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F Wang, K Rendahl, WC Manning, D Quiroz, M Coyne, SS Miller; Inhibition of Choroidal Neovascularization (CNV) by Adeno-Associated Virus (AAV) Mediated Expression of sFLT1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4602.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the anti-angiogenic activity of sFlt1 overexpressed in retinal pigment epithelium (RPE) using an AAV vector with a CMV promoter (AAV-CMV-sFlt1) in a rat model of CNV induced by AAV-VEGF. Methods: sFlt1 was cloned by RT-PCR based techniques from native human fetal RPE cells and packaged into AAV by a triple transfection protocol. Culture medium of 293 cells infected with AAV-sFlt1 was used to inhibit VEGF induced proliferation of human microdermal vascular endothelial cells (HMVEC). The total amount of sFlt1 protein in the conditioned media was determined by a sandwich ELISA. AAV-VEGF+AAV-sFlt1 was co-injected into the subretinal space (SRS) of one eye and AAV-VEGF+AAV-GFP was injected into the SRS of the contralateral eye of Sprague Dawley rats (1010-1011 particles in 2 µl). Pathologic changes in the retina were evaluated by electroretinogram (ERG) and histology. Results: HMVEC proliferation was inhibited by conditioned media from rAAV-sFlt1 infected 293 cells. VEGF (0.1 nM)-induced proliferation was completely inhibited by sFlt1 at 1 nM. Inhibition was also observed at 0.1 nM sFlt1. This inhibition was not seen with HMVEC cells treated with conditioned media from AAV-lacZ infected cells, or cells treated with recombinant VEGF alone (control). Six weeks after injection, ERGs were recorded simultaneously from both eyes of each animal. The ERG a- and b- waves amplitudes were increased about 100% in three animals using AAV-sFlt1. Three months after injection, quantification of CNV was made using histological sections. The extent of neovascularization in the AAV-sFlt1+AAV-VEGF injected eye was 39% smaller than that in AAV-GFP+AAV-VEGF injected eye. Conclusion: Preliminary results show that AAV mediated overexpression of sFlt1 in RPE can inhibit the development of CNV, suggesting that AAV-sFlt1 potentially could be used to inhibit CNV associated with the «wet» form of age-related macular degeneration (AMD).
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