Targets and tools: recent advances in the development of anti-HCV nucleic acids

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.     

Evaluation of synthetic oligonucleotides as inhibitors of West Nile virus replication

A series of synthetic oligonucleotide phosphorothioate 15-mers were generated against specific sequences in the West Nile virus RNA genome. These antisense oligonucleotides targeted (1) conserved features of the West Nile virus RNA genome that may be expected to lead to inhibition of virus replication since such features play essential roles in the virus lifecycle; (2) G-quartet oligonucleotides with potential facilitated uptake properties and that also targeted conserved sequences among a range of West Nile virus strains. Several formulations with significant in vitro antiviral activity were found. Among the active oligonucleotides were examples that targeted both C-rich RNA sequences of the West Nile RNA genome as well as recognized conserved sequences key to West Nile virus replication. Since the antiviral activity of the latter oligonucleotides diminished upon 2'-O-methyl substitution, it is likely that their activity involves RNase H-catalyzed RNA degradation. One G-rich oligonucleotide that did not target a West Nile virus RNA sequence also was found. These results suggest the potential of antisense strategies for the control of West Nile virus replication if the attendant problem of oligonucleotide delivery can be adequately addressed.     

Inhibition of RNA virus infections with peptide-conjugated morpholino oligomers

RNA virus infections cause immense human disease burdens globally, and few effective antiviral drugs are available for their treatment. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are nuclease resistant and water-soluble single-stranded-DNA-analogues that can enter cells readily and act as steric-blocking antisense agents through stable duplex formation with complementary RNA. Recently there have been a number of publications documenting sequence-specific and dose-dependent inhibition of non-retroviral RNA virus infections by PPMO in both cell culture and murine experimental systems. PPMO have suppressed viral titers by several orders of magnitude in cell cultures, and have reduced viral replication in and/or increased survivorship of mice experimentally infected with poliovirus, coxsackievirus B3, dengue virus, West Nile virus, Venezuelan Equine encephalitis virus, respiratory syncytial virus, Ebola virus and influenza A virus. Along with evaluating PPMO efficacy and toxicity, these studies also explored PPMO mechanism of action, pharmacologic properties and the generation and characterization of resistant virus. Effective PPMO target sites in viral RNA have included regions of highly conserved sequence thought to be important in the pre-initiation or initiation of translation, or in long-range RNA-RNA interactions involved in viral RNA synthesis. These studies provide guidance for the design of steric-blocking antisense agents against RNA viruses, insights into viral molecular biology and novel strategies for the development of antiviral therapeutics. The purpose of this review is to summarize notable findings from the reports documenting antiviral activity by PPMO, with a focus on the specific regions of viral RNA that provided the most effective targets for PPMO-based inhibition of viral replication.     

Future promise of siRNA and other nucleic acid based therapeutics for the treatment of chronic HCV

RNA interference (RNAi) is gaining favor as a potential therapeutic option for the treatment of Hepatitis C virus infections. RNAi, first discovered in plants, induces sequence specific degradation of messenger RNA following the introduction of short interference RNA (siRNA). RNAi is a natural defense mechanism used by plants to combat viral infections, and the discovery of RNAi activity in mammalian cells has prompted several drug companies to investigate and exploit RNAi based drugs as a potential therapy against HCV infections. A number of research groups have demonstrated that strong RNAi activity can be induced against HCV using synthetic siRNA duplexes as triggers, or by expressing short hairpin RNAs from plasmid or viral vectors. However, much work remains to improve delivery, maintain specificity and limit the development of virus resistance. HCV is capable of evading RNAi activity through the incorporation escape mutations within the siRNA target sequence, highlighting the importance of implementing strategies to limit the development of resistance. Other nucleic acid based therapies such as antisense oligonucleotides, RNA aptamers and ribozymes have also been considered for use as HCV therapeutics, and we will outline the potential opportunities and obstacles to their use as well as RNAi.     

