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C. Wu, E. Ivanova, Y. Zhang, Z.-H. Pan; AAV-Mediated Subcellular Targeting of Chop2-GFP in Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3298.
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© ARVO (1962-2015); The Authors (2016-present)
As a possible strategy to restore vision after the loss of photoreceptors in retinal degenerative diseases, we have previously demonstrated the ability of adeno-associated virus (AAV2)-mediated expression of channelodopsin-2 (Chop2) and halorhodopsin (HaloR) for restoring ON and OFF light responses in retinal ganglion cells (RGCs). For the approach of expressing Chop2/HaloR in RGCs, expression would be required in the somatodendritic region and minimized in the axon in order to maintain visual spatial processing. Additionally, achieving differentially targeted expression of Chop2 and HaloR could recreate the antagonistic center-surround receptive field in RGCs and further improve the visual spatial processing for the restored vision. In this study, we are screening protein sorting motifs for AAV-meditated targeting expression of Chop2-GFP in RGCs.
Subcellular targeting motifs were added to a channelopsin-2 (Chop2) and GFP (Chop2-GFP) fusion construct. The expression and function of the constructs were first examined in HEK 293 cells with fluorescence microscopy and patch-clamp recordings. AAV2 vectors with Chop2-GPF-motif constructs were produced and intravitreally injected into the eyes of adult C57BL/6J mice. Chop2-GFP targeted expression was assessed in whole-mount retinas by comparing the ratios of GFP fluorescence intensity of the dendrite, soma, and axon of subcellular targeting motifs to those of the control Chop2-GFP expression without a targeting motif.
Of the motifs tested so far, the myosin binding domain (MBD) motif from melanophilin led to increased expression in dendrites. The dendrite:soma ratio with the MBD motif is more than twice that of the control. The ankyrin binding domain (ABD) motif from sodium channel Nav1.6 resulted in increased expression in the axon initial segment (AIS) and decreased expression in dendrites. The AIS:soma ratio with ABD motif is more than four times that of the control while the dendrite:soma ratio is only half that of the control.
AAV-mediated expression of Chop2-GFP with the MBD and ABD motifs can preferentially target the somatodendritic compartment and the axon initial segment of RGCs, respectively. The addition of protein sorting motifs could be used for improving visual spatial processing for the Chop2/HaloR-based gene therapy in RGCs.
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