**6.1 Antisense oligonucleotides (AMO)**

Antisense inhibition of miRNA function has been an important tool for uncovering miRNA biology and potential therapeutics [31]. Synthetic oligonucleotides can be used therapeutically when miRNA dysregulation contributes to pathophysiology. These oligonucleotides are known as anti-miRNA oligonucleotides (miRNA inhibitors). To improve functional potency and to provide protection against nuclease degradation, they are often chemically modified [31, 32]. An ideal modification should increase binding affinity to the extent that specificity is compromised and should be non-toxic. There are four most common oligonucleotide modifications: (1) 2'-O-methyl groups, (2) phosphorothioate, (3) locked nucleic acid (LNA) anti-miRNA constructs, and (4) N,N-diethyl-4-(4-nitronaphthalen-1-ylazo) phenylamine (ZEN).

2'-O-methyl groups are the first generation of AMOs. 2'-O-methyl modifications can help AMOs to increase nuclease resistance and facilitate binding affinities to miRNA by the addition of an O-methyl group to the 2'-C atom. Phosphorothioate, compared to the 2'-O-methyl analogs, bonds at both the 3′ and 5′ ends to prevent nuclease degradation, and a 3′ cholesterol tail to help with cell uptake [33]. These modifications help the AMO to penetrate into tissues and

**99**

*Therapeutic Implication of miRNA in Human Disease DOI: http://dx.doi.org/10.5772/intechopen.82738*

LNA-AMOs [39].

**6.2 miRNA sponges**

phenomenon in human and animals.

**6.3 miRNA masking**

organs and significantly increase their half-lives in the target tissues. However, the first generation of AMOs have relative low potencies to be effective in animal model [34]. Second generation AMOs contain other modifications at the 2′ sugar position. Locked nucleic acid (LNA) modifications which are bicyclic nucleic acids that tether the 2'O to the 4'C via methylene bridge locking sugar into a 3′ endo conformation have been shown to have the best binding affinity and nuclease resistance. This group of AMO has been widely used in experimental animals [35–37]. Currently, the most advanced miRNA targeting therapy is SPC3649 (miravirsen), which is a locked nucleic acid-modified oligonucleotide antagonizing miR-122. This is the first miRNA-targeted drug to enter human clinical trials [38]. Despite LNA modifications have higher binding affinity, these modifications can lead to off-target effects which may cause toxicity *in vivo* [35]. Recently, a new compound called N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN), when is included at each end of the AMO, led to increased binding affinity to the miRNA and inhibited exonuclease degradation. Recent studies have shown that this group of AMOs (ZEN-AMOs) have higher potency and less toxicity than

miRNA sponges are transcripts that contain multiple (typically 4–10 separated by a few nucleotides) tandem-binding sites to a miRNA of interest and are transcribed from mammalian expression vectors. The use of miRNA sponges in mammalian cells was introduced by Ebert and colleagues [40]. miRNA sponges have been found to occur naturally as long non-coding RNA in plants and animals. Synthetic miRNA sponges are usually plasmid or viral vectors which contain tandemly arrayed miRNA binding sites, separated with a small nucleotide spacer and inserted into a 3'UTR of the reporter gene driven by an RNA polymerase II promoter [40, 41]. miRNA sponges have the ability to inhibit an entire family of miRNA by using the common seed sequence, and can therefore inhibit multiple miRNAs at once. Some of the endogenous circular RNAs have been founded to function as nature miRNA sponges. For example, circRNA7 has been shown to be functions as a miRNA sponge for miRNA-7 in the mouse tissues [42]. The authors have further demonstrated that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge [42]. These finding suggest that circRNA functioning as a miRNA sponge to regulate miRNA expression may be a common

MicroRNA-masking antisense oligonucleotide technology (miR-mask) is another strategy for miRNA-based therapeutics. In contrast to miRNA sponges, miR-masks consist of single-stranded 2'-O-methyl modified antisense oligonucleotides that are fully complementary to the expected miRNA binding site in the 3'-UTR of target mRNA [43]. A miR-mask does not directly interact with its target miRNA but binds to the binding site of that miRNA in the 3' UTR of the target mRNA by fully complementary mechanism. Therefore, the miR-mask blocks the access of its target miRNA to the binding site so as to rescue its target mRNA via blocking the action of its target miRNA. miR mask is designed to be fully complementary to the target mRNA sequence of a miRNA, which suggests that the antimiRNA action of a miR-mask is gene-specific. The strategy of miRNA masking has been used to disrupt miRNA function and involves masking the target site on target mRNA using a modified single-stranded RNA complementary to the target

*Therapeutic Implication of miRNA in Human Disease DOI: http://dx.doi.org/10.5772/intechopen.82738*

organs and significantly increase their half-lives in the target tissues. However, the first generation of AMOs have relative low potencies to be effective in animal model [34]. Second generation AMOs contain other modifications at the 2′ sugar position. Locked nucleic acid (LNA) modifications which are bicyclic nucleic acids that tether the 2'O to the 4'C via methylene bridge locking sugar into a 3′ endo conformation have been shown to have the best binding affinity and nuclease resistance. This group of AMO has been widely used in experimental animals [35–37]. Currently, the most advanced miRNA targeting therapy is SPC3649 (miravirsen), which is a locked nucleic acid-modified oligonucleotide antagonizing miR-122. This is the first miRNA-targeted drug to enter human clinical trials [38]. Despite LNA modifications have higher binding affinity, these modifications can lead to off-target effects which may cause toxicity *in vivo* [35]. Recently, a new compound called N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN), when is included at each end of the AMO, led to increased binding affinity to the miRNA and inhibited exonuclease degradation. Recent studies have shown that this group of AMOs (ZEN-AMOs) have higher potency and less toxicity than LNA-AMOs [39].

