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Daniyar Dauletbekov, Alun R Barnard, Mandeep S Singh, Robert E MacLaren; Assessment of endogenous silencing of a cone-specific gene (Cnga3) delivered to rods using an AAV2/5 vector with CBA promoter.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3333.
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© ARVO (1962-2015); The Authors (2016-present)
Although AAV retinal gene therapy has been applied safely in several clinical trials, success in terms of function has so far only been reported when using the ubiquitous (CBA) promoter. The issue of tissue specific gene expression is complex and has led to the design of cell-specific promoters and microRNA targeting sequences, but the concept of endogenous silencing of mRNA transcripts for genes expressed ectopically remains relatively uninvestigated. We previously noted that transduction of the Cnga3 knockout achromatopsia mouse retina with an AAV2/5.CBA.Cnga3 vector led to robust expression of Cnga3 protein, but this was restricted to cones. Although this might be explained by antibody specificity, we were interested to explore if there was any evidence of silencing of Cnga3 in transduced rods.
Triple knockout (TKO) mice: Gnat1-/- ; Cnga3-/-; Opn4 -/- were used in all experiments. A recombinant AAV2/5 viral vector containing a ubiquitous chicken beta-actin (CBA) promoter and murine Cnga3 cDNA was linked bicistronically to an internal ribosome entry site (IRES) to the gene encoding enhanced green fluorescent protein (GFP). The GFP served as a marker for successful translation of transgenic mRNA in rods and cones. Subretinal injections were performed on 3-6 week old TKO mice (5 x 10 11 genome particles). Cnga3 Immuno-staining was performed after 6-8 weeks.
Sections were taken through areas of injection and expression of Cnga3 protein was confirmed to be restricted to cone outer segments using immunostaining. Although expression of GFP was seen in both cones and surrounding rods, the mean ratio of GFP expressing cones to rods was 22% (n=7, 95% confidence interval 11-32%). This is significantly higher than the true ratio of these cells in this mouse model (<5%). Furthermore at the edges of transduced areas, GFP expression was seen in areas to be almost exclusively in cones, as rod transduction fell below threshold. Electroretinogram testing confirmed restored cone function.
These observations could be explained by a mild silencing effect of Cnga3 when expressed in rods. Assuming vector tropism is similar, the observed level of translated transgenic mRNA appears to be significantly higher than predicted in cones compared with rods. Further molecular analyses will be required to confirm this.
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