Cone photoreceptors fulfill critical functions such as central visual acuity, color vision, and photopic vision. Cones can be affected primarily or secondarily by retinal diseases that lead to severe visual deficits in the affected patients. Targeting gene expression to the cones, both for gene replacement therapy or for the synthesis of neurotrophic or cone survival factors, could provide rescue and sustained cone function. For these therapeutic investigations, dogs represent a valuable model for the development of cone-directed gene therapy, especially since two canine achromatopsic lines exist with either a genomic deletion (i.e. functional null) or missense mutation of the CNGB3
The work presented herein with human cone opsin promoters provides the groundwork for future cone-directed gene therapy in canine models, and complements recent studies of cone-directed gene expression in the rat, ferret, guinea pig, and primate retina.22,23
Compared to other viral vectors, rAAV provides many advantages, such as its ability to infect both mitotic and post-mitotic growth arrested cells with high efficiency, its ability to accept non-viral regulatory sequences, and the lack of any associated human disease.29
The specific cell targeting is based on the use of cell-type specific promoters, the site of inoculation, and the AAV serotype.30
Currently, nine AAV serotypes are widely available; their AAV capsid proteins influence the cellular tropism as well as the speed of onset and intensity of gene expression.30
The combination of the serotype 2 genome with capsid proteins from other serotypes, i.e., pseudotyped AAV vectors, allow the enhancement of vector transduction characteristics in the retina.31,32
For example, transduction of both RPE and photoreceptor cells can be achieved by subretinal injection of either AAV2 or AAV5; however, the efficiency of transduction appears much greater with AAV5, especially in photoreceptors.33,34
The aim of this project was to evaluate various human cone opsin promoters for their specificity and efficacy to target gene expression to the canine cone photoreceptors. We were able to successfully target gene expression to the canine L/M-cones using the human red cone opsin promoter PR2.1 in rAAV5. The expression of the reporter gene GFP was both specific and effective in that all L/M-cones evaluated histologically were indeed expressing the GFP, and no significant expression of the reporter gene was seen in other cell types. The PR2.1 promoter has recently been used for successful cone-directed gene therapy in a mouse model of achromatopsia caused by a mutation in the cone alpha-transducin gene.21
We were unable to detect expression of GFP when using PR0.5, the shortest version of the human red cone opsin promoter. Adding 3 copies of the 35-bp LCR to the PR0.5 promoter led to detectable GFP expression in the cones. However, the level of GFP expression was lower than with PR2.1, and expression in the canine L/M-cones was detected, almost exclusively, using by immunolabeling. Provided that these low expression levels are stable, such outcome may not be undesirable in cases where overexpression could be detrimental to the cell, and a lower expression level is desired. In addition, the use of a shorter promoter would allow the introduction of a longer gene into the rAAV vector construct.
Unfortunately, the blue cone opsin promoter HB569 did not lead to GFP expression in canine S-cones. Instead, this promoter led to the expression of GFP in a few L/M-cones, rods, and in the RPE. At this point it is unclear why this promoter is not effective in transducing canine S-cones, but targets expression to a subgroup of L/M-cones, rods and RPE cells. The application of the same promoter in rats showed that about 37% of the GFP positive cones were S-cones, about 13% were M-cones, and almost half of the GFP positive photoreceptors in rats were rods.24
The results in our dogs are even less specific as we were not able to identify a GFP positive S-cone. These results are surprising given that the HB569 sequence appears to align better with the S-cone promoter sequence of the dog (75% identity) than the rat (68% identity; data not shown). Modifications will have to be made to the S-cone promoter in the future in order to successfully target the canine S-cones.
Similar to the rat,22
S-cone-specific GFP expression was also not found with the PR2.1 promoter in the dog, even though Wang19
and Alexander and colleagues21
reported that the 2.1 kb human red cone opsin gene promoter directed reporter gene expression in both M- and S-cones in transgenic mice. However, in regards to cone-targeted therapies in higher primates or dogs, such a result is not problematic; S-cones make up only 7% of the human cone population within 4 mm of the foveal center and are missing in a zone about 100 µm (0.35 degrees) in diameter near the site of peak cone density (foveal tritanopia).35
In dogs, we estimated that S-cones make up 8–13% of the cones. Thus we have demonstrated that highly specific targeting of reporter gene expression is possible in the predominant class of cones in the dog, and the results suggest that cone directed gene replacement therapy should be possible with the PR2.1 or 3LCR-PR0.5 promoters.
Finally, we were able to target gene expression to cones in a canine rcd1-affected retina, an example of a disease where cones are secondarily affected by a primary rod disorder. These very preliminary results suggest that cone-targeted therapies may be possible in diseases, where cones are secondarily affected.
In summary, we successfully proved the principle of cone specific gene targeting in a large animal model. Our results open the door for cone-specific gene therapy in canine models of primary and secondary cone disease.