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Mark M Hassall, Michelle E. McClements, Alun R Barnard, Sher A Aslam, Robert E MacLaren; Cone opsins and Crx are gene therapy candidates for the revival of cone photoreceptors in an RP mouse model. Invest. Ophthalmol. Vis. Sci. 2017;58(8):351.
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© ARVO (1962-2015); The Authors (2016-present)
It has been observed that residual cone photoreceptors remain in human patients with retinitis pigmentosa (RP) long after cone photosensitivity is extinguished. We hypothesised that there is a loss of cone photosensitivity that precedes cone cell death, resulting in a subpopulation of ‘dormant’ cone photoreceptors that could be reactivated through molecular therapies. We aimed to correlate changes in cone-specific gene expression on qPCR with phenotypical changes in cone function in a mouse model of RP, thereby identifying candidates for subsequent gene therapy trials.
The Rhodopsin knockout (Rho-/-) mouse model of RP was examined using Optical Coherence Tomography (OCT), Scanning Laser Ophthalmoscopy (SLO) and Electroretinography (ERG). Five time-points for qPCR were then chosen correlating to: 1) early rod degeneration; 2) the peak of rod cell death on OCT; 3) the beginning of cone cell death on SLO; 4) the loss of cone photosensitivity on ERG and 5) an advanced stage of degeneration. The neural retina from Rho-/- mice and wild type controls were collected across these five time-points (n=5 per time point). Each retinal transcriptome was extracted and converted to cDNA. Primers for qPCR were designed to eight genes involved in the mouse cone phototransduction cascade: Opn1mws, Opn1sws, Arr3, Crx, Pde6h, Cnga3, Cngb3 and Gnat2. All primer sets were optimised to achieve 90-100% efficiencies and all target gene values were normalised to Bactin.
In the WT mouse, cone-specific gene expression levels were stable across all time points except for a gradual decline in Pde6h and a small decline in Opn1sws expression between 17 and 25 weeks of age. Crx revealed a steep increase in expression levels between 6 and 17 weeks with a further increase between 17 and 25 weeks. By contrast, in Rho-/- mice all genes assessed decreased in expression over time (p<0.05). The biggest overall decline was observed between 6 and 12 weeks and was particularly apparent in Pde6h (though this was equivalent to that observed in WT), Opn1sws and Crx.
The results of the gene expression analyses correlated with the phenotypic observations and suggested that Crx and cone opsins are appropriate candidates for future gene therapy attempts to restore photosensitivity in dormant cone cells in RP.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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