A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models.

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

Intravitreal antisense oligonucleotide sepofarsen in Leber congenital amaurosis type 10: a phase 1b/2 trial.

CEP290-associated Leber congenital amaurosis type 10 (LCA10) is a retinal disease resulting in childhood blindness. Sepofarsen is an RNA antisense oligonucleotide targeting the c.2991+1655A>G variant in the CEP290 gene to treat LCA10. In this open-label, phase 1b/2 ( NCT03140969 ), 12-month, multicenter, multiple-dose, dose-escalation trial, six adult patients and five pediatric patients received ≤4 doses of intravitreal sepofarsen into the worse-seeing eye. The primary objective was to evaluate sepofarsen safety and tolerability via the frequency and severity of ocular adverse events (AEs); secondary objectives were to evaluate pharmacokinetics and efficacy via changes in functional outcomes. Six patients received sepofarsen 160 µg/80 µg, and five patients received sepofarsen 320 µg/160 µg. Ten of 11 (90.9%) patients developed ocular AEs in the treated eye (5/6 with 160 µg/80 µg; 5/5 with 320 µg/160 µg) versus one of 11 (9.1%) in the untreated eye; most were mild in severity and dose dependent. Eight patients developed cataracts, of which six (75.0%) were categorized as serious (2/3 with 160 µg/80 µg; 4/5 with 320 µg/160 µg), as lens replacement was required. As the 160-µg/80-µg group showed a better benefit-risk profile, higher doses were discontinued or not initiated. Statistically significant improvements in visual acuity and retinal sensitivity were reported (post hoc analysis). The manageable safety profile and improvements reported in this trial support the continuation of sepofarsen development.

Leber congenital amaurosis as an initial manifestation in a Chinese patient with thiamine-responsive megaloblastic anemia syndrome.

Thiamine-responsive megaloblastic anemia syndrome (TRMA) is an autosomal recessive disorder, inherited by the defective SLC19A2 gene that encodes a high-affinity thiamine transporter (THTR-1). TRMA is characterized by the occurrence of classical triad manifestations including megaloblastic anemia, diabetes mellitus, and sensorineural deafness. In addition to the systemic manifestations, ophthalmic features can be present and include retinitis pigmentosa, optic atrophy, cone-rod dystrophy, maculopathy, and Leber congenital amaurosis. Here we report a 6-year-old boy presenting severe early-onset retinal dystrophy with the initial diagnosis of Leber congenital amaurosis, which followed for 12 years. Diabetes mellitus occurred 3 years after vision problem. Eosinophilic granuloma of the left scapula was confirmed at 13 years old. Whole-exome sequencing was performed to identify two novel compound heterozygous variants c.725dupC (p.Ala243Serfs*3) and c.121G>A (p.Gly41Ser) in SLC19A2 gene (NM_006996.3). Oral thiamine supplementation treatment was initiated at 13 years. This case demonstrates Leber congenital amaurosis can present as the first clinical feature before systemic manifestations. Phenotypic variety should be aware and multidisciplinary teamwork and regular follow-up are important for TRMA patient care.

Intraperitoneal chromophore injections delay early-onset and rapid retinal cone degeneration in a mouse model of Leber congenital amaurosis.

Highly expressed in the retinal pigment epithelium (RPE), the RPE-specific 65-kDa (RPE65) enzyme is indispensable to generate 11-cis-retinal (11cRAL), a chromophore for rhodopsin and cone photopigments. RPE65 deficiency can lead to Leber congenital amaurosis type 2 (LCA2), in which the isomerization of photobleached all-trans-retinal into photosensitive 11cRAL is blocked, ultimately causing severe retinal dysfunction and degeneration. The related mouse models, which are constructed through gene knockout or caused by spontaneous mutations, morphologically present with early-onset and rapid retinal cone cells degeneration, including loss of short-wavelength-sensitive cone opsins (S-opsins) and mislocalization of medium-wavelength-sensitive cone opsins (M-opsins). Studies have shown that routine Rpe65 gene replacement therapy, mediated by an adeno-associated virus (AAV) vector, can restore RPE65 protein. However, AAV transfection and Rpe65 transgene expression require at least one to two weeks, and the treatment cannot fully block the early-onset cone degeneration. To determine the feasibility of delaying cone degeneration before gene therapy, we investigated the impact of 11cRAL treatment in an early-age LCA2 retinal degeneration 12 (rd12) mouse model. Similar to human patients, the mouse model carries a spontaneous mutation in the Rpe65 gene, which results in disrupted endogenous 11cRAL regeneration. We found that RPE65 deficiency did not notably affect rodent retinal vessels. Under red light illumination, the rd12 mice were intraperitoneally injected with exogenous 11cRAL from postnatal day (P) 14 to P21. Three days after the last injection, a notable recovery of retinal function was observed using scotopic and photopic electroretinograms. Using optical coherence tomography and histological analyses of the deficient retinas, we found changes in the thickness of the photoreceptor outer segment (OS); this change could be rescued by early 11cRAL treatment. In addition, the treatment notably preserved M- and S-opsins, both of which maintained appropriate localization inside cone cells, as shown by the wild-type mice. In contrast, the age-matched untreated rd12 mice were characterized by retinal S-opsin loss and M-opsin mislocalization from the photoreceptor OS to the inner segment, outer nuclear layer, or outer plexiform layer. Notably, 11cRAL treatment could not maintain retinal function for a long time. Ten days after the last injection, the rod and M-cone electroretinograms significantly decreased, and S-cone responses almost extinguished. Our findings suggest that early 11cRAL treatment is useful for restoring retinal function and rescuing morphology in the rd12 mouse model, and the early-onset and rapid cone degeneration can be delayed before gene therapy.

Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin.

Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA.

Targeting arrestin interactions with its partners for therapeutic purposes.

Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds of G protein-coupled receptors, certain receptors of other types, and numerous non-receptor partners. Arrestins have no enzymatic activity and work by interacting with other proteins, often assembling multi-protein signaling complexes. Arrestin binding to every partner affects cell signaling, including pathways regulating cell survival, proliferation, and death. Thus, targeting individual arrestin interactions has therapeutic potential. This requires precise identification of protein-protein interaction sites of both participants and the choice of the side of each interaction which would be most advantageous to target. The interfaces involved in each interaction can be disrupted by small molecule therapeutics, as well as by carefully selected peptides of the other partner that do not participate in the interactions that should not be targeted.

Effect of an intravitreal antisense oligonucleotide on vision in Leber congenital amaurosis due to a photoreceptor cilium defect.

Photoreceptor ciliopathies constitute the most common molecular mechanism of the childhood blindness Leber congenital amaurosis. Ten patients with Leber congenital amaurosis carrying the c.2991+1655A>G allele in the ciliopathy gene centrosomal protein 290 (CEP290) were treated (ClinicalTrials.gov no. NCT03140969 ) with intravitreal injections of an antisense oligonucleotide to restore correct splicing. There were no serious adverse events, and vision improved at 3 months. The visual acuity of one exceptional responder improved from light perception to 20/400.

HDAC6 inhibition by tubastatin A is protective against oxidative stress in a photoreceptor cell line and restores visual function in a zebrafish model of inherited blindness.

Retinal diseases, such as hereditary retinitis pigmentosa and age-related macular degeneration, are characterized by the progressive loss of photoreceptors. Histone deacetylase 6 (HDAC6) is considered as a stress surveillance factor and a potential target for neuroprotection and regeneration. Overexpression of HDAC6 has been connected to neurodegenerative disorders, and its suppression may provide protection. Here we show that HDAC6 is constitutively present in the mouse retina, and in the cone-like mouse cell line 661W. In 661W cells HDAC6 inhibition by the specific inhibitor tubastatin A (TST) led to the acetylation of α-tubulin, which is a major substrate for HDAC6. After oxidative stress, exerted by hydrogen peroxide, TST promoted cell survival and the upregulation of heat-shock proteins HSP70 and HSP25 by activation of heat-shock transcription factor 1. Furthermore, in response to oxidative stress the redox regulatory protein peroxiredoxin 1 (Prx1) was modulated in 661W cells by HDAC6 inhibition. The peroxide reducing activity of Prx1 is dependent on its acetylation, which is mediated by HDAC6. Pre-incubation with TST prevented the inactivation of Prx1 and its preserved activity may exert protective effects in photoreceptor cells. To determine whether TST treatment has a therapeutic effect on visual function, the dyeucd6 zebrafish model of inherited sight loss was utilized. Zebrafish have developed as a suitable model system for pharmacological testing. In vivo application of TST caused the hyperacetylation of α-tubulin, indicating that HDAC6 is active in this model. Furthermore, TST was sufficient to rescue visual function and retinal morphology. Hence, HDAC6 inhibition and the regulation of peroxiredoxin activity may play a significant role in protecting retinal cells and in particular photoreceptors, which are exposed to high levels of reactive oxygen species derived from oxidative stress-induced injuries.

Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis.

UNLABELLED: RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations. SIGNIFICANCE STATEMENT: LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations.

Oral 9-cis retinoid for childhood blindness due to Leber congenital amaurosis caused by RPE65 or LRAT mutations: an open-label phase 1b trial.

