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Targets and Tools: Recent Advances in the Development of Anti-HCV Nucleic Acids C. Romero-López, F. J. Sánchez-Luque and A. Berzal-Herranz* Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla 18100, Granada, Spain Abstract: Hepatitis C virus (HCV), the major etiological agent of transfusion-associated non-A, non-B hepatitis, is a severe health problem affecting up to 3% of the world population. Since its identification in 1989, enormous efforts have been made to characterize the viral cycle. However, many details regarding the virus’ penetration of hepatocytes, its replication and translation, and the assembling of virions remain unknown, mostly because of a lack of an efficient culture system. This has also hampered the development of fully effective antiviral drugs. Current treatments based on the combination of interferon and ribavirin trigger a sustained virological response in only 40% of infected individuals, thus the development of alternative therapeutic strategies is a major research goal. Nucleic acid based therapeutic agents may be of some potential in hepatitis C treatment. In recent years, much effort has gone into the improvement of DNA and RNA molecules as specific gene silencing tools. This review summarizes the state of the art in the development of new HCV therapies, paying special attention to those involving antisense oligonucleotides, aptamers, ribozymes, decoys and siRNA inhibitors. The identification of potential viral targets is also discussed.
Keywords: HCV, hepatitis C, gene silencing, RNA-based inhibitors, ribozyme, antisense, aptamer, siRNA. GENERAL FEATURES Hepatitis C virus (HCV) infection is a major global health problem. More than 3% of the world population is affected (WHO data), although the incidence varies from one region to another. Once the virus has entered the body it infects the liver cells and begins to replicate. Some 15-25% of infected individuals develop effective barriers that finally resolve the infection, but the remainder suffers the long-term replication of the virus [1]. These patients usually remain asymptomatic for years before showing fibrosis that may, in some 30% of these cases, gradually progress to cirrhosis and even hepatocellular carcinoma. At this stage, the only treatment possible is a liver transplant; indeed, hepatitis C patients figure strongly on liver transplant waiting lists [2]. Current therapies, based on the combination of interferon (IFN) and ribavirin, are effective in only 40% of patients. The high mutation rate of the HCV genome and the virus’ ability to escape the immune system are partially responsible for this failure. Thus the identification of new targets and the search for fully effective antiviral compounds are major goals of HCV research. HCV belongs to the Hepaciviruses (family Flaviviridae), which include yellow fever virus (the classic flavivirus), bovine diarrhea virus (BVDV, a pestivirus) and GB virus. The HCV genome shows great variability which allows the identification of six genotypes [3] differing from one other by up to 30% in their nucleotide sequences (reviewed in [4]). Further subtypes and isolates of these different genotypes *Address correspondence to this author at the Instituto de Parasitología y Biomedicina “López-Neyra” CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla 18100, Granada, Spain; Tel: +34 958 18 16 48; Fax: +34 958 18 16 32; E-mail:
[email protected] 1871-5265/06 $50.00+.00
varying in their virulence have also been described. Viral genotype clearly affects the success of interferon therapy, although no clear correlation with virulence exists. In addition, the HCV population present in an infected patient is structured in terms of quasispecies. This term defines the closely related sequences of a heterogeneous viral population infecting a single individual [5]. Genomic sequence diversity is mainly due to the high viral replication rate and the mutations introduced by viral RNA-dependent RNA polymerase (RdRp) during the replication of the viral genome [6, 7]. Quasispecies structure has been associated with the failure of infected people to clear the virus and the subsequent development of a chronic infection [8]. It has been reported that the viral populations infecting immunosuppressed patients show a reduced probability of mutation compared to that seen in non-immunocompromised individuals [9-11]. A strong host immune response may encourage the appearance of resistant variants. HCV virions have a diameter of 55-65 nm [12] and can be detected in patient serum either complexed to immunoglobulins or low density lipoproteins, or as free viral particles [13]. The genetic material is bound to the core protein, forming an icosaedric capsid. This is in turn covered by a lipid envelope. A heterodimeric complex consisting of the viral glycoproteins E1 and E2, sticks out through this envelope like spikes [14]. The HCV genome is a 9600 nucleotide-long, single stranded positive RNA molecule (Fig. (1); [15-17]). It contains two highly conserved untranslatable regions (UTR) flanking the coding sequence, which play an essential role in viral translation and replication [18-21]. The viral proteins are synthesized as a single precursor that is then co- and post-translationally processed by both viral and cellular © 2006 Bentham Science Publishers Ltd.
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Fig. (1). Genetic organization and protein products of HCV. Above: the HCV genome, showing the 5’ and 3’ untranslated regions. The proposed secondary structure for the CRE domain (cis-response element) is also shown. IRES-dependent translation generates a 3011 amino acid-long polyprotein product (below) which is subsequently processed into structural and non-structural proteins. The proteolysis products and their functions are indicated.
proteases. The structural proteins include the capsid protein (C), p7 and the envelope proteins E1 and E2. The nonstructural products are involved in polyprotein processing (NS2, NS3, NS4A) and replication (NS4B, NS5A and NS5B). The success of HCV infection is conditioned by how well the virus avoids the immune host response. The initial defense reaction depends on the viral pathogen-associated molecular pattern (PAMP). For HCV, the PAMP includes the genomic RNA, the NS5A protein, the core protein, and even entire viral particles, all of which can be presented by dendritic cells (for a review, see [22]). PAMP presentation initiates the production of interferon and cytokines [23] in an autoregulatory fashion ([24]; reviewed in [22]). It has been reported that the NS3/4A protein acts as an antagonist of certain key factors in the induction of the immune response. This mainly involves a reduction in IFN production, thus inducing alterations in the pathways and complexes whose functioning is dependent on it. For example, antigen presentation is greatly reduced, meaning T cells cannot be stimulated. NS3/4A has also been related to a prolonged blocking of pro-apoptotic factor activity, providing a molecular link between HCV infection and the development
of hepatocellular carcinoma [25]. Similarly, the NS5A protein may activate IL-8 production, which interferes with IFN-mediated response pathways, and in conjunction with E2 it may block protein kinase R (PKR), an enzyme involved in the amplification of the immune response. The intensity of these effects differs depending on the genetic diversity of the infecting viral pool. Finally, HCV genotypes 1a and 1b have a smaller number of UU and AU sites than genotypes 2 or 3, impeding RNase L-mediated degradation of the HCV genome (this enzyme specifically cleaves nucleic acids at these sites; [26]). This provides another possibility for variation in terms of resistance to IFN since RNase L is activated by this molecule. The identification of HCV in 1989 by Houghton and coworkers [15] led to numerous studies aimed at deciphering its molecular biology, but advances in our knowledge regarding the viral cycle have been hindered by the lack of an efficient and stable culture system. However, recent advances in replicon and pseudoparticle production methods [27, 28] have allowed a more detailed understanding of HCV spread and replication to be grasped. In addition, the latest virion production strategies provide the starting point for the
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identification and evaluation of new viral targets and the development of novel antiviral drugs [29-31]. This review discusses the main features and functions of the HCV targets that are being explored, and the therapeutic agents presently in use. New treatments based on the use of nucleic acids, potentially very powerful anti-HCV agents, are discussed. VIRAL TARGETS The high mutation rate of the HCV genome is a key determinant in the appearance of variants resistant to many of the antiviral compounds tested to date. Over recent years, much effort has been spent on the identification of new therapeutic targets and the development of effective antiHCV drugs. The most common drug targets under study are the 5’ and 3’ untranslated regions (5’UTR, 3’UTR) and the NS3 and NS5B proteins; these are important in the viral cycle and their conservation rates are high. This section summarizes the main features and roles of these elements in the viral cycle. THE 5’ UNTRANSLATED REGION This is the most commonly chosen target for the design of HCV inhibitors based on viral nucleic acid chemistry. This 341 nt-long region is one of the most conserved of the entire HCV genome: sequence identity is estimated at around 85% for all viral isolates [32, 33]. Domains essential for the initiation of viral replication [20] and translation [18, 19] have been identified within the 5’ UTR. The synthesis of the HCV polyprotein is mediated by an internal ribosome entry site domain (IRES; from nt 40 to 370) in a cap-independent manner (Fig. (2); [34]). Thus, the 5’ sequence coding for the capsid protein participates in the initiation of translation and may modulate the efficiency of this IRES [35]. Moreover, it has recently been reported that the N-terminal core domain might interact with the 5’UTR region and regulate viral translation [36, 37]. The secondary structure of the 5’UTR region was originally proposed by Lemon and co-workers [38]. However, further structural and functional analyses prompted its redefinition to include several new motifs and interactions. The most commonly accepted conformation is shown in Fig. (2). The 5’UTR region is folded into several stem-loop motifs that define different functional domains. The establishment of tertiary interactions among them has been described. Its maintenance is critical for IRES activity [39]. - Domain I of the 5’UTR region is crucial for the replication of the HCV genome [20]. It modulates viral polyprotein translation but it is not essential for IRES-mediated functions [40, 41]. - The linker region between domains I and II appears to be an unstructured, single-stranded sequence, but in fact it takes part in long-range RNA-RNA interactions involving the nucleotides at positions G432-G438 [42]. The resulting duplex favors the creation of a large loop-like structure in which the IRES region is enclosed. Its formation might, in some way, modulate IRES-dependent protein synthesis. This is in good agreement with previous reports describing that mutations at this level affect IRES-dependent protein synthesis [43].
