Volume 19, Issue 2, Pages 260-265 (February 2011) Inhibition of Choroidal Neovascularization in a Nonhuman Primate Model by Intravitreal Administration of an AAV2 Vector Expressing a Novel Anti-VEGF Molecule  Michael Lukason, Elizabeth DuFresne, Hillard Rubin, Peter Pechan, Qiuhong Li, Ivana Kim, Szilard Kiss, Christina Flaxel, Margaret Collins, Joan Miller, William Hauswirth, Timothy MacLachlan, Samuel Wadsworth, Abraham Scaria  Molecular Therapy  Volume 19, Issue 2, Pages 260-265 (February 2011) DOI: 10.1038/mt.2010.230 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Intravitreal administration of AAV2-sFLT01 (~2 × 109 vg) to C57BL/6 mouse eyes resulted in significantly fewer laser burns (P < 0.001) with neovascularization than uninjected eyes or eyes treated with an equivalent amount of an AAV2 vector coding for an irrelevant transgene. “n” represents the number of eyes lasered and evaluated that contained between 4 and 6 laser burns per eye. Molecular Therapy 2011 19, 260-265DOI: (10.1038/mt.2010.230) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 The amount of sFLT01 was measured in the aqueous humor collected in-life at various time points by paracentesis from two NHP studies. Animals in study A were injected intravitreally with 2 × 108 vg or 2 × 109 vg of AAV2-sFLT01 vector. All animals in study B were injected intravitreally with 2 × 1010 vg of AAV2-sFLT01 vector. The limit of quantitation (LOQ) was set at the mean plus two standard deviations of all the aqueous humor values from untreated animals. Molecular Therapy 2011 19, 260-265DOI: (10.1038/mt.2010.230) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Anti-AAV2Ab (a) total IgG titers and (b) the neutralizing antibody titers in blood serum pre- and post- (either 6 or 15 weeks) AAV2 vector administration to the eye. Molecular Therapy 2011 19, 260-265DOI: (10.1038/mt.2010.230) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 A comparison between preadministration antibody titers to AAV2 and the level of sFLT01 transgene expression measured in the aqueous humor. The data are organized by increasing sFLT01 expression levels. The limit of quantitation was set at the mean plus two standard deviations of all the aqueous humor values from untreated animals (a) total IgG titers and (b) the neutralizing antibody titers. Molecular Therapy 2011 19, 260-265DOI: (10.1038/mt.2010.230) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Photomicrograph of NHP eye processed with in situ hybridization to detect sFLT01 transgene mRNA (red chromagen). (a) Transduced transitional epithelial cells (TE) in the pars plana. (b) Retinal ganglion cells (GC) and Müller cells (M) transduced in the peripheral retina. Molecular Therapy 2011 19, 260-265DOI: (10.1038/mt.2010.230) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Late fluorescein angiograms of NHPs that received AAV2 vectors in one eye compared with the animal's uninjected contralateral eye. The AAV2-sFLT01 treated eyes demonstrate markedly reduced leaking CNV compared to the uninjected eye. No reduction in fluorescein leakage was observed in the eye treated with an AAV2-Null vector compared to the uninjected contralateral eye (bottom panel). Molecular Therapy 2011 19, 260-265DOI: (10.1038/mt.2010.230) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions