Efficient expression of self-complementary AAV in ganglion cells of the ex vivo primate retina

[摘要] Purpose: To evaluate the efficiency ofself-complementary adeno-associated virus (scAAV)-mediated geneexpression of green fluorescent protein (GFP) or the allotopic humanND4 subunit of complex I in ganglion cells of the primate retina. Methods: ScAAV2 containing the cDNAencoding the humanized GFP or allotopic ND4 subunit of complex I underthe control of the cytomegalovirus (CMV) immediate early gene enhancerand short chicken beta-actin promoter-exon1-intron1 (CBA) was injectedinto the vitreous cavity of five primate eyes after enucleation.Following incubation in standard Dulbecco's Modified Eagle Medium(DMEM) culture media overnight at 37 °C with 5% CO2, retinalflat mounts were probed with monoclonal GFP or FLAG antibodiesovernight followed by counterstaining with anti-mouse IgG conjugated tocy2. For identification of retinal ganglion cells (RGCs), the retinalwhole mounts were also stained with a Brn3a or Thy1.2 (proteinexpressed in RGCs. domain) antibody, then counterstained with cy3 orcy2. Immunofluorescence and colocalization were assessed using confocalmicroscopy. Quantitative analysis of GFP, ND4FLAG, Brn3a, or Thy1.2expressing cells was performed using Image J software. Results: While the endogenousfluorescence of GFP was seen in a few retinal cells, GFP and ND4FLAGimmunofluorescence was plentiful. The immunosignals were restricted tothe inner retina and colocalized to slightly more than half of allcells expressing Brn3a or Thy1.2, suggesting efficient expression inRGCs. Conclusions: Our findings suggest thatthe hybrid CMV enhancer-CΒA promoter can play an efficient role intargeting primate RGCs following intravitreal gene delivery using thescAAV2 vector. Donated ex vivo primate eyes may serve as a model systemfor testing RGC expression before in vivo intravitreal injections ofthis and perhaps other AAV serotypes.

[学科分类] 生物化学/生物物理