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- Domain II of the 5’UTR region consists of two helical segments separated by a highly conserved internal loop [44, 45] thought to participate in long-range interactions with other viral RNA domains [39]. This loop may also provide a site for the association of translation initiation factors [46, 47]. The nucleotides of the apical loop of domain II define an essential location where protein and RNA ligands can attach [39, 48-50]. - Domain III of the 5’UTR region is the anchorage site for the eIF3 factor and the 40S ribosomal subunit [50, 51]. It is composed of several subdomains that fold into stem-loop structures able to interact with other domains of the IRES [39]. The IIIabc subdomain consists of three stem-loop elements that form a four-way junction involved in binding with the 40S ribosomal subunit. This is needed for eIF3 factor recruitment [51]. The subdomains IIId and IIIe form the main anchoring sites for the 40S ribosomal subunit [5153]. Because of their importance in IRES-dependent translation, they have been the subject of much research and are considered excellent candidates for HCV targeting. NMR studies have revealed the three dimensional structure of subdomains IIId and IIIe, clarifying their critical role in IRES conformation and the maintenance of its biological function [53, 54]. Subdomain IIId adopts a stem-loop structure interrupted by an internal E-loop closed by a highly conserved apical loop which folds into a U-turn structure. This exposes the nucleotides and favors their interaction with other viral sequences and proteins. The subdomain IIIe contains a highly conserved GAUA tetraloop associated with an efficient viral polyprotein synthesis [53]. An important feature of domain III is the formation of a conserved pseudoknot structure involving subdomain IIIf and a pyrimidine-rich region (positions 325-330; [55]). This pseudoknot has been proposed to interact directly with the 40S ribosomal subunit [50, 51]. It is also present in related viruses and has been associated with the regulation of translation initiation [55, 56]. - Domain IV of the 5’UTR region contains the AUG initiation codon at nucleotide 342 that is included in an apical loop enclosing a helical motif, the stability of which is inversely related to IRES translational efficiency [57]. As mentioned above, the maintenance of the IRES secondary and tertiary structure is critical in IRES-dependent translation. The different domains may behave as initiation factors able to capture proteins necessary for the synthesis of the viral precursor polyprotein. The 5’UTR region recruits eIF3 and the 40S ribosomal subunit to form a ribonucleoprotein complex that unleashes the formation of active 80S ribosomal systems, and therefore promotes polyprotein translation [50, 58]. The essential role of this region in the viral cycle and its high conservation rate make it a very attractive therapeutic target. THE 3’ UNTRANSLATED REGION This region is a 200-240 nt-long sequence located at the 3’ end of the HCV RNA genome. Its secondary structure is maintained across different viral isolates (Fig. (3); [59]). Mutational analysis has been used to decipher the biological role of the 3’ UTR [60]. Enzymatic and chemical analyses
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Fig. (2). Proposed secondary structure of the 5’UTR domain of HCV. Domains involved in the interaction with eIF3 factor and ribosomal subunit 40S are marked in boxes. The translation start codon is shown in bold.
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Fig. (3). Structural model proposed for the HCV 3’UTR segment. The different domains of the entire secondary structure are indicated.
have identified three different domains: the VR region, the polyU/C tract and 3’X region [61, 62]. The 3’UTR begins with a short and highly variable sequence, although it also contains a conserved motif and UG dinucleotide [63]. This domain is named VR (variable region) and it has been shown its implication in viral RNA synthesis using subgenomic replicon systems [21]. A polyU/C-rich tract of variable length and composition follows the VR sequence. This can interact with host proteins and regulate viral translation [64, 65]. Some of these host proteins have been identified, e.g., polypyrimidine tractbinding protein (PTB), heterogeneous nuclear ribonucleoprotein C (hnRNP C) and glyceraldehyde-3-phosphate dehydrogenase (GADPH), among others [66-68]. Finally, the 3’UTR region contains a 98 nt-long domain known as the 3’X region – one of the most conserved motifs within this region. It folds into three different stem-loop motifs – SL1, SL2 and SL3 [59] – and is involved in viral RNA replication [21, 69-71] and virion infectivity in vivo [60]. Its interaction with human ribosomal complexes has
also been reported, suggesting a potential role in the modulation of IRES-dependent translation [72]. It is important to note that this domain is exclusive to HCV (reviewed in [73]) and is an excellent candidate target for new antiviral drugs. THE NS3 PROTEIN One of the most extensively studied and best understood targets in anti-HCV therapy is the NS3 protein - a multifunctional molecule composed of two domains with serine protease and helicase activity [74, 75]. Protease activity is one of the preferred targets for the designers of new drugs. Protease has a key role in HCV infection since it is responsible for polyprotein processing during virus maturation. The host proteases first cleave the precursor to release the structural proteins. Secondly, virallyencoded NS2/NS3 proteases yield the mature NS3 protein [76] which initiates the cleavage of the remaining target sites in an Mg2+-dependent reaction [77]: first, cotranslationally at the NS3/NS4A junction, and then in the order NS5A-5B, NS4A-4B and NS4B-5A [78]. The cis cleavage is governed
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by the intrinsic folding of the polyprotein, which brings the protease domain and its substrate close together. The transreaction has more stringent requirements including the correct positioning of the protease and its polyprotein substrate (reviewed in [79]). The proteolytic processing mediated by NS3 is dependent on NS4A, a small polypeptide composed of only 54 aminoacids [80, 81]. The interaction between them maps to the 22 residues most N-terminal of the NS3 protein [82, 83] and induces several conformational changes affecting the catalytic residues, giving rise to a highly stable complex containing NS4A [84]. NS3 has no membrane anchoring domains itself, but its cofactor NS4A is an amphipathic peptide that can interact with membranes via a highly hydrophobic stretch of aminoacids to form a transmembrane helix. This allows the NS3/4A complex to be immobilized on the surface of the endoplasmic reticulum (ER; [85]), a requirement for the establishment of the replication complex. The structure of the protease-NS4A complex has been resolved by X-ray crystallography. This information has given rise to intensive efforts to identify potential targets for new antiviral drugs. As a result, a chymotrypsin-like folding with an exposed Zn-binding site located on the surface of the complex was detected [86]. Zinc ions (a requisite for catalytic activity; [87]), are coordinated via three cysteine residues in a long loop region that acts as a linker between two β-barrel domains and which contributes to the conformational integrity of the molecule as a whole (reviewed in [88]). This structural feature clearly differentiates NS3/4A from other members of the serine-protease family. Consequently, it is plausible that compounds affecting the coordination sphere of the metal ions could act as effective and specific inhibitors of protease activity. Research into the three dimensional structure of the protease-NS4A complex has also contributed to the identification of the substratebinding site. This is positioned in a wide, very exposed cleft (reviewed in [89]) and can bind a minimum 10 amino acidlong substrate. Competitive inhibitors based on the structure of the cleavable substrate have been designed (reviewed in [90]). The enzyme is also clearly inhibited by its reaction products [91], which opens a broad range of possibilities for the design of mimetic peptide drugs. The helicase activity of NS3 maps to its C-terminal end. This domain mediates the RNA duplex unwinding promoted by NTP hydrolysis during viral replication, suggesting that this helicase also has NTPase activity [92]. RNA duplex separation requires Mg2+ and is severely inhibited by monovalent ions [77]. Crystallography studies and mutational assays of the helicase domain have shown it to have a unique Y shape in which three domains are defined by each arm of the Y [9397]. Although separated by clefts, the different domains may interact with each other. This finding has led to the design of new helicase inhibitors that affect different functions simultaneously. The different NS3 activities show a high level of interdependence. It seems clear that NTPase capacity is necessary but not sufficient for helicase activity [97-100]. Moreover, NS3 helicase and protease activities are activated
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by polynucleotides, preferentially by poly(U) [101]. These observations suggest that genomic RNA modulates the interplay between the different functions of NS3 to help form a stable replication complex [101]. Recently, it has also been suggested that a relationship exists between the helicase and protease domains, and that this interaction positively modulates duplex unwinding and viral polyprotein processing [102, 103]. The above features have led to the proposal that NS3 may be an excellent target for the inactivation of HCV. The existence of several domains with different catalytic activities essential for HCV infection provides a good target series for possible drugs or combinations of drugs with different specificities. THE NS5B PROTEIN The NS5B protein is a key enzyme in the viral cycle because of its RNA-dependent RNA polymerase activity (RdRp). This enzyme is required for the synthesis of the negative and positive RNA strands during the replication of the genome. The NS5B protein is highly conserved across different HCV genotypes (and their subtypes and isolates), rendering it a potential target for the design of antiviral compounds with minimal side effects (reviewed in [104]). NS5B is produced as part of the polyprotein HCV precursor. Once it is processed by the NS3/NS4A complex, it is transported to the ER membranes and anchored there through the highly hydrophobic C-terminal end, the Nterminal domain facing the cytosol [105]. Here, NS5B is involved in the recruitment of non-structural HCV proteins for the formation of the replication complex, an indispensable step [106-108]. This association of HCV structural and non-structural proteins plus genomic RNA causes a deformation of the lipid layer in the ER membranes that can be appreciated by electron microscopy [106, 108, 109]. This alteration, known as a membranous web, is similar to the “sponge-like inclusions” detected in the liver of infected chimpanzees [110]. These macromolecular structures are components of a strategy developed by RNA viruses that afford them several advantages: the replication procedure is localized to a reduced cellular compartment, increasing the local concentration of the reaction substrates, the ER provides a solid support plus the lipids required for the process, and the viral genomic RNA is protected from degradation by cellular RNases. The NS5B protein has been extensively studied. Both its biochemical properties and the reaction it catalyses are well understood. Its RdRp activity was initially identified by the presence of a conserved motif in several enzymes with retrotranscriptase and polymerase functions [15]; this has subsequently been confirmed in vitro [111]. Its three dimensional conformation was resolved independently by a number of research groups [112-114]; it has the classic structure of other polymerases and retrotranscriptases, but with a number of unique features. Although NS5B folds to acquire a typical right hand shape with the palm, fingers and thumb domains clearly differentiated, it is distinguished from other polymerases by its more compact structure due to the two loops that span the fingers and the thumb and which enclose the catalytic core. This impedes conformational
