic on adaptive optics retinal imaging. These morphological rescue parameters correlated in part with the improvement in foveal-mediated vision after the treatment and adaptive optics imaging. Although the visual acuity improved only slightly at month 3, objective central cone evaluation with chromatic pupil campimetry showed an increase in the central sensitivity. In daily life, the patient reported her visional experience after the treatment as 'brighter'.

Conclusion: Rapid changes in the correlates of photoreceptor morphology after successful gene therapy in patients with LCA/EORD can be quantifiable on individual level.

Keywords: OCT; RPE65; case report; gene therapy; retinitis pigmentosa; voretigene neparvovec.

Introduction: Voretigene neparvovec-rzyl (Luxturna) was approved by the Australian Therapeutic Goods Administration on 4 August 2020 for the treatment of biallelic mutations in the RPE65 gene, a rare cause of congenital and adult-onset retinal dystrophy (predominantly Leber congenital amaurosis). Previous studies have shown that individuals who might participate in gene therapy trials overestimate clinical effect and underestimate risks. However, little is known about the perspectives of patients who may be offered approved gene therapy treatment for ocular conditions (as distinct from participating in clinical trials of gene therapy). The main objective of this study is to develop a tool to assess knowledge, attitudes and perceptions of approved and future genetic therapies among potential recipients of ocular gene therapy. In addition, we aim to assess the quality of life, attitudes towards clinical trials and vision-related quality of life among this cohort.

Methods and analysis: A new 'Attitudes to Gene Therapy for the Eye' tool will be developed following consultation with people with inherited retinal disease (IRD) and content matter experts. Australians with IRD or their guardians will be asked to complete an internet-based survey comprising existing quality of life and visual function instruments and items for the newly proposed tool. We expect to recruit 500 survey participants from patient support groups, the practices of Australian ophthalmologists who are specialists in IRD and Australian ophthalmic research institutions. Launch is anticipated early 2021. Responses will be analysed using item response theory methodology.

Ethics and dissemination: This study has received ethics approval from the University of Melbourne (#2057534). The results of the study will be published in a peer-reviewed journal and will be presented at relevant conferences. Organisations involved in recruitment, and the Patient Engagement Advisory committee will assist the research team with dissemination of the study outcomes.

Keywords: genetics; medical retina; vitreoretinal.

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15^R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15^R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15^R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9-cis retinal improved ERG amplitudes in Pcdh15^R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients.

Keywords: PCDH15; cell biology; exogenous retinoids; human; medicine; mouse; natural history; retinal degeneration; usher syndrome.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH₂) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH₂ with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH₂ derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4^-/-Rdh8^-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH₂ or other tested amino acid derivatives of Ret-NH₂. Ret-NH₂-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4^-/-Rdh8^-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

Purpose: To test the ability of a virtual reality (VR) orientation and mobility (O&M) protocol to serve a measure of functional vision for patients with inherited retinal degenerations (IRDs).

Methods: A VR-O&M protocol designed using a commercially available VR hardware was tested in normally sighted control subjects (n=7; ages 10-35yo; Average 22.5yo) and patients with RPE65-associated Leber Congenital Amaurosis (n=3; ages 7-18yo; Average 12.7yo), in two of them before and after gene therapy. Patients underwent perimetry and full-field sensitivity testing. VR-O&M parameters correlated with the visual dysfunction.

Results: Visual acuities in RPE65 patients were on average worse than 20/200, dark-adapted sensitivity losses >5 log units, and fields constricted between 20° and 40°. Before treatment, patients required ~1000-fold brighter environment to navigate, had at least x4 more collisions, and were slower both to orient and navigate compared to control subjects. Improvements in cone- (by 1-2 L.u.) and rod-mediated (by >4 L.u.) sensitivities post-treatment led to fewer collisions (at least by half) at ~100-fold dimmer luminances, and to x4 times faster navigation times.

Conclusion: This study provides proof-of-concept data in support for the use of VR-O&M systems to quantify the impact that the visual dysfunction and improvement of vision following treatments has on functional vision in IRDs. The VR-O&M was useful in potentially challenging scenarios such as in pediatric patients with severe IRDs.