BACKGROUND: Leber congenital amaurosis, caused by mutations in RPE65 and LRAT, is a severe form of inherited retinal degeneration leading to blindness. We aimed to assess replacement of the missing chromophore 11-cis retinal with oral QLT091001 (synthetic 9-cis-retinyl acetate) in these patients. METHODS: In our open-label, prospective, phase 1b trial, we enrolled patients (aged ≥6 years) with Leber congenital amaurosis and RPE65 or LRAT mutations at McGill University's Montreal Children's Hospital. Patients received 7 days of oral QLT091001 (10-40 mg/m(2) per day). We assessed patients at baseline and days 7, 9, 14, and 30, and then 2 months and every 2 months thereafter for up to 2·2 years for safety outcomes and visual function endpoints including Goldmann visual fields (GVF), visual acuity, and functional MRI assessment. We regarded patients as having an improvement in vision if we noted at least a 20% improvement in retinal area on GVF compared with baseline or a visual acuity improvement of five or more letters compared with baseline in two consecutive study visits (or any improvement from no vision at baseline). This study is registered with ClinicalTrials.gov, number NCT01014052. FINDINGS: Between December, 2009, and June, 2011, we enrolled and treated 14 patients aged 6-38 years who were followed up until March, 2012. Ten (71%) of 14 patients had an improvement in GVF areas (mean increase in retinal area of 28-683%). Six (43%) patients had an improvement in visual acuity (mean increase of 2-30 letters). Self-reported or parent-reported improvements in activities of daily living supported these findings. After 2 years, 11 (79%) patients had returned to their baseline GVF retinal area and ten (71%) had returned to baseline visual acuity letter values. Thus, three (21%) patients had a sustained GVF response and four (30%) had a sustained visual acuity response. Four patients had functional MRI scans, which correlated with visual response or absence of response to treatment. No serious adverse events occurred, although we noted transient headaches (11 patients), photophobia (11 patients), reduction in serum HDL concentrations (four patients), and increases in serum triglycerides (eight patients) and aspartate aminotransferase concentrations (two patients). INTERPRETATION: Non-invasive oral QLT091001 therapy is well tolerated, and can rapidly improve visual function in some patients with Leber congenital amaurosis and RPE65 and LRAT mutations. FUNDING: QLT, Foundation Fighting Blindness Canada, CIHR, FRSQ, Reseau Vision.

Pathophysilogical mechanism and treatment strategies for Leber congenital amaurosis.

Mutations in retinoid isomerase, RPE65, or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal recycling and cause Leber congenital amaurosis (LCA), the most severe retinal dystrophy in early childhood. We used Lrat (-/-), a murine model for LCA, to investigate the mechanism of rapid cone degeneration. We found that mislocalized M-opsin was degraded whereas mislocalized S-opsin accumulated in Lrat (-/-) cones before the onset of massive ventral/central cone degeneration. Since the ventral and central retina expresses higher levels of S-opsin than the dorsal retina in mice, our results may explain why ventral and central cones degenerate more rapidly than dorsal cones in Rpe65 (-/-) and Lrat (-/-) LCA models. In addition, human blue opsin and mouse S-opsin, but not mouse M-opsin or human red/green opsins, aggregated to form cytoplasmic inclusions in transfected cells, which may explain why blue cone function is lost earlier than red/green-cone function in LCA patients. The aggregation of short-wavelength opsins likely caused rapid cone degenerations through an ER stress pathway as demonstrated in both the Lrat (-/-) retina and transfected cells. Based on this mechanism, we designed a new therapy of LCA by reducing ER stress. We found that systemic injection of an ER chemical chaperone, tauroursodeoxycholic acid (TUDCA), is effective in reducing ER stress, preventing apoptosis, and preserving cones in Lrat (-/-) mice.

Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration.

Cone phototransduction and survival of cones in the human macula is essential for color vision and for visual acuity. Progressive cone degeneration in age-related macular degeneration, Stargardt disease, and recessive cone dystrophies is a major cause of blindness. Thyroid hormone (TH) signaling, which regulates cell proliferation, differentiation, and apoptosis, plays a central role in cone opsin expression and patterning in the retina. Here, we investigated whether TH signaling affects cone viability in inherited retinal degeneration mouse models. Retinol isomerase RPE65-deficient mice [a model of Leber congenital amaurosis (LCA) with rapid cone loss] and cone photoreceptor function loss type 1 mice (severe recessive achromatopsia) were used to determine whether suppressing TH signaling with antithyroid treatment reduces cone death. Further, cone cyclic nucleotide-gated channel B subunit-deficient mice (moderate achromatopsia) and guanylate cyclase 2e-deficient mice (LCA with slower cone loss) were used to determine whether triiodothyronine (T3) treatment (stimulating TH signaling) causes deterioration of cones. We found that cone density in retinol isomerase RPE65-deficient and cone photoreceptor function loss type 1 mice increased about sixfold following antithyroid treatment. Cone density in cone cyclic nucleotide-gated channel B subunit-deficient and guanylate cyclase 2e-deficient mice decreased about 40% following T3 treatment. The effect of TH signaling on cone viability appears to be independent of its regulation on cone opsin expression. This work demonstrates that suppressing TH signaling in retina dystrophy mouse models is protective of cones, providing insights into cone preservation and therapeutic interventions.

