**2.1 ASO's mechanism of action**

*Antisense Therapy*

to nucleases, as opposed to siRNA that need a carrier. Finally, for *in vitro* studies, siRNA is considered a better technology, since it's relatively easier to obtain a potent

In this review we will mainly focus on ASOs chemistry and mechanism of action. Indeed, since Zamecnik in 1978 used an ASO-like unmodified DNA sequence in cell culture, notable progress has been made in ASO pharmacology [3]. Currently, the efficacy of different ASOs is being studied in many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis but also in several cancer states. Numerous ASO-based therapeutics are being tested in clinical trials. In **Table 1** figures some of the ASO in clinical trial for cancer treatments. However, until now, only two ASOs have been approved by the US FDA to be used on humans, namely, fomivirsen (Vitravene),

siRNA since unmodified RNA works with high potency as opposed to ASOs.

**14**

**Table 1.**

*Registered clinical studies with ASO in cancer treatments in clinical trials.gov.*

Two major mechanisms contribute to the antisense activity. The first is that most ASOs are designed to activate RNase H, which cleaves the RNA moiety of a DNA–RNA heteroduplex and therefore leads to degradation of the target mRNA in the nucleus and cytoplasm. In addition, ASOs that do not induce RNase H cleavage can be used to inhibit translation by steric blockade of the ribosome in the cytoplasm [6]. When the ASOs are targeted to the 5′-terminus, binding and assembly of the translation machinery can be prevented [7]. Most mammalian RNAs undergo multiple post-transcriptional processing steps in the cell nucleus including addition of a 5′-cap structure, splicing and polyadenylation.

Regulation of RNA processing is another efficient mechanism in which ASOs can be utilized to regulate gene expression. Studies have been published documenting that ASOs can be used to destabilize pre-mRNA [8] and to regulate RNA splicing [9]. Another viable approach to reversibly 'switch' protein function is alternative splicing that generates RNA encoding antagonistic proteins (**Figure 1**). Whether RNaseH activity takes place favorably in the cytoplasm or in the nucleus is not well documented. However, most studies suggest that the most part of the inhibition takes place in the cytoplasm. Controversially, ASOs that targets pre-mRNA and are splice modulators are active in the nucleus.

#### **Figure 1.**

*ASOs mechanism of action. (1) In the absence of ASO, normal gene and protein expression is maintained. (2) Formation of ASO-mRNA heteroduplex in cytoplasm induces activation of RNase H, leading to mRNA degradation or (3) steric interference of ribosomal assembly. Alternatively, ASO can enter the nucleus and regulate mRNA maturation by (4) inhibition of 5*′ *cap formation, (5) inhibition of mRNA splicing and (6) activation of RNase H.*

#### **2.2 ASO's design**

There are several screening strategies to obtain potent ASO, such as using a computational algorithm in ASO design [10], mRNA walking [11], OD array [12] and RNase H mapping [13]. However, some of these approaches are labor intensive and require expensive automation equipment. Many factors can affect the strength and stability of the ASO-mRNA interaction, such as the mRNA secondary structure, thermodynamic stability and also the position of the hybridization site relative to functional motifs on the target RNA, such as the 5' CAP region or translational start site.

To find a highly potent ASO, the "hit rate" can be increased by considering four parameters during ASO design:

## *2.2.1 Prediction of the secondary structure of the RNA*

It is recognized that a secondary structure of the RNA accurately predicted, leads to effective ASO design [14, 15]. Some algorithm able to predict any single mRNA secondary structure and folding pattern are currently available, such as the *m*fold and the *s*fold program.

#### *2.2.2 Identification of preferable RNA secondary local structures*

In order for the ASO to be effective, it should target mRNA regions accessible to hybridization [16], such as joint sequences, internal loops, and hairpins of 10 or more consecutive nucleotides, usually located at the terminal end of the sequence [17]. It has been shown that highly conserved motifs are a good target of a potent ASO, whereas ASO targeting variable local motifs may induce non-sequence specific effects [18]. Therefore, the best approach to increase the 'hit rate' of potent ASO design is to target these conserved motifs among several optimal mRNA predicted secondary structures.

#### *2.2.3 Motifs searching and GC content calculation*

Although RNase activity is stimulated by the formation of the ASO-mRNA heteroduplex, leading to the mRNA degradation, it has been shown that this activity occurs independently of the ASO sequence, but is rather strongly correlated to the GC content which is also known to affect thermodynamic stability.

The perfect content of GC is still controversial; it is described that a strong ASO effect is observed with a percentage of 45–65% of G or C residues [10]; however, many of the ASOs used in therapy today do not have a GC content in this range.

#### *2.2.4 Binding energy prediction*

Thermodynamic energy is also important to a successful ASO design. Some available software can calculate thermodynamic properties between the target mRNA sequence and the ASO. To design a potent ASO, the binding energy between the ASO and mRNA should be DG37 ≥ −8 kcal/mol, whereas the energy for binding between ASOs should be DG37 ≥ −1.1 kcal/mol [19].

