An Engineered Zinc Finger Protein Transcription Factor Promoting Expression of Pigment Epithelium-Derived Factor Yields Long-Term Inhibition of Choroidal Neovascularization

Abstract

Purpose:: VEGF antagonists have shown success in treating neovascular AMD, however, repeat administrations are required to maintain efficacy. In addition, targeting VEGF alone may not yield maximal therapeutic effects, as other angiogenic factors also contribute to neovascularization. Engineered zinc-finger protein transcription factors (ZFP TF) can be designed to regulate the expression of essentially any endogenous gene. We sought to use engineered ZFP TFs to activate the transcription of the Pigment Epithelium-Derived Factor (Pedf) gene, which encodes a potent natural anti-angiogenic factor, and investigated whether long-term inhibition of choroidal neovascularization (CNV) can be achieved when the ZFP activator of PEDF was delivered by an Adeno-associated viral (AAV) vector.

Methods:: ZFP DNA binding domains (DBDs) were assembled to target the promoter of Pedf. The DBDs were linked to the activation domain of the NF-ΚB p65 subunit. The activities of these ZFPs were tested by transient transfection, followed by analysis for both mRNA and protein levels of PEDF. The effect of subretinal injection of an AAV vectors expressing the ZFP activator was tested in a mouse model of laser-induced CNV.

Results:: An engineered ZFP TF that activated PEDF expression (>20 fold) in transient transfection assays was identified. When delivered subretinally to mice using an AAV vector, this ZFP TF significantly activated transcription of PEDF (2-fold increase in Pedf mRNA, p=0.01) within posterior eye cups; it also reduced the size of laser-induced CNV by approximately 50% (p=0.004). Importantly, significant inhibition of CNV was maintained at 3 months and 6 months after a single subretinal injection.

Conclusions:: We have identified an engineered ZFP TF that successfully up-regulates the expression of PEDF in vivo and leads to the significant and long-term (6 months) suppression of laser-induced CNV in a mouse model. The level of PEDF produced from its endogenous gene is predicted to be more "physiological" than that produced from cDNA and its sustained up-regulation therefore may be better tolerated. As AAV vectors have been reported to maintain expression in the eye for greater than one year, these data suggest that potential therapies based on a ZFP activator of PEDF may be efficacious in combating CNV without repeat administrations.

Keywords: gene transfer/gene therapy • choroid: neovascularization