Suppression of Hepatitis C Viral Genome Replication with RNA-Cleaving

Dal-Hee Min and Dong-Eun Kim

Contents 1 Introduction ...... 430 2 Antiviral Antisense ...... 433 2.1 RNA-Cleaving Antisense Oligonucleotides: DNAzymes ...... 433 2.2 Chemical Modifications of Antisense Oligonucleotides ...... 434 3 Various Strategies for Delivery ...... 436 3.1 Oligonucleotide Delivery with Functional Polymers ...... 436 3.2 Oligonucleotides Delivery with Inorganic Nanomaterials ...... 437 4 Suppression of HCV Genome Replication with DNAzyme ...... 439 4.1 In Vitro Selection of DNAzymes that Cleave HCV RNA ...... 439 4.2 Inhibitory Effect of DNAzymes on HCV Replication in Hepatic Cells ...... 442 4.3 Delivery of DNAzyme with Iron Oxide Nanoparticles for HCV Knockdown 444 5 Conclusions ...... 447 References ...... 448

Abstract Downregulation of viral via oligonucleotide-based gene therapy is a potential strategy for the treatment of infection such as hepatitis C. Hepatitis C virus (HCV) is a small-sized, enveloped, positive-sense single-stranded RNA virus. As HCV has highly mutative properties and strong drug resistance, effective for HCV infection is currently unavailable. One of the potential therapeutic strategies for hepatitis C treatment is to cleave HCV RNA genome with proper antisense nucleic acids, thereby inhibiting virus replication in host. RNA-cleaving antisense oligodeoxyribozyme, known as DNAzyme, is an attractive therapeutic oligonucleotide which enables cleavage of mRNA in a sequence-specific

D.-H. Min Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea D.-E. Kim (*) WCU Program and Basic Research Laboratory, Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea e-mail: [email protected]

V.A. Erdmann and J. Barciszewski (eds.), From Nucleic Acids Sequences 429 to Molecular Medicine, RNA Technologies, DOI 10.1007/978-3-642-27426-8_17, # Springer-Verlag Berlin Heidelberg 2012 430 D.-H. Min and D.-E. Kim manner and thus silencing target gene. In this chapter, we discuss current status of functional antisense oligonucleotides that have been applied to inhibit HCV replica- tion in vitro and in vivo. In particular, the DNAzyme and the DNAzyme conjugated nanoparticle system are discussed in detail to demonstrate a successful usage of functional oligonucleotide and its delivery in vivo for further therapeutic application of functional oligonucleotides in the treatment of hepatitis C.

Keywords Antisense oligonucleotide • DNAzyme • Hepatitis C virus • Nano- material-based gene delivery • RNA cleavage

1 Introduction

Infection with hepatitis C virus (HCV) causes chronic hepatitis, if untreated, which can eventually lead to liver cirrhosis and hepatocellular carcinoma (Hoofnagle 2002). Although a combinational therapy of interferon-a and the analog (e.g., ribavirin) brought encouraging results, relatively poor efficacy and significant side effects are shown in over 50% of treated patients, not achieving stable virus clearance (McHutchison and Fried 2003; Shepherd et al. 2000). To date, intensive efforts have been directed to develop novel drugs against HCV; effective anti-HCV drug is not, however, available because of HCV’s high rate of mutation and drug resistance (Zein 2000). Thus, alternative HCV therapeutics in conjunction with the current therapy regimens will be needed in near future to treat HCV-infected patients (Walker et al. 2003). HCV is an enveloped virus with a positive-sense, single-stranded ~9,500 (nt) RNA genome that encodes a single long open reading frame, which is translated into a polyprotein including the core (C), envelope (E1, E2), and nonstructural (NS2 to NS5b) groups of (Choo et al. 1991; Kato et al. 1990) (Fig. 1a). Among different isolates of HCV with considerable variability, HCV genotypes 1a and 1b are most clinically relevant (Takamizawa et al. 1991). in the host is initiated under control of the internal ribosomal entry site (IRES) of 340 nt, which is located at the viral 50 untranslated region (50 UTR) (Tsukiyama-Kohara et al. 1992). HCV IRES is folded into a stable secondary structure and highly conserved among all HCV genotypes, which contains three distinct stem loops (II–IV) and a pseudoknot (Honda et al. 1999) (Fig. 1b). IRES directs the translational machinery to the initiator AUG codon, and mutations in various regions of the IRES cause deleterious effects in translation, which was proved in many in vitro studies. Since the IRES is unique in HCV RNA genome, which is distinguished from the cap-dependent translation of host mRNA, blocking of the IRES region with antisense oligonucleotides (AS-ODNs) could be exploited to achieve selective suppression of HCV (Honda et al. 1996). The IRES-mediated translation synthesizes HCV polyprotein, which is subse- quently processed into mature viral structural and nonstructural proteins by a series Suppression of Hepatitis C Viral Genome Replication 431 a

b

Fig. 1 (a) HCV RNA genome structure. Sites of proteolytic cleavage by NS3 are indicated by arrows.(b) Sequence and secondary structure of internal ribosomal entry site (IRES) located in the 50 UTR of HCV RNA genome of cotranslational and posttranslational cleavages by host signal peptidases (Hijikata et al. 1993; Mizushima et al. 1994) and two viral proteases: NS2-3 (Grakoui et al. 1993) and NS3 (Bartenschlager et al. 1993; Manabe et al. 1994; Tomei et al. 1993). Among the HCV nonstructural gene products, NS3 contains a trypsin-like serine 432 D.-H. Min and D.-E. Kim protease activity (see Fig. 1a for cleavage by NS3) and a helicase activity in the N- terminal and C-terminal, respectively (Yao et al. 1995). The viral protease NS3 has been identified as an attractive target for anti-HCV drugs, because its activity is indispensable for processing many of the nonstructural proteins of HCV. As an effective modality to treat the HCV, selective attenuation of the expression of viral genes is counted as one of the appealing antiviral strategies. As such, specific knockdown of the viral gene expression with functional nucleic acids has attracted considerable attention, which is regarded as gene therapy. Gene therapy generally refers to one of the therapeutic options to treat diseases caused by defects in gene expression by regulating gene expression at a posttranscriptional level (Dobson 2006; El-Aneed 2004; Labhasetwar 2005). To date, there have been tremendous efforts to utilize small functional oligonucleotides to specifically inhibit aberrant target genes, including short interfering RNA (siRNA) (McManus and Sharp 2002), antisense oligonucleotide (McMahon et al. 2011), (Lewin and Hauswirth 2001), and deoxyribozyme (DNAzyme) (Dass et al. 2008; Isaka 2007). One of the most significant advantages of using these agents for disease treatment is that almost all of the diseases caused by unregulated gene expression—including cancer and viral diseases—may be “treatable” by blocking synthesis through degrada- tion of the related mRNAs. Traditional drugs based on small organic compounds are limited in their function because they inhibit activities and/or protein–protein interactions by exerting on “already expressed proteins.” As a potential target for oligonucleotide-based gene therapy, pathogenic HCV RNA genome has been targeted by various antisense oligodeoxynucleotides (AS-ODNs) to selectively inactivate replication of the viral genome. Several groups have tested AS-ODNs that inhibit HCV viral genome replication and viral polyprotein synthesis both in vitro and in mice models (Alt et al. 1999; Brown-Driver et al. 1999;Hanecak et al. 1996;Limaetal.1997; Mizutani et al. 1995;SekiandHonda1995; Wakita and Wands 1994; Yao et al. 1995; Zhang et al. 1999). Phase I/II clinical trials on chronically HCV-infected patients with a phosphorothioate-modified antisense oligonucleotide (ISIS14803) were carried out but were stopped for reasons of lack of efficacy (McHutchison et al. 2006). Despite great potential of small oligonucleotides for disease treatment, delivery of the oligonucleotides remains the major obstacle to its therapeutic application because of its fast degradation by in physiological condition, inefficient cellular uptake, and lack of targeting capability. If AS-ODNs that were sensitive to degradation were used without appropriate delivery vehicle, low-affinity profiles toward their target and side effects were often observed in vivo (Crooke 2004). In addition, the therapeutic use of oligonucleotides is mainly challenged by delivery after systemic administration. The administered oligonucleotides need to travel through the bloodstream, out of the circulation, and act against the target cells. Then, it must find its target mRNA and be knocking out the message. Therefore, development of effective delivery vehicles is essential for successful oligonucleotide-based therapy. Notable delivery methods for small oligonucleotides among the ones developed to date will be discussed in the following section by showing successful demonstration in their strategy. Despite the drawbacks of the Suppression of Hepatitis C Viral Genome Replication 433