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changes occurring independently in each domain. It also has a protruding region involved in the correct positioning of the 3’ end of the viral genome. The NTPs and RNA substrate molecules access the active site through two positively charged channels. When the NTPs are correctly positioned, the template is fixed by interaction with the C-terminal domain. This induces the incorporation of a new NTP complementary to the template, causing the sliding back of the RNA substrate to the transcription initiation point, and thus promoting further RNA production [115]. De novo initiation is followed by RNA elongation, termination and the release of the newly synthesized RNA molecule. This mechanism is dependent on two Mg2+ ions and is primerindependent. In fact the only other requirement for the synthesis of the negative strand is the presence of GTP or ATP, which are needed for the initiation of the complementary nucleotides at the poly(U/C) stretch in the 3’UTR region [116, 117]. The substrate-binding cavity provides an attractive site for possible anti-HCV drugs. The ability of the NS5B enzyme to discriminate between HCV genomic RNA and external RNA templates has been much discussed. The binding of a recently discovered cisregulation element (CRE) to NS5B as well as several regions of the viral RNA suggests that it ensures viral rather than cellular RNA replication [118-120]. The need for other factors, however, cannot be ruled out. Because of the several steps that occur during RNA polymerization and the multiple needs of NS5B, this protein offers multiple targets for anti-HCV compounds. CURRENT THERAPEUTIC APPROACHES Most people with HCV fail to clear the virus and become chronically infected. Although the disease follows an asymptomatic course for at least 20 years in some 80% of cases, it finally causes liver injury. This involves severe fibrosis and necro-inflammatory symptoms in the initial stages, followed by cirrhosis and possibly hepatocellular carcinoma [2, 121]. The absence of any clinical symptoms during the early stage of infection despite persistent viral replication clearly suggests that HCV is not cytopathic on its own, and that the immune response plays a central role in liver injury. In both acute and chronic hepatitis C patients the T cell cytotoxic response is activated. If effective this can achieve viral clearance, but if incomplete, chronic viral replication occurs, accompanied by a permanent T cell cytotoxic effect which damages infected liver cells. A therapy is commonly regarded as successful when there is a sustained virological response, i.e., viral RNA is undetectable in the serum 24 weeks after treatment. Patients achieving this may show an improvement in their quality of life and have a favorable histological prognosis. However, some patients relapse. Treatment is usually recommended for patients with advanced chronic hepatitis C, for whom there is a high risk of serious liver injury. These patients show extensive fibrosis and their serum aminotransferase levels remain high. However, the clinical picture varies depending on the characteristics of the patient (e.g., whether he/she is an alcohol or drug abuser, or suffers autoimmune or cardio-
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pulmonary disease, depression or leukopenia. Pregnancy can also affect the clinical manifestations of infection [122]). Several therapeutic options exist, but only IFN, either alone or in combination with other drugs, has provided moderately positive results. IFN has been available since 1986 – in fact three years before the identification of HCV [123]. The molecular mechanism by which IFN operates is mediated by its binding to receptors on the surface of the target cells, which leads to a phosphorylation cascade involving different tyrosine-kinases. Finally, transcription factors are recruited to the nucleus where they induce the synthesis of a number of antiviral effector proteins (reviewed in [124]). Initially, monotherapy with IFN-α was the best anti-HCV treatment available, although less than 20% of patients showed a sustained virological response [125]. Combined therapy with ribavirin allowed the IFN doses used to be reduced (reviewed in [126]). Ribavirin is a guanosine analog with a broad antiviral spectrum that enhances the sustained virological response obtained with IFN treatment. Later, the development of new therapies using modified interferons led to a reduction in their systemic clearance, prolonging their half-life and reducing their antigenicity. Currently, the most popular treatment for chronic HCV infection is the combination of PEG-IFNα (polyethyleneglycol interferon-α) plus ribavirin. PEG-IFNα can be administered in two pharmacological forms: PEG-IFNα2a or PEG-IFNα2b (a smaller molecule than its counterpart with a shorter half-life; reviewed in [127]). Other modifications of IFN have been proposed to improve its bioavailability and pharmacokinetic properties, including conjugation with albumin, liposomes and polyamino groups (reviewed in [122]). The main limitation of this strategy is that about 60% of patients (mainly those infected with genotype 1) do not respond. The remaining 40% develop a sustained immune response against the viral infection favoring virion clearance from the plasma, though in most cases serious secondary effects are suffered (neuropsychiatric symptoms, influenzalike symptoms, hematological abnormalities, depression, cytopenia and anemia). Furthermore, combination therapy is expensive. There is therefore a great need to develop new therapeutic approaches that effectively block the different HCV genotypes and avoid the appearance of resistant quasispecies. PROTEASE NS3/4A INHIBITORS Blocking NS3 is an attractive strategy given the enzyme’s key role in viral infection and the success achieved against HIV protease (reviewed in [128]). NS3 provides three possible targets: the NS3/NS4A interaction site, the zinc binding site, and the association of the active complex with its substrate. The first two options have been provisionally discarded because of the difficulty of access for interfering compounds. The use of competitive inhibitors chemically similar to the substrate of the active complex is a more hopeful strategy; nevertheless, the absence of a well characterized catalytic site means success is unsure. The molecules designed so far have mostly been small mimetic peptides that bind to the core site where they block the catalytic reaction (reviewed in [88]). Some of these drugs have entered into clinical trials (reviewed in [129]). BILN
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2061 is a modified hexapeptide in which two amino acids are connected to form a macrocyclic structure (Boehringer Ingelheim; [130-133]). This design is based on the finding that the carboxy-terminal carboxylic acid of the proteolysis product acts as an affinity anchor, ensuring the specific binding of this compound to NS3/4A over other cellular proteases [130]. The main advantage of BILN 2061 lies in its oral administration route and minor side effect profile even at high doses. In patients treated with this drug, serum viral RNA levels are considerably reduced when the infecting virus is of genotype 1 (it is less effective against genotypes 2 and 3). However, research into this agent has been suspended since these reductions are transient [133]. In addition, the drug is cardiotoxic in animals and HCV escape mutants develop in culture systems [134]. An alternative substrate analogue is VX-950 (Vertex Pharmaceuticals; [135]). This peptidomimetic molecule has an α-ketoamide group that anchors it to a serine residue at the catalytic center. Preclinical assays have shown that viral RNA levels in subgenomic replicon systems are clearly reduced; the same result was obtained in transient infections. Phase Ib trials have just ended and the data show a reduction (though variable) in viral RNA levels in all patients treated. Other substrate analogs for NS3/4A are currently under investigation. To prevent the appearance of resistant viral variants, the combined use of several inhibitors with different specificities might be a good idea. POLYMERASE NS5B INHIBITORS The NS5B protein is another target under investigation. The compounds developed so far are mostly designed to block the polymerization of viral RNA, though the way they do this differs. These compounds can be classified as i) nucleoside analogs, ii) non-nucleoside analogs and iii) pyrophosphate mimics (reviewed in [129, 136]). The first class is the largest, and comprises substrate analogs that are inactive in their non-phosphorylated form. In the cytoplasm, phosphorylation is achieved by attaching a triphosphate group; this promotes the incorporation of the inhibitor to the enzyme’s active site, thus impeding the polymerization of viral RNA and releasing the incomplete copy. This approach has previously been employed in the inhibition of other virus, such as HIV, HBV and HSV. For HCV, it is currently exploited by the broad spectrum antiviral drug ribavirin. Its exact mechanism of action is a matter of discussion, although it would seem that its association with the active site induces the mis-incorporation of nucleotides, leading to a higher error rate [137]. In addition, ribavirin monophosphate is an effective inhibitor of inosine monophosphate dehydrogenase, an enzyme that participates in GTP synthesis. Its inhibition leads to the depletion of cellular GTP, which negatively affects viral RNA replication. As an alternative to ribavirin, NM283 (Valopicitabine; Idenix/Novartis), a 2-C-methylcitidine inhibitor of BVDV polymerase, is under consideration. This orally administered compound is currently being investigated in clinical trials and has been shown to block viral RNA polymerization [138]. The second class includes structurally diverse drugs that bind to the polymerase enzyme at sites different from the
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catalytic core, inducing conformational changes that finally distort the shape of the enzyme. This is the case of an orally administered compound developed by AKROS Pharma, JTK-003. This agent belongs to a family of molecules consisting of a benzimidazole core fused to a heterocyclic group. It acts as an allosteric inhibitor at the initiation of the polymerization step, impeding the synthesis of the complementary RNA strand by leaving the enzyme in an inactive conformational state (reviewed in [139]). This drug is currently being tested in clinical trials (phase II). The third class consists of inhibitors that interfere with the pyrophosphate binding site and which consequently block nascent RNA synthesis. Although their broad antiviral activity has been well documented, they have not yet been tested as anti-HCV drugs (reviewed in [88]). NUCLEIC ACID THERAPIES Direct action against the genetic information carried by the virus is one of the most promising anti-HCV strategies. Nucleic acids, particularly RNA, are excellent candidates for the development of antiviral agents. Moreover, advances in chemical synthesis techniques have improved the possibilities of developing therapeutic RNA molecules. However, the development of an effective nucleic acid therapeutic strategy is still pending. A number of aspects need to be taken into account in pursuing this goal [140142]: - Cell uptake of the inhibitor. The delivery of therapeutic nucleic acids to the inside of a target cell requires a transport system that allows the efficient penetration into the cell as well as the release of the nucleic acid from the endosomic vessels in which it must become encased [143]. The most popular mechanism involves the use of lipid vehicles (liposomes), though this is not without negative effects. A recently proposed technology based on nanoparticle engineering has opened up new possibilities (reviewed in [144]). - Target and inhibitor colocalization. This can be favored by using specific promoters that lead to the inclusion of specific signals into the inhibitor molecule, allowing for its specific subcellular localization [145]. - Active inhibitor concentration inside Appropriate concentrations must be reached.
the
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- Intracellular inhibitor stability. This is particularly important in the case of inhibitory RNA molecules, due mainly to the abundance of ribonucleases in the cytoplasm. This stability can be enhanced by incorporating nucleotides modified at certain positions during the chemical synthesis of RNA, thus reducing the latter’s eventual sensitivity to nucleases [146-151]. - Target genetic variability. The high genetic variability of RNA viruses, and therefore the relative ease of generation of resistant variants, is a major limitation of the use of nucleic acids as therapeutic agents. This might be overcome, however, by the combined use of several inhibitors with different specificities [152]. An alternative approach is the identification of highly conserved target sequences in the viral genome [153]. Recently, it has been described that the combination of different inhibitor domains in the same
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molecule may render substantially more specific molecules and it may avoid the generation of resistant variants [154158].
exon junctions. Antisense oligonucleotide inhibitors may also be used to block maturation processes or, if DNA-based, to induce target degradation mediated by RNase H (reviewed in [162]). These inhibitors have been traditionally designed on a trial and error basis; the production of optimized molecules is therefore rather difficult.
