**4. Perspectives**

Administration of oligonucleotides, including NATRE technology, is a unique therapeutic modality. Because the sequence of an oligonucleotide specifies the gene, one mRNA is selectively downregulated or upregulated (**Figure 7**). For example, administration of an *iNOS* sense oligonucleotide to endotoxemia/sepsis model rats showed little effects on endothelial NOS (eNOS) and neuronal NOS (nNOS) (unpublished data). Enzyme inhibitors (e.g., NOS inhibitors) generally have a broader specificity. Furthermore, it is rare that antibodies against oligonucleotides are raised.

NATRE technology is a powerful method to modulate *in vitro* and *in vivo* gene expression. Note that sense oligonucleotides to the mRNAs can be designed to inducible genes and many other genes. iNOS AS transcript was successfully administered to endotoxemia/sepsis model rats and improved their survival rate [38]. As shown in **Table 1**, many genes involved in inflammation are candidates suitable for clinical uses in the future. When a gene that is involved in diseases is selected, sense oligonucleotides to this gene can be easily designed and examined. Instead of sense oligonucleotides, antisense oligoribonucleotides (asORN) may be used to increase mRNA levels. Indeed, *IFN-A1* asORN inhibited the proliferation of Influenza virus in guinea pigs [46].

Sense oligonucleotides may apply to cancer, neurodegenerative disorders, and other diseases. For example, EPHA2 is over-expressed in various cancers, and the *EPHA2*

#### **Figure 7.**

*The NATRE technology using sense oligonucleotides. The principle of the NATRE technology is schematically shown. Potential therapeutic application using this technology is also shown.*

AS transcript may be involved in a subtype of breast cancer [26]. It is possible that sense oligonucleotides inhibit cancer progression and proliferation of cancer cells. Furthermore, the administration of *Bdnf*- and *Gdnf*-AntagoNATs to mice [44, 45] may facilitate the regeneration of neurons and glial cells in the central nervous system. When a gene that is involved in diseases is found, sense oligonucleotides to this gene can be designed. Furthermore, sense oligonucleotides can be designed in the genome of a pathogenic virus to inhibit viral multiplication.

Drugs and some constituents in functional foods and crude drugs of Japanese Kampo medicine mimic sense oligonucleotides by modulating mRNA stability [1]. When sodium salicylate reduced *iNOS* mRNA levels in hepatocytes, decreased mRNA stability was speculated [48]. It was reported that acetyl salicylate (aspirin) interacts with RNA by intercalating with the RNA duplex and destabilized the helix, resulting in a conformational change of the stem-loop structure of the RNA [49]. Because the drug-RNA interaction may affect mRNA stability, drugs and constituents may interfere with *iNOS* mRNA-AS transcript interactions like sense oligonucleotides. Indeed, several drugs or constituents in functional foods and crude drugs decreased the levels of both *iNOS* mRNA and AS transcript: dexamethasone (anti-inflammatory drug) [50], cucurbitacin B (triterpenoid in the fruit of *Momordica charantia*) [51], sakuranetin (flavonoid in the bark of *Prunus jamasakura*) [52], and standardized oligomerizedpolyphenol from *Litchi chinensis* fruit extract (OPLFE) [53]. The investigation of the drugs and constituents may clarify the mechanism of action of the sense oligonucleotide in future.
