Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors

PURPOSE. To develop a gene therapy system that specifically targets transgene expression to S-cones of the mammalian retina, the authors coupled recombinant AAV-mediated delivery with the use of a human blue-opsin (HB) promoter to drive expression. METHODS. Two regions of the HB promoter sequence, HB569 and HB996, were amplified from human DNA, cloned into an AAV vector cassette upstream of the green fluorescent protein (GFP) gene, and packaged into AAV2 and AAV5 capsids. Eyes of postnatal day (P) 40 to P48 Sprague-Dawley rats were subretinally injected with 2 L vector. Animals were humanely killed 2 to 3 weeks or 20 months after injection, and the pattern and persistence of GFP expression were analyzed in the treated retinas by immunohistochemistry, Western blotting, and RT-PCR. RESULTS. AAV5.HB.GFP vectors targeted photoreceptor transduction with an efficiency 20-fold higher than analogous serotype 2 vector. Both AAV5.HB.GFP vectors exhibited similar transduction efficiencies with patterns of GFP expression that did not vary depending on the size of the HB promoter used. Transgene expression was exclusively localized to photoreceptors of retinas treated with either vector. Furthermore, GFP expression was observed for at least 20 months. Dual GFP immunostaining with S-or M-opsin antibodies and GFP/PNA labeling revealed that cones coexpressing S-opsin/GFP or Mopsin/GFP constituted 37.5% Ϯ 8% and 13.5% Ϯ 3% of the GFP-positive photoreceptors, respectively, whereas rods constituted 49% Ϯ 5% of the GFP-positive photoreceptors. Because cones constitute approximately 1% of adult rat retinal photoreceptors, it was estimated that the relative transduction efficiency of AAV5.HB.GFP vectors was approximately 100:1 for cones versus rods. CONCLUSIONS. AAV5.HB.GFP vector injected into the subretinal space of Sprague-Dawley rats targeted gene expression to photoreceptor cells with an efficiency approximately 20-fold higher than that for AAV2.HB.GFP. Transgene expression regulated by the human blue cone-promoter persisted at least for 20 months. Cones coexpressing S-opsin and the GFP transgene appeared to prevail, confirming that in addition to having properties of the AAV serotype, the promoter choice is key to fine-tuning transgene delivery and expression in specific retinal cells. The system described here may be effective in a therapeutic setting in which strong S-cone transgene expression is required. (Invest Ophthalmol Vis Sci. 2006;47:3505-3513