- Access and binding to the target region. Choosing a target site in an RNA substrate is a major decision in the development of any inhibitory nucleic acid. The highly complex secondary and tertiary structure of RNA molecules can hamper the binding of the oligonucleotide to its specific target site. Thus, the structure of the substrate RNA whose inactivation is desired must be investigated. Great efforts have been made to develop ways of identifying accessible sites in target RNAs [159-161]. The advances being made in the design of inhibitory RNAs that can bind to densely structured regions may offer an alternative strategy [154, 155, 158].
High molecular stability and an efficient delivery system are important requirements of all antisense oligonucleotide strategies. Fig. (4) shows the most common substitutions used to increase DNA/RNA oligonucleotide half-life. The first generation of chemically modified inhibitors included phosphorothioate substitutions in which the non-bridging oxygen atoms in phosphate linkages were changed to sulfur [163, 164]. This type of modification has been widely used in gene silencing approaches since it confers high stability to antisense molecules and specifically estimulates RNase Hmediated cleavage when using DNA oligonucleotides. Phosphorothioate oligonucleotides are also very water soluble and show increased cellular uptake. The main disadvantages of this strategy include the greatly reduced affinity and specificity for the target sequence, and an enhanced, non-specific protein binding capacity which can be cytotxic (reviewed in [164]). Other possible substitutions at this level include methyl groups (giving rise to methylphosphonates) and amines (giving rise to phosphoramidites). Second generation molecules include substitutions with methyl, methylthioethyl, amine or fluor groups at the 2’ position [165]. These overcome the problems associated with phosphorothioate antisense molecules. The drawback is that 2’-modifications in DNA oligonucleotides lead to a failure to
In recent years, great progress has been made in the field of nucleic acid treatment and some of the above problems have been now successfully solved. This has led to the design of anti-HCV RNA and DNA drugs that are currently being assayed in clinical trials. ANTISENSE NUCLEIC ACIDS STRATEGIES Antisense oligonucleotides are short nucleic acid molecules whose sequence is complementary to an existing motif in a target RNA. They can be administered either directly by subcutaneous injection or as pharmacological compositions. Once inside the cell, these oligonucleotides bind to their target RNAs and interfere with their function. A number of targets have been routinely used: the promoter sequences, the translation initiation codon, and the intronA
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Fig. (4). Diagram showing chemical modifications incorporated into synthetic oligonucleotides. A) Phosphorothioate. B) 2’-O-methyl phosphorothioate. C) 2’-O-aminopropyl posphodiester. D) LNA/BNA (locked/bridged nucleic acids). E) Phosphoroamidite. F) Morpholino. G) PNA (peptide nucleic acid). H) HNA (hexitol nucleic acid). Figure adapted from [164].
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induce RNase H-mediated cleavage. The main effects of such agents differ according to the modification introduced. For example, the addition of a 2’-methyltioethyl group favors oligonucleotide binding to serum albumin, increasing the oligonucleotide’s in vivo distribution while also investing it with advantageous pharmacokinetic properties. However, the nuclease resistance of thus-modified oligonucleotides is reduced compared to those with other substitutions [163]. The last family of modified oligonucleotides is rather diverse, and includes zwitterionic oligonucleotides, LNAs (locked nucleic acids)/BNAs (bridged nucleic acids) [166], PNAs (peptide nucleic acids; [167]), morpholino [168] and HNAs (hexitol nucleic acids; [169]). The affinity of an oligonucleotide for its target is of prime importance [162]. This is determined by the number of hydrogen bonds between complementary bases and by the stacking of coplanar nucleotide stretches. For optimized activity, a minimum of 15-20 nucleotides complementary to the target sequence is required. However, this should never exceed 35-40 residues since the specificity determined by base pairing could be negatively affected. In the therapeutic context, an index describing the intracellular molecular properties of antisense oligonucleotides is essential. Known as the therapeutic index, this takes into account factors such as the non-specific interactions an oligonucleotide may undergo with molecules other than nucleic acid, etc. [162]. This index depends not only on the inhibitor’s sequence but also on the other chemical groups present. For example, antisense molecules carrying phosphorothioates tend to bind in a non-specific way to multiple proteins with low affinity [170]. The molecular mechanisms of action of antisense oligonucleotides are diverse (reviewed in [162]). They may act by occupying essential sites in target RNAs – sites required for capturing protein factors or for interaction with other nucleic acids. Such an interaction might impair key steps in the function of the RNA. Alternatively they may work by disrupting the structure of the target, thus impeding its activity, promoting its destabilization, and favoring its degradation by cellular nucleases or the RNase H-mediated system. The mechanism differs in each case according to the antisense target in the substrate RNA and to the composition of the inhibitor [163]. The antisense molecules developed for use in anti-HCV therapy have mainly been directed against the 5’UTR region, and more specifically against the IRES domain (Table 1; [151, 171-181]). The general aim of these approaches is to block the association of protein factors, such as the 40S ribosomal subunit or eIF3 to the IRES. Competition is developed between the inhibitor and these ligands for their binding sites (a high affinity and duplex rate formation are therefore desired features in these antisense molecule). Alternatively, binding of the antisense molecule may result in the disruption of IRES folding, which profoundly affects the initiation of translation. Targeting the 3’ end of the IRES region has been the preferred option for the design of antisense oligonucleotides [171-177, 179-182]. In all of these studies an efficient and specific inhibition of translation was
Berzal-Herranz et al.
Table 1. Antisense Oligonucleotides Against HCV
HCV Target Region
Efficiency
References
Interdomain I and II
+
a, m
Domain II
++
a, c, h, m
Domain III
+++
a, c, e, h, k, m
Domain IV
+++
a, b, c, e, f, g, h, i, m
5’-end core coding sequence
+++
a, b, c, d, g, h, j, l
NS3 protein coding sequence
+++
n
5’UTR region
a: [171]; b: [172]; c: [173]; d: [174]; e: [175]; f: [176]; g: [177]; h: [178]; i: [179]; j: [180]; k: [151]; l: [181]; m: [167]; n; [185]; +: less than 30% of reduction; ++: decrease in tested HCV functions between 30-70%; +++: high efficacious inhibitors, with more than 70% of efficiency in interfering with HCV targeted activities.
