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H.S. Zhang, S. Kachi, M. Kachi, P.–Q. Liu, H. Akiyama, N. Umeda, K. Yokoi, S. Hackett, P.D. Gregory, P.A. Campochiaro; Engineered Zinc Finger Protein Transcription factors as a Potential Therapy for Ocular Neovascularization . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1786.
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© ARVO (1962-2015); The Authors (2016-present)
Choroidal neovascularization (CNV) is the most common cause of severe vision loss in patients with age–related macular degeneration (AMD). Anti–angiogenesis therapies, especially those that target Vascular Endothelial Growth Factor (VEGF), have shown promise in treating neovascular AMD. However, targeting VEGF alone may not yield maximal therapeutic effects. Theoretically, zinc–finger protein transcription factors (ZFP TF) can be engineered to regulate the expression of any endogenous gene. We sought to test the effects in vitro and in vivo of two ZFPs, one designed, to repress the transcription of VEGF–A and one designed to activate the transcription of Pigment Epithelium Derived Factor (PEDF), a potent anti–angiogenic factor.
ZFP DNA binding domains (DBDs) were assembled to target the promoters of VEGF–A and PEDF. For VEGF–A repression or PEDF activation, the DBDs were linked to the repression domain of v–ErbA or the activation domain of NF–kB p65, respectively. The activities of these ZFPs were tested by transient transfection, followed by analysis for both mRNA and protein levels of VEGF–A and PEDF. The effect of intraocular injection of adeno–associated virus (AAV) vectors expressing ZFPs was tested in a mouse model of laser–induced CNV.
ZFPs that significantly activate PEDF expression (>20 fold) or repress VEGF–A expression (∼10 fold) in transient transfection assays were identified. When these ZFPs were packaged in AAV vectors and injected into mouse eyes, they regulated PEDF and VEGF–A expression and significantly reduced the area of CNV at Bruch’s membrane rupture sites by approximately 50% and 32%, respectively.
Engineered ZFP TFs can successfully regulate the expression of PEDF and VEGF–A in vivo and significantly suppress the development of CNV in a mouse model. Future studies will explore combined repression of VEGF and activation of PEDF. These data provide proof–of–concept for regulation of endogenous genes by gene transfer of ZFPs as a therapeutic approach for ocular diseases.
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