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Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA

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Abstract

Background & aims:

Hepatitis B virus (HBV) causes acute and chronic infections that may result in severe liver diseases. Animal models to study new treatment options in vivo have several drawbacks. Therefore, we were interested to establish a new small animal model in which HBV replication and especially new treatment options can be studied easily.

Methods:

Naked DNA of an HBV replication competent vector was transferred via tail vein into NMRI mice. HBV replication was studied in serum and liver of the animals. HBV replication was modulated by treatment through siRNA and nucleoside analogues.

Results:

Tail vein transfer of a HBV replication competent construct resulted in expression of HBV-specific transcripts in the liver, and up to 10% of hepatocytes became HBc- and HBsAg-positive. HBeAg, HBsAg, and viral DNA could be detected in the serum of the animals, followed by the induction of HBV-specific cellular immune responses. Nucleoside treatment of the mice resulted in reduced polymerase activity in the liver. Additionally, siRNA transfer in the animals led to a significant reduction of HBsAg and/or eventually HBeAg expression, which was dependent on the localization of the complementary sequence in the HBV genome.

Conclusions:

We have established a mouse model to study HBV replication and to investigate new and existing treatment approaches in vivo. Interestingly, siRNA seems a promising innovative treatment option to inhibit specifically HBV replication in vivo.

Section snippets

DNA constructs and siRNA

An HBV replication competent vector (pHBV 1.5) was used for all experiments as described earlier.1, 12 Twenty-one nucleotide siRNA was designed and purchased from Dharmacon Research Inc. in the ready-to-use option. Two different locations in the HBV genome were selected, located either in the coding region of the S-gene or the core (C) gene. The nucleotide mRNA target sequence in the S-gene is the following: 5′AAG CCU UAG AGU CUC CUG AGC-3′ (207-227) and in the core gene: 5′A AUU UGU UCA GUC

High-volume injection of a HBV replication competent vector results in serum and liver expression of HBV proteins

Earlier experiments indicated that high-volume injection of plasmid DNA results in liver transgene expression.16 We thus performed high-volume injection via tail vein of 20 μg of an HBV replication competent vector (pHBV 1.5) in mice and studied HBsAg and HBeAg serum expression. At least 3 animals were treated in parallel per time point indicated. Three and 4 days after pHBV 1.5 injection, maximal serum levels for HBsAg and HBeAg were found, respectively. At later time points, serum expression

Discussion

In the present study, we have developed a novel mouse model to study HBV replication in vivo. After transfer of an HBV replication competent vectors through high-volume tail injection, more than 10% of mouse hepatocytes became positive for HBV protein expression. Interestingly, further analysis indicated that viral capsids were assembled and began replicating the viral genome and were secreted into the serum of the animals as virions; thus, most components of the HBV life cycle can now be

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      Previous studies have shown that siRNA (21–25 nucleotides) directed against the core and/or HBx transcripts can inhibit HBV gene expression in HBV-infected cells and significantly decrease the levels of HBsAg, HBeAg, HBV RNA and HBV DNA [77]. Other studies also have highlighted potential synergism between siRNA and other anti-HBV treatment strategies, such as NAs [78]. Another recent and interesting study, by Li et al. [79], showed that a siRNA targeting the NLS of the HBcAg core protein can inhibit cccDNA recycling (intracellular amplification) [79].

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    Supported by a grant in the HepNet (to C.T. and M.P.M.) and the “fortüne-program” of the University of Tübingen, grant 1037-1-0 (to C.T.B.).

    1

    C. Klein and C.T. Bock contributed equally to this work.

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