*2.3.2 Second-generation ASO*

The second generation represents oligonucleotides in which the structural modification is not limited to the backbone linkage but additionally includes structural modifications of the ribose: ASO with 2'-O-alkyl modifications of the ribose were developed (**Figure 2b**). These modifications intend to improve binding affinity, to increase efficacy, to modulate the protein binding of oligonucleotides and to enhance nuclease resistance. 2'-O-Methyl (2'-OMe) and 2'-O-Methoxyethyl (2'-MOE) modifications of ASO-PTO are the most widely studied [28]. These second-generation ASOs are less toxic than PTO- modified ASOs and have a slightly enhanced affinity towards their complementary RNAs [29]. Moreover, an important aspect for these ASOs is that 2'modifications can reduce immunostimulatory effect [30]. However, 2'-OMe and 2'-MOE substitutions do not activate the RNase H to cleave the target mRNA, which decreases the efficacy of the ASO [31]. Indeed, mechanistic studies have been conducted to elucidate RNase H activity. They have shown that the flexibility of the ASO, the accessibility of the 2′-OH group of the RNA and the correct width of the minor groove of the ASO-RNA duplex are necessary for effective RNase H mediated mRNA degradation [32]. Since 2′-*O*-alkyl RNA ODs do not activate the RNase H, they inhibit mRNA expression only by a steric interference with translation. However, to increase its potency, an ASO should be able to induce cleavage by RNase H. Therefore, a chimeric ASO was developed (**Figure 2b**). It consists of a central 'gap' region containing 10 DNA or PTO DNA monomers and flanked on both sides (5'and 3'extremities) by approximatively 5 modified nucleotides such as 2′-OM or 2'-MOE (indicated by red and yellow regions of the OD in **Figure 2b**). This chimeric 'gapmer' ASO allows RNase H to sit in the central gap and to execute target-specific mRNA degradation; meanwhile, the flanking 2′-alkyl modified ends prevent nuclease cleavage of ASO. The 2′-*O*-MOE-PTO gapmers OGX-427 (Apatorsen) developed by Rocchi et al., directed against HSP27 and OGX-011 (Curtisen) against clusterin are currently undergoing clinical trials (**Table 1**). The ODs being tested clinically mainly incorporate relatively simple chemical modifications such as the PTO modifications and the 2′-alkyl modifications (first- and second-generation ODs).

**19**

*2.3.3.3 Locked nucleic acid*

*Antisense Oligonucleotides, A Novel Developing Targeting Therapy*

Third-generation ASOs have been developed to further improve nuclease resistance, increase binding affinity, and to enhance pharmacokinetics and biostability. They are characterized by chemical modifications of the nucleotide, and more precisely to its furanose ring [33]. Many modifications have been described, such as N3′-P5′ phosphoramidates, 2′-deoxy-2′-fluoro-β-D-arabino nucleic acid analogue (FANA), cyclohexene nucleic acids (CeNAs) and tricyclo-DNA (tcDNA), however, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO) and locked nucleic acid (LNA) are the three most studied third-generation ASOs [34, 35]. It should be noted that there is no single modification that covers all the desired properties for a modified ASO. As described before, chemical modifications can improve ASO-RNA hybridization affinity, enhance nuclease resistance, decrease toxicity and modulate pharmacokinetics. An optimal antisense can be designed depending on its use by mixing and matching the numerous chemical modifications accordingly.

PNA is a synthetic DNA mimic in which the deoxyribose phosphate backbone is replaced by polyamide linkages [36] (**Figure 2c**). PNAs are biologically very stable and have good hybridization properties. However, they do not activate mRNA cleavage via RNase activity, but rather block translation and thus protein expression, by forming sequence-specific duplex with mRNA, hence generating steric hindrance. Due to their neutral backbone, PNAs have low solubility and their cellular uptake remains a challenge for exploiting them as therapeutics antisense. PNAs delivery can be enhanced annealing a PNA strand with a negatively charged complementary oligonucleotide, and then enclosed the obtained duplex with a cationic lipid. To this end, PNAs are also conjugated to peptides for improving their cellular uptake [37, 38]. Furthermore, PNA can elicit antigen effects by hybridizing with double-stranded DNA [36, 39] resulting in transcriptional arrest. Substantial data have revealed the

effectiveness of PNA in gene silencing in various in vitro models [39, 40].

phase II clinical trials for restenosis, cancer and polycystic kidney disease.

Locked nucleic acids oligonucleotides figure among the most promising candidates developed the last few years. LNAs are chemically modified nucleotides with a ribose containing a methylene bridge between the 2′-oxygen and the 4′-carbon

PMOs are non-charged ASOs whose pentose sugar is substituted by a morpholino ring and the inter-nucleotide linkages are phosphoramidate bonds in place of phosphodiester bonds [41] (**Figure 2d**). PMOs avoid the RNase H recruitment; their effect is primarily mediated by steric interference of ribosomal assembly resulting in translational arrest. Synthetic PMOs are resistant to nucleases degradation in biological fluid. Because their backbone is uncharged, PMOs are unlikely to form unwanted interactions with nucleic acid-binding proteins [42]. PMOs do not enter mammalian cells easily in culture, but it has been shown that conjugating it with peptides such as arginine-rich peptide (ARP) can enhance its cellular uptake and antisense potency [43]. Antisense PMO oligonucleotide, have shown efficacy in animal models *in vivo* and in human clinical trials [44, 45]. Indeed, a PMO antisense agent is currently in

*2.3.3.2 Phosphoramidate morpholino oligomer*

*DOI: http://dx.doi.org/10.5772/intechopen.82105*

*2.3.3 Third-generation ASO*

*2.3.3.1 Peptide nucleic acid*