Strand-specific silencing of a picornavirus by RNA interference: evidence for the superiority of plus-strand specific siRNAs

RNA interference triggered by small interfering RNAs (siRNAs) can be used to effectively contain viral spread. Here, we report on the mechanism of action of siRNAs targeting the medically important coxsackievirus B3 (CVB-3) as a typical representative of viruses with a non-segmented RNA genome in positive-strand orientation. Antiviral siRNAs can be designed to target the genomic (+)-strand, the (-)-strand that occurs as a replication intermediate, or both. In the present study, two complementary and systematic approaches are presented providing direct evidence that silencing of the viral (+)-strand is the key to inhibit CVB-3: first, we used rational siRNA design to direct silencing activity specifically against either of the two viral strands. As a second approach, we employed siRNA containing modified nucleotides to render them specific for one of the virus RNAs. Experiments with infectious coxsackievirus revealed that the inhibitory efficiency correlates exclusively with the activity of the siRNAs directed against the viral (+)-strand. Our finding that only (+)-strand specific siRNAs exert significant antiviral potency may hold true for other RNA viruses with (+)-stranded genomes as well and may therefore be helpful in the development of efficient strategies to inhibit virus propagation.     

Recent Patents on development of nucleic acid-based antiviral drugs against seasonal and pandemic influenza virus infections

Influenza viruses are etiological agents of deadly flu that continue to pose global health threats, and have caused global pandemics that killed millions of people worldwide. The global crisis involving the avian H5N1 influenza provides compelling reasons to accelerate fast track development of novel antiviral drugs against the potential pandemic virus. The availability of neuraminidase inhibitors such as oseltamivir (tamiflu) improves our ability to defend against influenza viruses, but the incidences of tamiflu-resistance are rising rapidly. Nucleic acid-based antiviral drugs are promising classes of experimental antiviral drugs that have been shown in pre-clinical studies to be effective against seasonal and avian influenza viruses. The potency and versatility of these drugs make them potential candidates to be used in seasonal and pandemic influenza scenarios. The review will assess the recent patents, research and development of antisense oligonucleotides, small interfering RNA, immunomodulating RNA for the prevention and treatment of influenza infection.     

Progress in the development of nucleic acid therapeutics

Abnormal gene expression is a hallmark of many diseases. Gene-specific downregulation of aberrant genes could be useful therapeutically and potentially less toxic than conventional therapies due its specificity. Over the years, many strategies have been proposed for silencing gene expression in a gene-specific manner. Three major approaches are antisense oligonucleotides (AS-ONs), ribozymes/DNAzymes, and RNA interference (RNAi). In this brief review, we will discuss the successes and shortcomings of these three gene-silencing methods, and the approaches being taken to improve the effectiveness of antisense molecules. We will also provide an overview of some of the clinical applications of antisense therapy.     

Antisense strategies in degenerative joint diseases: sense or nonsense?

Oligodeoxynucleotides, ribozymes, and RNA interference make part of the antisense strategy, a new tool proposed to conquer cancer, viral infections, as well as cardiovascular and rheumatic diseases. The silencing effect of antisense strategies is both highly specific and potent - and only requires that the sequence of the target RNA is known. However, so far neither RNAi nor ribozymes have been approved for clinical use and only a single antisense agent is on the market. In the context of degenerative joint diseases, experimental data in the field of antisense strategies are still rare. Several studies from rheumatoid arthritis (RA), an inflammatory condition that leads to the progressive destruction of cartilage and bone within affected joints, however revealed promising results and taught us important lessons that might also be useful in therapeutic approaches for osteoarthritis (OA). To introduce these therapies in clinical practice, however, several hurdles still have to be overcome.     

RNA interference: its use as antiviral therapy

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more than just a response to exogenous genetic material. Small RNAs termed microRNA (miRNA) regulate cellular gene expression programs to control diverse steps in cell development and physiology. The discovery that exogenously delivered short interfering RNA (siRNA) can trigger RNAi in mammalian cells has made it into a powerful technique for generating genetic knock-outs. It also raises the possibility to use RNAi technology as a therapeutic tool against pathogenic viruses. Indeed, inhibition of virus replication has been reported for several human pathogens including human immunodeficiency virus, the hepatitis B and C viruses and influenza virus. We reviewed the field of antiviral RNAi research in 2003 (Haasnoot et al. 2003), but many new studies have recently been published. In this review, we present a complete listing of all antiviral strategies published up to and including December 2004. The latest developments in the RNAi field and their antiviral application are described.     

