Study On Microrna-122 Regulated Suicide Gene Therapy For Liver Cancer

Suicide gene therapy for hepatocellular carcinoma (HCC) using the adenovirus (Ad) vectors expressing herpes simplex virus thymidine kinase (TK) gene and ganciclovir (GCV) has shown promise in preclinical and clinical studies. However, this strategy was hampered by hepatotoxicity of Ad vectors and severe hepatic damage resulting from TK/GCV killing normal hepatocytes around the tumor. microRNA-122 (miR-122) is an abundant, liver-specific microRNA (miRNA) and found down-regulated or no expression in HCC. Using endogenous miR-122 to regulate TK expression may avoid killing the liver cells surrounding tumors by GCV treatment. Adeno-associated virus 8 (AAV8) is a new serotype and allows 100% transduction of hepatocytes and long-term gene expression without liver toxicity, but there is few data about the ability for recombinant AAV8 (rAAV8) vectors to transduce hepatocellular carcinoma in vivo.One aim of this research was to evaluate the regulation on transgene expression mediated by miR-122 and its ability of restricting the expression of therapeutic genes specifically in HCC rather than in normal liver cells to improve efficiency and safety of liver cancer suicide gene therapy; another aim was to investigate the ability for rAAV8 vectors to transduce HCC in vivo and the feasibility of applying to liver cancer gene therapy. First, sensor plasmids for detecting miRNA activities were constructed by adding miRNA target sequences to the 3'-untranslated region (3'UTR) of Gaussia princeps luciferase (Gluc) expression cassette. Sensor plasmids were delivered to mice by hydrodynamic tail vein (HTV) injection and the abilities for miR-122 and other miRNAs to regulate transgene expression in the liver were investigated. Then, the miR-122 regulated TK expressing plasmid was constructed by inserting miR-122 target sequences into the 3'UTR of TK gene and delivered to mice by HTV injection. The hepatotoxicity of TK/GCV system was evaluated by serum ALT, body weight and histopathological examination of liver. Then, Hepal-6 cells, a HCC cell line with down-regulated miR-122 expression were inoculated in liver to establish murine liver cancer models. rAAV8 or Ad vectors were given to tumor-bearing mice by intravenous or intratumoral injection and their transduction efficiencies to the liver and HCC were evaluated. Then, tumor-bearing mice were treated with intratumoral injection of different doses of Ad vectors expressing miR-122 regulated TK gene followed by treatment with GCV, and antitumor effects and liver toxicity were estimated.Our study showed delivering Gluc-based miRNA sensors to mice by HTV injection and then assaying Gluc activity in blood offered a sensitive and quantitative method for ex vivo monitoring of in vivo microRNAs in mouse liver. Results suggested CAG promoter-driven miRNA sensor was suitable for the long-term monitoring of endogenous miRNA activities such as miR-122, miR142 and miR-34a, as well as exogenously delivered miRNA. The Gluc expression in liver was inhibited significantly by insertion of the miR-122 target sequences (502.9-766.4-fold) rather than insertion of the miR-142 or miR-34a target sequences (6.8-10.8-fold). Moreover, the insertion of the miR-122 target sequences in the TK expression cassette avoided hepatotoxicity of GCV treatment.In our study, rAAV8 vectors demonstrated high efficiency of transgene expression in liver. However, rAAV8 vectors showed inefficiency of gene transfer to inoculated Hepal-6 HCC tissues either by intravenous or by intratumoral injection. Relative high transgene expression in HCC cells was acquired after intratumoral injection with Ad vectors, but there was higher tansgene expression in liver than tumor.Our study showed intratumoral injection with Ad vectors containing the miR-122 target sequences led to much higher tansgene expression in the tumor than the liver. Tumor-bearing animals received different doses of Ad vectors expressing TK by intratumoral injection and then treated with GCV. Both Ad vector bearing TK gene alone (Ad-TK) or with miR-122 target sequences (Ad-TK-122T) acquired viral dose-dependent antitumor effects. The liver toxicity was significantly elevated for Ad-TK, but no significant liver toxicity had been found for Ad-TK-122T, compared with the control vector Ad-Fluc (Fluc, Firefly luciferase). Intratumoral injection of Ad-TK-122T at dose of 1×1010 virus particles per mouse acquired significant antitumor effect without evident hepatotoxicity, but injection of Ad-TK at the same dose resulted in heavy hepatotoxicity and even death of mice.The research demonstrates that the strong regulation ability of miR-122 for transgene expression in the liver and the feasibility of utilizing miR-122 as a negative regulation element to prevent hepatotoxicity in liver-directed suicide gene therapy. miR-122 regulation can be used in intratumoral injection of Ad vector mediated TK/GCV system to acquire higher tolerable dose of vectors and enhance the safety of liver cancer suicide gene therapy. The research also demonstrates that rAAV8 is a robust vector for gene transfer to the liver. However, rAAV8 vectors show ineffective transduction for the experimentally inoculated HCC such as Hepa1-6 cells.