**5. Ribozymes**

**4. Antisense oligonucleotides**

146 Nucleic Acids - From Basic Aspects to Laboratory Tools

Antisense oligonucleotide, first recognized in 1978 by Zamecnik and Stevenson, is a small synthetic piece of DNA (usually 15–18 mer in length) that can bind complementary RNA by Watson-Crick base pairing. ASOs can target most RNA transcripts and have emerged as the ideal therapeutic agents for a broad number of diseases [52, 53]. Upon binding to their target, ASOs can modulate the intermediary metabolism of RNA by the recruitment of endogenous RNase H1 to interfere with RNA function [54]. Human RNase H1 is a ubiquitous enzyme that hydrolyzes the duplex formed between a DNA containing ssASO and target RNA through its N-terminus RNA-binding domain. In order to cleave the RNA in the duplex, the RNase H1 catalytic domain needs at least 5 consecutive DNA/RNA base pairs, and cleavage usually occurs within 7–10 nucleotides from the 5'-end of the RNA. After cleavage, the exposed phosphate on the 5'-end and hydroxyl on the 3'-end are recognized, and the RNA is subse‐ quently degraded by cellular nucleases. At some point after RNase H1 cleaves the RNA, the

Even though much progress has been made in the ASO field so far, there are still many questions that might result in nonspecific effects. One of the principle challenges for success is efficacious delivery to target organs. Because initial ASO molecules are either of low affinity or low membrane permeability, they suffered from poor solubility and rapid degradation by nucleases. In the field, many studies to improve the therapeutic potential of ASOs have focused on chemical modifications to either improve nuclease resistance, such as 2'-O-methoxyethyl (2'-MOE), or to facilitate cellular uptake, like phosphorothioate backbone that improves membrane penetration [55, 56]. Moreover, too many heparin-binding cell surface proteins have been identified to bind the phosphorothioate oligo with nanomolar affinity. The delivery of ASO drug, encapsulating with materials ranging from cationic lipids to dendrimers to alginate/

Over the past several years, antisense oligonucleotide-based targeted therapy has emerged rapidly. Interest in the field has ramped-up dramatically, as numerous ongoing clinical trials are evaluating the treatment effect on diseases with ASOs. Antisense oligonucleotide sodium LY2181308 (LY2181308), hybridizing to the human survivin mRNA, is well tolerated in patients with acute myeloid leukemia (AML). In combination with chemotherapy, LY2181308 does not cause additional toxicity, though 1/16 patients had incomplete responses, and 4/16 patients had cytoreduction [57]. Thus, future clinical trials are needed to further confirm its clinical benefit. In another open-label, parallel-group study, reducing factor XI levels by a second-generation antisense oligonucleotide FXI-ASO (ISIS 416858) is an effective method for prevention of postoperative venous thromboembolism. With respect to the risk of bleeding, FXI-ASO received once daily appeared to be safe [58]. In another phase II trial, compared with those who received placebo, the participants with Crohn's disease who received SMAD7 ASO Mongersen (formerly GED0301) had significantly higher rates of remission and clinical response [59]. Even more important, mipomersen, an antisense agent targeted to apolipopro‐ tein B, has recently received FDA (United States Food and Drug Administration) approval for the treatment of familial hypercholesterolemia (http://www.fda.gov/newsevents/newsroom/

ssASO is released and is available to reengage another transcript.

chitosan nanoparticles, has reached new heights of clinical acceptance [52].

Ribozymes, also termed catalytic RNA, are highly structured RNA sequences that can be engineered to specifically cleave target RNA molecules, similar to the action of protein enzymes. However, unlike protein ribonucleases, ribozymes cleave only at a specific location, using base-pairing and tertiary interactions to help align the cleavage site within the catalytic core. The general mechanism of ribozymes is as follows: a 2'-oxygen nucleophile attacks the adjacent phosphate in the target RNA backbone, resulting in cleavage products with 2', 3' cyclic phosphate and 5' hydroxyl termini [60].

Since ribozymes were accidentally discovered in 1982, it has been shown that RNA can act in at least two ways in biology: as genetic material and as a biological catalyst. Examples of ribozymes include the hammerhead ribozyme, the Leadzyme, and the hairpin ribozyme. In the last several years, crystal structures of these ribozymes have been determined, providing detailed views of the tertiary folds of these RNAs [60, 61], which would be modulated allosterically to increase specificity of ribozyme action.

Compared to other therapeutical RNAs such as siRNAs, the current therapeutic efficacy of ribozymes remains low due to their limited specificity, and structural instability [62]. And furthermore, the amount of free Mg2+ in the intracellular environment plays a critical limitation role for the catalytic activity [63]. To date, gene-therapy-based studies have focused upon developing strategies to stabilize ribozymes and transfect them into live cells. Rouge *et al.* reported the concept of ribozyme-spherical nucleic acid (SNA) conjugates and found that these conjugates could allow high cellular uptake of ribozymes, with favorable catalytic activity and stability [64]. Paudel et al. studied the effect of molecular crowding agents, like polyethylene glycol (PEG), on the folding and catalysis of ribozymes. They demonstrated that PEG favors the formation of the docked structure, which increases ribozymes' activity. In addition, Mg2+ induced folding in the presence of PEG occurs at concentrations ∼ 7-fold lower than in the absence of PEG [65].

Up to now, at least two clinical trials have positively showed the safety, feasibility, and longterm stability of using ribozymes targeted to different mRNAs, such as HIV (human immu‐ nodeficiency virus) elements [66] and VEGF-1 [67]. However, the transduction efficiency left room for improvement. In a phase II cell-delivered gene transfer clinical trial, 74 HIV-1 infected adults enrolled randomly received a tat/vpr specific ribozyme OZ1 or placebo. This study showed that OZ1-based gene therapy is safe, and has modest efficacy. In the future, modifi‐ cations would aim to increase the lymphocyte recovery in order to enhance the therapeutic effect [68]. Another phase II trial of RPI.4610, an antiangiogenic ribozyme targeting the VEGFR-1 mRNA, also demonstrated a well-tolerated safety profile but lacked the clinical efficacy, which results in precluding this drug from further development [69]. Thus, insuffi‐ cient success suggests that further investigation of allosteric regulation is essential to advance the drug development.
