Rose-comb was one of the chicken comb-variants first used by Bateson and Punnet in 1902 to demonstrate Mendelian inheritance in animals. Rose-comb is a monogenic trait that has been widely described in chickens. It is caused by a large structural rearrangement that leads to mis-expression of transcription factor MNR2 on chromosome 7. Rose-comb has pleiotropic effects in homozygous roosters, which is associated with poor sperm mobility. It was postulated that this is caused by the disruption of the CCDC108 gene located at the distal inversion breakpoint. In this study, we did the transcriptional profiling of combs and testes from Rose-comb Silky (RS) (R1/R1) and Beijing Fatty (BF) wild type chickens (r/r) using RNA-seq. We obtained 68,694,797 unique mapped reads and over 80% of the chicken genes were covered for each sample. In combs, we found that differentially expressed genes (DEGs) were significantly enriched in the retinol metabolism (RPE65, CYP26A1, and CYP26C1) and hedgehog-signaling pathway (PTCH1, GLI1, and HHIP), while genes related to cell differentiation and morphogenesis were down-regulated in R1/R1 chickens, suggesting that the transient expression of MNR2 might affect the expression of these genes and influence the development of comb tissue. For testes, DEGs were significantly enriched in the GO terms of binding activates and mitochondrial oxidation-reduction reactions. Our results suggested that the CCDC108 might be functionally related with mitochondrial oxidation-reduction reactions and caused subfertility of roosters. Compared with the genome average, the degree of expression variations within the inversion region did not show significant differences. However, DEGs near the breakpoints showed greater expression variance. Our results demonstrated that the large-scale rearrangements affected the gene expression only around the breakpoint in this case.

The in vitro induction of corneal epithelial cells (CECs) from human induced pluripotent stem cells (iPSCs) represents a new strategy for obtaining CE stem/progenitor cells for the surgical reconstruction of a diseased or injured ocular surface. The clinical promise of this strategy is considerable, but if the approaches' potential is to be realised, robust methods for the purification of iPSC-derived CE lineage cells need to be developed to avoid contamination with other cells that may carry the risk of unwanted side effects, such as tumorigenesis. Experiments conducted here revealed that during CEC isolation, CD200-negative selection using a cell sorter considerably reduced the contamination of the cell population with various non-CECs compared with what could be achieved using TRA-1-60, a conventional negative marker for CECs. Furthermore, CD200-negative sorting did not affect the yield of CECs nor that of their stem/progenitor cells. Single-cell gene expression analysis for CEC sheets obtained using CD200-negative sorting showed that all analysed cells were CE-lineage cells, expressing PAX6, delta-N p63, and E-cadherin. Non-CECs, on the other hand, expressed non-CEC genes such as FGFR1 and RPE65. CD200, thus, represents a robust negative marker for purification of induced CE lineage cells, which is expressed by undifferentiated iPSCs and non-CECs, including iPSC-derived neural and retinal cells.

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are inherited degenerative retinal dystrophies with vision loss that ultimately lead to blindness. Several genes have been shown to be involved in early onset retinal dystrophies, including CRB1 and RPE65. Gene therapy recently became available for young RP patients with variations in the RPE65 gene. Current research programs test adeno-associated viral gene augmentation or editing therapy vectors on various disease models mimicking the disease in patients. These include several animal and emerging human-derived models, such as human-induced pluripotent stem cell (hiPSC)-derived retinal organoids or hiPSC-derived retinal pigment epithelium (RPE), and human donor retinal explants. Variations in the CRB1 gene are a major cause for early onset autosomal recessive RP with patients suffering from visual impairment before their adolescence and for LCA with newborns experiencing severe visual impairment within the first months of life. These patients cannot benefit yet from an available gene therapy treatment. In this review, we will discuss the recent advances, advantages and disadvantages of different CRB1 human and animal retinal degeneration models. In addition, we will describe novel therapeutic tools that have been developed, which could potentially be used for retinal gene augmentation therapy for RP patients with variations in the CRB1 gene.

Keywords: crumbs homolog 1; gene therapy; leber congenital amaurosis; mouse model; retinitis pigmentosa.

Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called 'visual cycle'. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA). Despite the success of recent RPE65 gene therapy, follow-up studies show that patients continue to experience photoreceptor degeneration and lose vision benefit over time. In Lrat^-/- mouse model, mislocalized medium (M)-wavelength opsin was degraded, whereas mislocalized short (S)-wavelength opsin accumulated before the onset of cone degeneration. The mechanism for the foveal medium (M)/long (L)-wavelength cone photoreceptor degeneration in LCA is unknown. By crossing Lrat^-/- mice with a proteasome reporter mouse strain, this study showed that M-opsin-enriched dorsal cones in Lrat^-/- mice exhibit proteasome stress due to the degradation of large amounts of M-opsin. Deletion of M-opsin relieves the proteasome stress and completely prevents "M cone" degeneration in Lrat^-/-Opn1sw^-/- mice (a pure "M cone" LCA model, Opn1sw encoding S-opsin) for at least 12 months. These results suggest that M-opsin degradation-associated proteasome stress plays a major role in "M cone" degeneration in Lrat^-/- model. This finding may represent a general mechanism for "M cone" degeneration in multiple forms of cone degeneration due to M-opsin mislocalization and degradation. These results have important implications for the current gene therapy strategy for LCA that emphasizes the need for combinatorial therapies to both improve vision and slow photoreceptor degeneration.

Background/aims: Stargardt disease is a rare, inherited, degenerative disease of the retina that is the most common type of hereditary macular dystrophy. Currently, no approved treatments for the disease exist. The purpose of this study was to characterise the pharmacodynamics of emixustat, an orally available small molecule that targets the retinal pigment epithelium-specific 65 kDa protein (RPE65), in subjects with macular atrophy secondary to Stargardt disease.

Methods: In this multicentre study conducted at six study sites in the USA, 23 subjects with macular atrophy secondary to Stargardt disease were randomised to one of three doses of daily emixustat (2.5 mg, 5 mg or 10 mg) and treated for 1 month. The primary outcome was the suppression of the rod b-wave recovery rate on electroretinography after photobleaching, which is an indirect measure of RPE65 inhibition.

Results: Subjects who received 10 mg emixustat showed near-complete suppression of the rod b-wave amplitude recovery rate postphotobleaching (mean=91.86%, median=96.69%), whereas those who received 5 mg showed moderate suppression (mean=52.2%, median=68.0%). No effect was observed for subjects who received 2.5 mg emixustat (mean=-3.31%, median=-12.23%). The adverse event profile was consistent with prior studies in other patient populations and consisted primarily of ocular adverse events likely related to RPE65 inhibition.

Conclusion: This study demonstrated dose-dependent suppression of rod b-wave amplitude recovery postphotobleaching, confirming emixustat's biological activity in patients with Stargardt disease. These findings informed dose selection for a 24-month phase 3 trial (SeaSTAR Study) that is now comparing emixustat to placebo in the treatment of Stargardt disease-associated macular atrophy.

Keywords: clinical trial; drugs; electrophysiology; macula; retina.

Objectives: No therapeutic interventions are currently available for autosomal dominant retinitis pigmentosa (adRP). An RPE65 Asp477Gly transition associates with late-onset adRP, reduced RPE65 enzymatic activity being one feature associated with this dominant variant. Our objective: to assess whether in a proof-of-concept study, oral synthetic 9 cis-retinyl acetate therapy improves vision in such advanced disease.

Methods and analysis: A phase 1b proof-of-concept clinical trial was conducted involving five patients with advanced disease, aged 41-68 years. Goldmann visual fields (GVF) and visual acuities (VA) were assessed for 6-12 months after 7-day treatment, patients receiving consecutive oral doses (40 mg/m²) of 9-cis-retinyl acetate, a synthetic retinoid replacement.

Results: Pathological effects of D477G variant were preliminarily assessed by electroretinography in mice expressing AAV-delivered D477G RPE65, by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme- thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assays on RPE viability and enzyme activity in cultured cells. In addition to a mild dominant effect reflected in reduced electroretinographics in mice, and reduced cellular function in vitro, D477G exhibited reduced enzymatic RPE65 activity in vitro. In patients, significant improvements were observed in GVF from baseline ranging from 70% to 200% in three of five subjects aged 67-68 years, with largest improvements at 7-10 months. Of two GVF non-responders, one had significant visual acuity improvement (5-15 letters) from baseline after 6 months.

