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Elena V Olshevskaya, Sanford L. Boye, Kevin Tyler McCullough, Igor V Peshenko, Shannon Elizabeth Boye, Alexander M Dizhoor; Transgenic expression of human photoreceptor guanylyl cyclase GUCY2D with LCA1 mutations: diversity of physiological effects and localization in photoreceptors.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3180. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies evaluating the impact of mutations in human retinal guanylyl cyclase (GUCY2D) known to be associated with Leber congenital amaurosis (LCA1) revealed wide heterogeneity in the biochemical properties of the affected cyclase - some of the mutations completely inactivated RetGC1 and some did not . The purpose of this study was to test the mutant cyclase in vivo using AAV-mediated expression in mouse photoreceptors.
The activities of LCA1-linked RetGC1 mutants and their abilities to bind GCAP and RD3 were assessed in vitro and in cyto, using cultured HEK293 cells expressing fluorescently labeled RetGC1, GCAP1 and RD3. AAV-mediated expression of human wild type, R1091x, and S248W RetGC1  was evaluated in subretinally injected  RetGC1/RetGC2 double knockout (GCdKO) mice. ERG, immunofluorescence and biochemical properties of RetGC1s in the AAV-injected eyes were tested using the non-injected eyes as a control.
The R1091x and S248W RetGC1 retained the ability to bind GCAP1 and RD3 in cyto. R1091x, whose activity in vitro as a recombinant enzyme was reduced compared to wild type, was nonetheless able to effectively restore RetGC activity in GCdKO retinas in vivo and rescue both rod and cone ERG responses. In contrast, S248W RetGC1, which shows higher catalytic activity as a recombinant protein in vitro, failed to restore detectable cyclase activity in GCdKO retinas. Rod ERG responses also remained virtually undetectable and only traces of cone ERG responses could be identified. Immunostaining showed that the transgenic wild type and R1091x RetGC1 were strongly expressed in rod and cone outer segments with occasional mislocalization to the photoreceptor cell bodies, but the S248W only sparsely appeared in occasional cone outer segments and cell bodies and failed to accumulate in rod outer segments.
The S248W mutation does not affect biochemical properties of the RetGC1, but changes its normal expression and/or delivery to the outer segments in live mouse rods and cones. Surprisingly, the R1091x retains most of its ability to perform the cyclase function in vivo and therefore its role in causing LCA1 remains to be determined.References:  Jacobson et al. (2013) Human Molecular Genetics 22, 168-183;  Boye et al. (2013) Human Gene Therapy 24,189-202.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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