detected, in some cases up to 95%. These observations suggest that domain IV and its molecular environment might act as good targets for HCV inactivation. Of all the assayed antisense oligonucleotides, ISIS 14803 has probably shown the most promising results. This agent is a chemically modified DNA antisense oligonucleotide containing several thioate group substitutions [179, 180]. It shows complementarity to residues 345-365 of the HCV genome within domain IV of the IRES region containing the translation initiation codon. Preclinical investigations in cell cultures and mouse models have shown its promising ability to block HCV translation and replication [180]. These results have encouraged clinical trials of this agent (currently in phase II; [181, 182]). The initial phase I trials, which involved patients with chronic HCV infection, showed an approximately 10-fold reduction in serum HCV RNA levels that could be attained in some subjects. In the subsequent phase II clinical trials, reductions of up to four orders of magnitude in circulating viral particle levels were achieved. It is noteworthy that most of the patients treated with ISIS 14803 were infected with viral genotype 1, the most common and the most resistant to interferon therapy. During the course of these investigations, the generation of quasispecies with mutations within the IRES region was analyzed. Although several changes were observed, none affected the complementary residues to ISIS 14803 or their neighboring sequences, suggesting strong conservation constraints for this target site [182]. PNAs and LNAs have recently been successfully used to block HCV IRES-dependent translation [167]. PNAs are DNA/RNA analogs with a 2-aminoethylglycine backbone that confers high stability against nuclease degradation [183]. They also show high sequence specificity, discriminating targets with mismatched nucleotides [184]. LNA molecules incorporate modified nucleotides with a bridging methylene group between the 2’ and 4’ positions, inducing an increase in Tm by as much as 10ºC in the whole compound and restricting any conformational changes that might occur. The PNA and LNA molecules used were designed for targeting different conserved motifs within the
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HCV IRES, such as the interdomain I-II region, apical loop II, apical loop IIIb, domain IIIc, stem-loop IIId, the pseudoknot, and the sequences surrounding the translation start codon. The PNAs directed against the IIId domain, the pseudoknot motif and translation start codon effectively inhibited IRES-dependent translation (close to 80% in ex vivo assays). The LNAs significantly interfered with IRES function, although to a lesser extent. However, LNAs directed against motifs in the core protein coding sequence blocked protein synthesis by around 80%. The tested PNAs and LNAs probably function by interfering with the association of indispensable translation factors rather than by RNA destabilization. These results show the efficacy of these strategies and confirm the importance of these motifs as therapeutic targets. The NS3 coding region has also been used as a target [185]. Several phosphorothioate antisense DNA molecules have been designed, the most potent being those directed against a highly conserved sequence exposed in single stranded regions (according to secondary structure predictions). These molecules inhibit NS3 protein translation and greatly reduced the proteolysis product levels in a dosedependent manner in cell cultures. Protease inhibitors, in contrast, only appear to affect NS3 function. The promising results obtained with this strategy, which achieves the significant inhibition of several HCV functions, suggest that antisense oligonucleotides, either alone or in combination with conventional anti-HCV drugs, may have therapeutic potential. It should be noted that the appearance of resistant viral variants may be overcome by the combined use of different inhibitory oligonucleotides with different specificities. THE APTAMER STRATEGY Great expectations have been aroused by the therapeutic potential of aptamers [186]. Aptamers are small oligonucleotides with the ability to bind specific ligands in a highly efficient fashion [187, 188], a consequence of their sequence and structure. Their very small size (15 kDa, 25-40 nt) helps them cross the plasma membrane, a problem encountered with larger molecules such as antibodies. However, this property is also responsible for their rapid clearance from the serum. Although it can be overcome by complexing them to larger, inert compounds such as PEG or liposomes, this tends to neutralize the original advantage of being small [189-191]. The rational design of antisense RNAs restricts the number of structures that can be tested as possible anti-HCV agents. However, the development of in vitro selection methodologies has allowed these constraints to be overcome. They have allowed the identification of nucleic acids with a wide range of phenotypes (reviewed in [186, 192-194]). In vitro selection strategies for the isolation of aptamers are known as SELEX (Systematic Evolution of Ligands by EXponential enrichment; Fig. (5A)). The in vitro selection process is determined by three key features: i) the initial variability of the pool of nucleic acids, ii) the selection of variants with a desired phenotype, and iii) the amplification of molecules positive for that phenotype. Briefly, a heterogeneous pool of nucleic acids is subjected to a selection pressure designed to encourage the rescue of the
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required phenotype. Inactive molecules can be either discarded or analyzed. Active molecules can be selectively rescued and amplified yielding a pool that can be transcribed again and subjected to new selection rounds. This allows for the enrichment of the population with the active variants. The first step in a SELEX procedure (Fig. (5B)), is to prepare an initial population of DNA or RNA molecules with a number of variable nucleotides in their central positions. Fixed sequences are located at the ends of these molecules with the aim of facilitating annealing with specific primers in the later reverse transcription and amplification steps. Sequence variability provides a population of molecules with diverse structures allowing variants with different specificities and affinities to be generated. The entire pool is then incubated with the ligand. Two routes may then be followed to recover the ligand-bound aptamers; either the ligands are immobilized on a solid support via interactions through specific groups (e.g. biotin and streptavidin), or they are selectively captured via their possession of a particular domain or specific structure. The recovered aptamers are then amplified. The selected molecules can then either be subjected to further rounds of selection or tested. The process ends with the selection of the aptamers that fold into the consensus structure required for the selected function. This methodology was initially reported by two independent groups [187, 188] and successfully used to identify aptamers with a range of specificities ranging from different ions [195] to small ligands [187], peptides [196], proteins [188], cell organelles [197], viruses [198] and even whole cells [199]. The selection of aptamers for the development of new therapeutic agents has become popular in recent years. The possibility of blocking viral proteins or critical RNA regions is of great interest and has stimulated much research, though no pre-clinical or clinical trials have yet been performed. Modifications of aptamers, such as those mentioned above for antisense oligonucleotide, should also be tested. However, the maintenance of the secondary and tertiary inhibitory structure would be important. One of the most common targets for possible anti-HCV aptamers is the NS3 protein (Table 2; [200-207]). Initial research tried to identify aptamers directed against the whole protein [200, 201]. In vitro selection procedures were developed and used with various RNA populations resulting from the randomization of stretches of nucleotides differing in their length (12-18 nucleotides [201] and 120 nucleotides [200]). The selected aptamers showed conserved consensus motifs and structural similarities. They also bound efficiently and specifically to their targets. In all cases, the aptamers moderately blocked protease and/or helicase activity in in vitro assays, with maximum inhibition values close to 50% [200]. The authors of these studies indicated that this modest inactivation effect was probably due to a diffuse binding of the RNA molecule to the NS3 protein over multiple sites (although many other explanations are possible). It therefore seemed appropriate to identify those targeting only one of the two catalytic NS3 domains. Attempts were made to select specific aptamers targeting a truncated form of the NS3 protein bearing only the protease domain [202]. This strategy identified effective (up to 70%) inhibitors of protease activity in vitro. Further
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Fig. (5). A) In vitro selection of RNA molecules from a heterogeneous pool. Active and inactive RNA molecules are selectively separated depending on their phenotype. They are then reverse transcribed and amplified. New selection rounds can be undertaken after the transcription of the cDNAs corresponding to the selected variants. Figure adapted from [192]. B) SELEX (systematic evolution of ligands by exponential enrichment) strategy for in vitro selection of RNA aptamers directed against a specific ligand.
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Table 2. Aptamers Identified for Targeting HCV
HCV Target
Efficiency
References
Helicase domain
++ +++
a, e f, g, h
Protease domain
+ ++ +++
b a c, d, e, h
+++
i, j
Domain II
++
k, l
Domain III
++
m
Domain IV
n. d.
n
n. d.
n
NS3 Protein
NS5B Protein
5’UTR
3’UTR
a: [200]; b: [201]; c: [202]; d: [203]; e: [204]; f: [205]; g: [206]; h: [207]; i: [211]; j: {Bellecave, 2003 #1817}; k: [212]; l: [216]; m: [217]; n: [214]; detailed legends for +, ++ and +++ are given in Table 1; n. d., non determined.
analysis verified aptamer binding to occur at basic residues on the protease domain surface at a site other than the catalytic center [208]. This confirms the involvement of a non-competitive inhibition mechanism. The secondary structure of the selected aptamers was extensively investigated and a consensus structure eventually defined, involving three stem-loop domains giving rise to a three-way structure in which domains I and III appear essential to inhibitory activity [209]. This prompted the analysis of the ex vivo inhibitory activity of some of these inhibitors [203]. Several copies of the tested aptamers were cloned in tandem and controlled by a strong cytoplasmic promoter. The resulting cassette also incorporated a catalytic RNA domain derived from the HDV ribozyme, allowing the generation of aptamer monomers after transcription. NS3 protease activity was found to be effectively inhibited, in some cases by close to 100%. More recently, RNA aptamers targeting the helicase domain have also been identified by similar SELEX methods [205, 206]. These effectively block NS3 unwinding activity (with inactivation values of close to 90% in some cases) without affecting NTPase function. These aptamers act by competing with the substrate for the helicase domain and have been shown to fold into a structure similar to the 3’ end of the negative strand of the HCV genome, an area that specifically interacts with the NS3 helicase domain [210]. Finally, a new strategy has been developed: the so-called rational design of dual functional aptamers [207]. The inhibitors produced combine two originally separate aptamers [202, 206] with different ligand specificities – one specific for the helicase domain and one specific for the protease domain – linked by a polyU sequence. These compounds bind efficiently to both targets and prevent viral replication by inhibiting proteolytic polyprotein processing and genomic RNA unwinding. The NS5B protein is also an attractive candidate for targeting with aptamers. SELEX methodologies developed by a number of authors have been successfully used to
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identify DNA and RNA oligonucleotides with high affinities and specificities for HCV RdRp [211-213]. The selected aptamers all fold differently, but in general the results reported suggest that the acquisition of a stem-loop structure favors their binding to the target site. Moreover, misfolding of this conformation abolishes binding of these aptamers to NS5B protein in in vitro systems [211]. The binding site of aptamers within NS5B differs. For example, the inhibitors selected by Tomei and co-workers [211] showed affinity for a basic patch located on the surface of the thumb domain (i.e., not in the catalytic cleft), while the binding sites of those identified by Astier-Gin and coworkers [212] and Lai and co-workers [213] are unknown. However, it is clear that they interfere with the replication of several RNA templates, suggesting that these binding sites lie close to one another. This information led to the development of effective inhibitors and to the identification of potential therapeutic targets in the HCV polymerase enzyme. The 5’UTR region has also been frequently chosen as a target for aptamers designed to interfere with HCV translation [214-217]. Aptamers of this type were identified by using in vitro selection methods in which the RNA ligand consisted of isolated specific domains of the IRES region (II, III and IV; Fig. (2)). This allowed for the identification of consensus motifs within the aptamers that were complementary to unique sequences in the IRES domain mapping to apical loops. These consensus motifs and their anchoring sequences in the RNA substrate are shown in Table 2. It has been shown that the association between these inhibitors and the viral RNA is promoted by loop-loop interactions, sometimes between apical loops [215, 217]. Thus, the mechanism of action implies a first binding step resulting in the formation of a kissing complex. Natural antisense RNAs act through the formation of these reversible unions to rapidly and specifically regulate a number of biological processes [218]. This property is exploited by aptamers which have improved ligand-binding capacities compared to conventional antisense oligonucleotides – properties that translate into increased inhibitory activity. Aptamers developed against the IIId domain were found to block IRES-dependent translation by about 50% in an ex vivo system [217]. Toulmé and co-workers have reported a different kind of interaction between substrates and inhibitory RNA aptamers [214, 216]. The association starts with nucleotides in an apical loop of the target binding to the specific residues in an internal loop of the aptamer. Selected aptamers are known to bind effectively to their targets in apical loops in domains II and IV. These apical loop-internal loop (ALIL) interactions are known to occur in several natural systems, in which they effectively modulate a number of cellular processes (reviewed in [219]). These loop-loop interactions should be considered in rational RNA-based drug design. RIBOZYMES AS ANTI-HCV AGENTS In the early 1980s, work performed independently by the groups of Thomas Cech and Sidney Altman led to the
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discovery of catalytically active RNA molecules or ribozymes [220, 221]. These molecules have both target recognition and catalytic abilities. Since then, numerous natural catalytic RNA motifs have been described. Natural ribozymes normally cleave the RNA phosphodiester backbone. The only known exception is the ribosome. These reactions proceed in an intramolecular context (i.e., a cisreaction), except for those mediated by RNase P and ribosomes, are highly specific, and are governed by WatsonCrick interactions established between the ribozyme and the substrate RNA. Natural ribozymes have been extensively studied and the minimum domain sizes with catalytic activity have been determined. The sequential and structural requirements for this function are also known [222-225]. This has allowed the development of trans cleaving ribozymes. These molecules mimic protein enzymes, and are neither altered nor consumed during catalysis. Since the nucleotides involved in the catalytic reaction generally have no role in substrate recognition, ribozyme specificity can be easily modified to target any RNA molecule of interest. Numerous investigations have been performed using ribozymes against multiple viral and cellular RNAs (reviewed in [226]), proving the potential of these molecules as therapeutic tools. Traditionally, ribozymes have been classified into three different groups: introns, RNase P and small catalytic RNAs. The last of these includes four RNA motifs that catalyze a transesterification reaction yielding products with 5’hydroxyl and 2’,3’-phosphate termini. Of these, the hairpin and the hammerhead ribozymes have been the most widely used in gene inactivation assays because of their small size and their minimal substrate sequence requirements [227]. Hairpin ribozymes were first discovered in the negative strand of the satellite RNA associated with tobacco ringspot virus ([-]sTRSV; [224, 228]). These participate in RNA satellite replication: multimeric copies generated through the rolling circle mechanism are processed by the hairpin catalytic motif and subsequently ligated, producing circular, monomeric molecules [229-231]. The minimal catalytic domain of hairpin ribozymes is composed of a 50 nt-long RNA sequence that recognizes and cleaves an external RNA molecule (Fig. (6A)). The secondary structure of the ribozyme-substrate complex, as well as nucleotides important in catalysis, were elucidated by in vitro selection procedures and mutational assays [232235]. Two domains were identified in the ribozyme-substrate complex, A and B, both composed of two helices separated by an internal loop. A and B fold upon each other after the ribozyme-substrate interaction has occurred so that the loop residues stay close together. This conformational change is known as docking, and is essential for catalysis [236, 237]. The other nucleotides important in catalysis are mainly located in single-stranded regions. Specificity can be easily changed by introducing appropriate nucleotide substitutions in the ribozyme sequences involved in helices 1 and 2, to allow base pairing with the RNA molecule desired. In ribozyme substrates, a conserved G residue is present at position +1 [238], although this can be modified as long as a compensatory modification is made at position C25 in the ribozyme molecule [239]. This suggests a possible interac-
Berzal-Herranz et al.
