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B. S. Pawlyk, S. Khani, E. Kasperek, J. Young, O. V. Bulgakov, M. A. Adamian, X. Sun, A. J. Smith, R. R. Ali, T. Li; Expression Targeting of Rod and Cone Photoreceptors Using a Human Rhodopsin Kinase (GRK1) Enhancer/Promoter Delivered With an Adeno-Associated Vector. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1677.
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Rhodopsin kinase 1, also known as G protein-coupled receptor kinase 1 (GRK1), is a component of the light adaptation pathway expressed in both rods and cones. The purpose of this study is to develop highly active derivatives of the human GRK1 gene promoter suited for targeting exogenous genes for expression in rods and cones.
Human GRK1 promoter, its multimers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were linked up with luciferase or GFP reporter and the promoter activity assayed in WERI retinoblastoma or Crx/Sp1 supplemented HEK-293 cells. The GRK1 promoter derivative displaying the highest activity in vitro was integrated upstream of GFP in an adeno-associated viral (AAV2/5) construct and tested by subretinal injection in mice. GFP expression was assessed by fluorescence microscopy and the results compared with mouse eyes receiving a mouse opsin promoter-based GFP-AAV2/5 construct.
Among the derivatives tested in vitro, the promoter carrying the full extent of the conserved 5' untranslated sequence showed the highest relative activity in WERI and Crx/Sp1 cotransfected HEK-293 surpassing unmodified promoter constructs by up to 3 fold. Subretinal injection with an AAV construct carrying this segment drove robust GFP expression confined to the photoreceptor layer of the retina. Double-labeling with cone markers in a cone-rich rodent retina confirmed that GRK1 promoter drove GFP expression in cone photoreceptors. Compared to a short opsin promoter, the GRK1 promoter appeared to be similarly efficient but lead to a more uniform expression in rods. It was also more efficient in targeting cone photoreceptors. There was no GFP expression in the retinal pigment epithelium or the inner retina.
The human GRK1 promoter derivative extending into the 5'-UTR appears to be a highly active and specific promoter in photoreceptors. It is a promoter of choice to drive expression of transgenes in both rods and cones.
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