#### **2.3 ASO's chemical modification**

The use of unmodified ASOs is limited as they have an overall charged property that prevents them from getting through the cell membrane and are rapidly attacked by all types of intracellular endonucleases and exonucleases, usually via 3′-5′ activity

**17**

**Figure 2.**

*by chimeric ASO, and (c–e) third-generation ASO.*

*Antisense Oligonucleotides, A Novel Developing Targeting Therapy*

in biological fluid. In addition, the degradation products of phosphodiester ODs may be cytotoxic and also exert anti-proliferative effects. Moreover, some unspecific hybridization has been observed and finally for most applications immunostimulation has also been a matter of concern [20, 21]. This part will be further detailed in

Numerous chemical modifications have been developed to improve nuclease resistance, extend tissue half-life, reduce non-sequence-specific toxicity, as well as

First-generation ASOs are the ones having a phosphorothioate (PTO) modified backbone. These ASOs have a sulfur atom that substitutes the non-bridging oxygen atoms in the phosphodiester bond [22] (**Figure 2a**). PTO modification confers higher resistance to the ASO against nuclease degradation, leading to higher bioavailability. Additionally, they are highly soluble and have excellent antisense activity [23]. Finally, PTO-modified ASOs promote degradation of target mRNA by RNase H enzyme. Nevertheless, this modification may slightly reduce the affinity of the ASO for its mRNA target because the melting temperature (Tm) of the ASO-mRNA heteroduplex is decreased [20]. PTO modification is the most widely

*Chemical modifications of ASO. (a) First-generation PTO, (b) second generation, RNase H cleavage induced* 

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

to increase affinity and potency (**Figure 2**).

the toxicity chapter.

*2.3.1 First-generation ASO*

*Antisense Oligonucleotides, A Novel Developing Targeting Therapy DOI: http://dx.doi.org/10.5772/intechopen.82105*

in biological fluid. In addition, the degradation products of phosphodiester ODs may be cytotoxic and also exert anti-proliferative effects. Moreover, some unspecific hybridization has been observed and finally for most applications immunostimulation has also been a matter of concern [20, 21]. This part will be further detailed in the toxicity chapter.

Numerous chemical modifications have been developed to improve nuclease resistance, extend tissue half-life, reduce non-sequence-specific toxicity, as well as to increase affinity and potency (**Figure 2**).

### *2.3.1 First-generation ASO*

*Antisense Therapy*

**2.2 ASO's design**

parameters during ASO design:

*m*fold and the *s*fold program.

predicted secondary structures.

*2.2.4 Binding energy prediction*

**2.3 ASO's chemical modification**

*2.2.3 Motifs searching and GC content calculation*

between ASOs should be DG37 ≥ −1.1 kcal/mol [19].

There are several screening strategies to obtain potent ASO, such as using a computational algorithm in ASO design [10], mRNA walking [11], OD array [12] and RNase H mapping [13]. However, some of these approaches are labor intensive and require expensive automation equipment. Many factors can affect the strength and stability of the ASO-mRNA interaction, such as the mRNA secondary structure, thermodynamic stability and also the position of the hybridization site relative to functional motifs on

To find a highly potent ASO, the "hit rate" can be increased by considering four

It is recognized that a secondary structure of the RNA accurately predicted, leads to effective ASO design [14, 15]. Some algorithm able to predict any single mRNA secondary structure and folding pattern are currently available, such as the

In order for the ASO to be effective, it should target mRNA regions accessible to hybridization [16], such as joint sequences, internal loops, and hairpins of 10 or more consecutive nucleotides, usually located at the terminal end of the sequence [17]. It has been shown that highly conserved motifs are a good target of a potent ASO, whereas ASO targeting variable local motifs may induce non-sequence specific effects [18]. Therefore, the best approach to increase the 'hit rate' of potent ASO design is to target these conserved motifs among several optimal mRNA

Although RNase activity is stimulated by the formation of the ASO-mRNA heteroduplex, leading to the mRNA degradation, it has been shown that this activity occurs independently of the ASO sequence, but is rather strongly correlated to the

The perfect content of GC is still controversial; it is described that a strong ASO effect is observed with a percentage of 45–65% of G or C residues [10]; however, many of the ASOs used in therapy today do not have a GC content in this range.

Thermodynamic energy is also important to a successful ASO design. Some available software can calculate thermodynamic properties between the target mRNA sequence and the ASO. To design a potent ASO, the binding energy between the ASO and mRNA should be DG37 ≥ −8 kcal/mol, whereas the energy for binding

The use of unmodified ASOs is limited as they have an overall charged property that prevents them from getting through the cell membrane and are rapidly attacked by all types of intracellular endonucleases and exonucleases, usually via 3′-5′ activity

GC content which is also known to affect thermodynamic stability.

the target RNA, such as the 5' CAP region or translational start site.

*2.2.2 Identification of preferable RNA secondary local structures*

*2.2.1 Prediction of the secondary structure of the RNA*

**16**

First-generation ASOs are the ones having a phosphorothioate (PTO) modified backbone. These ASOs have a sulfur atom that substitutes the non-bridging oxygen atoms in the phosphodiester bond [22] (**Figure 2a**). PTO modification confers higher resistance to the ASO against nuclease degradation, leading to higher bioavailability. Additionally, they are highly soluble and have excellent antisense activity [23]. Finally, PTO-modified ASOs promote degradation of target mRNA by RNase H enzyme. Nevertheless, this modification may slightly reduce the affinity of the ASO for its mRNA target because the melting temperature (Tm) of the ASO-mRNA heteroduplex is decreased [20]. PTO modification is the most widely

#### **Figure 2.**

*Chemical modifications of ASO. (a) First-generation PTO, (b) second generation, RNase H cleavage induced by chimeric ASO, and (c–e) third-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 mechanisms [26].

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.