Translational relevance: A VR-O&M test will provide much needed flexibility, both in its deployment as well as in the possibility to test various attributes of vision that may be impacted by gene therapy in the setting of translational studies.

Precis: This study provides proof-of-concept data in support for the use of a virtual reality orientation and mobility test to quantify the impact of the disease and of treatments thereof on functional vision in inherited retinal degenerations.

Keywords: LCA; RPE65; gene therapy; mobility; orientation; virtual reality.

This review presents the phenotypic and genotypic profiles of a select group of inherited retinal diseases (IRDs) that are currently the focus of retinal gene therapy trials globally. Research progress in IRD treatment trials may soon lead to their availability in Australia and New Zealand, as either approved treatment or a clinical trial. The salient clinical characteristics of retinitis pigmentosa-the largest IRD category-are highlighted, with specific reference to RPE65-associated Leber congenital amaurosis, followed by other specific IRDs, namely choroideremia and ABCA4-associated Stargardt disease. These IRDs are selected based on their candidacy for gene therapy. Guidance on the clinical diagnostic tests that support each of these diagnoses will be presented. More broadly, the most useful structure and function measures to monitor IRD progression is discussed, along with the key assessments that offer differential diagnostic insight. This review is intended to be a clinical guide for optometrists, to assist in assessment and management of individuals who may be eligible for current and future gene therapies. A companion article in this issue will provide an overview of the basic principles of gene therapy and its development as a new treatment for inherited retinal diseases.

Keywords: Gene therapy; inherited retinal disease; retinal dystrophy; retinitis pigmentosa.

Retinal degenerative diseases are considered a major challenge all over the world, and stem cell therapy is a promising approach to restore degenerative cells due to RD. MSCs are multipotent stem cells found in a variety of tissues. They are capable of differentiating into various retinal cell types, so it can be a good candidate for various degenerative disorders like retinal degenerations. β-carotene is an antioxidant that could accelerate the stem cell differentiation while using the proper scaffold. In this study, we evaluated the effect of β-carotene on the differentiation potential of ciliary epithelium-derived MSCs isolated from mouse eyes on alginate-based scaffolds. MSCs were isolated from mouse ciliary epithelium, cultured in DMEM medium supplemented with 10% FBS, and identified by detecting their surface antigens. Three 3D culture systems, alginate, alginate/gelatin, and gelatin hydrogels were prepared, and their structures were checked via SEM. MSCs were cultured on 3D and 2D culture system scaffolds following treated with differentiation medium containing 50 μM β-mercaptoethanol, 1 × minimum essential medium-nonessential amino acids and 20% of knockout serum replacement and β-carotene. MSCs viability and differentiation ability were examined by MTT and ICC, respectively. The expression changes of several retinal specific genes (Nestin, RPE65, and Rhodopsin) were also evaluated by qPCR. Over 80% of cells isolated from mouse ciliary epithelium were positive for MSC-specific markers. The viability rates of MSCs grown on all alginate-based scaffolds were above 70%. MSCs cultured on alginate-based scaffold in the differentiation medium containing β-carotene expressed higher levels of rhodopsin protein compared to a 2D culture. Also, the expressions of Nestin, Rhodopsin, and RPE65 genes were upregulated in β-carotene-treated MSCs grown on alginate-based scaffolds. Our results indicate that the addition of β-carotene to the differentiation medium, along with applying alginate-based scaffolds, could induce higher differentiation in mouse ciliary epithelium-derived MSCs into specialized retinal cells.

Keywords: Cell scaffolds; Differentiation; Retinal cell; Stem cell.

Purpose: To evaluate the mononuclear cells in the subretinal exudate in Coats' disease.

Design: Retrospective case series.

Methods: Five enucleated globes and one cytology sample with Coats' disease and one case of chronic retinal detachment following repair of an open globe injury were examined immunohistochemically to identify the intraretinal and subretinal exudative cells. The two biomarkers were RPE65 for retinal pigment epithelium and CD163 for histiocytes, each tagged with different chromogens, yellow for pigment epithelium and purple for CD163+ monocytes/histiocytes. Expressions were sought of both biomarkers together or singly. A color shift to red in the cells' chromogenic reaction indicated the simultaneous presence of the two biomarkers.

