- Open Access
ESF-EMBO Symposium: Antiviral Applications of RNA Interference
© ter Brake et al; licensee BioMed Central Ltd. 2008
- Received: 04 July 2008
- Accepted: 18 September 2008
- Published: 18 September 2008
- RNAi Machinery
- Cellular miRNAs
- Flock House Virus
- Mammalian Virus
- RNAi Therapeutics
The first ESF-EMBO symposium on "Applications of antiviral RNA interference (RNAi)" was held in the spring of 2008 (5–10 april) in Sant Feliu de Guixols at the Costa Brava in Spain. Some 60 participants from the fields of RNAi research and virology came together to present their latest findings on RNAi-virus interactions, as well as the progress in the development of RNAi-based antiviral therapeutics. One of the big topics concerned the role of RNAi in natural antiviral defence mechanisms in mammals [1–3]. In addition, new solutions to improve the efficacy and safety of RNAi-based antiviral drugs were presented. The combined expertise of researchers studying RNAi in plants, insects and mammalian systems greatly stimulated the overall discussion. The meeting was funded by the European Science Foundation (ESF) in partnership with the European Molecular Biology Organisation (EMBO).
The first session of the meeting focused on RNAi technology. The most common strategies to induce RNAi are stable intracellular expression of short hairpin RNA (shRNA) or transient transfection of synthetic small interfering RNAs (siRNAs). Mark Kay from Stanford University discussed RNAi-based gene therapy approaches against virus-induced hepatitis using shRNAs. One of the problems of this approach is that the adeno associated virus vector used to deliver the shRNA-expression cassette can trigger an immune response. To solve this problem one could transiently suppress the immune response. However, a more elegant method to evade immunity is to select for less immunogenic vectors via capsid shuffling. This approach resulted in a 100.000 fold more effective vector. Another problem that was discussed was shRNA toxicity . Previously, it was shown that overexpression of virus-specific shRNAs in liver caused lethality in mice by saturation of Exportin 5 (Exp5), thus interfering with export and maturation of endogenous microRNAs (miRNAs). New data was presented that also implicated Ago2, the slicer in the RNA-induced silencing complex (RISC), as a rate limiting factor. Combined overexpression of Ago2 and Exp5 reduced toxicity associated with shRNA overexpression and enhanced shRNA knockdown activity.
Focusing on the RNAi mechanism, Mark Kay and his co-workers also asked the question why miRNA targets are only functional in the 3'UTR of the mRNAs and not in the open reading frame (ORF). Data was presented indicating that miRNA translational inhibition is affected by the speed of the translating ribosome. miRNA target sequences within ORFs can in fact become functional when translation is slowed down, e.g. when a miRNA target site is preceded by rare codons. Earlier, Lytle and co-workers also showed functionality of miRNA targets in 5'-UTRs of reporter genes, and concluded that any position on a target RNA may be mechanistically sufficient to repress translation .
Besides the use of viral vector systems for intracellular expression of RNAi-inducers, synthetic siRNAs are also considered highly effective candidate therapeutics. Joachim Engels (Goethe University Frankfurt) gave some background information about the chemical synthesis of siRNAs. Developments like the 2'-acetoxyethyl (ACE) RNA chemistry and the incorporation of modified, especially cationic, nucleotides form the basis for the synthesis of highly stable effective siRNAs. Jorgen Kjems (University of Aarhus) discussed some of the latest developments in the use of modified siRNAs. One of the major problems with synthetic siRNAs is their low stability in serum. A comprehensive study was conducted with many different chemistries at the 2'O ribose position such as aminoethyl and guanidinoethyl , and it was shown that siRNA half life and efficacy can be greatly enhanced by introducing modifications at specific positions both in the passenger and the guide strand of the siRNA. Off-target effects caused by the incorporation of the passenger strand in RISC were effectively avoided by design of a nicked passenger strand in the so called small internally segmented interfering RNA (sisiRNA) design. Furthermore, off-target effects could be avoided by incorporation of specific modifications in the guide strand of the siRNA. In addition, Kjems focussed on siRNA delivery systems and showed that nanoparticles based on chitosan were highly effective for siRNA delivery, particularly in the lungs.
An interesting novel technique termed RNAu was presented by Puri Fortes (University of Navarra) . RNAu is based on expression of U1 small nuclear RNA (snRNA) of which the 5' nucleotides 2–11 are modified to base-pair with a 10 nucleotide target within the 3' terminal exon of a gene of interest. Binding of the modified U1 snRNA inhibits polyadenylation, resulting in degradation of the transcript and gene knockdown. The U1 snRNA mechanism tolerates a single mismatch at positions 1, 2, 9 and 10, the central 6 nucleotides require perfect base-pairing but do allow a single G-U base-pair. The presence of multiple target sites within the 3' exon enhances inhibition, and a knockdown of gene expression of up to 700-fold can be achieved. Interestingly, when combined with RNAi, additive or even synergistic inhibition was obtained.
