**4.2 Antiviral ribozymes**

410 Myocarditis

component of the RISC, is located in the p-bodies (Sen & Blau, 2005). Its cytoplasm localization is critically important for anti-coxsackievirus action as this virus is replicated only in cytoplasm. In addition, in the case of RNAi, an endogenous cellular pathway is followed, which could explain the high efficiency with which siRNAs are able to reach 1000 times higher than the ASON in cleavage of the same target molecule (Bertrand et al., 2002; Grunweller et al., 2003). However, the limitations for RNAi are present (Hemida et al., 2010); similar to the ribozymes, the selection of the suitable target for binding is restricted, particularly for miRNA, as the search for effective targeting sites are often limited in the

CVB3, one of the most frequently used model systems for study of viral replication and pathogenesis, is also widely employed for evaluation of NA-based antiviral agents. The early investigations mainly focused on the application of the second and third generations of the ASONs. McManus and coworkers are one of the pioneer groups to study the potential possibility to inhibit CVB3 replication using ASONs. Their earliest work using regular ASONs to target the different sites of 5'UTR of CVB3 genome successfully mapped the IRES by *in vitro* translation inhibition assay (Yang et al., 1997). This study provided useful information for the design of ASON for inhibiting CVB3 replication *in vitro* and in mouse models. Later, they used PS-ASONs targeting the 5' and 3'UTRs as well as the start codon region and found that the oligomers targeting the 5' and 3' proximate ends of the CVB3 genome are the most effective candidates to inhibit viral replication in HeLa cells. Each of these two ASONs resulted in ~80% reduction of viral particle production, which is followed by the candidates targeting the IRES and the initiation codon region (A. Wang et al., 2001). The importance of these sites for ASON binding was further confirmed by *in vivo* evaluation using a murine myocarditis model, although the antiviral efficiency is not as high as that

To improve the stability of the oligomers, our group designed eight phosphorodiamidate morpholino oligomers (PMO) targeting both the sense and antisense strands of the CVB3 replication intermediate. To increase the efficiency of drug internalization, the PMO were conjugated to a cell-penetrating arginine-rich peptide. These modified ASONs were evaluated in HeLa cells and HL-1 cardiomyocytes in culture and in a murine myocarditis model (Yuan et al., 2006). One of the oligomers, designed to target a sequence in the 3' portion of the CVB3 IRES, was found to be especially potent against CVB3. Treatment of cells with this oligomer prior to CVB3 infection produced an approximately 3-log10 decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when this oligomer treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of this oligomer once prior to and once after CVB3 infection showed an ~2-log10-decreased viral titer in the myocardium at 7 days post infection and a significantly decreased level of cardiac tissue

In addition to the many ASON reports, another strategy using CpG containing oligodeoxynucleotide to activate antiviral immunity has been reported (Cong et al., 2007). The mechanism is that the C-type of CpG oligomer can induce anti-CVB3 activity in human peripheral blood mononuclear cells (PBMCs) through the induction of synthesis of natural

3'UTR of mRNA.

**4.1 Anti-CVB3 ASONs** 

**4. NA-based antivirals against CVB3 infection** 

obtained from *in vitro* evaluation (Yuan et al., 2004).

damage, compared to the controls (Yuan et al., 2006).

mixed interferons.

Ribozyme as an antiviral agent has been tested for many virus infections; however, report on anti-CVB3 has not been documented. Here, we will take HCV as an example to briefly discuss the potential application of ribozyme for the treatment of HCV infection, as many recent reports found that HCV is a new causal agent of myocarditis (Matsumori, 2005; Matsumori et al., 2006). To investigate the potential application of synthetic, stabilized ribozymes for the treatment of chronic HCV infection, Macejak *et al*. designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5'UTR of HCV RNA including the IRES (Macejak et al., 2000). It was shown that the inhibitory activity of ribozyme targeting site at nucleotide 195 of HCV RNA exhibited a sequence-specific dose response, required an active catalytic ribozyme core, and was dependent on the presence of the HCV 5'UTR. In an investigation of new genetic approaches on the management of this infection, six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome were isolated from a ribozyme library that was expressed using recombinant adenovirus vectors (Macejak et al., 2001). Treatment with synthetic stabilized anti-HCV ribozymes and vector-expressed HCV ribozymes has the potential to aid in treatment of patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome. Gonzalez-Carmona and colleagues used RNA transcripts from a construct encoding a HCV-5'-NCR-luciferase fusion protein to test four chemically modified HCV specific ribozymes in a cell-free system and in HepG2 or CCL13 cell lines. They found that ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in cell culture and may be suitable for the development of novel optimized hammerhead structures (Gonzalez-Carmona et al., 2006).