Results: The majority of the mononuclear cells in Coats' disease were CD163 (purple) positive, and a minority were RPE65 (yellow) positive. An intermediate number of cells were RPE65/CD163 positive (orange-red). The eye with a chronic retinal detachment had an equal distribution of CD163 positive and RPE65/CD163 positive cells.

Conclusions: The retinal pigment epithelium has several well-delineated phenotypes and functions. In normal visual physiology, the pigment epithelium supports the photoreceptors and participates in their renewal by phagocytosis of the tips of the photoreceptors. The expression of CD163, a feature of hematopoietically derived monocytes, together with RPE65 in the retinal pigment epithelium, supports differentiation toward histiocytes. Yellow staining detached pigment epithelial cells were rare. The presence of histiocytoid pigment epithelium at the level of Bruch's membrane probably also has implications for macular degeneration.

Keywords: Coats’ disease; histiocytes; macrophages; macular degeneration; monocytes; retina; retinal pigment epithelium; retinal telangiectasias; subretinal exudate.

The ability of iron to transfer electrons enables the contribution of this metal to a variety of cellular activities even as the redox properties of iron are also responsible for the generation of hydroxyl radicals (•OH), the most destructive of the reactive oxygen species. We previously showed that iron can promote the oxidation of bisretinoid by generating highly reactive hydroxyl radical (•OH). Now we report that preservation of iron regulation in the retina is not sufficient to prevent iron-induced bisretinoid oxidative degradation when blood iron levels are elevated in liver-specific hepcidin knock-out mice. We obtained evidence for the perpetuation of Fenton reactions in the presence of the bisretinoid A2E and visible light. On the other hand, iron chelation by deferiprone was not associated with changes in post-bleaching recovery of 11-cis-retinal or dark-adapted ERG b-wave amplitudes indicating that the activity of Rpe65, a rate-determining visual cycle protein that carries an iron-binding domain is not affected. Notably, iron levels were elevated in the neural retina and RPE of Abca4^-/- mice. Consistent with higher iron content, ferritin-L immunostaining was elevated in RPE of a patient diagnosed with ABCA4-associated disease and in RPE and photoreceptor cells of Abca4^-/- mice. In neural retina of the mutant mice, reduced Tfrc mRNA was also an indicator of retinal iron overload. Thus iron chelation may defend retina when bisretinoid toxicity is implicated in disease processes.

Keywords: Fenton reaction; bisretinoid; ferritin; high-performance liquid chromatography (HPLC); iron; light; lipofuscin; retina; retinal degeneration; retinal pigment epithelium.

Background: Voretigene neparvovec is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. In this study, we report on a novel and objective evaluation of a retinotopic photoreceptor rescue.

Methods: Seven eyes of five patients (14, 21, 23, 24, 36 years, 1 male, 4 females) with bi-allelic RPE65 mutations have been treated with voretigene neparvovec. The clinical examinations included visual acuity testing, dark-adapted full-field stimulus threshold (FST), dark-adapted chromatic perimeter (DAC) with a 30-degree grid, and a 30 degrees grid scotopic and photopic chromatic pupil campimetry (CPC). All evaluations and spectral domain optical coherence tomography were performed at baseline, 1 month and 3 months.

Results: All except the oldest patient had a measurable improvement of the rod function assessed via FST, DAC or scotopic CPC at 1 month. The visual acuity improved slightly or remained stable in all eyes. A cone function improvement as measured by photopic CPC was observed in three eyes. The gain of the dark-adapted threshold with blue FST and the DAC stimuli (cyan) average correlated strongly with age (R²>0.7). The pupil response improvement in the scotopic CPC correlated with the baseline local retinal volume (R²=0.5).

Conclusions: The presented protocols allow evaluating the individual spatial and temporal effects of gene therapy effects. Additionally, we explored parameters that correlated with the success of the therapy. CPC and DAC present new and fast ways to assess functional changes in retinotopic maps of rod and cone function, measuring complementary aspects of retinal function.

Keywords: diagnostic tests/investigation; dystrophy; genetics; retina; treatment other.