Thomas Hohn (University of Basel) introduced the mechanism of RNAi in plants and its interaction with viruses. Plants use RNAi as an antiviral defence in which viral replication intermediates in the form of dsRNA are processed by the Dicer-like enzyme (DCL) . Furthermore, plants can amplify the RNAi effect using RNA-dependent RNA polymerase (RdRP) and siRNAs as primers. The RNAi machinery in plants is rather complex with four DCL enzymes: DCL1 processes primary-miRNA (pri-miRNAs) with different product sizes depending on the substrate, DCL2-4 process dsRNA. DCL2 can compensate for deficiencies in the other DCL enzymes and yields a 22-nucleotide (nt) product, DCL3 and 4 produce 24 and 21-nt siRNAs, respectively. RNAi in plants can be triggered by DNA viruses and RNA viruses. For instance, Hohn showed that two DNA viruses, the Cabbage leaf curl virus (CaLCuV) and the Cauliflower mosaic virus (CaMV), triggered the synthesis of 21, 22 and 24-nt siRNAs, and the cytoplasmic RNA tobamovirus Oilseed rape mosaic virus (ORMV) triggered predominantly 21-nt siRNAs. Since the RNAi machinery in plants can act as a potent antiviral response, viruses in turn have evolved RNA silencing suppressors (RSS) as a countermeasure. For instance, the p19 protein from Tombusvirus can bind and neutralize siRNAs. Interestingly, the AC2 protein from Mungbean yellow mosaic virus-Vigna (MYMV) is not an RNAi suppressor itself, but apparently triggers the activation of an endogenous RSS activity.
Björn Krenz (University of Stuttgart) reported on the Abutilon Mosaic Virus, which was engineered as a versatile vector to deliver genes in to plants. It was subsequently employed to silence phytoene desaturase in Nicotiana benthamiana, demonstrating that this viral vector is a valuable tool for functional studies. Juan Antonio García (CNB-CSIC, Madrid) presented work on the cucumber vein yellowing Virus (CVYV), which is a member of the potyviridae. Remarkably, CVYV does not encode the silencing suppressor HCPro that is typical for potyviridae, but instead produces the P1a-b protein that is proteolytically processed into P1a and P1b instead of a single P1 protein. P1b is a serine protease that accumulates in infected plants and functions as an RSS. It contains a Zn-finger and LXKA basic motif, which are both required for RSS function. P1b binds siRNAs but also endogenous miRNAs, which affects the miRNA expression pattern of the host cell. In the plum pox virus, HCPro could be replaced by P1b, adding further proof that P1b is an RSS.
Kirsi Lehto (University of Turku) presented data on plant virus encoded RSS factors and their role in virus-induced disease. RSS genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. Depending on the species of the host plant the RNA silencing suppressors caused different disease phenotypes. In addition, the suppressors demonstrated different effects on crucifer-infecting Tobamovirus (crTMV) infections. Apparently, these suppressors act at different levels in the RNAi pathway, and interfere with miRNA function to variable degrees.
Olivier Voinnet (Institute de Biologie Moléculaire des Plantes, Strasbourg) showed that the interaction between host and pathogen is more complicated than simple defence and counterdefence mechanisms. Arabidopsis encodes for 10 different Ago genes, Ago1 minus plants are hypersensitive to viruses indicating that Ago1 is involved in antiviral responses. Previously, Ago1 was shown to act within the miRNA pathway. Thus, miRNA and antiviral pathways appear to converge. In addition to RNA silencing, resistance (R) genes are also involved in blocking virus replication in plants. These genes encode receptors that detect pathogens and activate strong defences similar to pattern-recognition receptors in mammals . It is becoming clear that genes involved in RNAi are in fact R genes that regulate the hypersensitive response (HR). HR causes apoptosis of the local region surrounding the infection thus preventing further viral spread. There is also evidence that HR factors are part of RISC. Although the antiviral function of RNAi in mammals is still debated, Olivier Voinnet extended the function of RNAi in plants to a defence against bacterial pathogens [22, 23]. Specific plant miRNAs are induced in response to bacterial pathogens that are detected via the flagellin receptor. Similar to viruses, bacteria also encode specific factors that are translocated to the plant cells to block the miRNA pathway. These effectors were identified and found to affect processing of Ago1. In this way, viral and bacterial infections can join forces and benefit from each others presence by a severe attack on the RNAi defence mechanism.
Recently, it has become clear that mammalian viruses interact with components of the host RNAi machinery. Viruses can express miRNAs to regulate the expression of cellular genes, or viral gene expression may be activated or repressed by cellular miRNAs. In addition, several viruses encode suppressors of RNAi. A separate session was dedicated to these complex interactions between viruses and the RNAi machinery.
