Developmental dynamics of cone photoreceptors in the eel

Background: Many fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins?Results: The Rh2 and Sws2 opsin sequences from the European Eel were isolated, sequenced and expressed in vitro for an accurate measurement of their lambda(max) values. In situ hybridisation revealed that glass eels express only rh2 opsin in their cone photoreceptors, while larger yellow eels continue to express rh2 opsin in the majority of their cones, but also have <5% of cones which express sws2 opsin. Silver eels showed the same expression pattern as the larger yellow eels. This observation was confirmed by qPCR (quantitative polymerase chain reaction).Conclusions: Larger yellow and silver European eels express two different cone opsins, rh2 and sws2. This work demonstrates that only the Rh2 cone opsin is present in younger fish (smaller yellow and glass), the sws2 opsin being expressed additionally only by older fish and only in <5% of cone cells

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.This study was funded by ProQR. iPSC work in the Cheetham lab is also supported by the Wellcome Trust, Fight for Sight, RP Fighting Blindness, and Moorfields Eye Charity

Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat

Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients

The regulation of the mammalian circadian system by the inner retina

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Dissecting a role for melanopsin in behavioural light aversion reveals a response independent of conventional photoreception.

Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA) is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT), melanopsin knockout (Opn4(-/-)) and rd/rd cl (melanopsin only (MO)) mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO) mice, lacking three elements deemed essential for conventional photoreception (Opn4(-/-) Gnat1(-/-) Cnga3(-/-)). Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.This study was funded by ProQR. iPSC work in the Cheetham lab is also supported by the Wellcome Trust, Fight for Sight, RP Fighting Blindness, and Moorfields Eye Charity

Antisense Therapy for a Common Corneal Dystrophy Ameliorates TCF4 Repeat Expansion-Mediated Toxicity

Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (R50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease