retinaldehyde and Leber-Congenital-Amaurosis

RPE65 isomerase, expressed in the retinal pigmented epithelium (RPE), is an enzymatic component of the retinoid cycle, converting all-trans retinyl ester into 11-cis retinol, and it is essential for vision, because it replenishes the photon capturing 11-cis retinal. To date, almost 200 loss-of-function mutations have been identified within the

Topics: Age of Onset; Amino Acid Substitution; Animals; Choroideremia; cis-trans-Isomerases; Clinical Trials, Phase I as Topic; DNA, Complementary; Enzyme Replacement Therapy; Female; Gene Knock-In Techniques; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Leber Congenital Amaurosis; Male; Mice; Mutation, Missense; Pedigree; Point Mutation; Proof of Concept Study; Protein Isoforms; Retinaldehyde; Retinitis Pigmentosa

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

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Electroretinography; Injections, Intraperitoneal; Leber Congenital Amaurosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinaldehyde; Tomography, Optical Coherence

Fatty acid transport protein 4 (FATP4), a transmembrane protein in the endoplasmic reticulum (ER), is a recently identified negative regulator of the ER-associated retinal pigment epithelium (RPE)65 isomerase necessary for recycling 11-

Topics: Animals; cis-trans-Isomerases; Cone Opsins; Disease Models, Animal; Diterpenes; Fatty Acid Transport Proteins; Humans; Leber Congenital Amaurosis; Mice; Mice, Knockout; Mutation; Retina; Retinaldehyde; Vision, Ocular

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.. 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).. 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.. 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.

Topics: Animals; Carrier Proteins; Cell Count; Cell Survival; cis-trans-Isomerases; Dark Adaptation; Disease Models, Animal; Electroretinography; Eye Proteins; Gene Knockout Techniques; Leber Congenital Amaurosis; Light; Mice; Mice, Knockout; Microscopy, Fluorescence; Opsins; Retinal Cone Photoreceptor Cells; Retinaldehyde; Rhodopsin

In this study, we investigated the expression of the gene encoding β-galactosidase (Glb)-1-like protein 3 (Glb1l3), a member of the glycosyl hydrolase 35 family, during retinal degeneration in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout (Rpe65(-/-)) mouse model of Leber congenital amaurosis (LCA). Additionally, we assessed the expression of the other members of this protein family, including β-galactosidase-1 (Glb1), β-galactosidase-1-like (Glb1l), and β-galactosidase-1-like protein 2 (Glb1l2).. The structural features of Glb1l3 were assessed using bioinformatic tools. mRNA expression of Glb-related genes was investigated by oligonucleotide microarray, real-time PCR, and reverse transcription (RT) -PCR. The localized expression of Glb1l3 was assessed by combined in situ hybridization and immunohistochemistry.. Glb1l3 was the only Glb-related member strongly downregulated in Rpe65(-/-) retinas before the onset and during progression of the disease. Glb1l3 mRNA was only expressed in the retinal layers and the RPE/choroid. The other Glb-related genes were ubiquitously expressed in different ocular tissues, including the cornea and lens. In the healthy retina, expression of Glb1l3 was strongly induced during postnatal retinal development; age-related increased expression persisted during adulthood and aging.. These data highlight early-onset downregulation of Glb1l3 in Rpe65-related disease. They further indicate that impaired expression of Glb1l3 is mostly due to the absence of the chromophore 11-cis retinal, suggesting that Rpe65 deficiency may have many metabolic consequences in the underlying neuroretina.

Topics: Aging; Animals; Animals, Newborn; Carrier Proteins; Choroid; cis-trans-Isomerases; Disease Models, Animal; Disease Progression; Down-Regulation; Eye Proteins; Gene Expression; Glycoside Hydrolases; Immunohistochemistry; In Situ Hybridization; Leber Congenital Amaurosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Degeneration; Retinal Pigment Epithelium; Retinaldehyde; RNA; RNA, Messenger

To determine the efficacy of intravitreal administration of 9-cis-retinal in restoring visual function in Rpe65-mutant dogs.. 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.. 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.. Our findings establish that 9-cis-retinoid therapy can restore visual function in a canine model of human disease resulting from RPE65 mutations.. 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.

Topics: Animals; Carrier Proteins; Dark Adaptation; Disease Models, Animal; Diterpenes; Dogs; Electroretinography; Eye Proteins; Female; Intravitreal Injections; Isomerism; Leber Congenital Amaurosis; Male; Mutation; Photoreceptor Cells, Vertebrate; Retinaldehyde; Retreatment; Vision, Ocular; Visual Acuity

Topics: Animals; Disease Models, Animal; Diterpenes; Dogs; Electroretinography; Eye Proteins; Intravitreal Injections; Leber Congenital Amaurosis; Mutation; Retinaldehyde; Vision, Ocular; Visual Acuity