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M.C. Mauck, J. Neitz, T.B. Connor, Jr., W.W. Hauswirth, M. Neitz; Longitudinal in vivo Characterization of Expression of Viral Delivered Genes for L–opsin and Green Fluorescent Protein in Cone Photoreceptors of Gerbils . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4071.
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Delivery of genes to retinal cones using recombinant adeno–associated virus (rAAV) has potential for treating vision loss. Here we developed methods for monitoring expression of viral delivered genes over time, in the same animal. We injected two viruses, one carrying a reporter gene whose expression can be monitored visually, and the other carrying an opsin gene whose expression could be monitored functionally. We chose the gerbil (Meriones unguiculatus) as the animal model system because our long–term interest is to use viral–gene delivery to modify cone vision. Gerbils have a well–developed cone system, with 11–14% of their photoreceptors being cones.
We used replication deficient serotype 5 rAAV to deliver the gene for green fluorescent protein (GFP) or for human long–wavelength sensitive opsin (L–opsin). In each rAAV vector, the gene (GFP or L opsin) was under control of the human X–chromosome opsin gene enhancer termed the Locus Control Region (LCR) and promoter. These regulatory elements were previously shown to direct expression of GFP selectively to rat M–cones (W. Hauswirth, unpublished results), and was expected to do the same in gerbil. Five ul (∼2x10 10 vector genomes) of a mixture of rAAV–GFP and rAAV–L–opsin were injected subretinally. Images were obtained immediately pre– and post–injection, and at ∼14 day intervals thereafter using the RetCamII under normal or fluorescence detection configurations. Spectral flicker electroretinograms were performed to assess cone function.
RetCamII images documented the locations and sizes of the blebs. GFP fluorescence was detected as early as 14 days post–injection. GFP fluorescence was clearly present 28–32 days after the injection (n=3) . Compared to 14 days, GFP fluorescence at 28–32 days was greater in intensity and covered a larger area of retina. Color fundus images showing the injection site and fluorescence images showing the GFP expression pattern were taken sequentially and superimposed using landmarks visible in both images. These demonstrated that the peak GFP fluorescence was at the site of injection. ERG results indicated the functional expression of the opsin.
The RetCamII provides a means of conducting longitudinal studies of GFP expression in the retina of living animals. Injecting a mixture of two viruses, one carrying GFP and the other carrying a therapeutic gene allows visual assessment of the retinal area expressing transgenes and function of the product of the co–delivered therapeutic genes.
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