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  • Title: Efficient expression of self-complementary AAV in ganglion cells of the ex vivo primate retina.
    Author: Koilkonda RD, Hauswirth WW, Guy J.
    Journal: Mol Vis; 2009 Dec 16; 15():2796-802. PubMed ID: 20019878.
    Abstract:
    PURPOSE: To evaluate the efficiency of self-complementary adeno-associated virus (scAAV)-mediated gene expression of green fluorescent protein (GFP) or the allotopic human ND4 subunit of complex I in ganglion cells of the primate retina. METHODS: ScAAV2 containing the cDNA encoding the humanized GFP or allotopic ND4 subunit of complex I under the control of the cytomegalovirus (CMV) immediate early gene enhancer and short chicken beta-actin promoter-exon1-intron1 (CBA) was injected into the vitreous cavity of five primate eyes after enucleation. Following incubation in standard Dulbecco's Modified Eagle Medium (DMEM) culture media overnight at 37 degrees C with 5% CO(2), retinal flat mounts were probed with monoclonal GFP or FLAG antibodies overnight followed by counterstaining with anti-mouse IgG conjugated to cy2. For identification of retinal ganglion cells (RGCs), the retinal whole mounts were also stained with a Brn3a or Thy1.2 (protein expressed in RGCs. domain) antibody, then counterstained with cy3 or cy2. Immunofluorescence and colocalization were assessed using confocal microscopy. Quantitative analysis of GFP, ND4FLAG, Brn3a, or Thy1.2 expressing cells was performed using Image J software. RESULTS: While the endogenous fluorescence of GFP was seen in a few retinal cells, GFP and ND4FLAG immunofluorescence was plentiful. The immunosignals were restricted to the inner retina and colocalized to slightly more than half of all cells expressing Brn3a or Thy1.2, suggesting efficient expression in RGCs. CONCLUSIONS: Our findings suggest that the hybrid CMV enhancer-CBetaA promoter can play an efficient role in targeting primate RGCs following intravitreal gene delivery using the scAAV2 vector. Donated ex vivo primate eyes may serve as a model system for testing RGC expression before in vivo intravitreal injections of this and perhaps other AAV serotypes.
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