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Mol Vis. 2009 Aug 21;15:1680-9.

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

Author information

1
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA. eivanova@med.wayne.edu

Abstract

PURPOSE:

The conversion of inner retinal neurons to photosensitive cells via viral mediated expression of channelrhodopsin-2 (ChR2) offers a new potential approach for the restoration of vision after photoreceptor degeneration. This study was conducted to evaluate the recombinant adeno-associated virus serotype 2 (rAAV2)-mediated long-term expression and safety of ChR2 in the mouse retina.

METHODS:

rAAV2 vectors carrying a fusion construct of channelopsin-2 (Chop2) and green fluorescent protein (GFP; Chop2-GFP) under the control of a hybrid cytomegalovirus early enhancer and chicken beta-actin (CAG) promoter were injected at different concentrations into the eyes of wild-type adult mice. The retinas were harvested up to 18 months after virus injection for immunostaining and electrophysiological studies. Injected mice were kept either under normal light conditions, or exposed to a strong blue light. The expression of GFP and the density of the cells in the ganglion cell layer (GCL) were examined.

RESULTS:

The expression of Chop2-GFP was stable for up to 18 months. Chop-GFP was observed predominantly in retinal ganglion as well as amacrine cells. At the highest virus concentration (6 x 10(12) GC/ml), up to 20% of the cells in the GCL were infected by the virus. At the lowest virus concentration (1 x 10(10) GC/ml), the expression was targeted to AII amacrine cells. The concentration of the virus, the light conditions, and the percentage of Chop2-GFP-positive cells had no effect on the density and, thus, on the survival of the cells in the GCL. Sufficient number of functional ChR2 channels were maintained in ganglion cells to drive robust membrane depolarization and spike firing in response to light.

CONCLUSIONS:

Expression of Chop2-GFP could be achieved in retinal neurons in vivo for the duration of the lifespan of mice. The expression of Chop2-GFP did not cause any detectable toxicity and cell death to neurons of the ganglion cell layer.

PMID:
19710944
PMCID:
PMC2730750
[Indexed for MEDLINE]
Free PMC Article

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