tion between G+1 and C25 during the docking process. The optimal substrate sequences for cleavage have also been also elucidated (Fig. (6A); [240]). Great efforts have been made to optimize hairpin ribozymes with the aim of developing more effective in vivo molecules. The substitution of U39 by C [232, 241] or the extension and stabilization of helix 4 achieves increased activity [242, 243]. Hammerhead ribozymes were first identified in plant pathogen RNAs [231, 244, 245]. Their in vivo function is similar to that of the hairpin ribozymes, although the details of the RNA ligation reaction remain unclear. Hammerheadlike structures have been detected in many cellular RNA molecules [246], and catalytic ability has been shown for three domains discovered in animal RNAs (in a transcript of a satellite DNA in newts [247], Schistosoma mansoni [248], and Dolichopoda spp. cave crickets [249]). The minimal motif required for catalytic activity was defined by deletion assays [245] [223, 250, 251], thus allowing the generation of a trans-cleaving system. In the secondary structure model proposed (reviewed in [252]), three helical elements can be distinguished connected by single-stranded regions with residues important for catalytic activity (Fig. (6B)). As for the hairpin ribozyme, the sequences responsible for substrate recognition can be modified to target different RNAs. The consensus sequence for the cleavage site in the RNA substrate has been defined as 5’-NHH↓ (N = any nucleotide; H = A, C or U; ↓ = cleavage site). The hammerhead ribozyme is probably the most studied, and it has been used extensively in gene inactivation assays (reviewed in [226]). Numerous reports suggesting the use of ribozymes as an HCV therapy have been published (see Table 3). The preferred target region has usually been the 5’UTR and the coding sequence of the 5’-core protein [253-258]. In pioneering work, Kay and co-workers [259] identified effective anti-HCV hammerhead ribozymes by using an in vitro selection procedure. Briefly, RNA obtained from HCVinfected livers was purified and incubated with a hammerhead ribozyme library, in which helix 1 and 3 were randomized. Specific cleavage products were detected, reverse transcribed, amplified and sequenced, and this allowed hammerhead ribozymes to be designed by inferring with the nucleotide sequence in helices 1 and 3 (employing sequences complementary to the identified cleavage site). This approach allowed the identification of the most easily processed sites in the target RNA, although the hammerhead ribozymes responsible for that cleavage were determined theoretically. The catalytic molecules obtained were directed against several positions in the 3’ end of the IRES region and the conserved sequences in the negative strand. For this, ribozymes were cloned in adenoviral vectors and their inhibitory activity tested in primary human hepatocytes obtained from infected patients. A mixture consisting of several inhibitors induced remarkable reductions in HCV RNA levels. In earlier work, Wu and co-workers [254] reported the use of hammerhead ribozymes targeting four positions in the IRES and core-coding protein sequence. The
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Fig. (6). A) Hairpin ribozyme. The sequence requirements for the complex to form between the catalytic hairpin motif and its substrate are shown. N = any nucleotide; Y = C or U; R = A or G; B = C, G or U; V = A, C or G; H = A, C or U. Important nucleotides for hairpin ribozyme activity are indicated in bold. Sequences in the J2/1 region important for cleavage are shown in box “a”. A and B represent domains of the ribozyme-substrate complex. J1/2-J4/3, single stranded regions. H1-H4, double stranded regions. The cleavage site is indicated by an arrow. Nucleotides composing catalytic domain are shown in capital letters. Lower-case represents a substrate molecule. Figure adapted from [226]. B) Hammerhead ribozyme. Sequence requirements for the hammerhead ribozyme cleavage activity when complexed with its RNA substrate. Nucleotides indispensable for the catalysis reaction are emphasized in bold. Capital letters represent the hammerhead ribozyme, the RNA substrate is indicated by lower-case letters. N = any nucleotide; Y = C or U; R = A or G; H = A, C or U. H1-H3, helix 1-helix 3. Table 3. Ribozymes Used for Cleaving HCV Genome
HCV Target Region
Efficiency
References
Domain II
+ ++
e c, e
Domain III
+ ++ +++
e b, c, e a, d, e, f
++ +++
b, c a
5’UTR
5’-end core coding sequence
a: [259]; b: [254]; c: [255]; d: [256]; e: [257]; f: [258]; +, ++ and +++, see Table 1.
production of these ribozymes in eukaryotic cells was achieved through the use of suitable expression vectors. A moderate reduction in IRES-dependent translation was achieved – reporter protein levels fell to around 50% in cells producing these ribozymes. Hairpin ribozymes have scarcely been used in HCV inactivation experiments, though the results obtained are comparable to those achieved with hammerhead catalytic RNAs [255]. As mentioned above, the successful use of inhibitory RNAs as therapeutic agents, particularly ribozymes, requires the improvement of intracellular stability, target accessibility
and association with the cleavage site etc. The results obtained with most ribozymes in initial assays were transient, so chemical modifications were introduced during their synthesis in an attempt to increase their half-life. Unlike in antisense systems, substitutions in the sequence of a catalytic RNA can severely affect its function, and several attempts have been made to identify modifications that can be made successfully without impairing catalytic activity [260]. Blatt and co-workers generated a stabilized hammerhead ribozyme specific for cleaving the HCV genome at nucleotide 195 [257]. This consisted of a modified molecule bearing 2’-O-methyl nucleotides, 2’deoxy-2’-C-allyl-uridine and phosphorothioate linkage modifications. In addition, deoxyabasic residues were incorporated at the 3’ end. This molecule was shown to be catalytically active in vitro and in ex vivo systems against chimeric HCV poliovirus, inhibiting viral RNA replication by around 80%. Moreover, 95% of viral replication was blocked when it was combined with IFN-α [258]. Pharmacokinetic analyses using mice were developed to investigate its half-life in serum and its tissue localization [256]. Ribozyme molecules were detected in hepatocyte cytoplasm 48 h after administration, demonstrating the feasibility of this strategy. These excellent results suggested this strategy may be a viable way of inactivating HCV. Preclinical and clinical assays were initiated with this socalled HeptazymeTM but after initially promising results these investigations were for some reason suspended.
