Abstract
Purpose: :
The aim of this study was to determine the retinal cell types transduced by recombinant adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 2 (AAV2) after subretinal injection in rabbits.
Methods: :
We constructed an Ad5 vector expressing Zs-Green (ZsG) and an AAV2 vector expressing green fluorescent protein (GFP), both under the control of a CMV promoter. Doses of 5x106 pfu and 2x109 DRP, respectively, in a volume of 20 µl were injected into the subretinal space of both eyes of male Dutch Belted rabbits (1.5-2 kg, n = 4/group). Subretinal PBS injections served as controls (20 µl/eye, n = 4). At 1-3 weeks post injection, rabbits were sacrificed. Following removal of the cornea and lens, the posterior eye cups were fixed in 4% PFA solution for 2 hours. Retina and RPE/choroid were dissected and mounted onto slides as flat whole mounts. ZsG and GFP were visualized by direct fluorescence microscopy or confocal microscopy. Some retinal and RPE/choroid whole mounts were processed for immunocytochemistry. Antibodies against RPE65, calbindin, PKC, GFAP, tyrosine hydroxylase (TH), neurofilament-L (NF), calretinin, and macrophage RAM-11 (MR) were used to identify cell types expressing ZsG or GFP.
Results: :
After injections of Ad5, ZsG was detected predominantly in the RPE around the injection site by fluorescent microscopy. After injections of AAV2, GFP expression was widespread in both the RPE and neural retina. Confocal microscopy confirmed Ad5-mediated ZsG expression in the RPE, and AAV2 mediated GFP expression in the RPE and inner retinal layers. Neither Ad5 nor AAV2 mediated expression in the photoreceptor layer. Immunocytochemistry identified RPE65-positive RPE cells which expressed ZsG in Ad5 injected retinas. In addition, AAV2 transduced various cell types expressing GFP, including neurons (labeled with calbindin, PKC, TH, HF and calretinin), glial cells (labeled with GFAP), and macrophages (labeled with MR).
Conclusions: :
Adenovirus serotype 5 and AAV serotype 2 vectors deliver reporter genes to the rabbit retina. Ad5 transduction is specific to RPE cells, while AAV2 transduces RPE cells as well as retinal neural cells except for photoreceptors. The reason for the tropism for specific retinal cell types and the optimal dose for expressing transgenes remains to be determined.
Keywords: adenovirus • retina • gene/expression