[Anti-HBV effect of nucleotide analogues on mouse model of chronic HBV infection mediated by recombinant adeno-associated virus 8].

We evaluated the anti-HBV effects of nucleotide analogues, Entecavir (ETV) and Lamivudine (LAM) targeting mouse model of HBV persistent infection with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV). Ninety percent (27 of 30 mice) of rAAVS-treated mice were chosen as mouse model. Four groups were orally administrated with different doses of ETV (1 mg/(kgd) or 0.1 mg/(kgd)) and LAM (500 mg/(kgd) or 100 mg/(kgd)) once a day for 10 days. The other two groups were set as normal saline treated and untreated control. We detected the levels of HBV DNA, HBeAg and HBsAg in sera at different time. Results indicate that HBV DNA level decreased significantly (P < 0.05) in drug-treated groups compared with normal saline group after drug administration. Fifteen days after the drug withdrawal, HBV DNA level rebounded back obviously (P < 0.05) in groups with low doses of ETV and LAM. However, there was no apparent change of HBeAg and HBsAg in the whole process among all groups. These results showed that our model could reflect the anti-viral effect of nucleotide analogues. This model can be a useful and convenient tool for anti-HBV drug discovery.

Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model.

OBJECTIVE: Intratumoral administration of adenoviral vector encoding herpes simplex virus (HSV) thymidine kinase (TK) gene (Ad-TK) followed by systemic ganciclovir (GCV) is an effective approach in treating experimental hepatocellular carcinoma (HCC). However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy. miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. METHODS: By inserting miR-122 target sequences (miR-122T) in the 3' untranslated region (UTR) of TK gene, we constructed adenovirus (Ad) vectors expressing miR-122-regulated TK (Ad-TK-122T) and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. RESULTS: Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. CONCLUSIONS: miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.

[Application of secretary luciferase labeled orthotopic transplant model of hepatocellular carcinoma to evaluate tumor response to interferon-beta gene therapy].

To establish an orthotopic transplant mouse model of hepatocellular carcinoma (HCC) labeled with secretary luciferase and to study its response to anti-tumor treatment with interferon-beta gene therapy. We labeled the murine hepatoma Hepal-6 cells with secretary Gaussia princeps luciferase (Gluc), and then injected Gluc labeled Hepal-6 cells intrasplenically in C57BL/6 mice. We monitored blood Glue to evaluate the tumor development and anti-tumor effects of hydrodynamic injection with interferon-beta expressing plasmid. We successfully established the orthotopic mouse model of HCC by intrasplenic injection of Glue labeled Hepal-6 cells. The Glue blood assay could reflect the amount of cancer cells in vivo, tumor progression, as well as anti-tumor effect of interferon-beta gene therapy. In conclusion, Gluc labeled orthotopic transplant mouse model of HCC can ex vivo real-time monitor the tumor development and tumor response to treatments.

Long-term ex vivo monitoring of in vivo microRNA activity in liver using a secreted luciferase sensor.

Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology. However, in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs. Sensors for miRNA were developed by adding miRNA target sequences to the 3'-untranslated region of Gaussia princeps luciferase (Gluc) mRNA. These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood. Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells, but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing. Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities. We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122, miR142 and miR-34a activities, as well as for exogenous miR-34a activity. Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.