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The improvement of the association of inhibitory RNA to their target sites has been – and remains – a major goal in the development of second generation ribozymes. In the early 1990s, modified hammerhead ribozymes targeting the HIV genome were reported [261, 262]. Known as catalytic antisense RNAs, helices 1 and 3 of these ribozymes were extended with the aim of increasing access to the target sequence. However, the duplexes generated were too stable, preventing ribozyme turnover. To overcome this, asymmetric hammerhead ribozymes were constructed by lengthening only one of the two binding arms [263]. This allowed the release of the cleavage product and consequently achieved the desired turnover. More recently, new catalytic antisense RNAs have been generated against HIV [155]. In an attempt to take advantage of the structural features existing in the target molecule, stable stem-loop structure domains were attached to the 3’ end of the ribozymes. These domains are composed of a sequence complementary to another stem-loop domain in the target molecule. The interaction between these domains enhanced ribozyme efficiency. Further, stable stem-loop structured sequences at the termini of RNA molecules have been reported to increase resistance to nuclease-mediated degradation [264]. The proposed mechanism of action of the new molecules resembles that of naturally occurring antisense-controlled systems, in which two stem-loop structural domains specifically and efficiently interact [218]. Viral inactivation assays showed up to 95% reductions in HIV RNA replication [157]. These excellent results lead the authors to extrapolate this model to the design of new anti-HCV inhibitors [158]. An innovatory in vitro selection procedure was designed to obtain new molecules targeting the IRES domain. An RNA library was constructed based on the previously described hammerhead ribozyme that cleaves HCV RNA at nucleotide 363 (HH363; [259]). The library RNAs all carried a 25 ntlong randomized region attached to the 3’ end of the ribozyme, whose function was to allow effective binding of the putative inhibitor RNAs to the HCV IRES domain. The method involved two consecutive selection steps (Fig. (7)). In the first stage, active molecules specifically binding the whole 5’UTR region were selected. With this aim, an RNA substrate containing only the first 356 nucleotides of the viral genome – therefore not cleavable by HH363 – was constructed (5’HCV-356). This was internally biotinylated and immobilized in a sepharose-streptavidin column. The RNA library molecules were incubated with 5’HCV-356 to allow specific binding through the variable 25 nt domain, which acted as an aptamer for the 5’UTR HCV region. The associated variants were recovered by denaturing the RNARNA interactions and then subjected to the second selection step. This consisted of the selective identification of those inhibitory RNAs that retained catalytic activity for processing an HCV RNA substrate molecule carrying the first 691 nucleotides of the viral genome (5’HCV-691). The latter RNA molecule was annealed through its 3’ end to a biotinylated probe and again immobilized in a sepharosestreptavidin column. The resulting pool from the first step was loaded onto the column and the molecules that actively cleaved the HCV RNA were eluted bound to their 5’ cleavage product. The non-active molecules remained bound to the substrate in the column. These selected variants were specifically reverse-transcribed and amplified. A fraction of
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the resulting cDNA was used for transcription and subjected to subsequent selection rounds. The remaining molecules were cloned and sequenced. This strategy allowed for the identification of chimeric RNA molecules composed of an aptamer and a catalytic domain. It is proposed that the anchoring site for the aptamer maps to conserved regions essential for IRES-dependent translation; these included the pseudoknot and domains II, III and IV [158]. This was the first report in which aptamer molecules targeting the pseudoknot structural motif of the IRES had been described. The newly obtained inhibitory RNAs efficiently blocked IRES-dependent translation in an in vitro translation system by up to 97%. Current investigations are focused on testing the ex vivo activity of these chimeric inhibitors. This innovative approach of combining in a single molecule the features of aptamers and ribozymes seems very promising since greater HCV inhibition values are achieved than with the ribozyme or aptamer on their own [158]. The achievements of recent years clearly suggest that the use of ribozymes as specific inhibitors has huge potential. Moreover, the solution of several problems associated with their in vivo use has led to their proposal as RNA-based therapeutic agents. The further development of second generation ribozymes will almost certainly aid in the identification of more effective anti-HCV molecules with improved binding properties and stability. HCV SILENCING BY siRNAs RNA duplexes can induce gene silencing through the activation of interference RNA pathways. This mechanism was first seen in animals in 1995 by Guo and Kemphues [265] who analyzed the role of the gene par-1 in the development of the worm Caenorhabditis elegans. Huge amounts of single stranded RNA (ssRNA) complementary to par-1 were introduced into the animal, with a highly evident gene silencing effect. This phenomenon was called interference and was confirmed three years later by Mello and co-workers [266]. In this study, double-stranded (ds) RNA molecules induced a stronger silencing effect than that previously observed with ssRNA. This led to the proposal that dsRNA was in fact responsible for the inhibition observed. RNA interference (RNAi) is an ancient, sequencespecific, dsRNA-mediated, post-transcriptional gene silencing mechanism. The process starts by the selective cleavage of dsRNA molecules into shorter molecules of 2128 nt by Dicer, an RNase III-like ribonuclease. These small molecules are known as siRNA (small interfering RNAs). siRNAs proceed from a large, double-stranded RNA molecule generated as an intermediate during viral replication, by a transposition mechanism or through transgene activity. Dicer carries one helicase and two RNase III-like domains [267]. These activities allow for the unwinding of the dsRNA molecule and its subsequent cleavage. The resulting siRNAs are incorporated into a ribonucleoprotein complex known as a RISC (RNA-induced silencing complex) to form siRNPs (small interfering ribonucleoproteins). Their activation exposes the siRNA antisense strand which guides sequence-specific RNA target selection by Watson-Crick base pairing. Cleavage of the target
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Fig. (7). In vitro selection procedure developed by Berzal-Herranz and co-workers. [158] for the identification of anti-HCV chimeric RNA molecules composed of an aptamer domain and a catalytic motif. S, streptavidin; B, biotin ligand; Pri1is and Pri2is, specific primers for cDNA synthesis and amplification; T7p, T7 promoter; PBS, primer binding site. See text for details.
molecule then occurs; degradation is usually rapid. In some organisms, a fraction of the processed products can be converted to dsRNA by an RdRp, giving rise to new interfering molecules that amplify the silencing effect.
An alternative mechanism involves the ligation of several antisense strands. This leads to the generation of a cRNA capable of hybridizing with target molecules, thus forming new dsRNA species that can be used by the interference
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machinery (transitive RNAi; [268]). Mismatches between a single-stranded siRNA and its target RNA can greatly decrease the efficiency of RNA degradation. This highlights the great sequence specificity of RNAi systems. The discovery of interference routes provided an alternative way to specifically silence RNA molecules. Several attempts have been made to develop siRNAs targeting highly conserved regions of the HCV genome (Table 4; [269-281]). Some have used coding sequences for non-structural proteins as target sites [269, 272, 274, 276, 279, 281]. In all cases in which the NS5B coding protein sequence was targeted [269, 272, 274, 276, 279, 281], effective inhibition of replication (by up to 90%) was achieved (as assessed in HCV replicon systems). Cells Table 4. siRNAs Targeting Conserved Sequences in HCV Genomic RNA
HCV Target Region
Efficiency
References
Domain I
+
i
Domain II
+ ++
c j
Domain III
+ ++ +++
c, d, i, j i c, i
Domain IV
+++
i, k
3’UTR
++
j
5’-end core coding sequence
++ +++
d c, h
E2 Protein coding sequence
+++
e
NS3 Protein coding sequence
+ ++ +++
b a d, e
NS4B Protein coding sequence
+ +++
a c
NS5A Protein coding sequence
+ ++ +++
a g c
NS5B Protein coding sequence
++ +++
a b, d, e, f
5’UTR
a: [269]; b: [272]; c: [274]; d: [276]; e: [279]; f: [281]; g: [271]; h: [270]; i: [273]; j: [278]; k: [280]; indications for +, ++ and +++ are shown in Table 1.
bearing replicon subgenomic HCV RNA were able to grow in the presence of the antibiotic neomycin. Anti-NS5B siRNAs transfection greatly reduced cell survival when the antibiotic was incorporated into the medium [272]. Moreover, reductions in the numbers of positive and negative RNA strands were detected. This confirmed the data obtained in studies using aptamers against polymerase, which led to their authors proposing that reductions in polymerase activity strongly affected HCV RNA synthesis
[211-213]. With respect to siRNAs, the observed effect was attributed to the direct degradation of the genomic RNA and, indirectly to a destabilization of the replication complex in which the HCV genome is included ([275] and references therein). Both explanations are possible and complementary. The appearance of siRNAs-resistant replicon clones was observed by the generation of point mutations that disturbed siRNA pairing at the target site [281]. However, this was circumvented by the combination of several siRNAs with different specificities. Contradictory results have been reported when the NS3 protease coding sequence has been used as an siRNA target [269, 272, 276, 279]. While Richardson and co-workers [272] detected only marginal reductions in HCV RNA levels in replicon culture systems, other authors [269, 276, 279] have reported the efficient inhibition of RNA synthesis (in some cases in a dose-dependent fashion [269]). The explanation for these differences could lie in the different target sites used. The presence of secondary and tertiary structures in the RNA molecule to be degraded may interfere with the access of the siRNP complex, impeding the activity of the RISC. Alternatively, the explanation may lie in the appearance of resistant variants as reported in replicon systems (reviewed in [28]). The sequences encoding the NS4B and NS5A proteins have also been targeted in HCV inactivation assays. Opposing conclusions were obtained when siRNAs targeting these genes were overproduced in replicon systems [269, 271, 274], confirming that the design of these inhibitory molecules is critical and subject to problems similar to those affecting other nucleic acid-based gene inactivation systems. The IRES region has been used as a target for siRNA molecules, mainly because of its high sequence conservation rate [270, 273, 274, 278, 280]. Both the surrounding sequences of the translation start codon and the pseudoknot structural motif have been found effective targets, with inhibition of IRES-dependent translation in replicon systems reaching almost 80% in some cases [273, 274]. Furthermore, in in vivo assays in mice, IRES activity was suppressed by up to 90% [280], suggesting the huge potential of this approach with respect to HCV inactivation. In addition, the combination of siRNA inhibitors targeting not only the HCV 5’UTR but also the coding sequences for essential cellular processes, leads to enhanced reduction of HCV RNA levels in replicon systems [278]. The induction of the IFN response is a determining factor in the blocking of viral gene expression by siRNAs, and has been extensively investigated [282-285]. The siRNA transfection of human cells involves the activation of the Jak-Stat pathway and a general dose-dependent upregulation of the genes whose expression is dependent on IFN [284]. This effect might be associated with the binding of siRNAs to PKR. Alternatively, it has been proposed that the enhancement of IFN-associated routes might occur through the incorporation of additional nucleotides from the promoter sequence [282]. Other possible limitations of this approach include the overproduction of siRNAs that might affect the interference machinery used in the regulation of cell processes. This could upset the internal equilibrium of the cell and alter the expression of essential genes not related to
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the target. Potential non-specific effects caused by siRNAs should be taken into account in the development of such interference strategies. Finally, the combined use of several antiviral strategies might be effective. It has recently been reported that simultaneous transfection with hairpin ribozymes directed against the 3’UTR HCV region and siRNAs targeting the IRES domain triggers the efficient inhibition (up to 90%) of HCV replication in subgenomic replicon systems [286]. In addition, IFN production was not detected after the overproduction of these inhibitors, demonstrating the specificity of this strategy. THE DECOY STRATEGY A strategy gaining increasing acceptance as antiviral agents is the use of small genomic viral elements specialized in binding protein factors essential to the infective cycle. These molecules, known as decoys, capture in trans factors essential to virus replication or translation. Several reports indicate that the depletion of these indispensable factors by decoys may be an effective antiviral strategy [287-294]. Therapeutic decoy molecules need to show high specificity and stability. Specificity is guaranteed by using oligonucleotides carrying sequences present in domains of the viral genome, while stability can be improved by incorporating chemical substitutions, as noted above. The main advantage of decoys is that the appearance of viral escape mutants becomes more difficult; a double modification of the RNA domain and the coding protein sequence affecting the primary structure would be required. Consequently, the use of decoys should not, at least in theory, be affected by genetic heterogeneity. Decoys have been used to interfere with HCV infection (Table 5). Specific domains in the IRES and CRE regions were chosen as targets for the inhibition of HCV replication and translation [293, 294]. The overproduction of domain III, and more specifically subdomains IIIe and IIIf, was shown in replicon systems to strongly prevent IRES-dependent translation as well as S5 protein binding – important for protein synthesis. No direct inhibition of replication was seen, suggesting a highly specific effect on IRES functionality. Other authors [294] have used isolated domains of the CRE (cis-response element) region as decoys. Recently, it was shown that this highly conserved domain (CRE) located Table 5. The Use of RNA Decoys for Capturing Essential Viral Factors
HCV Target Region
Efficiency
References
Domain II
+
a
Domain III
+++
a
5BSL3.1
+++
b
5BSL3.2
+++
b
5BSL3.3
+
b
+
b
5’UTR
CRE region
3’UTR
a: [293]; b: [294]; +, ++ and +++ are indicated in Table 1.