AS-ODNs and delivery issues, catalytic oligonucleotides such as DNAzymes, , and small interfering have attracted a particular attention at the present time (De Francesco and Migliaccio 2005). This review will focus exclu- sively on the recent DNAzyme strategies against HCV to give a practical insight toward functional AS-ODNs. Detailed study of isolation of DNAzymes that specifi- cally cleave HCV RNA genome and suppress HCV genome replication in cells and mice will be discussed.

2 Antiviral Antisense Oligonucleotides

2.1 RNA-Cleaving Antisense Oligonucleotides: DNAzymes

One of the strategies to inhibit HCV replication in cells is to cleave HCV RNA genome with either ribozyme or deoxyribozyme (Bartolome et al. 2004; Oketani et al. 1999; Shippy et al. 1999; Trepanier et al. 2006). For example, natural hammerhead ribozyme self-cleaving motif has been modified to bind and cleave the passenger RNA strand (Fig. 2A). The modified version of natural hammerhead ribozyme (trans-cleaving hammerhead ribozyme) was applied to cleave the target RNA in vitro (Choi et al. 2008; Ludwig et al. 1998). Ribozymes perform catalytic reactions with great precision through Watson–Crick base pairing with comple- mentary sequences. Because ribozymes can be encoded and transcribed from DNA, administration of ribozyme has been accomplished by elaborate vector designs used for overexpression of the ribozyme. Alternatively, efforts have also been made in the synthesis and delivery of ribozymes, not using expressible gene vectors. How- ever, ribozymes are not effective due to their short half-life and difficulty in large- scale synthesis. A decade later, similar but different class of RNA-cleaving oligonucleotides, , has entered into the stage of gene therapy tools because of their small size, ease of synthesis, and moderate resistance to chemical and enzymatic degradation (Steele et al. 2003). Deoxyribozymes, the RNA-cleaving short DNA , also named as DNA (DNAzymes), were derived by in vitro selection from a combinatorial library of DNA sequences that are capable of cleaving a short target RNA in a sequence-specific manner (Santoro and Joyce 1997). DNAzyme possessing a catalytic motif of “10-23” has a potential to bind and cleave any target RNA that contains a purine–pyrimidine junction (Santoro and Joyce 1997) (Fig. 2A), allo- wing far greater flexibility in choosing the target sites than hammerhead and hairpin ribozymes. The Watson–Crick base pairing of binding arms confers substrate recognition and binding of DNAzyme to any desired target site of RNA, which makes DNAzyme a powerful tool for gene inactivation strategies. DNAzymes offer several advantages, including cost-effectiveness, straightforward chemical modifi- cation, and relatively high stability in serum compared to RNA (Appaiahgari and Vrati 2007). 434 D.-H. Min and D.-E. Kim a

b Base Base Base O O O O N O Base O O O O -O -O OMe -O O Base P O Base P O Base P O Base N N S O O O O O O P N O O Base N O O O -O O -O OMe -O O P O P O P O Base N N S O O

(a) (b) (c) (d) (e)

Fig. 2 (A) RNA-cleaving antisense oligonucleotides: hammerhead ribozyme and 10-23 DNAzyme. (B) Chemically modified antisense oligonucleotides: (a) phosphorothioate, (b) 20-O- methyl oligoribonucleotides, (c) locked nucleic acids (LNA), (d) , (e) (PNA)

The potential utility of DNAzymes as effective gene therapy agents has been demonstrated by experiments in which pathogenic gene expression was modulated at the posttranscriptional level in various clinically relevant model systems in vitro and in vivo (Achenbach et al. 2004; Dass 2004). A number of research groups have utilized DNAzymes to selectively cleave and disrupt the function of target genes (Lu et al. 2005; Santiago et al. 1999; Wu et al. 1999). Despite the flexibility in potential cleavage sites for DNAzyme, selection and identification of DNAzymes that might be effective and optimal in binding and cleavage of the target mRNA have to be determined empirically. Efforts to predict RNA secondary structure and its influence on the binding of other nucleic acids through free-energy minimization algorithm were not accurate, particularly in long RNAs (Heale et al. 2005; Kretschmer-Kazemi Far and Sczakiel 2003; Luo and Chang 2004; Schubert et al. 2005; Westerhout and Berkhout 2007). Clearly, finding accessible regions for DNAzyme binding in the long stretch of target RNAs gives a major limitation to the effectiveness of DNAzymes. In addition to these challenges, like other oligonucleotides, the lack of a safe, efficient system for the delivery of DNAzymes to target disease sites and tissues remains a major obstacle to the clinical use of DNAzymes.

