Evaluation of the adeno-associated virus mediated long-term expression of channelrhodopsin-2 in the mouse retina

[摘要] Purpose: The conversion of inner retinalneurons to photosensitive cells via viral mediated expression ofchannelrhodopsin-2 (ChR2) offers a new potential approach for therestoration of vision after photoreceptor degeneration. This study wasconducted to evaluate the recombinant adeno-associated virus serotype 2(rAAV2)-mediated long-term expression and safety of ChR2 in the mouseretina. Methods: rAAV2 vectors carrying a fusionconstruct of channelopsin-2 (Chop2) and green fluorescent protein (GFP;Chop2-GFP) under the control of a hybrid cytomegalovirus early enhancerand chicken β-actin (CAG) promoter were injected at differentconcentrations into the eyes of wild-type adult mice. The retinas wereharvested up to 18 months after virus injection for immunostaining andelectrophysiological studies. Injected mice were kept either undernormal light conditions, or exposed to a strong blue light. Theexpression of GFP and the density of the cells in the ganglion celllayer (GCL) were examined. Results: The expression of Chop2-GFP wasstable for up to 18 months. Chop-GFP was observed predominantly inretinal ganglion as well as amacrine cells. At the highest virusconcentration (6×1012 GC/ml), up to 20% of the cells in theGCL were infected by the virus. At the lowest virus concentration (1×1010GC/ml), the expression was targeted to AII amacrine cells. Theconcentration of the virus, the light conditions, and the percentage ofChop2-GFP-positive cells had no effect on the density and, thus, on thesurvival of the cells in the GCL. Sufficient number of functional ChR2channels were maintained in ganglion cells to drive robust membranedepolarization and spike firing in response to light. Conclusions: Expression of Chop2-GFPcould be achieved in retinal neurons in vivo for the duration of thelifespan of mice. The expression of Chop2-GFP did not cause anydetectable toxicity and cell death to neurons of the ganglion celllayer.

[学科分类] 生物化学/生物物理