Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector

Abstract

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors.

Gene expression in intrahepatic tumors through DNA recombination by a replication-activated adenovirus vector

“…The modified vector used in this study contains the IR sequence that mediates predictable viral DNA rearrangement that leads to selective transgene expression in tumors depending on DNA replication. 13 Our study suggests that the E1-deleted adenovirus containing the IR sequence is superior to Ad.E1-deleted adenovirus in tumor-selective gene expression.…”

“…Recent studies suggest that DNA replication for E1-deleted adenovirus is supported by many human cancer cells, but is deficient in primary normal cells including fibroblast and liver. 13,19 Moreover, E1-deleted adenovirus replicates selectively in tumor cells though at low efficiency. The modified vector used in this study contains the IR sequence that mediates predictable viral DNA rearrangement that leads to selective transgene expression in tumors depending on DNA replication.…”

“…Notably, however, both vectors mediated viral DNA replication in rat hepatocyte WB cells, although viral DNA replication was absent in human fibroblasts, human mammary epithelium, normal human hepatocyte, and primary mouse hepatocytes (data not shown), which is consistent with previous studies. 13 The WB cell is an immortalized rat liver hepatocyte cell line, a normal diploid epithelial cell with doubling time equal to that of the cancer cells used in this study. Although it lacks the transformed phenotypes represented in most cancer cells, 20 it is possible that dysregulated cell growth may provide extra factors to activate viral DNA replication.…”

“…Ad.IR-BG vector containing the b-galactosidase gene under the control of RSV promoter was constructed as described previously. 13 Ad.Luc vector with CMV-Luciferase gene was originally constructed through a homologous recombination in bacteria using the vectors pAdEasy-1 and pAdTrack-CMV. Vectors were propagated by the Vector Core Facility of the University of Michigan (Ann Arbor, MI) according to the protocol described previously.…”

“…The systemic administration of the vector in a hepatic tumor model resulted in tumor-specific marker gene (b-gal) expression and, more importantly, prodrug conversion (9-aminocamptothecin glucuronide to 9-aminocamptothecin and 5FC to 5FU) when a secreted form of b-glucuronidase and a cytosine deaminase/uracil phosphoribosyltransferase were used as therapeutic genes. 13,14 In this study, we further investigate Ad.IR-BGmediated tumor-specific gene expression in liver cancer models for both primary hepatocellular carcinoma and colon carcinoma metastases by assessing the specificity and activation mechanisms of the replication-activated adenovirus-induced gene expression in comparison to the standard E1-deleted adenovirus vector.…”