Retinoids in Vision

University Of California-Irvine, Irvine CA

Investigators

Linked publications, trials & patents

Abstract

Vision is initiated when retinal photoreceptors respond to light via isomerization of the opsin-bound visual chromophore 11-cis-retinal (11cRAL), releasing all-trans-retinal and triggering signal-transduction that transmits visual information to the brain. Sustained vision requires continuous renewal of the chromophore, originally thought to occur exclusively through the canonical visual (retinoid) cycle. Recent research advances have identified alternative pathways for production of 11cRAL in the retinal pigment epithelium (RPE) and Müller cells and developed gene-therapy approaches to restore 11cRAL synthesis in the setting of disease. These new findings have raised key questions regarding the flow of 11cRAL and its metabolites within and between the RPE, photoreceptors, and Müller glia, as well as the long-term efficacy of gene therapy to correct visual cycle deficits. We will address these questions by using novel mouse models that enable study of subcellular distribution of retinoids with unprecedented resolution and chemical specificity, and by applying newly developed genome-editing technologies to compare the efficacy and longevity of gene alteration/correction versus currently available gene-augmentation techniques to rescue retinal-disease models related to retinoid metabolism. Aim 1: Elucidate the trafficking of 11-cis-retinoids within the neural retina. We developed a novel retinoid- trapping method to isolate retinol isomers within specific cells and subcellular compartments, enabling robust detection of these species by HPLC and in vivo two-photon microscopy, even when they are present as transient intermediates under physiological conditions. This approach will enable definitive resolution of hypothesized retinoid trafficking pathways that have, to date, not been possible to probe experimentally. Aim 2: Determine the efficacy of genome-editing versus gene-supplementation therapy in rd12 mice. Gene therapy is gaining prominence for treating deficiencies in visual-cycle enzymes. However, gene- augmentation therapy has several drawbacks, including limited durability and persistence of the dysfunctional gene. Genome editing can mitigate these shortcomings. We will compare the efficiency and durability of both treatments. Mice expressing the mutant Rpe65 gene will be treated at different ages with Rpe65-AAV vectors, or with virus-like particles (VLPs) delivering the genome editor; and therapeutic durability will be tested over time. Aim 3: Develop genome-editing therapy for Rpe65-associated autosomal dominant retinitis pigmentosa (adRP). We made an adRP mouse model that expresses the c.1430A>G (D477G) mutant of Rpe65. This mutation serendipitously introduces a protospacer adjacent motif (PAM), enabling us to use CRISPR-Cas9 to selectively inactivate the mutant gene and restore function. Alternatively, we will use prime editors to correct the RPE65 deficiency with a long substitution extending from the PAM sequence. Editors and guide RNA will be delivered using VLPs and lipid nanoparticles carrying Cas9-ribonucleoprotein cargo. We will optimize the editing efficiency and evaluate treatment efficacy in restoring normal function in this adRP disease model.