**3.2 Ribozymes**

406 Myocarditis

Fig. 1. Structures of certain nucleic acid analogs used to synthesize different generation ASONs. (A) unmodified deoxyribonucleotide. (B) Phosphorothioate modification of the phosphodiester backbone replaces the non-bridging oxygen with a sulfur atom. (C) Second generation ASONs with a 2'-akyl or 2'-methoxy ethyl groups further stabilize the molecule. (D) Phosphorodiamidate morpholino oligos have a modified backbone and modified sugar

are also less toxic than PS-ASONs (Cotten et al., 1991); however, the 2'-*O*-alkyl group simultaneously shields heteroduplexed ASON-RNA from RNase H and therefore cannot induce direct cleavage of the target RNA. These modified ASONs function mainly by blocking translation via steric hindrance of elongating ribosome. In order to retain the advantage of the RNAse H mechanism while still conferring some benefits of 2'-O-alkyl protection, chimeric oligos containing both 2' unmodified and 2'-modified DNAs, called gapmers, were conceived. Gapmers are typically end-modified, allowing a normal DNA-RNA heteroduplex to form mid-strand, although they may also consist of centre-modified

ring and are electrically neutral.

Ribozymes are catalytically active small RNA (~30-100 nts) molecules that act as enzymes to specifically cleave single strand RNA without the need of proteins. A major therapeutic advantage of ribozymes is the ability to make them *trans-acting* and to confer specificity to virtually cleave any target sequence (Peracchi, 2004). This can be achieved by fusing the ribozyme core sequence at the 5' and 3' ends with the sequences that are complementary to the target sequence. Of the nine groups of ribozymes, the hammerhead and hairpin ribozymes have received a great deal of attention (Scherer& Rossi, 2003). Hammerhead ribozymes, originally identified from plant viroid and viroid RNA, are composed of about 30 nts and have minimal requirements for the cleavage site, in which virtually any motif with the dinucleotide sequence UU, UC or UA can be targeted (Haseloff& Gerlach, 1992). For this reason, hammerhead ribozyme is very popular for the design of therapeutic ribozymes. On the other hand, the hairpin ribozyme has a more complex structure and requirements for target sequences, with a preference for GUC and cleavage occurring directly upstream of the G residue (Kore et al., 1998).

An advantage of ribozyme over ASON is its catalytic mode of action, which should in principle require a much lower concentration of ribozymes as compared to non-catalytic

Nucleic Acid-Based Strategies for the Treatment of Coxsackievirus-Induced Myocarditis 409

Synthetic siRNA, shRNA, ASON

Fig. 2. NA-based antiviral strategies for pathogens of viral myocarditis. Antiviral nucleic acids can either be transfected into cells or expressed intracellularly. ASONs hybridize to viral mRNA to induce RNase H-mediated cleavage of RNA strand of the DNA-RNA duplexes. Some modified ASONs cannot induce RNase H but they have a high affinity for the target and inhibit translation by steric hindrance of ribosome or splicing. Binding of ribozymes to the target sequence can trigger cleavage of the viral RNA. siRNAs

incorporated in the RISC target the viral RNA by perfect sequence complementation and induce cleavage of the target sequence by RNAse H activity of Ago protein. miRNAs (or AmiRNAs) target viral RNA by imperfect sequence complementation and induce gene silencing by destabilizing mRNAs (e.g., 3' deadenylation or 5' de-capping) and suppression of translation initiation and elongation. In addition, siRNAs can also target cellular genes

initiation factors on the mRNA or by disassociation of the premature translation initiation complexes. According to an alternative model, the mRNA destabilization is via miRNAinduced 3' end de-adenylation or 5' end de-capping, which results in degradation of mRNA after cleavage. Recently, some other mechanisms have been suggested. As review of the detailed mechanisms of RNAi action is beyond the scope of this article, the readers can refer to the recent reviews (Bartel, 2009; Carthew& Sontheimer, 2009; Q. Liu, Paroo, Z., 2010; Moazed, 2009). It should be pointed out that RNAi strategies for antiviral design have some advantages over the ASONs. Although they all cleave target mRNAs by RNase H, the modified ASON DNA induces activation of RNase H and cleavage of target sequence in nucleus, while the dsRNA functions primarily in cytoplasm. Ago2, the most important

(e.g., CAR and signal molecules) involved in viral entry and replication.

ASONs. In addition, chemical modifications of ribozyme can increase its stability and improve therapeutic potential (Gonzalez-Carmona et al., 2006; Jakobsen et al., 2007). Antiviral ribozymes have been extensively tested in different gene therapy settings (Haasnoot et al., 2007). On the other hand, ribozyme also has its limitation, which is that target site selections are limited due to sequence requirements at the cleavage site and to structural constrains that interfere with ribozyme function to a higher extent (Frese, 2006). Therefore, the selection of appropriate target sites is of utmost importance which can not be predicted but must rather be determined empirically and which depends on the particular ribozyme used.

