1000. Down-Regulating the Epithelial Glycoprotein-2 Promoter Utilizing Engineered Zinc Finger Protein Transcription Factors

Abstract

Top of pageAbstract Engineered zinc finger protein transcription factors (ZFP-TFs) are emerging as novel powerful tools to regulate endogenous gene expression. Zinc finger proteins consist of modular building blocks, with each domain recognizing 3-4 base pairs. Stitching these modular domains together results in the recognition of an extended DNA sequence, increasing the specificity of the target sequence. Specific amino acid positions within each domain recognize the DNA. Altering the amino acid residues at these positions therefore results in the recognition of different base pairs. A wide variety of effector domains can be attached to the DNA-binding zinc finger protein, for example to activate or inhibit gene transcription. In this study we utilized engineered ZFP-TFs to specifically inhibit the expression of the epithelial glycoprotein-2 (EGP-2). EGP-2 is expressed in the most prevalent types of carcinomas, including breast, ovarian, and colon cancer. Recently, others have demonstrated that down-regulation of EGP-2 using siRNA or ODNs resulted in a decrease in the migration, invasion, and proliferation potential of tumor cells. Therefore, inhibiting the expression of EGP-2 can provide a novel anti-cancer treatment for a broad range of tumor types. Down-regulating EGP-2 expression by targeting the mRNA requires the targeting of many molecules. Direct down-regulation of the EGP-2 expression at the DNA level using engineered ZFP-TFs can therefore be more effective. We engineered a zinc finger protein that specifically bound to a 9 base-pair target sequence in the EGP-2 promoter sequence. After attaching a gene-expression inhibition domain to the DNA-binding ZFP, co-transfection of EGP-2-positive cells with a plasmid expressing the engineered ZFP-TF and a plasmid containing the luciferase gene under the control of the EGP-2 promoter resulted in 60% inhibition of luciferase expression. To further increase the specificity for binding to the EGP-2 promoter, we engineered three different ZFP-TFs targeting 18 base pair sequences within the EGP-2 promoter. Co-transfection experiments demonstrated that the engineered ZFP-TFs caused up to 80% down-regulation of the EGP-2 promoter while no down-regulation of a non-target promoter was observed. Combining inhibition at the DNA level with inhibition at the RNA level using siRNA might even further enhance the down-regulation of EGP-2 promoter activity. In this study we have demonstrated that ZFP-TFs can be engineered to down-regulate the EGP-2 promoter, generating a novel therapeutic for the treatment of a broad variety of tumor types.