Mutations in ciliary proteins lead to a spectrum of debilitating disorders, collectively termed ciliopathies. Retinitis pigmentosa (RP) due to ciliary dysfunction in photoreceptors (PRs) is a commonly observed genetically heterogeneous retinal degenerative disease, for which there is no treatment. PRs (rods and cones) are highly polarized neurons with an outer segment, responsible for light detection, and an inner segment, which houses the protein production and trafficking machinery. The inner and outer segments are linked by a bridge-like structure called the connecting cilium (CC), which facilitates the transport of proteins and lipids to the outer segment. Our long term goal is to understand the modes of protein and lipid transport in PR cilia to better understand the pathogenesis of retinal ciliopathies, identify therapeutic targets and design suitable therapies. This application focuses on understanding the role of a major retinal ciliopathy protein, RPGR (RP GTPase regulator) in regulating protein trafficking via the CC of PRs. The RPGR gene expresses two major alternatively spliced isoforms: RPGRconst (constitutive; exons 1-19) and RPGRORF15 (exons 1-15+part of intron 15; ORF15). Our studies have revealed that RPGR isoforms are regulated both at the post-transcriptional and post-translational levels and are involved in overlapping yet distinct functions in regulating PR health. Building upon our progress during the funded period, we propose to understand RPGR function by examining precise roles of RPGR isoforms. We hypothesize that post-transcriptional and post-translational cross-talk between RPGRORF15 and RPGRconst isoforms modulates the resultant disease phenotype. The current application proposes to delineate the role of ORF15 in regulating alternative splicing of RPGR isoforms (Aim 1) and interrogate the interplay between RPGR isoforms in regulating PR phosphoinositide metabolism (Aim 2). All reagents, assays, model systems and equipments that are already on hand will be utilized. Under the first aim, we will perform transcriptional analysis of RPGRORF15 and RPGRconst in dermal fibroblasts from patients carrying RPGR mutations and identify specific splicing regulators that alter RPGR splicing. We will also determine the photoreceptor-specific regulation of RPGR splicing by subretinally injecting RPGR minigenes into mouse pups. Studies proposed in Aim 2 focus on delineating the role of isoform-specific post- translational modifications of RPGR in regulating phosphoinositide composition of PR outer segment. We will also determine the role of a phosphoinositide modulator in maintaining PR integrity and in altering the severity of RPGR-associated retinal degeneration. Our approach is significant because it focuses on understanding the mechanisms of one of the most severe and prevalent features of ciliopathies. Such knowledge will inform us about the mechanisms employed by the cilium for long-term functional and structural maintenance. These studies are not only relevant to RPGR mutations, but other forms of retinal ciliopathies that result in PR degeneration but spare the initial generation of the cilium. The innovative aspects of our research are: (i) utilization of fibroblasts derived from patients carrying RPGR mutations to assess the effect of disease mutations on the post-transcriptional regulation RPGR and (ii) validation of the results from patient cells in the context of PR-specific post-transcriptional regulation using subretinal injection of minigenes into mice. In addition, we propose novel studies to fill the knowledge gap regarding the function of the distinct RPGR isoforms using animal models and patient fibroblasts. These studies represent an innovative combination of conceptual and methodological approaches that will aid our understanding of severe retinal ciliopathies.

Public Health Relevance

The proposed research is relevant to public health because these studies are expected to increase the understanding of blindness due to retinal pathogenesis in RPGR-associated disease. In addition, we will gain new knowledge on the pathogenic mechanisms of many other retinal degenerative disorders that are part of pleiotropic disorders due to ciliary dysfunction, collectively termed ?ciliopathies?. Such knowledge will allow us to design better therapeutic strategies. Thus, the proposed research is relevant to the part of NIH?s mission that pertains to developing fundamental knowledge that will help reduce the burdens of human disability.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Special Emphasis Panel (ZRG1)
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Neuhold, Lisa
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University of Massachusetts Medical School Worcester
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Zhang, Wei; Li, Linjing; Su, Qin et al. (2018) Gene Therapy Using a miniCEP290 Fragment Delays Photoreceptor Degeneration in a Mouse Model of Leber Congenital Amaurosis. Hum Gene Ther 29:42-50
Khanna, Hemant (2018) More Than Meets the Eye: Current Understanding of RPGR Function. Adv Exp Med Biol 1074:521-538
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