Evaluation of Adeno-Associated Virus-Mediated Gene Transfer into the Rat Retina by Clinical Fluorescence Photography

Abstract

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.

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy

“…[15][16][17][18][19] rAAV-mediated expression of GFP persists at least 3 years after subretinal injection without significant toxicity and immune response, 42 and would therefore be a suitable delivery vector for long-term inhibition of ocular NV. In this present study, we demonstrated that the stable expression of rAAV-mediated sFlt-1 injected directly into the eye suppressed experimental corneal and choroidal NV.…”

“…The flat mounts were further embedded in optical cutting temperature compound (Sakara Finetek, Torrance, CA, USA), frozen and sectioned at [8][9][10][11][12] m. Real-time monitoring of GFP expression following subretinal injection was achieved by fluorescence fundus photography. 16 The percentage GFPpositive area within the injection area was determined from the fluorescence fundus photograph using image analysis software based on the Matrox Imaging Library (Canada). 16 …”

“…16 The percentage GFPpositive area within the injection area was determined from the fluorescence fundus photograph using image analysis software based on the Matrox Imaging Library (Canada). 16 …”

“…Adeno-associated virus (AAV)-mediated gene delivery enables the long-term expression of transgenes, [15][16][17][18][19] thus providing a potential solution for the long-term delivery of anti-angiogenic agents in the eye. However, one of the difficulties of ocular anti-angiogenic therapies is that several types of cells such as retinal pigment epithelial (RPE) cells, Muller cells, ganglion cells, the inner nuclear layer, macrophages and choroidal vascular endothelial cells have all been identified as potential contributors to VEGF upregulation.…”

“…16,19 In this study, AAV was delivered transclerally into the subretinal space near the equator (Figure 5a). Using fluorescence fundus photography, at least 60% of cells within the injection bleb, which covered an area of approximately one-quarter of the retina, were transduced by AAV-CMV.gfp (Figure 5b).…”