Use Of MiR-199to Control Oncolytic Adenovirus To Conditionally Replicate In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is a widespread cancer in our country,causingmillions deaths every year. The gene-virology therapy is one of promising strategy totreat for HCC. To this end, various viral vectors have been engineered to carryingtransgenes. Among them, oncolytic adenovirus has unique advantages. It can directlydestroy cancer cells, and mediate effective transgenic expression in the process of viralproduction as well. To date, a series of approaches have been developed to ensureoncolytic adenovirus to conditionally kill cancer cells. However, systemic administrationof adenoviruses can cause significant infection of hepatocytes and may lead to livertoxicity. Therefore, it remains a challenge to selectively target the cancer cells andleaving the normal cells, especial normal liver cells unharmed in the therapeutic use ofoncolytic adenovirus for HCC.MicroRNAs (miRNAs) are versatile, noncoding RNAs, which exertposttranscriptional regulation by targeting mRNAs through specific recognition of shortsequences, leading to decreased protein production. It has been well documented thatmiRNA is expressed in tissue-and differentiation state-specific patterns, and is greatlyassociated with cancer development. Because of its tissue-specific expression profile andshort targeting site, miRNA provides considerable flexibility in the design ofconditionally replicative adenoviruses. By introduction of miRNA target sequence intothe3'-untranslated region (UTR) of a key gene that has an essential role in viral growthand replication (e.g. E1A), the virus replication can be controlled by the tissue-specificendogenous miRNA, thus eliminating the unwanted pathology of wild-type adenovirus.The miR-199a/b-3p is highly expressed in hepatocytes and several types of tissues.Data have shown that miR-199a/b-3p is downregulated in almost all of primary HCCtissues and HCC cell lines, making it an ideal candidate for adenoviral regulation. In thisstudy, we have engineered an oncolytic adenovirus, SG7011199T, by introducing eightcopies of miR-199a/b-3p target sites into the3' untranslated region of E1A, a key geneassociated with adenoviral replication, to control the expression of the E1A gene,subsequently the replication and cytotoxicity of the virus. The results showed that theE1A expression (both RNA and protein levels) of the SG7011199Twas tightly regulatedaccording to the endogenous expression level of the miR-199a/b-3p gene. As contrastedwith wild-type adenovirus, the replication of SG7011199Twas distinctly inhibited in normal liver cells lines (i.e. L02) and ovarian cancer cell line (i.e. SK-OV3) expressedhigh level of miR-199a/b-3p, whereas was almost not disturbed in HCC cells (i.e. Hep3Band Huh7) with low level of miR-199a/b-3p. Consequently, the cytotoxicity ofSG7011199Tto normal liver cells was successfully decreased while its oncolytic activityto HCC cells was maintained in vitro and in mice with xenograft HCC tumor. Theseresults suggested that SG7011199Tmay be a promising anticancer agent or vector tomediate the expression of therapeutic gene, broadly applicable in the treatment for HCCand other cancers where the miR-199a/b-3p gene is downregulated.