May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Human Bestrophin Promoter Drives Rpe-Specific Gfp Expression in the Canine Retina
Author Affiliations & Notes
  • B. Zangerl
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • K. E. Guziewicz
    Ophthalmology, University of Zurich, Zurich, Switzerland
  • A. M. Komaromy
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • S. J. Lindauer
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • J. J. Alexander
    Ophthalmology, University of Florida, Gainesville, Florida
  • L. G. Glushakova
    Ophthalmology, University of Florida, Gainesville, Florida
  • W. W. Hauswirth
    Ophthalmology, University of Florida, Gainesville, Florida
  • G. M. Acland
    J.A. Baker Institute, Cornell University, Ithaca, New York
  • G. D. Aguirre
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • Footnotes
    Commercial Relationships  B. Zangerl, None; K.E. Guziewicz, None; A.M. Komaromy, None; S.J. Lindauer, None; J.J. Alexander, None; L.G. Glushakova, None; W.W. Hauswirth, None; G.M. Acland, None; G.D. Aguirre, None.
  • Footnotes
    Support  FFB, NEI 06855, NEI 17549, ONCE
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 1701. doi:https://doi.org/
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    • Get Citation

      B. Zangerl, K. E. Guziewicz, A. M. Komaromy, S. J. Lindauer, J. J. Alexander, L. G. Glushakova, W. W. Hauswirth, G. M. Acland, G. D. Aguirre; Human Bestrophin Promoter Drives Rpe-Specific Gfp Expression in the Canine Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1701. doi: https://doi.org/.

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

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Abstract

Purpose: : Canine multifocal retinophathy (cmr), caused by mutations in the bestrophin gene, is a large animal model for human Best disease. To begin the development of a gene therapy approach to treat cmr, a recombinant Adeno-Associated virus (rAAV) was made to express GFP under control of the human bestrophin promoter (hVMD2-GFP). Normal canine eyes were used to determine the level and specificity of expression.

Methods: : The VMD2 promoter was a gift from D. Zak. Normal dogs at 4 months of age had one eye injected in either the subretinal or sub-RPE space with 3.35 x1013 vg of hVMD2-GFP AAV2/1; the contralateral eye was either injected with saline or remained uninjected. Fundus exams were performed 2 and 4 weeks after injection, and eyes were collected after 4 weeks, fixed in 4% paraformaldehyde, and embedded in OCT. GFP expression was evaluated on 7µm sections with and without specific antibody.

Results: : The fundus exam revealed a small scar at the site of injection, but eyes were otherwise normal. After 4 weeks, ophthalmoscopy with a blue light source showed green fluorescence only with subretinally injected hVMD2-GFP. In sections, GFP expression was evident in the area corresponding to the hVMD2-GFP subretinal bleb by direct GFP fluorescence and antibody labeling. Sub-RPE injection of hVMD2-GFP yielded a significantly lower number of GFP expressing cells, and expression could only be detected by antibody labeling.

Conclusions: : The human bestrophin promoter drives stable and specific GFP expression in canine RPE cells after in vivo transduction with rAAV. Currently, we are evaluating rAAV mediated expression of wild-type bestrophin in place of GFP in the same construct. These results provide a basis for further therapeutic studies in the cmr dog which are of petential significance for therapy in human patients with Best disease.

Keywords: gene transfer/gene therapy • retinal pigment epithelium • adenovirus 
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