2.2 Chemical Modifications of Antisense Oligonucleotides

Oligonucleotides without chemical modifications are generally vulnerable to nucle- ase degradation in vivo. To overcome this problem, AS-ODNs have been Suppression of Hepatitis C Viral Genome Replication 435 chemically modified on the sugar and the backbone. As the first and second generation, AS-ODNs were chemically modified on backbone linkage with replacement of oxygen atom to sulfur (phosphorothioates) [Fig. 2B(a)], methyl group (methylphosphonates), or amines (phosphoramidates). Among these, the phosphorothioates have been the most successful in gene silencing due to their sufficient resistance to nucleases. Alternatively, chemical modification has been also applied to the position 20 of ribose with an alkoxy group (e.g., methyl group) [Fig. 2B(b)]. For example, antisense 20-O-methyl oligoribonucleotides targeting the HCV IRES were shown to inhibit translation of a HCV RNA genome in rabbit reticulocyte lysate (Tallet-Lopez et al. 2003). DNAzymes have been also modified using the phosphorothioate linkages to render the oligonucleotide more resistant to endogenous nucleases. However, such alterations resulted in several deleterious sequence-independent effects such as toxicity (Wahlestedt et al, 2000), immuno- logic responsiveness (Fluiter et al. 2003), and increased affinity for cellular proteins (Rockwell et al. 1997). The third generation of chemically modified oligonucleotides containing struc- tural elements has shown an efficient control of HCV replication in vitro, including locked nucleic acids (LNAs) (Laxton et al. 2011), peptide nucleic acids (PNAs) (Alotte et al. 2008), and morpholino ODN (McCaffrey et al. 2003). All of the modifications enhanced nuclease resistance without hampering specific binding to the target RNA sequence. As shown in Fig. 2B(c), LNA nucleotide has the ribose moiety modified with an extra bridge connecting the 20 oxygen and 40 carbon, which effectively increases affinity for complementary sequences (Braasch and Corey 2001). The LNA-incorporated oligonucleotides showed increased thermal stability of duplexes toward complementary DNA or RNA, stability toward 30 exonuclease degradation (Petersen et al. 2000). Recently, LNA-based ODN targeting the host microRNA miR-122 entered to the phase II clinical test as of late 2010 (Lanford et al. 2010). LNAs have been also applied to modify DNAzymes (Vester et al. 2002; Schubert et al. 2003) to increase binding affinity. LNA incorporation into DNAzymes, however, has been found to diminish both catalytic activity under single turnover conditions (Vester et al. 2002) and biological potency (Fluiter et al. 2005). Alternatively, [Fig. 2B(d)] are another promising ODNs that pos- sess favorable hybridization, nuclease stability, and toxicity profiles. Morpholino ODNs designed to target the HCV IRES prevented HCV IRES translation in a preclinical mouse model (McCaffrey et al. 2003). In addition, PNAs are nucleic acid analogs containing natural nucleoside bases on a pseudo-peptide backbone with strong affinity to DNA and RNA sequences (Nielsen 1997) [Fig. 2B(e)]. PNA ODN targeting HCV IRES sequence showed a strong inhibition of HCV IRES- driven translation in a rabbit reticulocyte lysate (Alotte et al. 2008). Despite its specific affinity to and strong knockout of the target RNA, PNA needs an appropri- ate transfection reagent to be delivered into the cell because of its charge neutrality and insolubility. 436 D.-H. Min and D.-E. Kim

3 Various Strategies for Oligonucleotide Delivery

An efficient and biocompatible delivery system for oligonucleotides is required for its successful clinical application. The carrier type can be classified into two categories—viral and nonviral approaches (Park et al. 2006). Viral vectors have proven their efficiency in the transfection, but there remains safety issue as a major concern of long standing. Nonviral approaches include various cationic lipids, polymers, proteins (e.g., protamine), and various inorganic nanomaterials (Fig. 3). This section will focus on nonviral approaches for oligonucleotide delivery with more in-depth discussion for nanomaterial-based delivery strategies which are recently explosively studied.

3.1 Oligonucleotide Delivery with Functional Polymers

Cationic Lipids. To date, various drugs have been formulated into liposomes which consist of cationic phospholipids to improve cellular uptake and pharmacokinetics (Fig. 3a). Liposomes can fuse with cell membranes and thereby induce efficient cellular entry of drugs. Commercially available phospholipids for oligonucleotide transfection (i.e., Lipofectamine 2000) are typically in the form of mixtures of cationic, fusogenic, and other types of lipids. The cationic lipid-based delivery systems have been extensively used to deliver oligonucleotides into mammalian cells (Li and Szoka 2007). Many successful demonstrations have been reported for oligonucleotide delivery assisted with cationic lipids in vitro and in vivo (Zimmermann et al. 2006; Khoury et al. 2006; Morrissey et al. 2005). However, cytotoxicity of cationic lipid itself should be overcome to be more widely useful as a therapeutic formula. Polymers. Polyethylenimine (PEI; Fig. 3b) is one of the most widely used cationic polymers for oligonucleotide delivery (Ge et al. 2004; Schiffelers et al. 2004; Grzelinski et al. 2006). PEI–oligonucleotide complexes are thought to elec- trostatically interact with cell surface and be internalized by endocytosis. There are a number of reports showing therapeutic applicability of PEI for oligonucleotide delivery in many disease models. One of the concerns on PEI is the high toxicity at raised concentrations. Several studies are reported on improving biocompatibility and reducing cytotoxicity of PEI by chemical modification. Other types of cationic polymers based on polyhistidine and polylysines are also demonstrated for their capability as oligonucleotide delivery vehicles. Proteins. Proteins presenting high cationic charge densities are also harnessed for oligonucleotide delivery (Choi et al. 2010). Protamine-antibody conjugate protein (Fig. 3c) was used for targeted siRNA delivery showing high efficacy (Song et al. 2005). Antibody served as a targeting moiety for receptor-specific binding to cells and protamine noncovalently adsorbed siRNA. This approach minimized adopting synthetic or artificial components in the system. Suppression of Hepatitis C Viral Genome Replication 437

H H H N N H 2 N * N

Cationic lipid Polymers (PEI)

+ + + NM + + + + + + + +

Inorganic nanoparticles Protein (Protamine) (NM: nanomaterials)

Fig. 3 Various strategies for oligonucleotide delivery designed for therapeutic applications

3.2 Oligonucleotides Delivery with Inorganic Nanomaterials

Recent rapid progress in nanotechnology accelerated the development of new interdisciplinary research areas. Nanomedicine refers to the use of nanomaterials to devise medical innovations in a wide range of medical applications from drug discovery and delivery to biomarker discovery and diagnostics (Sekhon and Kamboj 2010). Specifically, inorganic biomedicine bridges inorganic nanomaterials and biomedicine by actively utilizing unique physical and chemical properties of proper inorganic nanomaterials to biomedical applications. Com- monly used inorganic nanomaterials (Fig. 3) include gold nanoparticle (AuNP) (Patel et al. 2010), quantum dot (QD) (Han et al. 2001), and magnetic nanoparticle (MNP) (Brown et al. 2005). When gold, silver, and even semiconductors are made in very small sizes, usually below hundreds of nanometers, they no longer behave in 438 D.-H. Min and D.-E. Kim ways they did in bulk. These nanomaterials possess unique physicochemical and optical properties which are beneficial for various biological applications including oligonucleotide delivery. For example, AuNP induces surface plasmon resonance for facile optical detection, and semiconductor nanoparticles present strong fluo- rescence with very narrow emission spectra at different wavelength depending on their size. One of the important advantages of using inorganic nanomaterials in drug delivery is that multifunctionality can be modularly rendered into the delivery system by introducing targeting , imaging probes (often times, nanoparticle itself serves as an imaging agent), multiple drugs, and triggered drug releasing system into/onto the pertinent nanomaterials while controlling size and shape of delivery vehicle. To date, DNA has been readily conjugated to inorganic nano- materials which can form structures of a limited number of particles or aggregates of these particles (Deng et al. 2005; Zhao et al. 2006). Since DNA can act as a scaffold due to its potential to form organized microstructures, the development of DNA delivery vehicles with various inorganic nanoparticles presents a facile and feasible challenge. AuNPs can be relatively easily synthesized by chemical reduction of chloroauric acid. Tuning synthetic condition generates AuNPs with various sizes ranging from a few to hundreds nanometers. Due to well-developed surface modification techniques, attachment of biofunctional molecules onto AuNP surface can be easily performed via specific interaction between thiol functional groups and gold surface. The Mirkin group (Giljohann et al. 2009) and the Rotello group (Ghosh et al. 2008) are leading groups in the bioapplication of AuNP. AuNPs coated with oligonucleotides were used to deliver oligonucleotides into mammalian cells with appreciable efficiency (Ghosh et al. 2008). It is believed that nanosized objects can be easily engulfed into cells with different degrees depending on the shape, relative size, surface charge, and coating material. Low cytotoxicity of AuNP made it very attractive nanomaterial for in vivo application. QD is one of the semiconductor nanomaterials made of cadmium selenide or cadmium telluride (Xing and Rao 2008). Nowadays, QD is gradually replacing conventional organic dyes in some bioprobing experiments due to bright fluores- cence, narrow emission spectra, broad excitation wavelength, and lack of photobleaching even under prolonged irradiation (Smith et al. 2009). QD is reported to show gene silencing when introduced to cells in the form of QD–oligonucleotide complex whereby location of the drug delivery vehicle can be monitored by imaging of QD fluorescence in situ (Derfus et al. 2007). However, safety of QD is an issue to be solved since UV irradiation is reported to induce the release of extremely toxic cadmium metal from QD (Derfus et al. 2004). MNP has been popularly used for MRI contrast agent. MNP is considered to be much safer than other nanomaterials such as QD for in vivo application. Dextran- coated iron oxide MNP is one of the commonly used MNPs for MRI, which is approved by United States Food and Drug Administration (Sekhon and Kamboj 2010). The MNP exhibits long circulation time and enhances MRI visualization of tumors. There have been efforts to use MNP–oligonucleotide complex to achieve Suppression of Hepatitis C Viral Genome Replication 439

