788. Engineered Zinc Finger Protein Transcription Factors as a Potential Therapy for Choroidal Neovascularization

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 maynot yield maximal therapeutic effects. Zinc-finger protein transcription factors (ZFP TF) can be engineered to regulate the expression of virtually any endogenous gene. We sought to test the effects in vitro and in vivo ZFPs designed to repress the transcription of VEGF-A and those 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-κB p65, respectively. The activities of these ZFPs were tested by transient transfection. ZFPs that significantly activate PEDF expression (>20 fold) or repress VEGF-A expression (∼10 fold) in transient transfection assays were identified. Adeno-associated virus (AAV) vectors that express a ZFP activator of PEDF and a ZFP repressor of VEGF-A were produced, and their in vivo efficacies were tested by intraocular injection in a mouse model of laser-induced CNV. AAV-PEDF activator and AAV-VEGF repressor regulated PEDF and VEGF-A expression and reduced the area of CNV at Bruch's membrane rupture sites by approximately 50% and 32%, respectively.These results demonstrate that 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, they provide proof-of-concept for using engineered ZFP TFs as a therapeutic approach for ocular diseases. Future studies will explore combined repression of VEGF and activation of PEDF. Because PEDF can antagonize the activities of a variety of growth factors by inducing apoptosis of proliferating endothelial cells, simultaneously activating PEDF and repressing VEGF-A could achieve more potent inhibition of angiogenesis than methods targeting VEGF alone. Moreover, because highly efficient delivery to the eye and sustained ZFP expression can be achieved using AAV vector, long-term therapeutic benefit is possible with a single administration.