#### **3.3 RNAi-based strategies**

The term of RNA interference (RNAi) refers to a cellular process by which a double-strand RNA (dsRNA) sequence specifically inhibits the expression of a gene. This very efficient process of posttranscriptional gene silencing (PTGS) was discovered first in plant (Napoli et al., 1990) and served as a protection against viruses and genetic instability arising from transposons (Bartel, 2004). Accumulated evidence suggests that RNAi also plays a role in the antiviral defense mechanism in mammalian cells (Bennasser et al., 2005; Berkhout& Jeang, 2007; Cullen, 2006; Lecellier et al., 2005). These findings fuel the interests of the researchers to use RNAi not only for study of gene regulation but also for antiviral drug development (Lecellier et al., 2005; Otsuka et al., 2007).

The specificity of RNA silencing is mediated by small RNAs called short interfering RNAs (siRNA) and microRNA (miRNA). Both types of RNAs are generated by members of the Dicer family. This group of class III endoribonucleases cleaves double stranded non-coding RNA into fragments with a length of 21-25 nts. For siRNA, the long dsRNA or transgeneexpressed short hairpin RNA (shRNA) is cleaved by Dicer. These RNAs are assembled into a multi-component complex, known as the RNA-induced silencing complex (RISC), which incorporates a single strand (antisense strand) of the siRNA serving as a guide sequence to silence the target gene (Hannon, 2002; Tomari& Zamore, 2005) (Fig. 2). For miRNA, this endogenous gene regulator is processed from primary RNA (priRNA) transcripts of noncoding regions or introns of protein-coding polymerase II transcripts. They are processed by RNase III Drosha to approximately 70-nt long pre-miRNAs, which are transported into cytoplasm by exportin-5 and are cleaved by Dicer to become the functional miRNA. Similar to siRNA, they also form a RISC with Argonaut proteins (having RNse H activity) and bind to their target mRNAs. The modes of actions of siRNA and miRNA depend on the degree of complementation between the siRNA or miRNA and their target sequences. siRNAs usually target coding regions by complementary base-paring and induce sequence-specific cleavage of mRNA substrate (Caudy et al., 2003); however, miRNA preferentially recognize target sequences in the 3'UTR of mRNAs and this target site is often in multi-copy (Brennecke et al., 2005; Grimson et al., 2007; Krek et al., 2005; Lewis et al., 2003). The binding of the miRNA often takes place with an incomplete homology, although a perfect base-pairing in the seed region (positions nt 2-8 from 5' end of the antisense strand) of miRNA forms the core of interaction. Depending on the complete or partial homology between the miRNA and mRNA, the result can be cleavage of the target mRNA or repression of translation (Fig. 2) (Doench et al., 2003; Parker et al., 2005).

The precise mechanisms of RNAi-mediated suppression of gene expression have been studied extensively and made significant progress. The proposed mechanisms include the translation suppression by blocking the binding and scanning of ribosome and other

ASONs. In addition, chemical modifications of ribozyme can increase its stability and improve therapeutic potential (Gonzalez-Carmona et al., 2006; Jakobsen et al., 2007). Antiviral ribozymes have been extensively tested in different gene therapy settings (Haasnoot et al., 2007). On the other hand, ribozyme also has its limitation, which is that target site selections are limited due to sequence requirements at the cleavage site and to structural constrains that interfere with ribozyme function to a higher extent (Frese, 2006). Therefore, the selection of appropriate target sites is of utmost importance which can not be predicted but must rather be determined empirically and which depends on the particular

The term of RNA interference (RNAi) refers to a cellular process by which a double-strand RNA (dsRNA) sequence specifically inhibits the expression of a gene. This very efficient process of posttranscriptional gene silencing (PTGS) was discovered first in plant (Napoli et al., 1990) and served as a protection against viruses and genetic instability arising from transposons (Bartel, 2004). Accumulated evidence suggests that RNAi also plays a role in the antiviral defense mechanism in mammalian cells (Bennasser et al., 2005; Berkhout& Jeang, 2007; Cullen, 2006; Lecellier et al., 2005). These findings fuel the interests of the researchers to use RNAi not only for study of gene regulation but also for antiviral drug