Inorganic nanomaterial

Fluorescent dye

Oligonucleotides

Targeting agent

Cell

Fig. 4 A structure of multifunctional nanoparticle target in cultured cells and in vivo (Agrawal et al. 2009). This approach allows “noninvasive imaging” of the delivery vehicles in live objects to track localization, accumulation, and secretion of the MNP–oligonucleotide complexes. Scientists have developed MRI probes with better performance as a contrast agent by tuning sizes or composition of MNPs (Jang et al. 2009). Multi- functional MNPs have been well explored to present targeting moiety, drug, and imaging agent in one nanoparticle (Huh et al. 2005; McCarthy and Weissleder 2008) (Fig. 4). Chemical functionalization of MNP with antibodies, peptides, proteins, organic fluorescent dyes, and oligonucleotides could allow the strategy for the multifunctional MNP.

4 Suppression of HCV Genome Replication with DNAzyme

4.1 In Vitro Selection of DNAzymes that Cleave HCV RNA

AS-ODNs that direct their target by Watson–Crick base pairing need to be designed by avoiding the underlying secondary structure of target RNA, which hampers accessibility of AS-ODNs. Because of unpredictable steric and topological constraints of long stretch of RNA, knowledge of the location of unpaired loop does not guarantee effective hybridization sites (Lima et al. 1997; Matveeva et al. 1997). To overcome this problem, pools of 10-23 DNAzymes were constructed, which possess randomized annealing arm sequence (Dz pools I and II in Fig. 5), and screened for accessible sequences in full-length transcript of HCV NS3 RNA as a target RNA (Lee et al. 2010). The Dz pool I contains oligonucleotides (58 nt) consisting of a central 10-23 catalytic core motif (15 nt) flanked by two binding 440 D.-H. Min and D.-E. Kim

Fig. 5 DNAzymes used as random sequences pool for in vitro selection of DNAzymes that cleave target RNA arms of randomized sequences, and the Dz pool II contains defined sequences for PCR amplification at both 50/30 ends of Dz pool I sequence. Sequence diversity was created by randomizing sequences of two arms totaling 16 nt (8 and 7 Ns plus 1 purine). The fixed terminal sequences of Dz pool II were designed to use PCR amplification of the DNAzymes that are bound to the target RNA substrate in the absence of magnesium. Selection of accessible cleavage sites by DNAzyme was performed using com- binatorial approach as shown in Fig. 6, which is precisely described elsewhere (Lee et al. 2010). The cleaved RNA products from either Dz pool I or II were subjected to primer extension and analyzed on sequencing gels in comparison with the corresponding dideoxy sequencing ladder, and the cleavage sites were identified. Eighteen cleavage sites were identified in the HCV NS3 target RNA (Table 1). The positions of effective DNAzymes in the full-length target RNA tended to be clustered into groups separated by regions containing unreactive sites. In many cases, relatively active target sites such as cleavage sites 1,476–1,488, 904–911, 670–681, and 468–475 nt (Table 1) were very close or even overlapping. This result indicates that these DNAzyme target sites may form an accessible single-stranded loop in the RNA secondary structure (Lee et al. 2010). Eight DNAzyme-working sites were chosen out of eighteen identified cleavage sites based on the band intensity of cleavage products in the sequencing gel, and the Suppression of Hepatitis C Viral Genome Replication 441

Fig. 6 Schematic of DNAzyme selection procedure by using the randomized DNAzyme pools [see the reference for detail; Lee et al. (2010)] corresponding DNAzymes were synthesized (Table 1). Cleavage of the target RNA by these synthesized DNAzymes was examined individually. Products of RNA cleavage reaction were observed with the Dz681 and Dz904 targeting on 681st and 904th base of the NS3 RNA, respectively (Fig. 7a). The RNA cleavage activity was more prominent with a shorter RNA substrate (500 nt) of HCV NS3 RNA fragment (501st base to 1,000th base, Fig. 7b). Potential cleavage sites for DNAzymes identified after the screening process mainly reside at a localized portion of the 442 D.-H. Min and D.-E. Kim

Table 1 Cleavage sites in the HCV NS3 RNA screened with the DNAzyme library [adapted from Lee et al. (2010)] RNA sequence Cleavage DNAzyme sequencea site (base) 50UGGCAUGCAIUGUCAGCU 30 1,488 – 50AUCAUGGCAIUGCAUGUC 30 1,484 (Dz1488)50GACATGCAggctagctacaacgaGCCATGAT 30 50CCAAAUUCAIUCAUGGCA 30 1,476 – 50UAGGAGCCGIUCCAAAAU 30 1,434 (Dz1434)50ATTTTGGAggctagctacaacgaGGCTCCTA 30 50ACGGGCCAAICACCCCUG 30 1,407 (Dz1407)50CAGGGGTGggctagctacaacgaTGGCCCGT 30 50GGUUGCCCGIUCUGCCAG 30 1,191 – 50GCGGGCUUAICCUGAAUA 30 1,168 – 50UGUAACACAIUGUGUCAC 30 911 – 50ACUGUAACAICAUGUGUC 30 909 – 50GAUUGACUGIUAACACAU 30 904 (Dz904)50ATGTGTTAggctagctacaacgaAGTCAATC 30 50GACAUCUCAIUUUUCUGC 30 717 – 50GCAAAGCCAIUCCCCAUU 30 681 (Dz681)50AATGGGGAggctagctacaacgaGGCTTTGC 30 50CUUCUACGGICAAAGCCA 30 673 – 50CCCCUUCUAICGGCAAAG 30 670 (Dz670)50TTTTGCCGggctagctacaacgaAGAAGGGG 30 50ACCCCAAUAIUCGAGGAG 30 627 – 50CGCCCUACAICAUCAUAA 30 475 (Dz475)50TTATGATGggctagctacaacgaGTAGGCGC 30 50CCGGGGGGGICCUACGAC 30 468 (Dz468)50GTCGTAGGggctagctacaacgaGCCCCCGG 30 50ACCCCAACAIUCAGAACU 30 375 – NA NA (DzI)50GCATCAAAggctagctacaacgaTGTGTAGA 30 50GCAAAGCCAIUCCCCAUU 30 681 (mtDz681)50AATGGGGAggctatgtacaacgaGGCTTTGC 30 50GCAAAGCCAIUCCCCAUU 30 681 (asDz681)50AATGGGGAGGCTTTGC 30 aDNAzyme sequences (name in parenthesis) are designed to be complementary to sequences flanking the selected target cleavage site. The 10-23 catalytic motif is indicated in lower case and the sequence for the target binding arms is in upper case

