659. Development of Designed Zinc Finger Protein Transcriptional Regulators for Use in Multi-Factor Therapeutic Angiogenesis

Abstract

Gene therapy-mediated revascularization strategies offer considerable promise in the treatment of cardiovascular disease. To date, over two dozen studies have reported that gene-based delivery of angiogenic factors can enhance blood flow and/or tissue function in preclinical models of limb and heart ischemia. However, although several programs have advanced into clinical trials, it has been noted that natural angiogenesis proceeds via the coordinate action of multiple factors; therefore strategies utilizing two or more angiogenic factor genes may realize enhanced therapeutic benefit1. In consideration of this possibility we are developing a panel of designed zinc finger protein (ZFP) transcriptional activators for regulation of multiple different angiogenic factor genes. In previous studies we had described the development of pro-angiogenic ZFPs that function via activation of VEGF-A2,3,4. Here we have extended this approach to develop regulators of three additional factors: Ang-1, FGF-5 and VEGF-C. These studies involved: (i) DNase I mapping of accessible locus regions; (ii) design and assembly of ZFPs targeting sites within accessible and/or functional promoter regions; and (iii) ZFP screening for activation of gene expression. By this approach, we have identified ZFPs that activate Ang-1, FGF-5 and VEGF-C by up to 14-fold in cells from both human and mouse. Furthermore, by targeting a sequence common to the promoters of VEGF-A and VEGF-C we have produced a ZFP that coordinately activates both of these genes. The use of designed ZFPs to regulate a patient's own endogenous genetic loci offers a number of advantages over cDNA-based gene therapy approaches, including the capacity to upregulate all splice variants of a therapeutic gene2,3,4, the option for ligand-regulatable protein expression using a single introduced transgene5, and transgene size economy (a critical feature for multi-factor therapies with vectors of limited insert capacity). We anticipate that these ZFPs will provide powerful reagents for the development of multi-factor strategies for therapeutic angiogenesis.