Driven by the energy of a photon, the visual pigments in rod and cone photoreceptor cells isomerize 11-cis-retinal to the all-trans configuration. This photochemical reaction initiates the signal ...transduction pathway that eventually leads to the transmission of a visual signal to the brain and leaves the opsins insensitive to further light stimulation. For the eye to restore light sensitivity, opsins require recharging with 11-cis-retinal. This trans-cis back conversion is achieved through a series of enzymatic reactions composing the retinoid (visual) cycle. Although it is evident that the classical retinoid cycle is critical for vision, the existence of an adjunct pathway for 11-cis-retinal regeneration has been debated for many years. Retinal pigment epithelium (RPE)-retinal G protein-coupled receptor (RGR) has been identified previously as a mammalian retinaldehyde photoisomerase homologous to retinochrome found in invertebrates. Using pharmacological, genetic, and biochemical approaches, researchers have now established the physiological relevance of the RGR in 11-cis-retinal regeneration. The photoisomerase activity of RGR in the RPE and Müller glia explains how the eye can remain responsive in daylight. In this review, we will focus on retinoid metabolism in the eye and visual chromophore regeneration mediated by RGR.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered ...DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.
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•Engineered virus-like particles (eVLPs) overcome three bottlenecks to protein delivery•DNA-free eVLPs efficiently deliver gene editing proteins with minimal off-target editing•Base editor eVLPs reduced serum Pcsk9 levels 78% following 63% liver editing in mice•Base editor eVLPs improved visual function in a mouse model of genetic blindness
Engineered, DNA-free virus-like particles efficiently deliver gene editing proteins, have minimal off-target effects, can be applied in vivo to deliver base editors to multiple organs, and are used to improve visual function in a mouse model of genetic blindness.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Precision genome editing in the eye Suh, Susie; Choi, Elliot H; Raguram, Aditya ...
Proceedings of the National Academy of Sciences - PNAS,
09/2022, Volume:
119, Issue:
39
Journal Article
Peer reviewed
Open access
CRISPR-Cas-based genome editing technologies could, in principle, be used to treat a wide variety of inherited diseases, including genetic disorders of vision. Programmable CRISPR-Cas nucleases are ...effective tools for gene disruption, but they are poorly suited for precisely correcting pathogenic mutations in most therapeutic settings. Recently developed precision genome editing agents, including base editors and prime editors, have enabled precise gene correction and disease rescue in multiple preclinical models of genetic disorders. Additionally, new delivery technologies that transiently deliver precision genome editing agents in vivo offer minimized off-target editing and improved safety profiles. These improvements to precision genome editing and delivery technologies are expected to revolutionize the treatment of genetic disorders of vision and other diseases. In this Perspective, we describe current preclinical and clinical genome editing approaches for treating inherited retinal degenerative diseases, and we discuss important considerations that should be addressed as these approaches are translated into clinical practice.
Genome-editing technologies have ushered in a new era in gene therapy, providing novel therapeutic strategies for a wide range of diseases, including both genetic and nongenetic ocular diseases. ...These technologies offer new hope for patients suffering from previously untreatable conditions. The unique anatomical and physiological features of the eye, including its immune-privileged status, size, and compartmentalized structure, provide an optimal environment for the application of these cutting-edge technologies. Moreover, the development of various delivery methods has facilitated the efficient and targeted administration of genome engineering tools designed to correct specific ocular tissues. Additionally, advancements in noninvasive ocular imaging techniques and electroretinography have enabled real-time monitoring of therapeutic efficacy and safety. Herein, we discuss the discovery and development of genome-editing technologies, their application to ocular diseases from the anterior segment to the posterior segment, current limitations encountered in translating these technologies into clinical practice, and ongoing research endeavors aimed at overcoming these challenges.
Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, ...resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.
Background
Skin is the organ most extensively exposed to light of a broad range of wavelengths. Several studies have reported that skin expresses photoreceptive molecules called opsins. However, the ...identity and functional role of opsins in the human skin remain elusive. We aim to summarize current scientific evidence on the types of opsins expressed in the skin and their biological functions.
Methods
A primary literature search was conducted using PubMed to identify articles on dermal opsins found in nonhuman animals and humans.
Results
Twenty‐two articles, representing, however, a non‐exhaustive selection of the scientific papers published in this specific field, met the inclusion criteria. In nonhuman animals, opsins and opsin‐like structures have been detected in the skin of fruit fly, zebrafish, frog, octopus, sea urchin, hogfish, and mouse, and they mediate skin color change, light avoidance, shadow reflex, and circadian photoentrainment. In humans, opsins are present in various skin cell types, including keratinocytes, melanocytes, dermal fibroblasts, and hair follicle cells. They have been shown to mediate wound healing, melanogenesis, hair growth, and skin photoaging.
Conclusion
Dermal opsins have been identified across many nonhuman animals and humans. Current evidence suggests that opsins have biological significance beyond light reception. In nonhuman animals, opsins are involved in behaviors that are critical for survival. In humans, opsins are involved in various functions of the skin although the underlying molecular mechanisms remain unclear. Future investigation on elucidating the mechanism of dermal opsins will be crucial to expand the therapeutic benefits of photobiomodulation for various skin disorders.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Photoisomerization of the 11-cis-retinal chromophore of rod and cone visual pigments to an all-trans-configuration is the initiating event for vision in vertebrates. The regeneration of ...11-cis-retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11-cis-retinal also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein–coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11-cis-retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Müller glia, whereas mouse RGR is expressed in RPE but not in Müller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The vitamin A derivative 11-
retinaldehyde plays a pivotal role in vertebrate vision by serving as the chromophore of rod and cone visual pigments. In the initial step of vision, a photon is absorbed ...by this chromophore resulting in its isomerization to an all-
state and consequent activation of the visual pigment and phototransduction cascade. Spent chromophore is released from the pigments through hydrolysis. Subsequent photon detection requires the delivery of regenerated 11-
retinaldehyde to the visual pigment. This
conversion is achieved through a process known as the visual cycle. In this review, we will discuss the enzymes, binding proteins and transporters that enable the visual pigment renewal process with a focus on advances made during the past decade in our understanding of their structural biology.
Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been ...expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.
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