Using RNAi to Treat Other Viruses

Although many previous studies on RNAi-mediated inhibition have focused on HIV-1, there is a growing body of data addressing the inhibition of other animal and human viruses. These include RNA viruses such as hepatitis C virus (HCV), poliovirus, Semliki Forest virus (SFV), influenza virus A, rhesus rotavirus (RRV), and Rous sarcoma virus (RSV), and DNA viruses such as human papillomavirus type 16 (HPV-16) and hepatitis B virus (HBV). In most of these studies, the RNAi machinery was directly targeted towards the viral RNA using synthetic siRNAs.

Hepatitis induced by HBV or HCV is a major health problem. At present, hundreds of millions of individuals are infected worldwide. Although there is an effective vaccine against HBV, it is only useful for the prevention of viral infection. There is no vaccine for HCV. Hepatitis caused by these two viruses has therefore been an important target for potential RNAi therapy.

The first demonstration of RNAi efficacy against a virus in vivo involved the hydrodynamic co-delivery of an HBV replicon and an expression unit encoding an anti-HBV shRNA in mice. HBV is a member of the family Hep-adnaviridae and has a 3.2-kb circular dsDNA genome. During infection, four RNAs are transcribed, which encode the coat protein (CP), polymerase (P), surface antigen (S), and transactivator of transcription (X). HBV production in Huh-7 cells was shown to be reduced by up to 20-fold through the transfection of a vector-expressing shRNA against the X mRNA (Shlomai and Shaul 2003).

Inhibition of HBV in the liver of mice was achieved through the co-trans-fection of HBV DNA and shRNA-expressing plasmids (McCaffrey et al. 2003), which resulted in a six fold decrease in the amount of secreted HBV surface antigen in the serum. This small-animal model of human infectious disease shows that it is possible to use RNAi as a potent antiviral therapy in mammals.

HCV is a major cause of chronic liver disease, which can lead to liver cirrhosis and hepatocellular carcinoma (Reed and Rice 2000). The HCV genome is a positive-strand RNA molecule with a single open reading frame encoding a polyprotein that is processed post-translationally to produce at least 10 proteins. HCV is a member of the family Flaviviridae and has a (+) single-stranded (ss)RNA genome.

Subgenomic and full-length HCV replicons that replicate and express HCV proteins in stably transfected human hepatoma-derived Huh-7 cells have been used to study the effects of various antiviral drugs (Lohmann, et al. 1999; Pietschmann, et al. 2001; Ikeda et al. 2002). Several groups have now tested the efficacy of the siRNA-mediated inhibition of replicon function using these systems (Kapadia et al. 2003; Randall et al. 2003; Wilson et al. 2003). These replicons support HCV RNA transcription and protein synthesis, but do not produce infectious viruses.

siRNAs targeted against sequences in the viral non-structural proteins NS3 and NS5B have been shown to cause profound (up to 100-fold) inhibition of HCV replicon function in cell cultures (Kapadia et al. 2003; Randall et al. 2003; Seo et al. 2003; Wilson et al. 2003). Furthermore, the internal ribosomal-entry site (IRES) in the well-conserved 5' UTR of the HCV RNA has also been a good target. Both siRNAs and shRNAs have been reported to inhibit HCV replicon function in cells (Seo et al. 2003; Wilson et al. 2003; Yokota et al. 2003; Hamazaki et al. 2005).

In another in vivo study, siRNAs were used to treat fulminant hepatitis induced by an agonistic Fas-specific antibody in mice (Song et al. 2003b). Fasmediated apoptosis of hepatocytes can be triggered by HBV and HCV infection. Infusing siRNAs targeting Fas mRNAs into the tails of the mice blocked this self-destructive inflammatory response of the liver. These findings indicate that major hurdles remain before this therapy can be applied to humans.

As with HIV therapeutics, delivery of the siRNAs or shRNA vectors is the main challenge for the successful treatment of HCV.

Generally, human influenzal lesions are local infections that remain in the upper portion of the respiratory tract and do not proceed to pneumonia. Nevertheless, in high-risk patients, cases of influenzal pneumonia have been reported in which expansion of the virally infectious focus is observed in a pulmonary lesion. Furthermore, such cases are often accompanied by a secondary bacterial pneumonia. The influenza virus belongs to the family Orthomyxoviridae and has a (-) ssRNA genome. Its genome is composed of eight separate segments. The proteins encoded by the eight segmented genes include HA and NA, as well as the Ml and M2 membrane proteins, which are located on the surface of the envelope. Furthermore, a nucleoprotein complex (RNP) is located at the center of the virus and is composed of the gene RNA, three RNA polymerase subunits (PB1, PB2, and PA) and a nucleoprotein (NP). A non-structural protein (NS) is synthesized from the eighth segmented gene. Amantadine and rimantadine are known antiviral agents for the influenza A virus; however, neither drug can cope with mutants and both have strong side effects (Atmar et al. 1990; Wang et al. 1993). Treatment by an inactivated vaccine has also been attempted; however, the vaccine cannot sustain antibody productivity for a long period and, thus, cannot completely prevent the spread of infection (Hirota et al. 1996).

Ge et al. (2003) showed that siRNAs targeting conserved regions (PA and NP) of the influenza genome inhibited virus production in cell culture and in embryonated chicken eggs. Furthermore, RNAi mediated by PA-, NP-, and PB1-specific siRNAs or shRNAs expressed from DNA vectors prevented and treated influenza A virus infection in mice (Ge et al. 2004). In addition, Tompkins et al. (2004) showed that the administration of influenza-specific siRNAs decreased lung virus titers and protected mice from lethal challenge by a variety of influenza A viruses, including the potential pandemic subtypes H5 and H7. This specific inhibition of influenza virus replication requires homology between the siRNAs and gene targets, and is not the result of IFN induction by dsRNAs. For therapeutic applications against the influenza A virus, the siRNAs can be administered via intranasal or pulmonary routes. RNAi is more potent than the antisense approach (Mizuta et al. 1999), and the evaluation of this technology as a treatment for the influenza virus through human clinical trials is expected to take place in the near future.

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