*3.2.5 Conjugation of sense oligonucleotides*

When modified, but non-conjugated oligonucleotides are introduced in the body, they are transferred to the liver and kidney. To improve *in vivo* delivery to an organ or a tissue, sense oligonucleotides are often conjugated at their ends with cell-penetrating arginine-rich peptides and cell-permeable hydrophobic molecules [32, 33]. Cell-penetrating arginine-rich peptides are derived from the Tat protein of human immunodeficiency virus (HIV)-1 [35] and synthetic arginine oligomer peptides (Arg6); and cell-permeable hydrophobic molecules are cholesterol [36] and C12 spacer.

The conjugation does not affect the potency of *iNOS* oligonucleotides to decrease *iNOS* mRNA expression [18]. When an *iNOS* sense oligonucleotide conjugated to these molecules was introduced into hepatocytes, all conjugated sense oligonucleotides were as effectively decreasing *iNOS* mRNA levels as the non-conjugated sense oligonucleotide [18]. Appropriate conjugation may facilitate the delivery of sense oligonucleotides to target tissues or organs (see Section 3.7).

#### **3.3 Regulation of mRNA levels by sense oligonucleotides in culture cells**

Because the modification of sense oligonucleotides is essential, modified sense oligonucleotides were used for the introduction to cells. The *in vitro* effects of NATRE technology (including AntagoNAT technology) on mRNA levels in the cells are summarized in **Table 1**.


#### **Table 1.**

*Examples of the in vitro effects of NATRE technology on mRNA levels.*

## **3.4 Administration of sense oligonucleotides to animals**

When NO is excessively produced by iNOS in hepatocytes and Kupffer cells (resident macrophages) of the liver, it leads to multiple organ failure [38]. Endotoxemia model rats with hepatic failure are often used to evaluate drugs. These model rats are prepared either by intravenous injection of D-galactosamine (GalN) and LPS [38–40], or by LPS injection after partial hepatectomy [38, 41]. These rats resemble the animals suffering from sepsis or septic shock.

After optimization of the sequence and modification of *iNOS* sense oligonucleotides in hepatocytes, the best sense oligonucleotide was administered into the endotoxemia model rats [38]. When the sense oligonucleotide was intravenously injected with GalN and LPS to rats, the survival rate was markedly increased, and apoptosis in the hepatocytes markedly decreased in the sense oligonucleotide-treated rats [38].

Because LNA is efficiently accumulated in the liver [34], the LNA-modified *iNOS* sense oligonucleotide may function in the liver where the *iNOS* gene is highly expressed. Taken together, NATRE technology using *iNOS* sense oligonucleotides may be applicable to treat sepsis and septic shock.

#### **3.5 AntagoNAT technology**

To increase mRNA levels by modulating the mRNA-AS transcript interactions, an *AntagoNAT* oligonucleotide has been used, which is an antagonist to an AS transcript (NAT) and defined as a single-stranded antisense oligonucleotide to a specific AS transcript [37]. Each AntagoNAT oligonucleotide contained a mixture of OmeNAs and LNAs.

When an AS transcript overlaps with its corresponding mRNA, the AntagoNAT is identical to a sense oligonucleotide. Therefore, AntagoNAT technique is very close to NATRE technology. Both technologies use sense oligonucleotides to knockdown AS transcript. However, NATRE technology has been applied to decrease mRNA levels, whereas AntagoNAT technology has been applied to increase mRNA levels.

It has been reported that AntagoNAT-mediated knockdown of brain-derived neurotrophic factor (*Bdnf*) and glial-derived neurotrophic factor (*Gdnf*) AS transcripts resulted in increased levels of *Bdnf* and *Gdnf* mRNAs in HEK293T cells [37]. The underlying mechanism may be similar to those indicated in **Figure 5**, or to other mechanisms that were previously mentioned [7].

AntagoNAT oligonucleotides can be administered to animals. When *Bdnf*-AntagoNAT was intracerebroventricularly delivered, the *Bdnf* mRNA levels increased in the mouse brain [37]. Collectively, the *Bdnf*-AntagoNATs functioned *in vitro* and *in vivo*, although it increased the *Bdnf* mRNA levels.

AntagomiR (antagomir), which is a synthetic oligonucleotide complementary to a microRNA, is used to sequester endogenous microRNA [42]. Each antagomir sequence is identical to a specific mRNA and similar to several mRNAs that share microRNA-binding sites (seed sequences). Therefore, antagomirs are another type of sense oligonucleotides. When microRNA is involved in the mRNA-AS transcript interactions, the antagomir technology may be applied to analyze these interactions. See an example in [23].

### **3.6 Comparison with other methods**

The mechanisms of two conventional technologies, i.e., antisense and siRNA technologies [11], are schematically shown (**Figure 6**).

*The Natural Antisense Transcript-Targeted Regulation Technology Using Sense Oligonucleotides… DOI: http://dx.doi.org/10.5772/intechopen.108281*

**Figure 6.**

*Mechanisms of conventional mRNA knockdown technologies. (A) Antisense technology. The mRNA that hybridizes with an antisense oligonucleotide is digested by RNase H1. (B) siRNA technology. siRNA recruits Argonaut (Ago) proteins to form RISC, which destroys mRNA. See details in the text.*

## *3.6.1 Antisense technology*

A single-stranded antisense oligonucleotide hybridizes with an mRNA and forms a local DNA:RNA hybrid. RNase H1 recognizes DNA:RNA hybrids and selectively digests the RNA strand, leading to the degradation of the mRNA. Therefore, the antisense oligonucleotides should be DNA. The presence of an AS transcript is not essential for this method.

## *3.6.2 siRNA technology*

siRNA is a synthetic double-stranded RNA, and one strand of the siRNA ( i.e., guide strand) is complementary to a target mRNA. Typical siRNA consists of 19 base pairs and 2-nt 3′ overhangs. siRNA interacts with Argonaut (Ago) proteins to form RNA-induced silencing complex (RISC) and then binds to the target mRNA. The guide strand of siRNA hybridizes with the mRNA (especially, 3′UTR) in the RISC, resulting in degradation of the target mRNA. The other RNA strand (i.e., passenger strand) is destroyed during the RISC formation. This mechanism mimics mRNA degradation by microRNA.

As mRNA knockdown methods, NATRE technology using sense oligonucleotides is compared with conventional methods, i.e., antisense technology and siRNA technology (**Table 2**). Other than these technologies, there are various oligonucleotide technologies that are applied to therapies of disease.


#### **Table 2.**

*Comparison of the methods to knockdown mRNAs.*
