Cone-specific expression using a human red opsin promoter in recombinant AAV.
OBJECTIVE
To determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector.


METHODS
An rAAV vector was constructed that contains a 2.1kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472bp mouse rod opsin promoter, previously shown to drive photoreceptor-specific expression, was also used. Each recombinant virus was injected into the subretinal space of rat, ferret or guinea pig eyes. GFP expression was analyzed 4-6 weeks after injection microscopically.


RESULTS
The human 2.1kb cone opsin gene upstream sequence targeted GFP expression only to a subset of photoreceptors. Cone-specific expression was shown by co-localization of GFP fluorescence and cone-specific opsin antibody staining. Additionally, in rats, expression was specific for L/M-cones whereas no S-cones exhibited GFP fluorescence. The efficiency of rAAV mediated cone transduction surrounding the injection site was high since every L/M-cone antibody-staining cone was also positive for GFP expression.


CONCLUSIONS
The human red/green opsin gene promoter used in this study is sufficient to direct efficient cone-specific gene expression in several mammalian species, suggesting that key cell-type specific regulatory elements must be broadly conserved in mammals. These observations have significance in devising gene therapy strategies for retinal dystrophies that primarily affect cones and point toward a way to functionally dissect the cone opsin promoter in vivo.
Li
Qiuhong
Q
Department of Ophthalmology, Box 100284, JHMHSC, College of Medicine, University of Florida, Gainesville, FL 32610-0284, USA. qli@eye.ufl.edu
Timmers
Adrian M
AM
Guy
John
J
Pang
Jijing
J
Hauswirth
William W
WW
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D005289:Ferrets; D018390:Gene Targeting / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008322:Mammals / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity
D017299:Rod Opsins; C095855:long-wavelength opsin; D049452:Green Fluorescent Proteins