Chemical chaperone TUDCA preserves cone photoreceptors in a mouse model of Leber congenital amaurosis.

PURPOSE: Mutations in either retinoid isomerase (RPE65) or lecithin-retinol acyltransferase (LRAT) lead to Leber congenital amaurosis (LCA). By using the Lrat(-/-) mouse model, previous studies have shown that the rapid cone degeneration in LCA was caused by endoplasmic reticulum (ER) stress induced by S-opsin aggregation. The purpose of this study is to examine the efficacy of an ER chemical chaperone, tauroursodeoxycholic acid (TUDCA), in preserving cones in the Lrat(-/-) model. METHODS: Lrat(-/-) mice were systemically administered with TUDCA and vehicle (0.15 M NaHCO(3)) every 3 days from P9 to P28. Cone cell survival was determined by counting cone cells on flat-mounted retinas. The expression and subcellular localization of cone-specific proteins were analyzed by western blotting and immunohistochemistry, respectively. RESULTS: TUDCA treatment reduced ER stress and apoptosis in Lrat(-/-) retina. It significantly slowed down cone degeneration in Lrat(-/-) mice, resulting in a ∼3-fold increase in cone density in the ventral and central retina as compared with the vehicle-treated mice at P28. Furthermore, TUDCA promoted the degradation of cone membrane-associated proteins by enhancing the ER-associated protein degradation pathway. CONCLUSIONS: Systemic injection of TUDCA is effective in reducing ER stress, preventing apoptosis, and preserving cones in Lrat(-/-) mice. TUDCA has the potential to lead to the development of a new class of therapeutic drugs for treating LCA.

Light prevents exogenous 11-cis retinal from maintaining cone photoreceptors in chromophore-deficient mice.

PURPOSE: To determine the effect of light/dark cycles on the cones of 11-cis retinal-treated RPE65/rhodopsin double knockout (Rpe65(-/-)Rho(-/-)) mice. Studies have shown that cones degenerate in chromophore-deficient mouse models for Leber Congenital Amaurosis (LCA), but exogenous supplementation of the native 11-cis retinal chromophore can inhibit this degeneration, suggesting that 11-cis retinal could be used as a therapeutic agent for preserving functional cones in patients with LCA. However, these treated mice were maintained in the dark. METHODS: 11-cis Retinal was introduced into Rpe65(-/-)Rho(-/-) mice at postnatal day 10 as a single subcutaneous injection mixed with a basement membrane matrix. The mice were maintained in either normal light/dark cycles or constant dark conditions. Fluorescence microscopy was used to assess retinal morphology. Cone cell survival was determined by counting cone opsin-containing cells on flat-mounted P30 retinas. Cross-sections of P21 mouse retina were used to assess cone cell integrity by visualizing opsin localization. Cone function was determined by electroretinography (ERG). RESULTS: Previous studies have shown that 11-cis retinal-treated mice lacking RPE65 and raised in constant dark have higher cone photoreceptor cell number, improved cone opsin localization, and enhanced cone ERG signals when compared with untreated mice. However, in this study the authors show that 11-cis retinal-treated Rpe65(-/-)Rho(-/-) mice raised in cyclic light did not show the improvements seen with the dark-reared mice. CONCLUSIONS: Thus, 11-cis retinal by itself, as well as other agents that form photosensitive pigments, will not be good therapeutic candidates for preserving cones in LCA.

Improvement of visual performance with intravitreal administration of 9-cis-retinal in Rpe65-mutant dogs.

OBJECTIVE: To determine the efficacy of intravitreal administration of 9-cis-retinal in restoring visual function in Rpe65-mutant dogs. METHODS: Intravitreal injection of 9-cis-retinal was administered in 1 eye of 7 Rpe65-/- dogs at a range of ages. Electroretinogram analysis and testing of visual performance was used to evaluate outcomes after a single injection and in 2 dogs after a second injection in the same eye. RESULTS: In 5 of 7 injected dogs, 9-cis-retinal injection resulted in increased rod electroretinogram responses and improved functional vision. Three injected dogs exhibited increased 33-Hz flicker amplitudes characteristic of cone-mediated responses. Electroretinogram improvement was no longer evident by week 10 postinjection in 1 dog monitored over time. A second injection of 9-cis-retinal was performed in the same eye of 2 of the 7 dogs and also resulted in rescue of visual function. CONCLUSION: Our findings establish that 9-cis-retinoid therapy can restore visual function in a canine model of human disease resulting from RPE65 mutations. CLINICAL RELEVANCE: These positive proof-of-principle results provide support for the development of intravitreal devices for sustained delivery of 9-cis-retinal as a therapy for conditions resulting from failure of the visual cycle.