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Mark Christian Butler, Aaron R Morawski, Mohammed Zuber, Jack M Sullivan; Toward a Novel Gene Therapy for Dry Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3315.
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Rod photoreceptor rhodopsin (RHO) is a rational target for gene-based suppression in a therapeutic strategy for dry age-related macular degeneration (dAMD). The bulk of outer segment mass phagocytized by the RPE is RHO and bis-retinoid accumulation is expected to be directly proportional to the RHO levels. We are investigating hammerhead ribozymes (hhRz) against RHO as a candidate dAMD therapeutic strategy.
Ribozymes, targeting an accessible region in RHO mRNA, and RHO RNA were simultaneously transcribed in vitro and cleavage activity determined by PAGE with appropriate controls. Double knockout (ABCA4, RDH8) mice on C57BL/6N background (dKO) and C57BL/6N controls were grown in 10 lux or in 350 lux cyclic white light (12hr:12hr). dKO mice have acute elevation of all-trans-retinal with light exposure and accumulate toxic bis-retinoids under regular growth. A historical broad band (peak 528 nm, width 66 nm) retinal light damage (LD) device and a modern narrow band (peak 505 nm, width 30 nm) LD device delivered doses of light with varying spectral overlap with the RHO absorption band. Ultrahigh resolution spectral domain optical coherence tomography (OCT) for mice (Bioptigen Inc) measures outer nuclear layer (ONL) thickness and outer segment length, electroretinography (ERG) assesses photoreceptor function, and difference spectroscopy quantifies RHO levels.
HhRzs targeting RHO mRNA and embedded in an engineered adenoviral VAI scaffold were able to cleave target mRNA at the expected location in vitro. HhRz catalytic core mutation obviated cleavage, and the VAI scaffold RNA without hhRz also failed to cleave target as additional control. Ribozymes are cloned into AAV vectors for preclinical therapeutic testing. dKO mice grown at 10 lux and exposed to 1700 lux in broad or narrow band LD devices undergo marked ONL thinning by OCT and ERG a-wave and b-wave loss whereas C57BL/6N control mice show stability of ONL and ERG 2 weeks after LD. dKO mice grown chronically at 350 lux show greater sensitivity to LD in the narrow band LD device compared to the broad band device at 2 wks post LD by both OCT and ERG. Rhodopsin levels are measurable by difference spectroscopy to correlate with the effects.
Light-adaptation impacts LD sensitivity in dKO mice and may relate to photostasis. The dKO model with LD is a useful paradigm to test RHO hhRzs as a candidate therapy for dAMD.
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