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Astra Dinculescu, Yoshikazu Imanishi, Frank M. Dyka, Jianwen Liu, Issam McDoom, Xuan Liu, Hanna Västinsalo, Reetta Jalkanen, Eeva-Marja Sankila, William W. Hauswirth; Clarin-1 Expression In The Retina Using AAV Mediated Gene Delivery. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3336.
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Human Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by clarin-1 (CLRN1) gene mutations leading to progressive retinitis pigmentosa and sensorineural hearing loss. Here, we employ rAAV vectors for studies aimed at identifying which retinal cells express clarin-1 in the mouse retina.
Three AAV type 8-733 (Y-F mutation at AAV capsid position 733) vectors containing 1, 2 or 3 kb of proximal mouse clarin promoter sequence driving mCherry reporter gene expression were injected into wild-type mouse eyes via a subretinal approach. Treated eyes were analyzed fundoscopically and histologically to determine the retinal pattern of fluorescent mCherry expression. In addition, the effects of a scAAV8-733 CBA vector expressing a hemagglutinin (HA)-tagged human clarin-1 (hClarin) on retinal structure and function were examined following subretinal injection.
The 1kb clarin promoter vector led to strong mCherry expression in both photoreceptor and RPE cells. Subretinal delivery of scAAV8-733 vector expressing hClarin (1010 vector genomes per injection) under control of a CBA promoter led to significant photoreceptor degeneration as evidenced by morphological and ERG analyses. This toxic effect was not seen with weaker vectors, such as conventional AAV2 expressing hClarin under the same CBA promoter. We also tested a 1:1000 dilution of the above scAAV8-733 (CBA)-hclarin vector up to 3 months after injection and found no ERG loss.
Reporter mCherry expression patterns when driven by mouse clarin promoter sequences suggest that photoreceptors are the main source of clarin expression in the adult mouse retina. Additionally, there appears to be a critical threshold for Clarin expression, above which photoreceptor cell death occurs. Since it is known that endogenous Clarin expression is likely to be low, it may therefore be prudent to employ therapeutic vectors at titers that produce physiologic levels of this protein to avoid overexpression toxicity.
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