RNA stretch. Therefore, a strategy to identify DNAzyme target sites together with in vitro RNA cleavage assay demonstrates its usefulness in design of RNA-cleaving oligonucleotides targeted to a long-stretched RNA (Lee et al. 2010).

4.2 Inhibitory Effect of DNAzymes on HCV Replication in Hepatic Cells

The selected DNAzymes were examined for inhibition of HCV genome replication in a system, which mimics persistent infection by harboring HCV genome (Lee et al. 2010). HCV permissive hepatoma cell line Huh-7 was transfected with HCV subgenomic replicon RNA that was transcribed from HCV replicon (pFK-I389neo/NS3-30/5.1) (Lohmann et al. 2001) and placed under antibiotic selective pressure. Cell viability under G418 selection pressure reflects ongoing replication of HCV replicon in these cells. Each DNAzyme was transfected into the HCV replicon-transfected cells, and the cell viability was assessed 48 h after transfection in G418-containing media. As shown in Fig. 8, cell viability was significantly reduced in HCV replicon cells treated with four Suppression of Hepatitis C Viral Genome Replication 443

1 1,543 500 Target RNA 1,000

5’ HCV NS3 gene 3’

Dz 681 Dz 904

ab

M C1 C2 Dz1484 Dz1434 Dz1407 Dz904 Dz670 Dz681 Dz468 Dz475 C1 C2 Dz1484 Dz1434Dz1407 Dz904 Dz670 Dz681 Dz468 Dz475 Substrate RNA Substrate RNA (500 bs) (1.54 kbs)

Fig. 7 HCV RNA cleavage by the selected DNAzymes in vitro. Cleavage of (a) the full-length HCV NS3 RNA and (b) 500-nt fragment of NS3 RNA

DNAzymes such as Dz904, 681, 670, and 468 (P < 0.005). As a control, a DNAzyme with irrelevant sequence that does not match NS3 target gene (DzI) was also tested. DzI did not show a significant efficacy against HCV replicon cells. The other control DNAzyme (mtDz681), which abrogated RNA cleavage activity by changing two nucleotides in the catalytic motif (see Fig. 5) but retaining the same binding arm sequences as of Dz681, was tested for cell viability of the HCV replicon cells. In addition, another variant of the most efficient DNAzyme (Dz681), which contains only the binding arm sequences of Dz681 without the catalytic motif (asDz681), was assayed to address if the target binding site is also available for conventional antisense oligodeoxynucleotide (ODN). Both mtDz681 and asDz681 significantly decreased the cell viability of the HCV replicon-transfected cells by 23% and 40%, respectively (Fig. 8a), suggesting that antisense ODN against the same target site as for Dz 681 is also effective. In order to determine whether the selected DNAzymes can inhibit the propaga- tion of the HCV replicon by eliminating the HCV RNA and protein expression, analysis and RT-PCR were performed (Lee et al. 2010). In the Western blot analysis for the NS3 expression, Dz681 showed the most potent inhibition of NS3 expression among 10 ODNs tested including the Dz681 variants (mtDz681 and asDz681) (Fig. 8b). At 48 h posttransfection with Dz681, levels of HCV NS3 protein were substantially decreased, when compared with the constitu- tive expression of b-actin (internal control), which was also observed with its antisense version (asDz681). Other DNAzymes including the control DNAzyme (DzI) that was ineffective in the cell viability decrease such as Dz475, 1407, 1434, and 1484 have failed to show significant decrease in the level of HCV NS3 protein in cells. Overall, the extent of inhibition of NS3 protein expression by each DNAzyme is correlated with the result of the cell viability test. Semiquantitative RT-PCR was also performed with HCV NS3-specific primers to further examine the downregulation of viral RNA by HCV NS3-specific DNAzymes. As shown in Fig. 8b, the HCV NS3 RNA expression was decreased by DNAzymes, Dz904, 670, 444 D.-H. Min and D.-E. Kim

a

P < 0.005

∗ ∗

*

DzI Dz904Dz670 Control Dz1488Dz1434 Dz681Dz468 Dz475 mtDz681asDz681 Dz1407 b

C I mtDz681asDz681 Dz1488 Dz1434 Dz1407 Dz904 Dz670 Dz681Dz468 Dz475 NS3 protein β-actin Western analysis

NS3 mRNA β-actin RT-PCR analysis

Fig. 8 (a) Cell viability assay to assess the efficacy of DNAzymes on suppression of HCV genome replication in Huh-7 cells. (b) Effect of DNAzymes on inhibition of target gene NS3 expression, protein, and mRNA [Recap from Lee et al. (2010)] and 681, which is consistent with the results of Western blot analysis and cell viability test. Interestingly, antisense ODN (asDz681) targeting the Dz681 binding site less inhibited an expression of viral NS3 RNA than that of NS3 protein. We speculate that inhibition of target protein expression was more pronounced than that of the corresponding RNA due to a working mechanism of antisense ODN in cells, in which antisense ODN reduces protein expression by hindering of ribosomal assembly and translation in addition to RNase H degradation of RNA.

4.3 Delivery of DNAzyme with Iron Oxide Nanoparticles for HCV Gene Knockdown

After the RNA-cleaving DNAzyme targeting the HCV NS3 RNA genome was identified, an iron oxide nanoparticle-based system has been recently developed for Suppression of Hepatitis C Viral Genome Replication 445