The specificity of RNA silencing is mediated by small RNAs called short interfering RNAs (siRNA) and microRNA (miRNA). Both types of RNAs are generated by members of the Dicer family. This group of class III endoribonucleases cleaves double stranded non-coding RNA into fragments with a length of 21-25 nts. For siRNA, the long dsRNA or transgeneexpressed short hairpin RNA (shRNA) is cleaved by Dicer. These RNAs are assembled into a multi-component complex, known as the RNA-induced silencing complex (RISC), which incorporates a single strand (antisense strand) of the siRNA serving as a guide sequence to silence the target gene (Hannon, 2002; Tomari& Zamore, 2005) (Fig. 2). For miRNA, this endogenous gene regulator is processed from primary RNA (priRNA) transcripts of noncoding regions or introns of protein-coding polymerase II transcripts. They are processed by RNase III Drosha to approximately 70-nt long pre-miRNAs, which are transported into cytoplasm by exportin-5 and are cleaved by Dicer to become the functional miRNA. Similar to siRNA, they also form a RISC with Argonaut proteins (having RNse H activity) and bind to their target mRNAs. The modes of actions of siRNA and miRNA depend on the degree of complementation between the siRNA or miRNA and their target sequences. siRNAs usually target coding regions by complementary base-paring and induce sequence-specific cleavage of mRNA substrate (Caudy et al., 2003); however, miRNA preferentially recognize target sequences in the 3'UTR of mRNAs and this target site is often in multi-copy (Brennecke et al., 2005; Grimson et al., 2007; Krek et al., 2005; Lewis et al., 2003). The binding of the miRNA often takes place with an incomplete homology, although a perfect base-pairing in the seed region (positions nt 2-8 from 5' end of the antisense strand) of miRNA forms the core of interaction. Depending on the complete or partial homology between the miRNA and mRNA, the result can be cleavage of the target mRNA or

ribozyme used.

**3.3 RNAi-based strategies** 

development (Lecellier et al., 2005; Otsuka et al., 2007).

repression of translation (Fig. 2) (Doench et al., 2003; Parker et al., 2005).

The precise mechanisms of RNAi-mediated suppression of gene expression have been studied extensively and made significant progress. The proposed mechanisms include the translation suppression by blocking the binding and scanning of ribosome and other

Fig. 2. NA-based antiviral strategies for pathogens of viral myocarditis. Antiviral nucleic acids can either be transfected into cells or expressed intracellularly. ASONs hybridize to viral mRNA to induce RNase H-mediated cleavage of RNA strand of the DNA-RNA duplexes. Some modified ASONs cannot induce RNase H but they have a high affinity for the target and inhibit translation by steric hindrance of ribosome or splicing. Binding of ribozymes to the target sequence can trigger cleavage of the viral RNA. siRNAs incorporated in the RISC target the viral RNA by perfect sequence complementation and induce cleavage of the target sequence by RNAse H activity of Ago protein. miRNAs (or AmiRNAs) target viral RNA by imperfect sequence complementation and induce gene silencing by destabilizing mRNAs (e.g., 3' deadenylation or 5' de-capping) and suppression of translation initiation and elongation. In addition, siRNAs can also target cellular genes (e.g., CAR and signal molecules) involved in viral entry and replication.

initiation factors on the mRNA or by disassociation of the premature translation initiation complexes. According to an alternative model, the mRNA destabilization is via miRNAinduced 3' end de-adenylation or 5' end de-capping, which results in degradation of mRNA after cleavage. Recently, some other mechanisms have been suggested. As review of the detailed mechanisms of RNAi action is beyond the scope of this article, the readers can refer to the recent reviews (Bartel, 2009; Carthew& Sontheimer, 2009; Q. Liu, Paroo, Z., 2010; Moazed, 2009). It should be pointed out that RNAi strategies for antiviral design have some advantages over the ASONs. Although they all cleave target mRNAs by RNase H, the modified ASON DNA induces activation of RNase H and cleavage of target sequence in nucleus, while the dsRNA functions primarily in cytoplasm. Ago2, the most important

Nucleic Acid-Based Strategies for the Treatment of Coxsackievirus-Induced Myocarditis 411

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).

RNAi-mediated antiviral strategies can achieve much higher efficiency than ASONs. Thus, recent studies have focused on the design and evaluation of anti-CVB3 siRNAs. This group of small double-stranded RNAs, as a silencer of target gene expression, can virtually inhibit any genes of virus and cell if the site of targeting within the gene is unique. Thus, the target search for anti-CVB3 siRNAs is not only concentrating on CVB3 genome but also extending

CVB3 genome harbors many *cis*-acting sequence elements for viral transcription and translation, such as the 5' and 3' UTRs, the IRES and other segments for binding of transcription and translation initiation factors. In addition, the viral genome also encodes many essential enzymes for CVB3 multiplication, such as proteases 2A and 3C as well as the RNA-dependent RNA polymerase 3D. These structures are rationale targets for design of anti-CVB3 siRNAs. This hypothesis has been tested by a number of groups. The earlier selection of the siRNA targets was focused on CVB3 protease 2A. Almost at the same time, two groups independently found that inhibition of 2A protease by specific siRNAs significantly reduced CVB3 replication. The first group by Yuan et al., evaluated five siRNAs targeting the 5'UTR, AUG start codon, VP1, 2A and 3D, respectively and found that siRNA targeting 2A (nts 3543-3561) showed strongest anti-CVB3 activity in HeLa cells, resulting in 92% reduction of viral replication and siRNAs targeting VP1, 3D and the 5'UTR

to the host cellular genes required for viral infection or replication.

**4.2 Antiviral ribozymes** 

**4.3 Anti-CVB3 siRNAs** 

**4.3.1 Targeting the CVB3 genome** 

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 3'UTR of mRNA.