New therapeutic strategies in the treatment of hepatitis B virus infection

Principally, because of the association of the chronic carrier state with the development of cirrhotic liver disease and hepatocellular carcinoma, chronic hepatitis B infection is a public health problem of global significance. In the main, therapy for chronic hepatitis B is limited to the use of alpha interferon for a limited number of chronic hepatitis B virus (HBV) carriers who have chronic hepatitis with active viral replication. The development of antiviral nucleoside analogues for the herpes viruses and human immunodeficiency virus (HIV) has resulted in the identification of several compounds which also have activity against HBV. Unfortunately, these agents have not been associated with the clearance of hepatitis B infection, but rather only the suppression of active infection while the patient is receiving medication. In addition, the development of drug-resistance to these agents by the virus will most likely limit their long-term efficacy. Gene therapy has recently been applied to HBV both in vitro and in vivo. This has included the use of antisense oligodeoxynucleotides and RNA, ribozymes, dominant negative mutants and therapeutic HBV vaccines. These newer therapeutic modalities may hold promise as effective treatments for chronic hepatitis B, but to date, have been limited by the problem of delivery to the target cell population or infected organ in vivo. Combination nucleoside analogue therapy may also provide an important treatment modality for chronic hepatitis B, although this will require further investigation.     

Molecular strategies to inhibit the replication of RNA viruses

There are virtually no antiviral drugs available for the treatment of infections with RNA viruses. This is particularly worrisome since most of the highly pathogenic and emerging viruses are, and will likely continue to be, RNA viruses. These viruses can cause acute, severe illness, including severe respiratory disease, hemorrhagic fever and encephalitis, with a high case fatality rate. It is important to have potent and safe drugs at hand that can be used for the treatment or prophylaxis of such infections. Drugs approved for the treatment of RNA virus infections (other than HIV) are the influenza M2 channel inhibitors, amantadine and rimantadine; the influenza neuraminidase inhibitors, oseltamivir and zanamivir, and ribavirin for the treatment of infections with respiratory syncytial virus and hepatitis C virus. The molecular mechanism(s) by which ribavirin inhibits viral replication, such as depletion of intracellular GTP pools and induction of error catastrophe, may not readily allow the design of analogues that are more potent/selective than the parent drug. Highly pathogenic RNA viruses belong to a variety of virus families, each having a particular replication strategy, thus offering a wealth of potential targets to selectively inhibit viral replication. We here provide a non-exhaustive review of potential experimental strategies, using small molecules, to inhibit the replication of several RNA viruses. Other approaches, such as the use of interferon or other host-response modifiers, immune serum or neutralizing antibodies, are not addressed in this review.     

Antisense oligonucleotides targeting the RNA binding region of the NP gene inhibit replication of highly pathogenic avian influenza virus H5N1

The H5N1 avian influenza virus (AIV) causes widespread infections in bird and human respiratory tracts, and vaccines and drug therapy are limited in their effectiveness. Recent studies of AIV structures have been published and provide new targets for designing antiviral drugs such as antisense oligonucleotides (AS ODNs), which effectively inhibit gene replication. In this study, we designed and synthesized three AS ODNs (NP267, NP628, NP749) that were specific for the RNA binding region of nucleoprotein (NP) based on AIV structure. Results showed that all three AS ODNs could inhibit viral replication in MDCK cells. The NP628 showed the best antiviral effect of all through viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, the liposome mediated NP628 could partially protect the mice from a lethal H5N1 influenza virus challenge. Moreover, the NP628 group had a lower viral titer and lung index in the infected mice when compared with the viral control. Our results showed that AS ODN targeting of the AIV NP gene could potently inhibit AIV H5N1 reproduction, thus, formulating a candidate for an emergent therapeutic drug for the pathogenic H5N1 influenza virus infection.     

Antisense oligonucleotides targeting the RNA binding region of the NP gene inhibit replication of highly pathogenic avian influenza virus H5N1

The H5N1 avian influenza virus (AIV) causes widespread infections in bird and human respiratory tracts, and vaccines and drug therapy are limited in their effectiveness. Recent studies of AIV structures have been published and provide new targets for designing antiviral drugs such as antisense oligonucleotides (AS ODNs), which effectively inhibit gene replication. In this study, we designed and synthesized three AS ODNs (NP267, NP628, NP749) that were specific for the RNA binding region of nucleoprotein (NP) based on AIV structure. Results showed that all three AS ODNs could inhibit viral replication in MDCK cells. The NP628 showed the best antiviral effect of all through viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, the liposome mediated NP628 could partially protect the mice from a lethal H5N1 influenza virus challenge. Moreover, the NP628 group had a lower viral titer and lung index in the infected mice when compared with the viral control. Our results showed that AS ODN targeting of the AIV NP gene could potently inhibit AIV H5N1 reproduction, thus, formulating a candidate for an emergent therapeutic drug for the pathogenic H5N1 influenza virus infection.     