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB, encode TRs; THRB2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65-/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL+ cells. Cone survival was significantly improved in Rpe65-/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2-/- and Rpe65-/- /Thrb2-/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Thyroid hormone (TH) signaling has been shown to regulate cone photoreceptor viability. Suppression of TH signaling with antithyroid drug treatment or by targeting iodothyronine deiodinases and TH receptors preserves cones in mouse models of retinal degeneration, including the Leber congenital amaurosis Rpe65-deficient mice. This work investigates the cellular mechanisms underlying how suppressing TH signaling preserves cones in Rpe65-deficient mice, using mice deficient in type 2 iodothyronine deiodinase (Dio2), the enzyme that converts the prohormone thyroxine to the active hormone triiodothyronine (T3). Deficiency of Dio2 improved cone survival and function in Rpe65-/- and Rpe65-deficiency on a cone dominant background ( Rpe65-/-/ Nrl-/-) mice. Analysis of cell death pathways revealed that receptor-interacting serine/threonine-protein kinase (RIPK)/necroptosis activity was increased in Rpe65-/-/ Nrl-/- retinas, and Dio2 deficiency reversed the alterations. Cell-stress analysis showed that the cellular oxidative stress responses were increased in Rpe65-/-/ Nrl-/- retinas, and Dio2 deficiency abolished the elevations. Similarly, antithyroid drug treatment resulted in reduced RIPK/necroptosis activity and oxidative stress responses in Rpe65-/-/ Nrl-/- retinas. Moreover, treatment with T3 significantly induced RIPK/necroptosis activity and oxidative stress responses in the retina. This work shows that suppression of TH signaling reduces cellular RIPK/necroptosis activity and oxidative stress responses in degenerating retinas, suggesting a mechanism underlying the observed cone preservation.-Yang, F., Ma, H., Butler, M. R., Ding, X.-Q. Deficiency of type 2 iodothyronine deiodinase reduces necroptosis activity and oxidative stress responses in retinas of Leber congenital amaurosis model mice.

VEGF is a crucial stimulator for choroidal neovascularization (CNV). Our aim was to develop a reproducible and valid treatment-naive quiescent CNV (i.e. without signs of exudation and with normal visual acuity) rat model by subretinal injection of an adeno-associated virus (AAV)-VEGFA165 vector. The CNV development was longitudinally followed up in vivo by scanning laser ophthalmoscopy/optical coherence tomography, fluorescein and indocyanine green angiographies and ex vivo by electron microscopy (EM) and immunohistochemistry. In total, 57 eyes were analysed. In vivo, a quiescent CNV was observed in 93% of the eyes six weeks post-transduction. In EM, CNV vessels with few fenestrations, multi-layered basement membranes, and bifurcation of endothelial cells were observed sharing the human CNV features. Human VEGF overexpression, multi-layered RPE (RPE65) and macrophages/activated microglia (Iba1) were also detected. In addition, 19 CNV eyes were treated up to three weeks with bevacizumab. The retinal and the CNV lesion thickness decreased significantly in bevacizumab-treated CNV eyes compared to untreated CNV eyes one week after the treatment. In conclusion, our experimental CNV resembles those seen in patients suffering from treatment-naive quiescent CNV in wet-age related macular degeneration, and responses to short-term treatment with bevacizumab. Our new model can, therefore, be used to test the long-term effect of new drugs targeting CNV under precisely defined conditions.

Keywords: Age related macular degeneration; Angiogenesis; Bevacizumab; Choroidal neovascularization (CNV); Electron microscopy; VEGF.