139
at the 3’ end of the NS5B coding sequence was essential for the formation of a stable replication complex and the initiation of RNA negative strand synthesis [119, 120, 295]. Three domains folding in stem-loop structures were detected and analyzed for their importance in viral replication. Two of these, 5BSL3.1 and 5BSL3.2, were found to be indispensable for RdRp activity, probably through the anchoring of the replication complex (including the NS5B protein; [119, 295]). Moreover, the 5BSL3.2 domain was found to be involved in the establishment of a kissing-loop interaction with an apical loop of the X-tail in the 3’UTR region. Therefore, it is thought that the overproduction of RNA species containing CRE domains might offer a way of combating HCV. These molecules would act as decoys, sequestering the viral polymerase and consequently interfere with viral replication. Molecules corresponding to domains 5BSL3.1 and 5BSL3.2 were shown to effectively inhibit RNA synthesis in different replicon systems, indicating that the different polymerases involved can be targeted despite the small variations in their nucleotide sequences. The adenoviral-mediated production of decoy molecules decreased the number of RNA viral copies by about 50% [294]. TOXICOLOGY AND PHARMACOKINETICS Pharmacokinetic analyses of clinically-used ribozymes and antisense oligonucleotides have been undertaken [182, 256, 296-299]. Injection is the preferred method for administering these agents, although others have been studied in animals. Several injection routes are possible: intraperitoneal, intravenous, intradermal and subcutaneous. The last two options are most favorable to the systemic distribution of the inhibitor, while subcutaneous injection favors its bioavailability [163]. Both antisense and ribozymes are found at higher concentrations close to the infusion site, the size of this area being directly proportional to the dose administered. At the systemic level, therapeutic nucleic acid molecules mainly accumulate in the liver, kidney, spleen, lung and, to a much lesser extent, in the brain, depending on their backbone composition, the number of substitutions the molecules carry, and the positions of the latter (reviewed in [163]). The dynamics of this accumulation are affected neither by the length nor sequence of these molecules. All these oligonucleotides are degraded within cells by enzymatic action at their non-modified positions. They are excreted in the urine 1-5 days after administration [182, 300]. RNA/DNA-based inhibitors are usually considered nontoxic to the receiving organism since nucleic acids are ubiquitous. However, toxic effects can occur, either because of the inhibitor interacting with the target (specific toxicity) or with related or non-related targets (non-specific toxicity; reviewed in [301]). Nucleic acid-based inhibitors show strong sequence and structural specificity, therefore secondary effects due to interactions with non-target RNA molecules ought not to be expected. However, the chemical modifications introduced into these molecules to increase their stability and bioavailability can provoke a number of undesired responses: these must be investigated before any potential therapeutic agent enters clinical trials. Animals models (e.g., primates or rodents) can be helpful, but the
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results obtained in one do not necessarily extrapolate to another (e.g., rodents are more sensitive than primates to RNA/DNA therapeutic agents) or, therefore, to humans. The most common toxicity symptoms are dose-dependent and include splenomegaly, lymphoid hyperplasia and diffuse mononuclear cell infiltration. In preliminary investigations performed in HCV-infected patients, antisense therapy has been associated with mild-moderate adverse effects such as thrombocytopenia, hyperglycemia, hypotension, fever and fatigue, and at high doses with hepatocellular degeneration. At the injection site, erythema and pain have been also described (for a review, see [163]). These effects have been most commonly reported with antisense agents, though probably because they have been the most commonly tested nucleic acid-based agents in clinical trials. These toxicities appear to be due to the composition of the molecules’ backbones and the modifications they carry, rather than to their nucleotide sequence. Nevertheless, CpG sequences do appear to exert a toxic effect. Recipient defense systems seem to associate these with a bacterial infection, unleashing a non-specific immune response [302]. Among the different chemical modifications described to induce toxicity, phosphorothioate substitutions should be emphasized. The harm these can cause is directly dependent on the number of modified positions. A severe consequence reported is the activation of the complement cascade, which can lead to cardiovascular collapse (reviewed in [163, 303]). These findings also suggest that care should be taken in the use of aptamers, which are similar to antisense molecules. In the case of ribozymes and siRNA agents, more extensive analyses have been performed. No severe toxic effects have been detected to date, and they seem to be well tolerated.
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zation is, however, necessary, possibly including the combination of nucleic acids-based therapies with conventional drugs. This may enhance the antiviral effects achieved, impede the generation of resistant HCV variants, and reduce the doses required, thus helping to control the appearance of adverse effects. ACKNOWLEDGEMENTS The work in our laboratory is supported by grants from the Spanish MEC (Ministerio de Educación y Ciencia; BMC2003-00669) and the Junta de Andalucía (CVI-265) to A.B-H. C.R-L was the recipient of a CSIC-I3P program fellowship funded by European Social Funds. F.J-L. is the recipient of a fellowship from the MEC. ABBREVIATIONS ALIL
=
Apical loop-internal loop
BNAs
=
Bridged nucleic acids
BVDV
=
Bovine diarrhea virus
CRE
=
Cis-response element
cRNA
=
Ribonucleic acid copy
DNA
=
Deoxyribonucleic acid
dsRNA
=
Double stranded ribonucleic acid
eIF3
=
Eukaryotic initiation factor 3
ER
=
Endoplasmic reticulum
GADPH
=
Glyceraldehyde 3-phosphate dehydrogenase
HCV
=
Hepatitis C virus
HIV
=
Human immunodeficiency virus
IL
=
Interleukin
HNAs
=
Hexitol nucleic acids
hnRNP C
=
Heterogeneous nuclear ribonucleoprotein C
IFN
=
Interferon
IRES
=
Internal ribosome entry site
LNAs
=
Locked nucleic acids
CONCLUDING REMARKS
NMR
=
Nuclear magnetic resonance
The possibility of using nucleic acids as gene inactivation tools has been extensively investigated over the last 15 years, and the results show they can be useful in the treatment of several diseases, including hepatitis C. In vitro selection techniques are powerful tools that have been widely used for the isolation of ever more specific and active inhibitors. These strategies have identified aptamers and ribozymes that efficiently interfere with essential steps in HCV replication. Others combining different functional domains can efficiently inhibit HCV IRES-dependent translation, and posi-tive results have been obtained in clinical trials. Advances in chemical synthesis could help the clinical use of these molecules become more generalized.
NS
=
Non-structural
NTPase
=
Nucleotide triphosphatase
NTPs
=
Nucleotides triphosphate
PAMP
=
Pathogen-associated molecular pattern
PBS
=
Primer binding site
PEG
=
Polyethylenglycol
PKR
=
Protein kinase R
PNAs
=
Peptide nucleic acids
Although the majority of the toxic events described are attributable to the chemical modification of nucleic acid agents, the latter are necessary for optimal drug activity. Further investigations are needed to identify new modifications or optimized molecules with high stability and functionality but without toxic effects. These results should be considered preliminary and should be compared with those for other conventional drugs. New strategies combining generic antiviral compounds with innovative molecular therapy approaches could be developed for a reduced toxicity and increased effectiveness.
The evidence available to date clearly suggests that this technology could have a promising future. Further optimi-
PTB protein =
Polypyrimidine tract-binding protein
RdRp
RNA-dependent RNA polymerase
=
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Infectious Disorders - Drug Targets 2006, Vol. 6, No. 2
RISC
=
Ribonucleic acid induced silencing complex
[24]
RNA
=
Ribonucleic acid
[25] [26]
RNAi
=
Ribonucleic acid interference
RNase
=
Ribonucleasa
SELEX
=
Systematic evolution of ligands by exponential enrichment
[28] [29]
SL
=
Stem-loop structure
[30]
siRNA
=
Small interfering RNA
[31]
siRNP
=
Small interfering ribonucleoprotein
ssRNA
=
Single stranded ribonucleic acid
[32]
sTRSV
=
Satellite tobacco ringspot virus
[33]
T7p
=
T7 promoter
UTR
=
Untranslated region
VR
=
Variable region
WHO
=
World Health Organization
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