**3. Small interfering RNA**

dose of Dz13 (10, 30, or 100 ìg) [29]. Followed-up over four weeks, c-Jun expression is reduced in all nine participants. Meanwhile, Dz13 could significantly promote apoptosis and stimulate inflammatory and adaptive immune responses in the tumors. Among the participants, five patients have a reduction in histological tumor depth. These results indicated that Dz13 possibly could represent a future treatment option for BCC prior to excision by surgery.

Atopic eczema Phase IIa Ongoing EUCTR2013-001091-38-DE

**Target Disease Phase Trial ID Refs.**

LMP1 Nasopharyngeal carcinoma Phases I/II Completed NCT01449942 [27, 28]

GATA-3 Asthma Phases I Completed NCT01470911 [30-33]

ACTRN12610000162011

NCT01554319 NCT01577953 NCT01743768 EUCTR2012-003570-77-DE NCT02079688

> NCT02129439 DRKS00006087

ACTRN12613000302752 [29]

Phases I Completed Phase I/Ib Ongoing

Phases I Completed Phases I Completed Phases II Completed Phase IIa Completed Phases I Completed

Phases IIa Ongoing

Phases I/II Ongoing Phase IIa Pending

c-Jun

Nodular basal-cell carcinoma Melanoma with satellite or in-transit metastasis

142 Nucleic Acids - From Basic Aspects to Laboratory Tools

Atopic dermatitis

Ulcerative colitis Chronic obstructive pulmonary disease

**Table 2.** Clinical trials of DNAzymes in anti-diseases therapy

The transcription factor GATA-3 plays an important role in the regulation of Th2-mediated immune mechanisms such as in allergic bronchial asthma, and the DNAzyme hgd40 has been shown to specifically and selectively reduce expression of GATA-3 mRNA. Turowska *et al.* found that hgd40 is evenly distributed in inflamed asthmatic mouse lungs within minutes after single dose application, and could slowly eliminate from lung tissue with the goal to minimize accumulation and to ensure continued exposure for efficacy [32]. Safety pharmacology studies showed that with no observable adverse event, hgd40 has a highly favorable toxicity profile when administered by aerosol inhalation at the therapeutic doses [33]. With good safety and tolerability in the phase I program [31], a randomized, double-blind, placebo-controlled, multicenter clinical trial of hgd40 was conducted in patients with allergic asthma, who had biphasic early and late asthmatic responses after laboratory-based allergen provocation [30]. After each study drug administered by inhalation once daily for 28 days, hgd40 significantly attenuates both late and early asthmatic responses and improves lung function. Moreover, the

Th2-regulated inflammatory responses are also attenuated.

Small interfering RNA (siRNA), first discovered in plants and *Caenorhabditis elegans* and later in mammalian cells, is a member of a family of noncoding RNAs (ncRNAs) that affect and regulate gene transcriptional and posttranscriptional silencing [34]. This sequence-specific gene-silencing phenomenon could cause mRNA to be effectively broken down after tran‐ scription, resulting in no obvious translation. SiRNA represents an emerging therapeutic approach against diseases for *in vivo* and *in vitro* studies, and along with novel drug delivery techniques, the challenge of siRNA-based therapeutics is only now being optimized. These discoveries led to a surge in interest in harnessing siRNA for biomedical research and drug development.

### **3.1. The possible mechanisms and challenges of siRNAs**

SiRNAs, synthetic mediators of RNA interference (RNAi), are basically dsRNA molecules designed specifically to silence expression of target genes. Cytoplasmic dsRNA molecules are considered unusual and are substrate for endonuclease Dicer, an RNase III family member. Vertebrate-specific TAR (HIV trans-activator RNA) RNA-binding protein (TRBP) and protein kinase R-activating protein (PACT) help Dicer to identify and dice dsRNA into about 21 bp fragments with 2 nucleotides overhangs at each end, generating the siRNA. Then recognized by an important enzyme Argonaute 2 (AGO2), siRNA of 21-23 nucleotides are incorporated into an RNA-induced silencing complex (RISC). RNA helicases unwind the double-stranded siRNA. The sense strand of the double-stranded siRNA is cleaved during the formation of the RISC complex, and the antisense strand guides RISC to the complementary target mRNA, which is rapidly degraded by RISC (Figure 2) [35, 36].

Though siRNAs can efficiently silence target gene expression in a sequence-specific manner, many challenges, including rapid degradation, poor cellular uptake, off-target effects and immune response, need to be addressed in order to carry these molecules into clinical trials [37, 38]. For example, Chung *et al*. illustrated the underappreciated off-target effects of siRNA gene knockdown technology. Hepatitis C Virus (HCV) depends on a core MOBKL1B (Mps one binder kinase activator-like 1B)–NS5A peptide complex to complete its life cycle. However, without the absence of MOBKL1B, siRNA of MOBKL1B still has off-target inhibitory effects on virus replication [39]. Researchers have tried to develop modified method to reduce the disadvantages. By using the default parameters in siDirect 2.0 Web server (http://siDir‐ ect2.RNAi.jp/), at least one qualified siRNA for >94% of human mRNA sequences in the RefSeq database can be designed [40]. In addition, chemical modifications have been shown to protect siRNAs from nuclease degradation without interfering with siRNA-silencing efficiency [37]. Thus, improvements in rational design strategies might have the potential to make the siRNAs

**Figure 2.** The process of siRNA-mediated degradation of target mRNA in eukaryotic cells. siRNA is recognized by AGO2 and incorporated into the RISC. After that, RNA helicases unwind the double-stranded siRNA, and the anti‐ sense strand guides RISC to the complementary targeted mRNA, which is cleaved by RISC and rapidly degraded.

more effective in the near future and to open the door to development of highly effective and safe therapeutics for clinical applications.

