467. Cationic Liposome-Based Delivery of DNA Modulating Enzymes to Specifically Regulate Gene Expression in Carcinoma Derived Cell Lines

Abstract

Gene therapy so far has not fulfilled its promise, mainly because efficiency and specificity of both the gene transferred and the transferring device are not sufficient enough. A new and potentially powerful way to treat diseases is to interfere with the endogenous gene expression of the gene of interest using DNA modifying enzymes, such as restriction enzymes and DNA-methyltransferases. These enzymes are commonly used in a wide range of genomic analysis, and by altering their specificity by coupling of gene-specific oligonucleotides or peptide-nucleic acids their specificity can be programmed at will. The delivery of these enzymes to the cell, however, remains a problem. Using the cationic liposomal device SAINT, we succeeded in the delivery of a wide range of proteins including the restriction enzyme PvuII and the methyltransferase M.SssI, which displayed functionality upon delivery, e.g. DNA fragmentation and silencing of transcription. We focused on the use of the methyltransferase enzyme M.SssI to silence the transcription of genes expressed by the human EGP-2 promoter. The epithelial glycoprotein 2 (EGP-2), also known as Ep-CAM or the pancarcinoma associated protein 17-1A, is expressed at high levels on the surface of most carcinomas. Despite the fact that EGP-2 is also expressed on normal simple epithelial tissue, EGP-2 is regarded as an attractive target for anti-cancer treatment strategies. We used this promoter to drive expression in an EGP-2 transgenic mouse model (Cancer Research, 2001) and to drive suicide genes to induce carcinoma specific cell death (submitted). Here we show that methylation is one of the mechanisms involved in the regulation of this carcinoma-specific promoter and that we can silence expression of genes driven by this promoter upon profection of the M.SssI enzyme. This is the first step in proofing that by targeting DNA manipulating enzymes expression of endogenous genes involved in diseases can be altered.