452. Adenoviral Gene Therapy Using CRAd Targeted to Cells with Hyperactive Wnt/β-Catenin Signaling

Abstract

During cell division, the genomic methylation pattern is faithfully maintained by DNA methyltransferase-1 (DNMT-1), which is responsible for transferring a methyl group to 5-position of the cytosine ring. Methylation occurs mostly during DNA replication and the expression of DNMT-1 is tightly regulated with the cell cycle. Previously, it has been demonstrated that although both cycling and growth-arrested cells transcribe DNMT-1 mRNA at a similar rate, the mRNA is detectable only in the cycling cells. The 3′ untranslated region (3′ UTR) of DNMT-1 mRNA contains copies of adelylated-and uridylate-rich elements (AREs), which can regulate stability of its own message as well as a heterologus β-globin mRNA in a growth-dependent manner. Because cancer cells proliferate much faster than most normal adult somatic cells, we hypothesized that cancer cell selective mRNA-stabilization and transgene expression could be achieved by using the DNMT-1 3 ′UTR ligated to a therapeutic gene or viral replicative gene. To test the hypothesis, the human DNMT-1 3′UTR (5090–5408) was amplified by RT-PCR from RNA prepared from the human melanoma cancer Mel-624 cell line and inserted into the luciferase reporter plasmid pGL-3 promoter. The effect of the DNMT-1 3′ UTR insertion was evaluated by comparing the relative luciferase activity in tumor (prostate cancer:PC-3,LnCap; glioma: U118, U87, U251; melanoma: Mel624 and Mel888; colorectal cancer: HCT116) and three normal cell lines (BEAS-2B, HUVAC, HTB-125). In the presence of DNMT1 3′ UTR, luciferase activity was 5-7 fold higher in all the tumor cell lines compared to the normal cells. Therefor, we generated a conditionally replicating adenovirus where the viral E1A gene, which is essential for viral replication, is ligated to the 318bp 3′-untranslated region of the DNMT-1 mRNA (AdDNMT1). In vitro studies with AdDNMT1 virus on a variety of normal and tumor cell lines have shown that productive infection and viral replication is restricted to rapid growing tumor cells. Early (E1A), late (fiber) viral protein expression and lytic replication was not observed in normal primary cell lines tested. These result supports the possibility of targeting tumor cells by using DNMT-1 3′UTR in the context of abnormal growth.