Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates.

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

VEGF-induced choroidal damage in a murine model of retinal neovascularisation.

BACKGROUND/AIMS: Photoreceptor-specific upregulation of vascular endothelial growth factor (VEGF) in a transgenic mouse model (Kimba) of retinal neovascularisation induces retinal vascular damage which appears similar to that in diabetic retinopathy. Here we have determined whether the choroidal vasculature is also affected in Kimba. METHODS: Kimba mice were assessed with fundus fluorescein angiography for mild, moderate or severe retinal vascular leakage prior to preparation of choroidal corrosion casts for quantitative analysis using scanning electron microscopy. VEGF was located immunohistochemically. RESULTS: Choroidal abnormalities included microaneurysms, constriction, shrinkage and dropout in the capillaries and tortuosity and loops in the arteries and veins which were similar to those observed in corrosion casts of the human choroid in diabetes. Similar to human diabetes, choroidal neovascularisation was not observed. The severity of choroidal damage correlated with the extent of retinal vascular leakage. In addition to the expected presence of VEGF in photoreceptors, VEGF was also detected in the pigment epithelium and choroid in the transgenic mice. CONCLUSION: We show that elevated retinal VEGF levels trigger pathophysiological changes in the choroid. We suggest that therapies to prevent vascular damage in diabetes must target both the retinal and choroidal vasculatures.

Generation of transgenic mice with mild and severe retinal neovascularisation.

AIM: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. METHODS: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. RESULTS: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. CONCLUSIONS: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.