Fig. 9 Schematic of the multifunctional iron oxide nanoparticle for DNAzyme delivery (Dz DNAzyme, MPAP myristoylated polyarginine peptide, Cy5.5 fluorescent dye, CPP cell- penetrating peptide) [Recap from Ryoo et al. (2012)]

the delivery of DNAzymes to treat hepatitis C (Ryoo et al. 2012). In this study, the iron oxide nanoparticles (MNs) were designed to have a magnetic core, coated with dextran, and are conjugated to the DNAzyme (Dz681) targeting the HCV NS3 RNA, a near-infrared fluorescent dye (Cy5.5), and a cell-penetrating peptide (CPP) that aids in membrane translocation (Fig. 9). The conjugated Cy5.5 dye enables tracking of the therapeutic nanoparticle in vitro and in vivo using fluorescence imaging, and the iron oxide core can be used for tracking via noninvasive magnetic resonance imaging. The fluorescent dye Cy5.5 was conjugated to the aminated MNs using Cy5.5 N-hydroxysuccinimide ester. Cy5.5–MNs were conjugated to thiolated Dz and a myristoylated polyarginine peptide (MPAP; myristic acid-ARRRRRRRC), using a cross-linker, sulfosuccinimidyl-6-(30-(2-pyridyldithio)-propionamido)- hexanoate (sulfo-LC-SPDP). MPAP which acts as a cell-penetrating peptide was used to enhance the efficiency of cellular uptake (Nelson et al. 2007). In the MN, linked to both Dz and MPAP (Dz–MPAP–MN), Dz and MPAP were attached via a disulfide linkage which can be cleaved in reducing environments, such as that in the cytoplasm (Saito et al. 2003). Uptake of MN, Dz–MN, and Dz–MPAP–MN by the HCV replicon cells was evaluated by Cy5.5 fluorescence microscopy 12 h after MN treatment. Cells transfected with the HCV subgenomic replicon RNA containing luciferase reporter 0 gene (pFK-I389neo/NS3-3 /5.1) (Lohmann et al. 2001) were used for assessing inhibition of HCV genome replication in cells by luciferase activity measurement and Western blot analysis. NS3-silencing efficiency of Dz-conjugated nanoparticles transfected in Huh-7 Luc-Neo cells was analyzed in vitro by Western blot analysis of HCV NS3 expression. The Dz-conjugated nanoparticles efficiently inhibited the HCV NS3 replication in cultured Huh-7 Luc-Neo cells. In addition, luciferase (reporter gene) assays of Huh-7 Luc-Neo cells treated with Dz–MPAP–MN indi- cate dose-dependent downregulation of the NS3 target gene. As shown in Fig. 10, Dz–MPAP–MN was most efficiently internalized and accumulated, primarily at the perinuclear region in the Huh-7 cells, whereas MN and Dz–MN were rarely observed inside the cells. Thus, the addition of the MPAP cell-penetrating peptide to Dz–MN greatly increases the extent of cellular uptake and NS3 knockdown. To examine the efficacy of the Dz-conjugated nanoparticles in vivo, mice were injected subcutaneously with Dz–MPAP–MN-treated Huh-7 replicon cells 446 D.-H. Min and D.-E. Kim

No treat MN Dz-MN Dz-MPAP-MN

Cy5.5/Hoechst 33342

Fig. 10 Fluorescence images of Huh-7 Luc-Neo cells after treatment with various magnetic iron oxide nanoparticle (MN) species (Blue Hoechst 33342-stained nuclei, red Cy5.5-conjugated MNs). Cellular uptake efficiency was much higher for the MPAP-conjugated MN than for the MN species lacking MPAP. Scale bar is 20 mm

a b c

d

Fig. 11 Analysis of Dz–MPAP–MN efficacy in vivo and biodistribution of Dz–MPAP–MN. (a) Subcutaneous injection of the HCV replicon cells treated with Dz–MPAP–MN into mice reduced the luciferase signal, indicating diminished HCV replication in vivo. (b–d) Biodistribution of Dz–MPAP–MN after intravenous administration to mice; (b) a whole-body image showing intense Cy5.5 fluorescence in the area of the liver, (c) fluorescence images of the extracted organs show that the particles accumulate primarily in the liver, (d) bright field and fluorescence images of the extracted liver tissue slice are shown. Cellular uptake and accumulation of the intravenously injected Dz–MPAP–MNs were observed in both hepatocytes and Kupffer cells [Recap from Ryoo et al. (2012)]

(Ryoo et al. 2012). Although the ideal test for the in vivo efficacy of drug-loaded nanoparticles would use a virus-infected animal model, small animals (e.g., mice) are not susceptible to infection by human , such as HIV-1 and HCV. Indeed, Suppression of Hepatitis C Viral Genome Replication 447

HCV infects only humans and chimpanzees (Bukh 2004). In fact, generating xenograft tumors of the HCV replicon Huh-7 cells was not straightforward; the subcutaneously injected cells rapidly lost all of their luciferase activity, within 4 days, because the host rejection process rapidly eliminated the replicon RNA (Zhu et al. 2006). As shown in the whole mouse image in Fig. 11a, the cells co- injected with Dz–MPAP–MN exhibited a lower luciferase signal than the controls, suggesting that Dz–MPAP–MN may effectively downregulate its target gene in vivo. For practical application of systemically introduced nanoparticles, the particles should accumulate in the liver, the site of Dz function. When mice were injected with Dz–MPAP–MN via their tail veins, the particles were found exclu- sively in the liver within 30 min after injection, as shown by Cy5.5 fluorescence imaging of the nanoparticles and ex vivo imaging after organ extraction (Fig. 11b, c). Examination of liver tissue sections by fluorescence microscopy showed that the injected particles were taken up by both hepatocytes and Kupffer cells (liver macrophages) in the liver. The accumulation of the particles in both types of cells, rather than just in the Kupffer cells, is an encouraging sign for their potential use for hepatitis C treatment (Fig. 11d).

5 Conclusions

RNA-cleaving DNAzymes are attractive functional AS-ODNs as therapeutic candidates, because (1) their ability to cleave RNA substrates in a sequence- specific manner can be used to silence target genes, (2) they are much more stable than ribozymes, and (3) they are less expensive than siRNA molecules. Since a key challenge for clinical application of functional AS-ODNs as an antiviral agent is to develop an efficient and safe delivery system, several nonviral delivery systems for oligonucleotides have been developed. Among them, inorganic nanoparticles are well characterized as DNA delivery and imaging agents. As an example for a usage of DNAzyme against RNA virus such as HCV, DNAzymes possessing randomized annealing arm sequence has been used to identify accessible cleavage sites in the target RNA, HCV NS3 RNA that encodes viral helicase and protease. When the selected DNAzyme was transfected into the hepatoma cells harboring the HCV subgenomic replicon, the DNAzyme efficiently inhibited HCV RNA replication by reducing the expression of HCV NS3. Therefore, the HCV genomic RNA-cleaving DNAzyme is a new tool for viral gene modulation, which can mitigate the chronic HCV replication in vivo, if combined with an appropriate delivery vehicle. To this end, the DNAzyme delivery system that harnesses well-characterized iron oxide nanoparticles was developed and exhibited effective silencing of expression of the HCV NS3 gene in mice. Thus, the presented study is an updated information regarding a demonstration of an effective design and validation of functional DNAzyme against viral RNA genome and its delivery system with a high potential for future clinical use in the treatment of hepatitis C. 448 D.-H. Min and D.-E. Kim

Acknowledgments This work was supported by NRF grants (2010-0019306, 2011-0016385) and the WCU project (R33-10128) funded by the MEST, Republic of Korea.

