**3.1 Antisense oligonucleotide (ASON)**

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., 2000).

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 al., 1996).

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

Nucleic Acid-Based Strategies for the Treatment of Coxsackievirus-Induced Myocarditis 407

ASON flanked by normal DNA, or more commonly, phosphorothioate-linked DNA so as to capitalize on the advantages of both types of modifications (Turner et al., 2006). Both designs also reduce the polyanionic side effects of the phosphorothioate modification (Monia et al., 1993). 2'-O-alkyl modified ASONs and mixed backbone gapmer ASONs

Third generation ASONs are phosphorodiamidate morpholino oligonucleotides (PMOs). PMOs are nonionic DNA analogues originally proven in loss of function knockdown studies in developmental systems such as zebrafish. The success and limitations of their usage have been recently reviewed comprehensively (Amantana& Iversen, 2005; Heasman, 2002). PMOs have an altered structure in which the ribose is replaced by a morpholine moiety and phosphorodiamidate (O-PONH2-O) linkers are used instead of phosphodiester bonds. Thus PMOs are resistant to digestion by nucleases and are electrically neutral, a property that reduces nonspecific interactions with intracellular proteins. Morpholinos form base pairs with target sequences, but the binding ability is no greater than binding of analogous DNA and RNA oligomers, necessitating the use of relatively long 25-base oligomers for antisense inhibition. In addition, PMO-RNA hybrids do not activate RNase H. Therefore, the mechanism by which the PMOs inhibit protein synthesis is via binding the critical mRNA elements, such as the mRNA 5'UTR or the start codon region, to prevent ribosomes from binding or scanning. Alternatively, PMOs may occupy the mRNA splice recognition site to block the normal posttranscriptional processing required for synthesis of the functional protein. A good example is the report on therapeutic application of PMOs to correct

aberrant splicing of mutated -globin precursor mRNA (Lacerra et al., 2000).

caused by PS-ASON, which contains negative oxygen atoms.

directly upstream of the G residue (Kore et al., 1998).

**3.2 Ribozymes** 

The limitations of PMOs are their low cellular uptake levels as compared to unmodified ASONs. To address this shortcoming, PMOs can be conjugated to certain positive peptide carriers such as arginine-rich HIV-TAT and *drosophila* antennapedia sequences (Cardarelli et al., 2007). Because PMOs have a standard phosphodiester linkage and are uncharged, the addition of a positive peptide conjugate does not cause the same aptamer interaction as that

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

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

represent a second generation of ASON.

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 ring and are electrically neutral.

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 ASON flanked by normal DNA, or more commonly, phosphorothioate-linked DNA so as to capitalize on the advantages of both types of modifications (Turner et al., 2006). Both designs also reduce the polyanionic side effects of the phosphorothioate modification (Monia et al., 1993). 2'-O-alkyl modified ASONs and mixed backbone gapmer ASONs represent a second generation of ASON.

Third generation ASONs are phosphorodiamidate morpholino oligonucleotides (PMOs). PMOs are nonionic DNA analogues originally proven in loss of function knockdown studies in developmental systems such as zebrafish. The success and limitations of their usage have been recently reviewed comprehensively (Amantana& Iversen, 2005; Heasman, 2002). PMOs have an altered structure in which the ribose is replaced by a morpholine moiety and phosphorodiamidate (O-PONH2-O) linkers are used instead of phosphodiester bonds. Thus PMOs are resistant to digestion by nucleases and are electrically neutral, a property that reduces nonspecific interactions with intracellular proteins. Morpholinos form base pairs with target sequences, but the binding ability is no greater than binding of analogous DNA and RNA oligomers, necessitating the use of relatively long 25-base oligomers for antisense inhibition. In addition, PMO-RNA hybrids do not activate RNase H. Therefore, the mechanism by which the PMOs inhibit protein synthesis is via binding the critical mRNA elements, such as the mRNA 5'UTR or the start codon region, to prevent ribosomes from binding or scanning. Alternatively, PMOs may occupy the mRNA splice recognition site to block the normal posttranscriptional processing required for synthesis of the functional protein. A good example is the report on therapeutic application of PMOs to correct aberrant splicing of mutated -globin precursor mRNA (Lacerra et al., 2000).

The limitations of PMOs are their low cellular uptake levels as compared to unmodified ASONs. To address this shortcoming, PMOs can be conjugated to certain positive peptide carriers such as arginine-rich HIV-TAT and *drosophila* antennapedia sequences (Cardarelli et al., 2007). Because PMOs have a standard phosphodiester linkage and are uncharged, the addition of a positive peptide conjugate does not cause the same aptamer interaction as that caused by PS-ASON, which contains negative oxygen atoms.
