326. Zinc Finger Protein Targeted Epigenetic Gene Regulation: Toward Directed Long-Term Gene Control

Abstract

Zinc finger protein transcription factors (ZFP TFs) are capable of regulating the expression of any endogenous gene, potentially providing direct therapeutics for the treatment of disease. To extend this approach we have coupled the versatility and specificity of the ZFP DNA binding domain with non-classical effector domains implicated in long-term, epigenetic control of gene expression. The use of epigenetic mechanisms of gene regulation has multiple applications in ZFP TF-driven gene therapy, including: (i) long-term correction of the disease state (beyond the duration of expression of the ZFP TF), (ii) heritable, stable modification of gene expression in stem cells for cell-based therapies and regenerative medicine and (iii) the direct application to disease caused by the mis-regulation of epigenetic states such as cancer progression. Using a designed ZFP DNA binding domain that recognizes a defined site in the promoter of the VEGF-A gene (a gene often over-expressed in cancer) we have shown that targeting of the catalytic domain from human DNA methyltransferase 3A (DNMT3A) resulted in potent, gene-specific transcriptional repression. Crucially, this repression function was lost when an enzymatically inactive mutant of the DNMT3a domain was employed. To determine directly whether DNA methylation was concomitant with repression of the target gene, stable cell lines expressing the ZFP-DNMT3A or the catalytically null mutant version were analyzed for VEGF-A promoter specific DNA methylation within the vicinity of the ZFP binding site. Expression of the wild type ZFP-DNMT3A fusion resulted in both DNA methylation of the VEGF-A promoter, as detected by methylation-sensitive restriction enzyme analysis, and it's transcriptional repression. Significantly, expression of the mutant DNMT fusion did not influence the methylation status of the promoter, and no repression function was observed. Thus, an engineered ZFP is shown to be able to specifically address a catalytically active DNA methylation domain to a predetermined target gene, and result in both the methylation of the promoter and its transcriptional silencing. These data demonstrate that epigenetic modifications can be targeted using ZFP TFs, and raise the possibility of long term, and potentially heritable gene expression changes for therapeutic use.