References

Achenbach JC, Chiuman W, Cruz RP et al (2004) DNAzymes: from creation in vitro to application in vivo. Curr Pharm Biotechnol 5:321–336 Agrawal A, Min DH, Singh N et al (2009) Functional delivery of siRNA in mice using dendriworms. ACS Nano 3:2495–2504 Alotte C, Martin A, Caldarelli SA et al (2008) Short peptide nucleic acids (PNA) inhibit hepatitis C virus internal entry site (IRES) dependent translation in vitro. Antiviral Res 80:280–287 Alt M, Eisenhardt S, Serwe M et al (1999) Comparative inhibitory potential of differently modified antisense oligodeoxynucleotides on hepatitis C virus translation. Eur J Clin Invest 29:868–876 Appaiahgari MB, Vrati S (2007) DNAzyme-mediated inhibition of Japanese encephalitis virus replication in mouse brain. Mol Ther 15:1593–1599 Bartenschlager R, Ahlborn-Laake L, Mous J et al (1993) Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 67:3835–3844 Bartolome J, Castillo I, Carreno V (2004) Ribozymes as antiviral agents. Minerva Med 95:11–24 Braasch DA, Corey DR (2001) (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol 8:1–7 Brown AB, Mahmood U, Cortes ML et al (2005) Magnetic resonance imaging and characteriza- tion of spontaneous lesions in a transgenic mouse model of tuberous sclerosis as a model for endothelial cell-based transgene delivery. Hum Gene Ther 16:1367–1376 Brown-Driver V, Eto T, Lesnik E et al (1999) Inhibition of translation of hepatitis C virus RNA by 2-modified antisense oligonucleotides. Antisense Nucleic Acid Drug Dev 9:145–154 Bukh J (2004) A critical role for the chimpanzee model in the study of hepatitis C. Hepatology 39:1469–1475 Choi WH, Choi BR, Kim JH et al (2008) Design and kinetic analysis of hammerhead ribozyme and DNAzyme that specifically cleave TEL-AML1 chimeric mRNA. Biochem Biophys Res Commun 374:169–174 Choi YS, Lee JY, Suh JS et al (2010) The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine. Biomaterials 31:1429–1443 Choo QL, Richman KH, Han JH et al (1991) Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 88:2451–2455 Crooke ST (2004) Progress in antisense technology. Annu Rev Med 55:61–95 Dass CR (2004) Deoxyribozymes: cleaving a path to clinical trials. Trends Pharmacol Sci 25:395–397 Dass CR, Choong PF, Khachigian LM (2008) DNAzyme technology and cancer therapy: cleave and let die. Mol Cancer Ther 7:243–251 De Francesco R, Migliaccio G (2005) Challenges and successes in developing new therapies for hepatitis C. Nature 436:953–960 Deng Z, Tian Y, Lee SH et al (2005) DNA-encoded self-assembly of gold nanoparticles into one- dimensional arrays. Angew Chem Int Ed 44:3582–3585 Derfus AM, Chan WCW, Bhatia SN (2004) Probing the cytotoxicity of semiconductor quantum dots. Nano Lett 4:11–18 Derfus AM, Chen AA, Min DH et al (2007) Targeted quantum dot conjugates for siRNA delivery. Bioconjug Chem 18:1391–1396 Dobson J (2006) Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery. Gene Ther 13:283–287 Suppression of Hepatitis C Viral Genome Replication 449

El-Aneed A (2004) An overview of current delivery systems in cancer gene therapy. J Control Release 94:1–14 Fluiter K, ten Asbroek AL, de Wissel MB et al (2003) In vivo tumour growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides. Nucleic Acids Res 31:953–962 Fluiter K, Frieden M, Vreijling J et al (2005) Evaluation of LNA modified DNAzymes targeting a single nucleotide polymorphism in the large subunit of RNA polymerase II. Oligonucleotides 15:246–254 Ge Q, Filip L, Bai A et al (2004) Inhibition of influenza virus production in virus-infected mice by RNA interference. Proc Natl Acad Sci USA 101:8676–8681 Ghosh P, Han G, De M et al (2008) Gold nanoparticles in delivery applications. Adv Drug Deliv Rev 60:1307–1315 Giljohann DA, Seferos DS, Prigodich AE et al (2009) Gene regulation with polyvalent siRNA- nanoparticle conjugates. J Am Chem Soc 131:2072–2073 Grakoui A, McCourt DW, Wychowski C et al (1993) A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci USA 90:10583–10587 Grzelinski M, Urban-Klein B, Martens T et al (2006) RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts. Hum Gene Ther 17:751–766 Han M, Gao X, Su JZ et al (2001) Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 19:631–635 Hanecak R, Brown-Driver V, Fox MC et al (1996) Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes. J Virol 70:5203–5212 Heale BS, Soifer HS, Bowers C et al (2005) siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res 33:e30 Hijikata M, Mizushima H, Akagi T et al (1993) Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67:4665–4675 Honda M, Brown EA, Lemon SM (1996) Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2:955–968 Honda M, Beard MR, Ping LH et al (1999) A phylogenetically conserved stem-loop structure at the 50 border of the internal ribosome entry site of hepatitis C virus is required for cap- independent viral translation. J Virol 73:1165–1174 Hoofnagle JH (2002) Course and outcome of hepatitis C. Hepatology 36:S21–S29 Huh YM, Jun YW, Song HT et al (2005) In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals. J Am Chem Soc 127:12387–12391 Isaka Y (2007) DNAzymes as potential therapeutic molecules. Curr Opin Mol Ther 9:132–136 Jang JT, Nah H, Lee JH et al (2009) Critical enhancements of MRI contrast and hyperthermic effects by dopant-controlled magnetic nanoparticles. Angew Chem Int Ed 48:1234–1238 Kato N, Hijikata M, Ootsuyama Y et al (1990) Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci USA 87:9524–9528 Khoury M, Louis-Plence P, Escriou V et al (2006) Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor alpha in experi- mental arthritis. Arthritis Rheum 54:1867–1877 Kretschmer-Kazemi Far R, Sczakiel G (2003) The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res 31:4417–4424 Labhasetwar V (2005) Nanotechnology for drug and gene therapy: the importance of understand- ing molecular mechanisms of delivery. Curr Opin Biotechnol 16:674–680 Lanford RE, Hildebrandt-Eriksen ES, Petri A et al (2010) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327:198–201 450 D.-H. Min and D.-E. Kim

