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Mark Hassall, Michelle McClements, Alun R Barnard, Sher A Aslam, Robert E MacLaren; Assessment of adjunctive gene therapy to dormant cones in end-stage retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2018;59(9):989. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
It is well known that residual ‘dormant’ cone photoreceptors remain in patients with Retinitis Pigmentosa (RP), long after cone photosensitivity is extinguished. It has also been shown by others that behavioural light responses may be restored by expressing Halorhodopsin (a bacterial chloride channel) in these cells. We sought to determine if a human protein normally present in cones might achieve the same aim. We first determined which genes encoding proteins in the phototransduction cascade were down regulated in dormant cones of the rhodopsin knockout mouse retina and, secondly, established if over-expressing these genes might restore some light responses.
Opn1 and Crx were found to be significantly downregulated compared with other cone genes. AAV2/8.CAG.CRX.WPRE.pA (AAV.CRX) and AAV2/8.CAG.OPN1LW.WPRE.pA (AAV.OPN) vectors were created. Rho-/- mice that expressed GFP in cones (n=105) received subretinal injections at PND21. Treated eyes received 1.5µL of either AAV.CRX (1x10^9, 1x10^8 or 1x10^7 gc), AAV.OPN (1x10^8 or 1x10^7gc) or PBS sham. Mice were repeatedly assessed over the subsequent four months using OCT, cSLO, ERG and OMR. Eyes were collected at multiple timepoints (PND42, 63, 105 and 135) for qPCR, IHC or WB.
Both vectors expressed transgene mRNA at PND42. The AAV.CRX eyes showed CRX staining in the nuclei of photoreceptors and the inner retina; expression appeared dose-dependent. The AAV.OPN eyes showed OPN1LW in the outer segments and RPE cells. Neither AAV construct improved cone-pathway ERG photosensitivity 3 weeks after treatment (p<0.001), despite transgene expression. Neither AAV treatment significantly prolonged ERG photosensitivity beyond PND84 (p<0.001), at which time cone function is extinguished in the untreated eye. OMR function in photopic conditions at PND98 was also not improved by either treatment (p<0.05). No cone survival benefit was shown. The higher doses of both vectors were associated with retinal damage and loss of the outer retina.
CRX and OPN1LW were found to be downregulated in the cone photoreceptors in end-stage Rho-/- mice. Re-expression of these genes did not however lead to improved cone function on ERG or OMR, nor prolonged cone survival. The positive responses to Halorhodopsin may therefore represent the fact that a channel protein does not need second messenger systems, which may also be compromised in cones in end-stage RP.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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