Duckhyang Shin

Optimization of linear double-stranded RNA for the production of multiple siRNAs targeting hepatitis C virus

RNA interference (RNAi)-based gene silencing possesses great therapeutic potential for inhibiting replication of human viruses such as hepatitis C virus (HCV). However, one of the putative limitations for its use as a therapy is the rapid emergence of escape variants. These contain deletions or mutations within the viral genome sequences complementary to the small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) being used for treatment. As a potential solution to this problem, we constructed an expression system for duplex RNAs harboring two siRNA units using convergent H1 and U6 Pol III promoters. Here, the length and orientation of the transcript, tandem siRNA (tsiRNA), were optimized to be processed by the intracellular ribonuclease Dicer into functional siRNAs targeting different sequences. Assessment in transfected cells indicates that the length of the tsiRNA duplex (40-42 base pairs) is more critical for both siRNA-producing capacity and gene silencing activity than the orientation of each siRNA unit. In Huh7 cells replicating full-length HCV RNA, expression of length-optimized tsiRNA inhibited viral protein levels as efficiently as a single 21-nucleotide siRNA-expression construct, without affecting miRNA maturation or induction of an interferon response. We verified that the anti-viral activity of tsiRNA was achieved by precise cleavage of two target sites. A distinct advantage of this strategy is that each side of the optimized linear duplex RNA could enter into the Dicer-mediated processing machinery, thus likely providing more equal and efficient production of multiple siRNAs required for reducing the chance of viral escape.

Hepatic siRNA delivery using recombinant human apolipoprotein A-I in mice.

Apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein (HDL), plays a key role in reverse cholesterol transport from peripheral tissues to liver or steroidogenic organs. Class B, type 1 scavenger receptor (SR-BI) is abundantly expressed in these target tissues and recognizes apo A-I of HDL for selective cholesteryl ester uptake. Recently, we reported the liver-targeting potential of plasma-derived apo A-I and the efficient delivery of therapeutic small interfering RNAs (siRNA) assembled with cationic liposome and apo A-I. In this study, we expressed and purified recombinant human apo A-I (rhapo A-I), low endotoxin grade, from an Escherichia coli expression system. The liver-targeting property of rhapo A-I was compared to that of plasma-derived apo A-I. Using a hepatitis C virus mouse model, intravenous administration of virus-specific siRNA with liposome and rhapo A-I significantly inhibited viral protein expression, demonstrating great promise for its use in clinical applications.

Comparison of immunogenicity of cell-and egg-passaged viruses for manufacturing MDCK cell culture-based influenza vaccines.

While cell culture-based technology has been recently used for manufacturing influenza vaccines, currently available seed viruses are mostly egg-derived reassortants that are egg-adapted to achieve high virus growth in eggs. For use as viruses for cell culture-based influenza vaccine manufacturing, egg-adapted viral seeds may undergo several passages in manufacturing cell lines. However, the suitability of such cell-passaged viruses for vaccine production remains largely unelucidated. In this study, influenza viruses produced in suspension Madin-Darby canine kidney (MDCK) cell cultures were compared to those produced in embryonated hens eggs for manufacturing MDCK cell culture-based influenza vaccines through comparability studies of virus productivity and vaccine immunogenicity. The results indicate no change in the amino acid sequence of the main antigens, including hemagglutinin (HA) and neuraminidase (NA), of cell-passaged viruses after three passages in suspension MDCK cells. In lab-scale (3-L) single-use bioreactors, suspension MDCK culture supernatants inoculated with cell-passaged viruses were found to show higher virus productivity, suspension MDCK culture supernatants inoculated with egg-passaged viruses, in respect to the HA titers and HA contents determined by single radial immunodiffusion. Finally, comparable hemagglutination inhibition and influenza-specific IgG titers were determined in the mice immunized with cell culture-based vaccines produced with cell- or egg-passaged viruses. These results indicate that MDCK cell-passaged viruses from egg-adapted viruses, as well as egg-derived seed virus, are suitable for MDCK cell culture-based influenza vaccine production.

Membrane-based hybridization capture of intracellular peptide nucleic acid.

Peptide nucleic acid (PNA) is a chimeric oligonucleotide with nucleotide-derived bases and a peptide backbone. Compared with natural nucleotides, PNA has several advantages, including improved stability and high sequence discrimination during duplex formation. Despite its potential for therapeutic application, analysis technologies have not been generalized, mainly due to ambiguous physiochemical properties resembling those of nucleic acids as well as protein. Here we present a PNA detection method: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer to a Western blotting membrane and then hybridization with a radiolabeled oligonucleotide probe. This method is useful for evaluating the quality of synthetic PNA and determining its intracellular localization.

1003. Inhibition of HBV Replication In Vivo by HBx-Specific siRNA

Top of pageAbstract Hepatitis B virus (HBV) causes acute and chronic hepatitis that could result in severe liver diseases such as cirrhosis and hepatocellular carcinoma. Because of the restricted host range of HBV, it has been difficult to study HBV replication in vivo and to test in vivo efficacy of possible antiviral therapeutics. In this study, we reproduced a naturally occurring HBV infection in mice by simply injecting HBV genome and also studied the effects of an HBV-specific short interfering RNA (siRNA) targeted to the X gene using this mouse model system. The viral replication cycle of HBV was resulted from the hydrodynamic transfection with the fragmented form of infectious HBV genome in immunocompetent mice. HBV envelope (HBe), HBV surface (HBs) antigens and viral DNA could be detected in the serum of the animals, followed by the induction of HBV-specific immune responses which mimics acute infection of HBV in humans. Also, the immunocompromized mice injected with the infectious HBV genome supported the prolonged replication of HBV mimicking the chronic infection of HBV in humans. In both systems, injection of siRNA targeted to the X gene region, where four major HBV transcript overlap, led to the dramatic inhibition of HBV replication up to 99% in the treated mice demonstrating the potential utility of HBx-specific siRNAs as anti-viral inhibitors for the treatment of chronic viral hepatitis. In summary, these mouse model systems offered opportunities to test in vivo efficacy of HBx-specific siRNAs for control of HBV replication.