**1. Introduction**

402 Myocarditis

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Viral myocarditis is the most common heart disease in infants, children, young adults and pregnant women. Although a number of viruses from different genera, such as adenovirus, hepatitis C virus (HCV), parvoviruses and cytomegalovirus have been reported to cause myocarditis (Bowles et al., 2003; Kindermann et al., 2008; Kuhl et al., 2005a; Kuhl et al., 2005b; Kyto et al., 2005; Mahrholdt et al., 2006; Matsumori, 2005; Matsumori et al., 2006), coxsackievirus, particularly coxsackievirus B3 (CVB3), is generally considered the primary etiological agent of myocarditis (Blauwet, 2010; Kuhl et al., 2005a; Mahrholdt et al., 2006). CVB3 infection of the heart is often persistent and enters the chronic phase, leading to dilated cardiomyopathy (DCM)(Andreoletti et al., 2009; L. T. Cooper, Jr., 2009; Kuhl et al., 2005b; Yajima& Knowlton, 2009), a squelae of viral myocarditis characterized by ventricular chamber dilation, increased wall thickness, weaker beating and abnormal heart function. Patients with DCM eventually develop into congestive heart failure.

To date, there is no clinically proven specific treatment available for viral myocarditis and DCM. Patients with DCM eventually need heart transplantation as the final treatment (Schultz et al., 2009). The managements for viral myocarditis are usually supportive therapies, such as improvements in cardiophysiology with medicine used to treat other kinds of heart diseases, and application of non-specific antiviral agents to decrease the viral load. The former measurements include administration of angiotensin-converting enzyme inhibitor or angiotensin receptor blockade, beta-adrenergic blockade, diuretics, etc (Dennert et al., 2008; Rose, 2009; Schultz et al., 2009); the later measurements include application of type I interferons or nucleotide analogs such as ribavirin, which is reviewed elsewhere (Blauwet, 2010; Dennert& Crijns& Heymans, 2008; Schultz et al., 2009). If myocarditis was caused by an autoimmune disorder, it would be appropriately treated by immunosuppression (Rose, 2009; Schultz et al., 2009). However, the effectiveness of treatment with immunosuppressive therapies has not reached a consensus amongst different studies. This can probably be attributed to the difficulty of confirmation and diagnosis of the etiology and pathogenesis of myocarditis. Thus it is very important to distinguish infectious and autoimmune disease since the same methods of treatment will not be optimal for both forms of heart muscle diseases. The diagnostic gold standard is endomyocardial biopsies with the histological Dallas criteria, in association with new

Nucleic Acid-Based Strategies for the Treatment of Coxsackievirus-Induced Myocarditis 405

attachment of CVB3 particles to CAR, the receptor changes conformation to form the viral A-particle, a product of the interactions between CVB3 and CAR, which then allows for the release of viral RNA into host cells and begins viral translation and transcription. The observation that soluble CAR can function as a virus trap leading to inactive A-particles has been suggested as a strategy for CVB3 therapy (Pinkert et al., 2009; Werk et al., 2009; Yanagawa et al., 2004). Depending on the different combination of viral strains and mouse models in study of CVB3 infection, a CVB3 co-receptor called decay accelerating factor (DAF, CD55) is sometimes also necessary for CVB3 entry of the host cells (Freimuth et al., 2008; Shafren et al., 1997). Thus, genes encoding CAR and DAF are important candidates for

ASON is probably the earliest NA-based antiviral agent developed. They are designed to bind a complementary sequence in the target mRNA to form RNA-DNA heteroduplexes. These double-stranded hybrid sequences are recognized by RNase H, which digests the RNA strand in the duplex, releasing the ASON to bind another target and so on, effectively silencing the encoded gene (Walder & Walder, 1988). Certain ASONs are not capable of activation of RNase H; instead they inhibit gene translation by steric competition with the translational machinery. In addition, ASONs, if bound to pre-mRNA at intron-exon junctions, can disrupt mRNA splicing (Munroe, 1988). Furthermore, ASONs can also disrupt RNA trafficking by occupying protein-RNA interaction sequences necessary for correct intracellular localization. For example, hnRNP A2 response element (A2RE) is identified as a key sequence required for the trafficking of myelin basic protein (Shan et al.,

