AAV–mediated expression of VEGF peptides inhibits retinal neovascularization (NV) in a Mouse Model of Retinopathy of Prematurity (ROP)

Abstract

Abstract: : Purpose: Previous studies have shown that exon 6 and exon 7 derived VEGF peptides block VEGF binding to its receptors and inhibit VEGF–induced angiogenesis in vitro. The purpose of this study is to evaluate whether AAV–mediated expression of these VEGF peptides can inhibit retinal NV in a mouse model of ROP. Methods: Three AAV vectors were constructed that each contain the CBA (chicken ß–actin) promoter and the sequence encoding VEGF exon 6 (amino acid 121–132), exon 6(amino acid 125–136) or exon 7 (includes amino acid 22–44 of exon 7 and the first cysteine of exon 8). An immunoglubin kappa secretory signal sequence was fused in frame at the 5’ end of each VEGF sequence. A woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was included at the 3’ end of each VEGF sequence to enhance expression. Postnatal day 2 (P2) neonatal mice were injected in one eye with the AAV vector expressing each VEGF peptide. The mice were placed in a 73% oxygen chamber from P7 to P12, and then returned to room air for another 5 days. At P17, the extent of retinal NV was evaluated by comparing the number of vascular endothelial nuclei immediately above the inner limiting membrane in retinal sections spanning the whole eye in virus injected and contralateral uninjected or PBS injected eyes. Results: The number of vascular endothelial nuclei was reduced 50–90% in each of the AAV vector injected eyes compared to partner control eyes in the ROP mouse. Care was taken to avoid any vascular endothelial cells associated with persistent hyaloid vasculature. Conclusions: The results indicate that these VEGF peptides are antagonists of neovascularization in vivo and may be useful in treating angiogenesis–associated retinal diseases in human.

Keywords: gene transfer/gene therapy • retinal neovascularization • retinopathy of prematurity