Purpose: This study aims to demonstrate the possibility of detecting segmental uniparental isodisomy (iUPD) using a next-generation sequencing gene panel by reporting a Leber congenital amaurosis (LCA) case caused by a homozygous pathogenic variant in RPE65 (c.1022 T > C:p.Leu341Ser) inherited exclusively from the proband's mother.Methods: Samples from the trio (proband, mother, and father) were sequenced with a next-generation sequencing (NGS) retinopathy gene panel (224 genes) and the VCF file containing all variants was used in order to determine single nucleotide variant (SNV) counts from each sample across all chromosomes.Results: Trio analysis showed that of 81 Chr1 inherited variants 41 were exclusively maternal, including 21 homozygous. The other 40 variants were common to both parents. On remaining autosomal chromosomes (Chr2-22) 645 inherited variants were found, 147 of them were exclusively maternal and 132 exclusively paternal. Based on these NGS data, it was possible to note that the proband's chromosomes 1 are more similar to his mother's chromosome 1 than his father's, suggesting the pathogenic homozygous variant found in this patient was inherited exclusively from the mother due to uniparental maternal isodisomy.Conclusions: This study presents a secondary analysis pipeline to identify responsible variants for a phenotype and the correct inheritance pattern, which is a critical step to the proper and accurate genetic counseling of all family members. In addition, this approach could be used to determine iUPD in different Mendelian disorders if the sequencing panel identifies variants spread throughout the genome.

Keywords: RPE65 gene; Uniparental isodisomy; diagnosis; gene panel; next-generation sequencing.

Purpose: Timely detection and multidisciplinary management of RPE65-related inherited retinal disorders (IRDs) can significantly improve both disease management and patient care. Thus, this Narrative Medicine (NM) project aimed to investigate the evolution of the care pathway and the expectations on genetic counseling and gene therapy by patients, caregivers, and healthcare professionals.

Patients and methods: This project was conducted between July and December 2020, involving five Italian eye clinics specialized in IRDs, targeted pediatric and adult patients, their caregivers, attending retinologists and multidisciplinary healthcare professionals. Narratives and parallel charts, together with a sociodemographic survey, were collected through the project webpage. In-depth interviews were conducted with Patient Association (PA) members and multidisciplinary healthcare professionals. All data were entered into the Nvivo Software for coding and analysis.

Results: Three pediatric and five adult patients with early-onset RPE65-related IRDs as well as eight caregivers were enrolled; 11 retinologists globally wrote 27 parallel charts; in-depth interviews were done with five multidisciplinary healthcare professionals and one PA member. Early diagnosis remains challenging, and patients reported to have changed up to 10 healthcare professionals before accessing their specialized center. Despite the oftentimes lack of awareness of patients and caregivers on the purpose of genetic testing, participants generally consider gene therapy as a therapeutic chance and a historic breakthrough for the management of RPE65-related IRDs. Well-organized networks to support the patient's referral to specialized centers - as well as a proper communication of the clinical and genetic diagnosis and the multidisciplinary approach - emerge as crucial aspects in facilitating an early diagnosis and management and a timely initiation of the rehabilitation pathway.

Conclusion: The project investigated the RPE65-related IRDs care pathway while integrating the different perspectives involved through NM. The analysis explored the patient's pathway in Italy and confirmed the need for a well-organized network and multidisciplinary care while highlighting several preliminary areas of improvement in the management of RPE65-related IRDs.

Keywords: IRDs management; diagnosis; inherited retinal dystrophies; multidisciplinary; patient’s pathway.

Human induced pluripotent stem cells (hiPSCs) generated from patients and the derivative retinal cells enable the investigation of pathological and novel variants in relevant cell populations. Biallelic pathogenic variants in RPE65 cause early-onset severe retinal dystrophy (EOSRD) or Leber congenital amaurosis (LCA). Increasingly, regulatory-approved in vivo RPE65 retinal gene replacement therapy is available for patients with these clinical features, but only if they have biallelic pathological variants and sufficient viable retinal cells. In our cohort of patients, we identified siblings with early-onset severe retinal degeneration where genomic studies revealed compound heterozygous variants in RPE65, one a known pathogenic missense variant and the other a novel synonymous variant of uncertain significance. The synonymous variant was suspected to affect RNA splicing. Since RPE65 is very poorly expressed in all tissues except the retinal pigment epithelium (RPE), we generated hiPSC-derived RPE cells from the parental carrier of the synonymous variant. Sequencing of RNA obtained from hiPSC-RPE cells demonstrated heterozygous skipping of RPE65 exon 2 and the introduction of a premature stop codon in the mRNA. Minigene studies confirmed the splicing aberration. Results from this study led to reclassification of the synonymous variant to a pathogenic variant, providing the affected patients with access to RPE65 gene replacement therapy.