*2.3.3 Third-generation ASO*

*Antisense Therapy*

mechanisms [26].

*2.3.2 Second-generation ASO*

performed chemical modification of ASOs for loss-of-function studies *in vitro* and *in vivo* for gene target identification and validation. These data have led to the introduction of PTO ASOs into clinical therapeutic trials. Indeed, Fomivirsen, a currently FDA approved drug for clinical use, is a first-generation PTO-modified ASO [24]. Despite the fact the PTO modifications are the most widely used ODs, they have many properties which render them suboptimal antisense effector molecules. The PTO backbone is known to induce sequence-independent effects attributable to its length dependent high affinity for various cellular proteins. However, this seemingly negative property of PTO ASOs to interact with certain proteins proved to be advantageous for the pharmacokinetic profile. Their binding to plasma proteins protects them from filtration and is responsible for an increased serum half-life [25]. Finally, these oligonucleotides can interact with components of the innate immune system such as Toll-like receptors (TLRs), inducing an immune response and triggering cytokines expression and other genes coding for the nonspecific defense

Nevertheless, to try to solve the several non-specific problems [27], new chemical modifications have been developed. Modifications to the base, sugar and backbone have been identified that increase binding affinity for the target RNA.

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

**18**

second-generation ODs).

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.
