In juvenile macular degeneration (JMD) (e.g. Stargardt, Best) and common dry age-related macular degeneration (dAMD), cellular and biochemical debris accumulates within and beneath the retinal pigment epithelium (RPE). These diseases reflect, in part, naturally occurring circadian shedding of rod and cone photoreceptor (PR) outer segment tips, and phagocytosis and lysosomal digestion by RPE cells. In the human parafovea, where dAMD starts, a single RPE cell underlies about 30-35 rods and a few cones. Due to RPE digestive limitations excess age-related materials called lipofuscin (LF) accumulate in RPE phagolysosomes. LF contains protein, lipid and carbohydrate components and contributes to sub-RPE deposits (flecks, drusen) seen in JMD and dAMD. LF has a brilliant autofluorescence under blue light excitation due to the dominant presence of toxic bis-retinoids (TBR) such as pyridinium salts (A2E) and retinaldehyde dimers (RetDi). These derive from covalent reaction, in PR outer segments, of two molecules of all-trans-retinal (ATR), resulting from visual pigment bleaching, with a single molecule of the membrane aminolipid phosphatidyl-ethanolamine. LF pigments accumulate with age in normals, and by age 30 are quantitated by fundus autofluorescence. Accumulation of TBRs in RPE cells reflects the normal daily accumulation of ATR from the visual cycle, with bleaching and regeneration of rod and cone opsins, integrated over many years. In JMD and dAMD TBR accumulation rates are accelerated. A2E and the more potent RetDi exert many toxic effects on the RPE cells and directly promote apoptosis. Accumulation of A2E/Ret-Di precedes spatial geographic loss of RPE cells and overlying PRs in dAMD/JMD. TBRs are well validated molecular targets for therapy of dAMD/JMD. Our hypothesis is that dAMD/JMD can be treated by reducing time-dependent accumulation of TBRs in PR and RPE cells. The rationale is that steady-state reductions in TBRs would decrease time-integrated (agerelated) toxicity and maintain viable RPE cells longer into life, which would act to preserve overlying PRs, maintain central vision due to cones, and slow or halt emergence of macular geographic atrophy. The long term objective is to develop a safe and effective gene therapy for dAMD/JMD. The objective of the proposed experiments is to use hammerhead ribozymes (hhRz) or RNA interference (shRNA) as genetic tools to knockdown (KD) expression of key proteins in rod PR or RPE cells that quantitatively contribute to daily accumulation of TBRs. This novel strategy is tested in a mouse model of dAMD/JMD (ABCR-/-//RDH8-/- double knockout), which has an outer retinal degeneration related to TBR accumulation. The strategy is: 1) reduce rhodopsin (RHO) in PRs to constrain ATR formation that results mostly from rod pigment bleaching, and reduce the stress imposed on the RPE to digest rod outer segment material (90% RHO), and 2) constrain retinoid cycle regeneration rates by reduction of 11-cis-retinol isomerase (RPE65) in RPE. By reducing the amount of RHO that forms and bleaches in rod PRs, daily ATR production is reduced under normal lighting. As ATR is a direct substrate, reduction in the rate of TBR accumulation is expected. The expected outcome is that reduction of these targets will rescue TBR-mediated retinal degeneration in the mouse model, with preserved photopic sensitivity (cones use a Mller cell retinoid visual cycle) at the expense of slight scotopic sensitivity loss (< -0.3 log).
Specific Aims are:
Aim 1. Further optimize the lead candidate hhRz for RHO and develop stronger hhRz and shRNA expression constructs for mouse RPE65.
Aim 2. Test for toxicity of candidate gene therapeutic agents after subretinal or intravitreal rAAV delivery in a mouse model humanized for normal RHO.
Aim 3. Test for rescue of retinal degeneration after subretinal or intravitreal rAAV delivery of therapeutic candidates in the double knockout (ABCR-/-//RDH8-/-) mouse model of dAMD/JMD, through knockdown of RHO or RPE65, measured as changes in the intrinsic rat

Public Health Relevance

Potential Impact on Veterans Health. AMD is the most common form of visual disability in the elderly and is a health care crisis. There are 4 million people in the US with severe visual disability due to AMD, and this number is expected to triple by the year 2020. There are 10-15 million others who have early forms of the disease. AMD prevalence increases exponentially over the age of 55 and, with our aging society, there are many millions of American Veterans who will suffer from visual loss due to AMD. I see Veterans in my Retinal and Macular Degeneration clinic and can attest to the suffering that AMD causes. Nonspecific vitamin support is the only approved therapy for dry AMD, the most common form (80-85%). As yet, there is no specific therapy for dry AMD, which promotes profound visual disability in about 20% of people. This proposed study is a preclinical test of a novel gene therapy strategy for dry AMD. Success in a mouse model is a first essential step toward human clinical trials, which could be conducted in the VA Healthcare System.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Neurobiology F (NURF)
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VA Western New York Healthcare System
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Froebel, Beau R; Trujillo, Alexandria J; Sullivan, Jack M (2017) Effects of Pathogenic Variations in the Human Rhodopsin Gene (hRHO) on the Predicted Accessibility for a Lead Candidate Ribozyme. Invest Ophthalmol Vis Sci 58:3576-3591
Yau, Edwin H; Butler, Mark C; Sullivan, Jack M (2016) A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets. Exp Eye Res 151:236-55
Butler, Mark C; Sullivan, Jack M (2015) A Novel, Real-Time, In Vivo Mouse Retinal Imaging System. Invest Ophthalmol Vis Sci 56:7159-68
Flynn, Erin; Ueda, Keiko; Auran, Emily et al. (2014) Fundus autofluorescence and photoreceptor cell rosettes in mouse models. Invest Ophthalmol Vis Sci 55:5643-52
Sullivan, Jack M; Yau, Edwin H; Taggart, R Thomas et al. (2012) Relieving bottlenecks in RNA drug discovery for retinal diseases. Adv Exp Med Biol 723:145-53
Sullivan, Jack M; Yau, Edwin H; Kolniak, Tiffany A et al. (2011) Variables and strategies in development of therapeutic post-transcriptional gene silencing agents. J Ophthalmol 2011:531380