Evaluation of adeno-associated virus-mediated gene transfer into the rat retina by clinical fluorescence photography.
The purpose of this study was to evaluate recombinant adeno-associated virus (AAV) as an in vivo gene transfer vector for the retina and to explore the possibility of monitoring the expression of green fluorescent protein (GFP) using a noninvasive method. Rats were injected subretinally with rAAV-gfp or rAAV-lacZ. Strong expression of the reporter gene in a circular area surrounding the injection site was observed in retinal whole mounts and tissue sections. Higher magnification revealed that cells demonstrating high levels of green fluorescence were hexagonal in shape, indicating they were retinal pigment epithelium (RPE) cells. Histological observation of retinal sections demonstrated that recombinant AAV specifically transduced RPE cells. Ten animals were injected with rAAV-gfp for longitudinal studies and the fluorescence was monitored by retinal fluorescence photography. The GFP signal was detected in 100% of the animals as early as 2 weeks postinjection and remained present throughout the experimental period of 4 months. After 2 weeks, a gradual increase in the number of transduced cells occurred before reaching maximal levels of GFP expression at 8 weeks. This was followed by a small decrease over 4 weeks before reaching stable expression at 16 weeks. Our results demonstrated that rAAV efficiently transduces rat RPE cells and that retinal fluorescence photography is suitable for monitoring GFP expression. By using this noninvasive technique, we demonstrated that repetitive measurements of GFP expression in vivo in the rAAV-gfp-transduced retina are possible. This study demonstrated that retinal fluorescence photography is a potent tool for studying AAV-mediated gene delivery in the retina.
Rolling
F
F
Lions Eye Institute, Perth, Western Australia, Australia. rolling@cyllene.uwa.edu.au
Shen
W Y
WY
Tabarias
H
H
Constable
I
I
Kanagasingam
Y
Y
Barry
C J
CJ
Rakoczy
P E
PE
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D018014:Gene Transfer Techniques / Q000592:standards*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D010781:Photography; D051381:Rats; D011995:Recombination, Genetic; D012160:Retina / Q000378:metabolism*; D014161:Transduction, Genetic
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D009355:Neomycin