Due to major problems including instability, non-specific delivery and unwanted side effects of the ASONs, the structure of this molecule has been modified extensively at different components (i.e., the bases, sugar or phosphate backbone) and has entered its third generation (Fig. 1). The first generation of chemical modification was designed to enhance nuclease resistance of ASON in serum (Stein et al., 1997). The representative of such is the phosphorothioate (PS) oligonucleotide (ON), in which one of the non-bridging oxygen atoms in the phosphodiester bond is replaced by sulfur, intended to prevent cleavage by nucleases. Early antiviral PS-modified ASONs exhibited the antisense properties of phosphodiester ASONs, such as the ability to induce RNase H activation, while showing enhanced stability *in vitro* for up to 48 hours (Hoke et al., 1991); reviewed in (Kurreck, 2003). One notable property of PS-ASON is their tendency to form aptamers, i.e., nonspecific interactions with proteins due to its negative charge. This is disadvantageous intracellularly because aptamer interactions can impede ASON interaction with its intended target, and hence its function. Conversely, the tendency for PS-ASONs to bind serum proteins albumin and alpha-2 macroglobulin in circulation actually improves their bio-distribution throughout the body *in vivo* and prevents them from being cleared for excretion (Crooke et

Another strategy to increase the stability of ASONs is the addition of alkyl groups at the 2' position of the ribose. 2'-*O*-methyl (OMe) and 2'-*O*-methoxy-ethyl (MOE) substitutions sterically shield the backbone from nuclease access, and also increase affinity to the target, shown by increased Tm, thus stabilizing the duplex (Cotten et al., 1991). 2'-*O*-alkyl ASONs

study of viral tropism and rationale targets for antiviral drug design.

**3. NA-based antiviral strategies 3.1 Antisense oligonucleotide (ASON)** 

2000).

al., 1996).

immunohistochemical and viral PCR analyses of cardiac tissues (L. T. Cooper et al., 2007). In case of confirmed autoimmune-related disease and lack of detectable viral infection, an immunosuppressive treatment combining corticoids and azathioprine may be beneficial to the patients (Frustaci et al., 2003). However, if the disease is primarily caused by viral infections, more specific antiviral agents would be the ideal drugs of choice. In recent years, the search for such antiviral drugs has become a new trend in drug development for treatment of viral myocarditis. The strategies for developing such antivirals include i) screening chemical compounds, such as Pleconaril, capable of interacting with picornavirus (particularly human rhinovirus) antireceptor to block viral entry of the host cells (Groarke& Pevear, 1999; Kaiser et al., 2000; Reisdorph et al., 2003), ii) application of herb medicine to reduce viral load or boost immune responses to limit viral replication (Si et al., 2007; Y. F. Wang et al., 2009), iii) development of small peptide inhibitors of viral proteases to block CVB3 replication cycle (Maghsoudi et al., 2010) and iv) production of recombinant soluble protein of coxsackievirus-adenovirus receptor (CAR) fused to a human immunoglobulin (sCAR-Fc) to block coxsackievirus B3 entry (Pinkert et al., 2009; Werk et al., 2009; Yanagawa et al., 2003; Yanagawa et al., 2004). Another very attractive and promising trend in drug development is the nucleic acid (NA)-based approach to target viral genome or cellular genes to block viral translation and transcription. These strategies include design and synthesis of antisense oligonucleotide (ASON), ribozyme, short interfering RNA (siRNA) and artificial microRNA (miRNA). In this chapter we will focus our discussion on the recent state of this group of antiviral agents for the treatment of myocarditis caused by CVB3 and other viruses that have been recently reported as causal agents of myocarditis.
