Suppression of Hepatitis C Viral Genome Replication with RNA-Cleaving Deoxyribozyme Dal-Hee Min and Dong-Eun Kim Contents 1 Introduction ................................................................................. 430 2 Antiviral Antisense Oligonucleotides ...................................................... 433 2.1 RNA-Cleaving Antisense Oligonucleotides: DNAzymes ........................... 433 2.2 Chemical Modifications of Antisense Oligonucleotides . .. ... .. ... .. ... .. ... .. ... .. 434 3 Various Strategies for Oligonucleotide 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 Gene Knockdown 444 5 Conclusions ................................................................................. 447 References ....................................................................................... 448 Abstract Downregulation of viral genes via oligonucleotide-based gene therapy is a potential strategy for the treatment of virus 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 antiviral drug 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 nucleoside 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 nucleotides (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 proteins (Choo et al. 1991; Kato et al. 1990) (Fig. 1a). Among different isolates of HCV with considerable nucleotide variability, HCV genotypes 1a and 1b are most clinically relevant (Takamizawa et al. 1991). Translation in the host cytoplasm 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 cell mRNA, blocking of the IRES region with antisense oligonucleotides (AS-ODNs) could be exploited to achieve selective suppression of HCV gene expression (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), ribozyme (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 protein synthesis through degrada- tion of the related mRNAs. Traditional drugs based on small organic compounds are limited in their function because they inhibit enzyme 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 nucleases in physiological condition, inefficient cellular uptake, and lack of targeting capability. If AS-ODNs that were sensitive to nuclease 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 tissue 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