April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Self-Complementary Double-Stranded AAV2 Drives Rapid and Efficient Expression of GFP in Ganglion Cells of the Primate Retina
Author Affiliations & Notes
  • R. D. Koilkonda
    Opthalmology, Bascom Palmer Eye Institute, Miami, Florida
  • W. W. Hauswirth
    Opthalmology, University of Florida, Gainesville, Florida
  • J. Guy
    Opthalmology, Bascom Palmer Eye Institute, Miami, Florida
  • Footnotes
    Commercial Relationships  R.D. Koilkonda, None; W.W. Hauswirth, None; J. Guy, None.
  • Footnotes
    Support  RO1 EY 017141, R24 EY018600, R01 EY 07982, P30 EY014801
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3009. doi:
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      R. D. Koilkonda, W. W. Hauswirth, J. Guy; Self-Complementary Double-Stranded AAV2 Drives Rapid and Efficient Expression of GFP in Ganglion Cells of the Primate Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3009.

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

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Abstract

Purpose: : Last year the authors of abstract #4514 "Tracking Transfection of Macaque Retinal Ganglion Cells With AAV2 Viral Vectors; In vivo Imaging Reveals Differences Between Two Promoters" presented data suggesting intravitreal injections of single-stranded (ss)AAV serotype 2 using the chicken beta-actin promoter (CBA) did not efficiently drive expression of GFP in RGCs, but rather in foveal cones. A pubmed review has not disclosed any studies of intravitreal AAV mediated gene expression in primates. Since intravitreal gene delivery to RGCs is central to our mitochondrial gene therapy of patients with Leber Hereditary Optic Neuropathy (LHON) and optic neuritis, the present study is to evaluate the efficiency of the CBA promoter successfully used in rodents to drive the downstream reporter GFP gene in the rhesus macaque eye and further to study the localization of the expressed protein.

Methods: : Self-complementary dsAAV2 containing the cDNA encoding the green fluorescent protein (GFP) under the control of the CBA promoter (AAV-CBA-GFP) was injected into the vitreous cavity of two eyes after enucleation, from a 4-year-old male rhesus macaque. Retinal flat mounts were probed with monoclonal GFP antibodies overnight followed by counterstaining with anti-mouse IgG conjugated to cy2. For identification of RGCs, the retinal whole mounts were also stained with Brn3a antibody, then counterstained with cy3. Immunofluorescence and co-localization were assessed using confocal microscopy. Quantitative analysis of GFP and Brn3a expressing cells was performed using the Image J software.

Results: : While GFP autoflorescence was not seen in the flat mounted primate retinal specimens, GFP immunofluorescence was plentiful. GFP expressing cells co-localized exclusively with those expressing Brn3a. Quantitative analysis revealed the mean percentage of GFP-positive cells was 43+7.2 (mean + SD) relative to the mean percentage for Brn3a-positive RGCs that was 56.6+7.2. Therefore, the efficiency of the CBA promoter in driving GFP expression in RGCs of ex vivo primate retina was 77%.

Keywords: gene transfer/gene therapy • retinal culture • ganglion cells 
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