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Zhuo-Hua Pan, Qi Lu, Tushar Ganjawala, JrGang Cheng; AAV-Mediated Expression Targeting of Retinal Rod Bipolar Cells with An Optimized mGluR6 Promoter. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2638.
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© ARVO (1962-2015); The Authors (2016-present)
AAV-mediated expression of microbial rhodopsins in surviving inner retinal neurons is a promising approach to restoring vision after retinal degeneration. Targeting ChR2 to specific bipolar cells is particularly appealing; but AAV-mediated targeted gene expression to specific bipolar cells, especially administered by intravitreal injection, poses a challenge. The use of a 200 bp enhancer of mGluR6 promoter and a basal SV40 promoter has been shown to be able to target ChR2 to ON bipolar cells by electroporation (Lagali et al., 2008). However, AAV-mediated delivery of ChR2 to ON bipolar cells with the same construct was achieved only via subretinal injection (Doroudchi et al., 2011). In this study, we aimed to develop mGluR6 promoter constructs for AAV-mediated gene delivery to specific retinal bipolar cell type(s) via intravitreal injection.
A series of AAV2 expression cassettes were constructed with the combination of various sequences of mGluR6 promoter, the 200 bp mGluR6 enhancer, and intron sequences of mGluR6 carrying a transgene of mCherry or ChR2-GFP. AAV vectors were produced by packaging the expression cassettes into AAV serotype 2 with an Y444F capsid mutation and were injected intravitreally into the eyes of C57BL/6J mice at age of approximately one month. The expression was examined one month after viral injection.
The constructs containing the 200 bp mGluR6 enhancer and a ~1 kb mGluR6 promoter sequence resulted in selective targeting of the transgenes to bipolar cells, predominantly rod bipolar cells (RBCs). The expression of the transgenes in RBCs was significantly enhanced with the use of a shortened mGluR6 promoter sequence and the inclusion of mGluR6 intron 3 and 4. The transduction efficiency was viral concentration dependent. At the concentration of ~1 x 1013 vg/ml, the expression of the transgenes in RBCs was observed across the entire retina with the highest density in the peripheral regions, where the majority of RBCs could be transfected.
We developed optimized mGluR6 promoter constructs that can achieve AAV-mediated targeted gene expression predominantly in RBCs administered by intravitreal injection. The ability of AAV-mediated targeted gene delivery to RBCs via intravitreal injection could facilitate the development of optogenetic-based therapies for vision restoration as well as genetic manipulation in RBCs in vitro and in vivo.
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