Conclusion: Families with D477G variant have been identified in Ireland, the UK, France, the USA and Canada. Effects of single 7-day oral retinoid supplementation lasted at least 6 months, possibly giving visual benefit throughout remaining life in patients with advanced disease, where gene therapy is unlikely to prove beneficial.

Keywords: clinical trial; degeneration; genetics; retina; treatment medical.

Molecular-genetic mechanisms of regeneration of adult newt (Pleurodeles waltl) retina were studied. For the first time, a comparative analysis of the expression of regulatory genes Pax6, Otx2, and Six3 and Fgf2 genes encoding signal molecules was performed in the native retinal pigment epithelium (RPE) and retina and at successive stages of retina regeneration. Cell differentiation types were determined using genetic markers of cell differentiation in the RPE (RPE65) and the retina (betaII-tubulin and Rho). Activation of the expression of neurospecific genes Pax6 and Six3 and the growth factor gene Fgf2 and suppression of activation of the regulatory gene Otx2 and the RPE65 were observed at the stage of multipotent neuroblast formation in the regenerating retina. The expression of genes Pax6, Six3, and Fgf2 was retained at a later stage of retina regeneration at which the expression of retinal differentiation markers, the genes encoding betaII-tubulin (betaII-tubulin) and rhodopsin (Rho), was also detected. We assume that the above regulatory genes are multifunctional and control not only transdifferentiation of RPE cells (the key stage of retina regeneration) but also differentiation of regenerating retina cells. The results of this study, demonstrating coexpression of Pax6, Six3, Fgf2, betaII-tubulin, and Rho genes, provide indirect evidence for the interaction of regulatory and specific genes during retina regeneration.

OBJECTIVE

To investigate unusual changes in the basal surface of the retinal pigment epithelium (RPE) cell layer in aging Rpe65 -/- and wild-type mice.

METHODS

The retinas of Rpe65 -/- and wild-type mice of different ages-6 weeks and 3, 6, 12-13 and 16 months-were examined by electron microscopy.

RESULTS

There was an age-related increase in the width of the basement membrane of both Rpe65 -/- and wild-type mice which was associated with loss of basal infoldings of the plasma membrane of the RPE cells and protrusions of basement membrane material deep into the cytoplasm of these cells. These changes were evident at 6 months of age in RPE65 -/- mice and became extensive at 1 year of age. Similar changes occurred in wild-type mice but were less extensive and were only evident after 1 year of age.

CONCLUSIONS

There is an age-dependent abnormality that develops at the basal surface of murine RPE cells, which resembles some of the changes observed in human age-related macular degeneration. These changes occur earlier in life and are more extensive in Rpe65 mutant mice.

In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.

Retinal pigment epithelium (RPE) acts as an outer blood-retinal barrier that limits the access of circulating xenobiotics to the eye. In addition, the RPE limits posterior elimination of intravitreally injected drugs to circulation. Thus, permeation in the RPE has a significant effect on ocular pharmacokinetics. The RPE is also a potentially important drug target in age-related macular degeneration. Therefore, the cell models of the RPE are important tools in ocular drug development, but poor availability and problems in reproducibility limit the use of primary RPE cell cultures. Furthermore, the best and widely used human cell line ARPE19 requires specialized culture conditions and a long time for cellular differentiation. In this paper, we describe a cell population arisen from the ARPE19 culture, with fast differentiation and improved barrier properties. This cell line, LEPI, forms clear microvilli and rapidly displays RPE-like cobblestone morphology after subculture in simple culture conditions. The LEPI cells show RPE-specific functions and expression of RPE65, ezrin, and BEST1 proteins. On filter, the LEPI cells develop tighter barrier than the ex vivo bovine RPE-choroid: permeability coefficients of beta-blockers (atenolol, nadolol, timolol, pindolol, metoprolol, betaxolol) ranged from 0.4 × 10^-6 cm/sec to 2.3 × 10^-6 cm/sec depending on the drug lipophilicity. This rapidly differentiating cell line will be an asset in ocular studies since it is easily maintained, it grows and differentiates quickly and does not require specialized culture conditions for differentiation. Thus, this cell line is suitable for both small scale assays and high throughput screening in drug discovery and development.

RPE65-associated Leber congenital amaurosis (LCA) is one of highly heterogeneous, early onset, severe retinal dystrophies with at least 130 gene mutation sites identified. Their pathogenicity has not been directly clarified due to lack of diseased cells. Here, we generated human-induced pluripotent stem cells (hiPSCs) from one putative LCA patient carrying two novel RPE65 mutations with c.200T>G (p.L67R) and c.430T>C (p.Y144H), named RPE65-hiPSCs, which were confirmed to contain the same mutations. The RPE65-hiPSCs presented typical morphological features with normal karyotype, expressed pluripotency markers, and developed teratoma in NOD-SCID mice. Moreover, the patient hiPSCs were able to differentiate toward retinal lineage fate and self-form retinal organoids with layered neural retina. All major retinal cell types including photoreceptor and retinal pigment epithelium (RPE) cells were also acquired overtime. Compared to healthy control, RPE cells from patient iPSCs had lower expression of RPE65, but similar phagocytic activity and VEGF secretion level. This study provided the valuable patient specific, disease targeted retinal organoids containing photoreceptor and RPE cells, which would facilitate the study of personalized pathogenic mechanisms of disease, drug screening, and cell replacement therapy.

To screen RPE65 in 187 families with Leber congenital amaurosis (LCA).

Sanger sequencing and/or targeted exome sequencing was employed to identify mutations in the RPE65 gene, and intrafamilial cosegregation analysis if DNA was available. In silico analyses and splicing assay were used to evaluate the variants' pathogenicity.

Genetic analysis revealed 15 mutations in RPE65 in 14 pedigrees, including one splice-site mutation, one frameshift mutation, three nonsense mutations, and ten missense mutations. Of the mutations identified in RPE65, seven are novel associated with LCA, including five missense variants (c.124C>T, c.149T>C, c.340A>C, c.425A>G, and c.1399C>G) and two indel (insertions or deletions) variants (c.858+1delG and c.1181_1182insT). In vitro splicing assay was performed to evaluate the functional impact on RNA splicing of novel mutations if two of three in silico analyses were predicated to be non-pathogenic at the protein level. Among these 15 variants, 14 were classified as 'pathogenic variants,' and a variant (c.124C>T) was 'variants with uncertain significance' according to the standards and guidelines of the American College of Medical Genetics and Genomics.

Mutations in RPE65 were responsible for 11 of the cohort of 187 Chinese families with LCA, which expands the spectrum of RPE65-related LCA in the Chinese population and potentially facilitates its clinical implementation.

BACKGROUND

Leber congenital amaurosis (LCA) is a visual disease which is caused by RPE65 mutations and results in retinal degeneration and severe vision loss in early infancy. According to previous researches, mutations of the RPE65 gene account for 16% of all cases of LCA. This study aimed to identify RPE65 gene mutations in Chinese patients with LCA.

METHODS

We recruited 52 sporadic patients from Peking University Third Hospital in 2016 and applied Sanger sequencing to identify variants among exons responsible for the disease. The genomic DNAs from blood leukocytes of these patients were isolated, and the entire coding region of the RPE65 gene was amplified by polymerase chain reaction. We then determined the sequence of RPE65 using ABI 3100 Genetic Analyzer.

RESULTS

Our study identified that only 1 out of the 52 patients with LCA carried the previously unreported homozygosis missense mutation c1174A>C (T392P) of the RPE65 gene. However, the mutation was associated with the disease phenotype and not detected in 100 normal controls.

CONCLUSIONS

Though we identified a novel missense mutation in the RPE65 gene that causes LCA, our result indicates that RPE65 mutations may not play a major role in the LCA patients in China since only 1 out of the 52 patients carried mutation in the RPE65 gene.

Background: This research aimed to establish recommendations on the clinical and genetic characteristics necessary to confirm patient eligibility for gene supplementation with voretigene neparvovec.

