May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Expression Targeting of Rod and Cone Photoreceptors Using a Human Rhodopsin Kinase (GRK1) Enhancer/Promoter Delivered With an Adeno-Associated Vector
Author Affiliations & Notes
  • B. S. Pawlyk
    Ophthalmology, The Berman-Gund Laboratory for the Study of Retinal Degenerations, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • S. Khani
    Ophthalmology, Biochemistry, Ross Eye Institute, State University of New York at Buffalo, Buffalo, New York
  • E. Kasperek
    Ophthalmology, Biochemistry, Ross Eye Institute, State University of New York at Buffalo, Buffalo, New York
  • J. Young
    Ophthalmology, Biochemistry, Ross Eye Institute, State University of New York at Buffalo, Buffalo, New York
  • O. V. Bulgakov
    Ophthalmology, The Berman-Gund Laboratory for the Study of Retinal Degenerations, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • M. A. Adamian
    Ophthalmology, The Berman-Gund Laboratory for the Study of Retinal Degenerations, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • X. Sun
    Ophthalmology, The Berman-Gund Laboratory for the Study of Retinal Degenerations, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • A. J. Smith
    Division of Molecular Therapy, Institute of Ophthalmology, University College London, London, United Kingdom
  • R. R. Ali
    Division of Molecular Therapy, Institute of Ophthalmology, University College London, London, United Kingdom
  • T. Li
    Ophthalmology, The Berman-Gund Laboratory for the Study of Retinal Degenerations, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships B.S. Pawlyk, None; S. Khani, None; E. Kasperek, None; J. Young, None; O.V. Bulgakov, None; M.A. Adamian, None; X. Sun, None; A.J. Smith, None; R.R. Ali, None; T. Li, None.
  • Footnotes
    Support NIH Grants EY010309, P30 EY14104, the Foundation Fighting Blindness, and the Foundation for Retinal Research
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1677. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: 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.

Methods:: 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.

Results:: 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.

Conclusions:: 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.

Keywords: gene transfer/gene therapy • photoreceptors 
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