Regulation of adenovirus replication by miR-199 confers a selective oncolytic activity in hepatocellular carcinoma

Oncolytic virotherapy represents a growing field of experimental cancer therapy. For safe and effective virotherapy, restricted tissue expression and replication of the virus is desirable. Various methods have been developed to achieve such restricted expression. They included the engineering of viral genomes through the insertion of tissue-specific promoters or genes encoding for tissue specific binding proteins. Here, we employed a new approach based on the use of microRNAs (miRNAs) to achieve tumor-specific viral expression and replication. miRNAs are approximately 22-nucleotide (nt)- long non-coding RNAs that are able to bind the 3’ untranslated regions (UTRs) of homologous target mRNAs and causing either their degradation or translation inhibition. Since miRNA are differentially expressed in cancer versus normal cells, it is theoretically possible to make virus expression restricted to cancer cells in a miRNA-dependent manner. Several studies have shown that miR-199 is significantly down-regulated in primary hepatocellular carcinoma (HCC) tissue and HCC cell lines. With this notion in mind, we developed a conditionally replication-competent oncolytic adenovirus, Ad-199T, by introducing four copies of miR-199 target sites within the 3′ UTR of the E1A gene, which is essential for adenovirus replication. In vitro studies of the properties of Ad-199T virus revealed that E1A expression was indeed tightly regulated both at RNA and protein levels depending upon the expression of miR-199. Consequently, Ad-199T could replicate in the HCC derived cells HepG2, negative for miR-199 expression, while its replication was strictly controlled in HepG2-199 cells, which were engineered to express high level of miR-199. A replication-competent miRNA independent Ad-Control was also generated,. Thus, these in vitro studies proved that cytotoxicity of Ad-199T was effective in HCC derived cells, which lacks expression of miR-199, and could be successfully controlled in cells that express miR-199 at high level. To assess in vivo properties of Ad-199T, we tested an orthotopic tumor model. HepG2 cells were implanted in the liver of newborn B6D2 mice. The cells could survive at least one week in this environment, enough for testing in vivo properties of Ad-199T. These studies revealed that intrahepatic delivery of Ad-199T led to virus replication in HepG2 derived xenograft tumors and a faster removal of cancer cells. Conversely, Ad-199T replication was not detected in normal, miR-199 positive, liver parenchyma. These results demonstrate that Ad-199T is a conditionally replicative adenovirus (CRAd) miR-199 dependent, with antitumor activity in vivo. This system allows replication of the oncolytic virus in HCC cells and, at the same time, tightly control replication in normal liver tissues, thus avoiding or reducing hepatotoxicity.