## **6.2 miRNA sponges**

*Antisense Therapy*

let-7c, let-7f, and miR-200c were significantly decreased in pancreas cancer patients whereas miR-486-5p and miR-451 were significantly increased in those patients compared with the normal people, which suggests that these miRNAs can be served as signature biomarkers for pancreas cancer [30]. Additionally, circulating miRNAs can be employed in diagnostic strategies to detect for alterations associated with either disorder affliction or treatment response. In the miRNA-based therapeutics, the miRNA expression is altered in different diseases and it is now feasible to correct miRNA expression by injecting miRNAs similar to the use of antisense mRNAs and RNAi. For example, because the activation of onco-miRNA genes could cause development of cancer, artificial antisense miRNAs could be synthesized and used

There are at least two main strategies to target miRNA expression for prevention and potential treatment of disease. The first strategies is the use of oligonucleotides or virus-based constructs to either directly block the expression of a disease-

associated signature miRNA or to directly substitute for the loss of expression of the miRNA. The second strategies is indirectly employing drugs to alter miRNA expression by targeting their transcription and processing. Blocking miRNA expression can be achieved by the use of antisense oligonucleotides, miRNA sponges, miRNAmask and small RNA inhibitors. Restoring downregulated miRNA expression can be achieved by using synthetic miRNA (miRNA mimic) or by inserting genes coding for miRNA into viral constructs. At the transcriptional level, small-molecule miRNA inhibitors can be employed to prevent the transitions from DNA transcript to pri-miRNA and pre-miRNA. Antisense oligonucleotides can be employed at the mature miRNA level to induce degradation or revert the mature miRNA into a duplex form with the antisense oligonucleotide. At the functional level, miRNA masks can bind complementarily to the 3' UTR region of target mRNA, competing for bindings with endogenous miRNAs for the specific target. miRNA sponges can be employed to bind target miRNA via complementary mRNA binding sites,

Antisense inhibition of miRNA function has been an important tool for uncovering miRNA biology and potential therapeutics [31]. Synthetic oligonucleotides can be used therapeutically when miRNA dysregulation contributes to pathophysiology. These oligonucleotides are known as anti-miRNA oligonucleotides (miRNA inhibitors). To improve functional potency and to provide protection against nuclease degradation, they are often chemically modified [31, 32]. An ideal modification should increase binding affinity to the extent that specificity is compromised and should be non-toxic. There are four most common oligonucleotide modifications: (1) 2'-O-methyl groups, (2) phosphorothioate, (3) locked nucleic acid (LNA) anti-miRNA constructs, and (4) N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-

2'-O-methyl groups are the first generation of AMOs. 2'-O-methyl modifications can help AMOs to increase nuclease resistance and facilitate binding affinities to miRNA by the addition of an O-methyl group to the 2'-C atom. Phosphorothioate, compared to the 2'-O-methyl analogs, bonds at both the 3′ and 5′ ends to prevent nuclease degradation, and a 3′ cholesterol tail to help with cell uptake [33]. These modifications help the AMO to penetrate into tissues and

to inhibit their targeted miRNAs to treat or prevent the cancer.

decreasing expression levels of target miRNAs (see **Figure 1**).

**6.1 Antisense oligonucleotides (AMO)**

**6. miRNA therapeutics: strategies**

**98**

phenylamine (ZEN).

miRNA sponges are transcripts that contain multiple (typically 4–10 separated by a few nucleotides) tandem-binding sites to a miRNA of interest and are transcribed from mammalian expression vectors. The use of miRNA sponges in mammalian cells was introduced by Ebert and colleagues [40]. miRNA sponges have been found to occur naturally as long non-coding RNA in plants and animals. Synthetic miRNA sponges are usually plasmid or viral vectors which contain tandemly arrayed miRNA binding sites, separated with a small nucleotide spacer and inserted into a 3'UTR of the reporter gene driven by an RNA polymerase II promoter [40, 41]. miRNA sponges have the ability to inhibit an entire family of miRNA by using the common seed sequence, and can therefore inhibit multiple miRNAs at once. Some of the endogenous circular RNAs have been founded to function as nature miRNA sponges. For example, circRNA7 has been shown to be functions as a miRNA sponge for miRNA-7 in the mouse tissues [42]. The authors have further demonstrated that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge [42]. These finding suggest that circRNA functioning as a miRNA sponge to regulate miRNA expression may be a common phenomenon in human and animals.

#### **6.3 miRNA masking**

MicroRNA-masking antisense oligonucleotide technology (miR-mask) is another strategy for miRNA-based therapeutics. In contrast to miRNA sponges, miR-masks consist of single-stranded 2'-O-methyl modified antisense oligonucleotides that are fully complementary to the expected miRNA binding site in the 3'-UTR of target mRNA [43]. A miR-mask does not directly interact with its target miRNA but binds to the binding site of that miRNA in the 3' UTR of the target mRNA by fully complementary mechanism. Therefore, the miR-mask blocks the access of its target miRNA to the binding site so as to rescue its target mRNA via blocking the action of its target miRNA. miR mask is designed to be fully complementary to the target mRNA sequence of a miRNA, which suggests that the antimiRNA action of a miR-mask is gene-specific. The strategy of miRNA masking has been used to disrupt miRNA function and involves masking the target site on target mRNA using a modified single-stranded RNA complementary to the target

sequence [44, 45]. The miRNA-masking method, in which only specific mRNA is masked, may lead to more specific and safer therapeutic strategies.