Methods: An expert steering committee comprising an interdisciplinary panel of Italian experts in the three fields of medical specialisation involved in the management of RPE65-associated inherited retinal disease (IRD) (medical retina, genetics, vitreoretinal surgery) proposed clinical questions necessary to determine the correct identification of patients with the disease, determine the fundamental clinical and genetics tests to reach the correct diagnosis and to evaluate the urgency to treat patients eligible to receive treatment with voretigene neparvovec. Supported by an extensive review of the literature, a series of statements were developed and refined to prepare precisely constructed questionnaires that were circulated among an external panel of experts comprising ophthalmologists (retina specialists, vitreoretinal surgeons) and geneticists with extensive experience in IRDs in Italy in a two-round Delphi process.

Results: The categories addressed in the questionnaires included clinical manifestations of RPE65-related IRD, IRD screening and diagnosis, gene testing and genotyping, ocular gene therapy for IRDs, patient eligibility and prioritisation and surgical issues. Response rates by the survey participants were over 90% for the majority of items in both Delphi rounds. The steering committee developed the key consensus recommendations on each category that came from the two Delphi rounds into a simple and linear diagnostic algorithm designed to illustrate the patient pathway leading from the patient's referral centre to the retinal specialist centre.

Conclusions: Consensus guidelines were developed to guide paediatricians and general ophthalmologists to arrive at the correct diagnosis of RPE65-associated IRD and make informed clinical decisions regarding eligibility for a gene therapy approach to RPE65-associated IRD. The guidelines aim to ensure the best outcome for the patient, based on expert opinion, the published literature, and practical experience in the field of IRDs.

Keywords: Gene therapy; Inherited retinal diseases; RPE65; Voretigene neparvovec.

We report the molecular basis of the largest Tunisian cohort with inherited retinal dystrophies (IRD) reported to date, identify disease-causing pathogenic variants and describe genotype-phenotype correlations. A subset of 26 families from a cohort of 73 families with clinical diagnosis of autosomal recessive IRD (AR-IRD) excluding Usher syndrome was analyzed by whole exome sequencing and autozygosity mapping. Causative pathogenic variants were identified in 50 families (68.4%), 42% of which were novel. The most prevalent pathogenic variants were observed in ABCA4 (14%) and RPE65, CRB1 and CERKL (8% each). 26 variants (8 novel and 18 known) in 19 genes were identified in 26 families (14 missense substitutions, 5 deletions, 4 nonsense pathogenic variants and 3 splice site variants), with further allelic heterogeneity arising from different pathogenic variants in the same gene. The most common phenotype in our cohort is retinitis pigmentosa (23%) and cone rod dystrophy (23%) followed by Leber congenital amaurosis (19.2%). We report the association of new disease phenotypes. This research was carried out in Tunisian patients with IRD in order to delineate the genetic population architecture.

IRDs are one of the leading causes of visual loss in children and young adults. Mutations in over 271 genes lead to retinal dysfunction, degeneration and sight loss. Though no cure exists, gene augmentation therapy has brought hope to the field. This systematic review sought to assess the efficacy of available gene therapy treatments for IRDs. Databases and public resources were searched for randomised controlled trials (RCTs) and non-randomised studies of interventions (NRSIs). Standard methodological procedures were used, including a risk-of-bias assessment. One RCT and five NRSIs were assessed, all for adeno-associated virus two (AAV2)-mediated treatment of RPE-specific 65 kDa (RPE65)-associated LCA (Leber congenital amaurosis). Five outcomes were reported for meta-analyses. Modest improvements in visual acuity, ambulatory navigation/mobility testing or central retinal thickness was observed. There was significant improvement in red and blue light full-field stimulus testing (FST) (red light risk ratio of 1.89, treated v control, p = 0.04; and blue light risk ratio of 2.01, treated v control, p = 0.001). Study design assessment using a ROBIN-I tool (Cochrane Library) showed risk-of-bias judgement to be "low/moderate", whilst there were "some concerns" for the RCT using a RoB-2 tool (Cochrane Library). Although comparison by meta-analysis is compromised by, amongst other issues, a variable amount of vector delivered in each trial, FST improvements demonstrate a proof-of-principle for treating IRDs with gene therapy.

Keywords: IRDs; Leber congenital amaurosis (LCA); RCT; clinical trial; full-field stimulus testing (FST); gene therapy; mobility; multi-luminance mobility testing (MLMT); retinitis pigmentosa; visual acuity (VA).