Laxton C, Brady K, Moschos S et al (2011) Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense targeting hepatitis C virus internal ribosome entry site. Antimicrob Agents Chemother 55:3105–3114 Lee B, Kim KB, Oh S et al (2010) Suppression of hepatitis C virus genome replication in cells with RNA-cleaving DNA enzymes and short-hairpin RNA. Oligonucleotides 20:285–296 Lewin AS, Hauswirth WW (2001) Ribozyme gene therapy: applications for molecular medicine. Trends Mol Med 7:221–228 Li W, Szoka FC Jr (2007) Lipid-based nanoparticles for nucleic acid delivery. Pharm Res 24:438–449 Lima WF, Brown-Driver V, Fox M et al (1997) Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity. J Biol Chem 272:626–638 Lohmann V, Korner F, Dobierzewska A et al (2001) Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75:1437–1449 Lu ZX, Ye M, Yan GR et al (2005) Effect of EBV LMP1 targeted DNAzymes on cell proliferation and . Cancer Gene Ther 12:647–654 Ludwig J, Blaschke M, Sproat BS (1998) Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17. Nucleic Acids Res 26:2279–2285 Luo KQ, Chang DC (2004) The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem Biophys Res Commun 318:303–310 Manabe S, Fuke I, Tanishita O et al (1994) Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3. Virology 198:636–644 Matveeva O, Felden B, Audlin S et al (1997) A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures. Nucleic Acids Res 25:5010–5016 McCaffrey AP, Meuse L, Karimi M et al (2003) A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 38:503–508 McCarthy JR, Weissleder R (2008) Multifunctional magnetic nanoparticles for targeted imaging and therapy. Adv Drug Deliv Rev 60:1241–1251 McHutchison JG, Fried MW (2003) Current therapy for hepatitis C: pegylated interferon and ribavirin. Clin Liver Dis 7:149–161 McHutchison JG, Patel K, Pockros P et al (2006) A phase I trial of an antisense inhibitor of hepatitis C virus (ISIS 14803), administered to chronic hepatitis C patients. J Hepatol 44:88–96 McMahon KM, Mutharasan RK, Tripathy S et al (2011) Biomimetic high density lipoprotein nanoparticles for nucleic acid delivery. Nano Lett 11:1208–1214 McManus MT, Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3:737–747 Mizushima H, Hijikata M, Tanji Y et al (1994) Analysis of N-terminal processing of hepatitis C virus nonstructural protein 2. J Virol 68:2731–2734 Mizutani T, Kato N, Hirota M et al (1995) Inhibition of hepatitis C virus replication by antisense oligonucleotide in culture cells. Biochem Biophys Res Commun 212:906–911 Morrissey DV, Lockridge JA, Shaw L et al (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 23:1002–1007 Nelson AR, Borland L, Allbritton NL et al (2007) Myristoyl-based transport of peptides into living cells. Biochemistry 46:14771–14781 Nielsen PE (1997) Peptide nucleic acid (PNA) from DNA recognition to antisense and DNA structure. Biophys Chem 68:103–108 Oketani M, Asahina Y, Wu CH et al (1999) Inhibition of hepatitis C virus-directed gene expression by a DNA . J Hepatol 31:628–634 Park TG, Jeong JH, Kim SW (2006) Current status of polymeric gene delivery systems. Adv Drug Deliv Rev 58:467–486 Patel PC, Giljohann DA, Daniel WL et al (2010) Scavenger receptors mediate cellular uptake of polyvalent oligonucleotide-functionalized gold nanoparticles. Bioconjug Chem 21:2250–2256 Suppression of Hepatitis C Viral Genome Replication 451

Petersen M, Nielsen CB, Nielsen KE et al (2000) The conformations of locked nucleic acids (LNA). J Mol Recognit 13:44–53 Rockwell P, O’Connor WJ, King K et al (1997) Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynu- cleotides. Proc Natl Acad Sci USA 94:6523–6528 Ryoo SR, Jang H, Kim KS et al (2012) Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown. Biomaterials 33: 2754–2761 doi:10.1016/j.biomaterials.2011.12.015 Saito G, Swanson JA, Lee KD (2003) Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities. Adv Drug Deliv Rev 55:199–215 Santiago FS, Lowe HC, Kavurma MM et al (1999) New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat Med 5:1264–1269 Santoro SW, Joyce GF (1997) A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 94:4262–4266 Schiffelers RM, Ansari A, Xu J et al (2004) Cancer siRNA therapy by tumor selective delivery with -targeted sterically stabilized nanoparticle. Nucleic Acids Res 32:e149 Schubert S, Gul DC, Grunert HP et al (2003) RNA cleaving “10-23” DNAzymes with enhanced stability and activity. Nucleic Acids Res 31:5982–5992 Schubert S, Grunweller A, Erdmann VA et al (2005) Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J Mol Biol 348:883–893 Sekhon BS, Kamboj SR (2010) Inorganic nanomedicine–part 1. Nanomedicine 6:516–522 Seki M, Honda Y (1995) Phosphorothioate antisense oligodeoxynucleotides capable of inhibiting hepatitis C virus gene expression: in vitro translation assay. J Biochem 118:1199–1204 Shepherd J, Waugh N, Hewitson P (2000) Combination therapy (interferon alfa and ribavirin) in the treatment of chronic hepatitis C: a rapid and systematic review. Health Technol Assess 4:1–67 Shippy R, Lockner R, Farnsworth M et al (1999) The hairpin ribozyme. Discovery, mechanism, and development for gene therapy. Mol Biotechnol 12:117–129 Smith AM, Mohs AM, Nie S (2009) Tuning the optical and electronic properties of colloidal nanocrystals by lattice strain. Nat Nanotechnol 4:56–63 Song E, Zhu P, Lee SK et al (2005) Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol 23:709–717 Steele D, Kertsburg A, Soukup GA (2003) Engineered catalytic RNA and DNA: new biochemical tools for drug discovery and design. Am J Pharmacogenomics 3:131–144 Takamizawa A, Mori C, Fuke I et al (1991) Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol 65:1105–1113 Tallet-Lopez B, Aldaz-Carroll L, Chabas S et al (2003) Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation. Nucleic Acids Res 31:734–742 Tomei L, Failla C, Santolini E et al (1993) NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol 67:4017–4026 Trepanier J, Tanner JE, Momparler RL et al (2006) Cleavage of intracellular hepatitis C RNA in the virus core protein by deoxyribozymes. J Viral Hepat 13:131–138 Tsukiyama-Kohara K, Iizuka N, Kohara M et al (1992) Internal ribosome entry site within hepatitis C virus RNA. J Virol 66:1476–1483 Vester B, Lundberg LB, Sorensen MD et al (2002) LNAzymes: incorporation of LNA-type monomers into DNAzymes markedly increases RNA cleavage. J Am Chem Soc 124:13682–13683 Wahlestedt C, Salmi P, Good L et al (2000) Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc Natl Acad Sci USA 97:5633–5638 Wakita T, Wands JR (1994) Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. In vitro model for selection of target sequence. J Biol Chem 269:14205–14210 452 D.-H. Min and D.-E. Kim

Walker MP, Appleby TC, Zhong W et al (2003) Hepatitis C virus therapies: current treatments, targets and future perspectives. Antivir Chem Chemother 14:1–21 Westerhout EM, Berkhout B (2007) A systematic analysis of the effect of target RNA structure on RNA interference. Nucleic Acids Res 35:4322–4330 Wu Y, Yu L, McMahon R et al (1999) Inhibition of bcr-abl oncogene expression by novel deoxyribozymes (DNAzymes). Hum Gene Ther 10:2847–2857 Xing Y, Rao J (2008) Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging. Cancer Biomark 4:307–319 Yao ZQ, Zhou YX, Feng XM et al (1995) Specific inhibition of hepatitis b virus gene expression by an antisense oligonucleotide in vitro. Acta Virol 39:227–230 Zein NN (2000) Clinical significance of hepatitis C virus genotypes. Clin Microbiol Rev 13:223–235 Zhang H, Hanecak R, Brown-Driver V et al (1999) Antisense oligonucleotide inhibition of hepatitis C virus (HCV) gene expression in livers of mice infected with an HCV-vaccinia virus recombinant. Antimicrob Agents Chemother 43:347–353 Zhao W, Gao Y, Kandadai SA et al (2006) DNA polymerization on gold nanoparticles through rolling circle amplification: towards novel scaffolds for three-dimensional periodic nanoassemblies. Angew Chem Int Ed 45:2409–2413 Zhu Q, Oei Y, Mendel DB et al (2006) Novel robust hepatitis C virus mouse efficacy model. Antimicrob Agents Chemother 50:3260–3268 Zimmermann TS, Lee AC, Akinc A et al (2006) RNAi-mediated gene silencing in non-human primates. Nature 441:111–114