#### **3.2. SiRNAs delivery systems**

Delivery of siRNAs to target tissues is impeded by many barriers at different levels. As possible drugs in the near future, targeted delivery of siRNAs provides remarkable opportunities for accelerating RNAi-based high-performance treatments. The success of siRNAs-based delivery systems may be dependent upon uncovering a delivery route and sophisticated delivery carriers. In this regard, Fujita *et al.* have reported a powerful platform (PnkRNA™ and nkRNA®) to promote naked RNAi approaches through inhalation without delivery vehicles in lung cancer xenograft models. This modified local drug delivery system could offer a promising strategy for enhancing RNAi effects in cancer therapy [41]. In addition, with high binding specificity, nucleic acid aptamer represents a different promising tool for selective delivery siRNAs to cancer cells or tissues, resulting in increasing the therapeutic efficacy as well as reducing toxicity [42]. Likewise, the latest studies in using cell-penetrating peptides (CPPs) combined with molecular cargos, including liposomes, polymers, nanoparticles, and so on, have indicated that for the delivery of siRNAs, the combination strategy can remit the reduced internalization efficiency caused by neutralization [43]. However, each transfection process needs to be optimized because of cell density, siRNA concentration, transfection reagents, etc.

### **3.3. Application of siRNAs – From the bench to the clinic**

more effective in the near future and to open the door to development of highly effective and

**Figure 2.** The process of siRNA-mediated degradation of target mRNA in eukaryotic cells. siRNA is recognized by AGO2 and incorporated into the RISC. After that, RNA helicases unwind the double-stranded siRNA, and the anti‐ sense strand guides RISC to the complementary targeted mRNA, which is cleaved by RISC and rapidly degraded.

Delivery of siRNAs to target tissues is impeded by many barriers at different levels. As possible drugs in the near future, targeted delivery of siRNAs provides remarkable opportunities for accelerating RNAi-based high-performance treatments. The success of siRNAs-based delivery systems may be dependent upon uncovering a delivery route and sophisticated delivery carriers. In this regard, Fujita *et al.* have reported a powerful platform (PnkRNA™ and nkRNA®) to promote naked RNAi approaches through inhalation without delivery vehicles in lung cancer xenograft models. This modified local drug delivery system could offer a promising strategy for enhancing RNAi effects in cancer therapy [41]. In addition, with high binding specificity, nucleic acid aptamer represents a different promising tool for selective delivery siRNAs to cancer cells or tissues, resulting in increasing the therapeutic efficacy as well as reducing toxicity [42]. Likewise, the latest studies in using cell-penetrating peptides (CPPs) combined with molecular cargos, including liposomes, polymers, nanoparticles, and so on, have indicated that for the delivery of siRNAs, the combination strategy can remit the

safe therapeutics for clinical applications.

144 Nucleic Acids - From Basic Aspects to Laboratory Tools

**3.2. SiRNAs delivery systems**

The discovery of RNA interference (RNAi) was approximately 20 years ago, and opened up a new mechanism for gene-silencing therapeutics. Kim *et al.* evaluated the inhibition effect on Notch1 expression by siRNA, and found that Notch1-targeted-siRNA could result in retarded progression of inflammation, bone erosion, and cartilage damage in collagen-induced arthritis (CIA) mice by efficiently inhibiting the expression of Notch1 in mRNA level [44]. Cao *et al.* demonstrated that after silencing the expression of vascular endothelial growth factor (VEGF) by siRNA, the number of living cells on the gel and the mucosa thickness are significantly decreased *in vivo*, which indicated siRNA-targeting VEGF may be useful as a convenient therapeutic option for chronic rhinosinusitis [45]. Similarly, VEGF-siRNA decreases the vesselforming ability and exhibited no testable cytotoxicity by significantly decreasing the expres‐ sion of VEGF mRNA and protein [46].

To date, given the progress of basic research, there are examples of clinical trial projects based on RNAi technology against cancer and other diseases. SiRNA therapeutics is now well poised to enter the clinical formulary as a new class of drugs in the near future. In an open-label phase I/IIa study in the first-line setting of fifteen patients with nonoperable locally advanced pancreatic cancer (LAPC), an siRNA drug (G12D) against KRAS, a Kirsten ras oncogene homolog from the mammalian ras gene family, is well tolerated, safe, and demonstrated a potential therapeutic efficacy to the patients enrolled, when combined with chemotherapy. However, five participants experienced serious adverse events [47]. In addition, a recent systematic analysis of a new RNAi therapeutic agent based on cationic lipoplexes containing chemically stabilized siRNAs, called Atu027, which silences expression of protein kinase N3 in the vascular endothelium in patients with advanced solid tumors. In one case of 24 patients, the study showed that Atu027 is tolerated up to 0.180 mg/kg, and no obvious dose-dependent toxicities are observed [48]. Likewise, the results from another case of 34 patients showed that Atu027 is safe in patients with advanced solid tumors, with 41% of patients having stable disease for at least 8 weeks [49]. Also, because SYL040012 is an siRNA designed to specifically silence β adrenergic receptor 2 (ADRB2) currently under development for glaucoma treatment *in vivo* and *in vitro* [50], a phase I clinical trial of SYL040012 with 30 healthy subjects having intraocular pressure (IOP) below 21 mmHg was conducted [51]. This trial found that admin‐ istration of SYL040012 over a period of 7 days significantly reduced IOP values regardless of the dose used, was well tolerated locally and had no local or systemic adverse events. Thus, taken together, these clinical studies conducted on siRNAs in the past few years indicate that safe and effective target gene knockdown is achievable. Though targeting any individual gene might lead to unanticipated clinical toxicity that could stop the development of any individual siRNA drug, we anticipate a rapid expansion of clinical trials for multiple clinical indications.