RNA-based therapeutic strategies for cancer

Recent progress in the field of RNA therapeutics has highlighted the potential of using RNA-based strategies for the treatment of human cancer. Emerging technologies such as small interfering RNA (siRNA) to trigger RNA interference (RNAi) and catalytic RNA molecules, called ribozymes, are being developed to modulate expression of genes to either block tumourigenesis itself, inhibit tumour growth or prevent metastasis. Delivery of mRNA or vectors based on positive-strand RNA viruses such as alpha viruses, picornaviruses and flaviviruses have also found applications in the development of cancer vaccines and for apoptosis of tumour cells. These approaches should help overcome some of the drawbacks of viral vectors used in the majority (~ 60%) of clinical trials for cancer gene therapy, including potential malignant transformation due to insertional mutagenesis with retroviral delivery and pre-existing immune responses to adenoviral proteins. In this review, the advantages and disadvantages of RNA-based therapeutic strategies and their potential use in cancer treatments will be compared.     

2-5A antisense treatment of respiratory syncytial virus

Although a prominent cause of upper and lower respiratory tract disease in infants and the elderly, clinical options for treatment of respiratory syncytial virus (RSV) infections remain limited. Historically, attempts to develop vaccines have been unsuccessful, and rapid viral mutation rates have stifled development of several small molecule-based antiviral agents. Thus, targeted approaches to block RSV replication, including humanized monoclonal antibodies and nucleic acid-based strategies (antisense and RNA interference), have emerged as potentially viable drug development options.     

Inhibition of multiple strains of Venezuelan equine encephalitis virus by a pool of four short interfering RNAs

RNA interference, mediated by short interfering RNAs (siRNAs), has been shown to have activity against a wide range of viruses and is a promising new antiviral therapy. Using multiple siRNAs that target conserved areas of the genome allows for increased chances of antiviral activity against different viral strains and also helps to prevent the emergence of escape mutants. In this study, four siRNAs were designed to target areas of conserved sequence between divergent strains of Venezuelan equine encephalitis virus (VEEV). A pool of these siRNAs inhibited the replication of all six strains of VEEV tested. A single nucleotide mismatch at the extreme 3' end of one of the siRNA sense strands did not affect antiviral activity but other mutations were not tolerated. Two strains of VEEV were tested for their abilities to overcome the inhibitory effects of RNA interference following 10 consecutive incubations in the presence of siRNAs. One strain remained susceptible throughout the course of the experiment but the other strain became resistant to the activity of siRNAs. Sequence analysis of the siRNA target sites in this strain showed that no mutations had been generated, indicating that the virus may had become resistant in some other manner. In the absence of effective antiviral drugs and vaccines to combat VEEV infection, these siRNAs offer a potential new therapeutic approach but, as with all antimicrobial agents, caution needs to be exercised with respect to the generation of resistance.     

Norovirus gastroenteritis, increased understanding and future antiviral options

Increasing epidemiological data indicate that norovirus is an important cause of acute gastroenteritis. Norovirus gastroenteritis is difficult to control owing to its widespread nature and the lack of a suitable antiviral agent or a vaccine to prevent infection. Despite the difficulty in cultivating noroviruses, significant advances in understanding the genomic structure, individual viral proteins, RNA replication strategy, and virus-host interaction of the virus have been made. These advancements provide new strategies in the development of antiviral agents against norovirus, including the inhibition of viral attachment to host cells through carbohydrate receptors, inhibition of viral protease and polymerase functions, and interference in viral replication. This review summarizes new developments in the treatment of norovirus gastroenteritis and discusses future research directions.     

Synthetic oligonucleotides: Useful molecules? A review.

Specific inhibition of mammalian genes is possible through the use of antisense oligonucleotides (AS ODNs) or ribozymes. These strategies have led to a better understanding of several cellular and molecular mechanisms, among which cancer development. Recently, these strategies have been applied also for therapeutical purposes in diseases such as AIDS and cancer. In some of these therapeutical trials the antisense strategy is combined with gene transfer technology: the AS ODN or the ribozyme are expressed within the cell by the use of adenoviral or retroviral vectors. However, many difficulties have still to be overcome before ODNs and ribozymes can be used routinely in the clinic.