Goran Akusjarvi (Uppsala University) presented data on how adenovirus interacts with the RNAi/miRNA pathways. He showed that the structured non-coding virus-associated RNAs (VA RNA I and II) are processed by Dicer and incorporated into RISC. Although only 2–5% of the total amount of the VA RNAs is diced, up to 80% of all RISC complexes contain VA-derived si/miRNAs late in infection . Of these, ~80% stem from VA RNAII, which is expressed at much lower levels than VA RNAI. Besides this VA RNAII bias, there also appears to be a strand bias for incorporation into RISC. Data was presented that this bias may arise from two different transcription initiation sites that are used during VA RNA expression. Puri Fortes (University of Navarra) presented data that blocking of the adenoviral VA miRNAs results in a decrease in viral titer, suggesting that VA miRNAs control the expression of genes whose expression affects adenovirus production. This group has also identified several putative targets for these miRNAs using a combination of bioinformatic approaches and microarray analysis. How these targets affect the viral cycle remains to be established.
A major problem with antiviral approaches against HIV-1 is the emergence of escape variants. Similar to the emergence of drug resistant mutations, RNAi resistant mutations have also been described . Thus, for the development of effective RNAi-based therapies against escape-prone viruses, the main objective is to effectively suppress virus replication while preventing the selection of resistant variants. In case of HIV-1 this is further complicated by the large heterogeneity of viral sequences within a patient. Miguel Angel Martinez (irsiCAixa Foundation, Barcelona) described two approaches aimed at preventing viral escape. First, one could counteract escape mutations against a specific siRNA by including second generation siRNAs that are directed against these specific mutants. In addition, one could also inhibit the virus with multiple siRNAs generated in vitro from Dicer-cleaved long dsRNA.
Karin Metzner (University of Erlangen) addressed the problem of HIV-1 resistance against regular antiviral drugs. It was proposed to use RNAi to specifically suppress these escape variants. Combining 3TC, a nucleoside Reverse Transcriptase inhibitor, with an siRNA directed against the most common 3TC-resistance mutation (Met184Val), proved to be effective in cell culture infections. Targeting essential cellular co-factors could be a valid approach to avoid RNAi resistance but also a way of defining new therapeutic targets. Eduardo Pauls (irsiCaixa Foundation, Barcelona) showed that targeting of αV integrin and β5 integrin with siRNAs could inhibit HIV-1 replication. This inhibition was not at the level of virus entry, reverse transcription or integration but appeared to block transcription from the HIV-1 long terminal repeat promoter. However, siRNAs were used in all of the above-mentioned approaches, and siRNA delivery in patients is still a major bottleneck.
Olivier ter Brake (University of Amsterdam) presented results on the development of an RNAi-based gene therapy for HIV-1. A single treatment with a lentiviral vector expressing a single shRNA results in stable induction of RNAi. In a combinatorial approach, four antiviral shRNAs were expressed from a single lentiviral vector. In a T cell line containing a single vector copy per cell, HIV-1 replication could be effectively controlled for up to 40 days, while escape mutants emerged in control single shRNA cell lines. This result highlights the therapeutic potential of such an approach. However, safety aspects still require intensive investigation. A pilot study was performed in a humanized mouse model in which Rag2-/-γc-/- irradiated newborn mice are engrafted with shRNA-transduced human haematopoietic stem cells. Development of the immune system was not affected by constitutive shRNA expression, although a slightly reduced engraftment efficiency of the transduced cells was observed. Furthermore, sequence-specific inhibition of HIV-1 replication was demonstrated in CD4+ T cells from this mouse .
Jens Kurreck (University of Stuttgart) presented data on RNAi-mediated inhibition of Coxsackie B3 virus (CoxB3). Using reporter constructs and virus he showed that only the plus-stranded RNAs can be targeted by the siRNAs. In addition, Kurreck showed that it is difficult to induce efficient RNAi knockdown when viral sequences are targeted that have complex RNA secondary structures.
Rainer Wessely (Munich University of Technology) gave an overview of CoxB3 involved in viral heart disease. siRNAs against CoxB3 were effective both in vitro and in an in vivo mouse model, yielding a 2–3 log reduction in virus replication. However, virus resistance was observed already after the first infection cycle, indicating that combinatorial RNAi approaches are required for effective and durable suppression. In an alternative approach, Sandra Pinkert (Charité, Berlin) demonstrated that CoxB3 can efficiently be inhibited in neonatal rat cardiomyocytes by vector mediated delivery of shRNA expression cassettes against the virus genome or its receptor, the coxsackievirus-adenovirus receptor (CAR). A soluble variant of CAR fused to the Fc domain of a human immunoglobulin had an even more potent antiviral effect suggesting that it might be worth to combine the different approaches.
Carolyn Coyne (University of Pittsburgh) uses RNAi to investigate entry of enteroviruses into polarized endothelial cells. Recently, she used a large scale screen to identify genes involved in entry of CoxB3 and poliovirus. One of the hits, the Yes kinase was characterized in more detail by low molecular weight inhibitors and its knockdown or inhibition was found to prevent entry of CoxB3 (but not of poliovirus) into human bone marrow endothelial cells.
Alexander Karlas (Max-Planck-Institute for Infection Biology, Berlin) reported on the use of RNAi against influenza virus A. siRNAs modified with locked nucleic acids (LNA) and delivered by chitosan were found to be efficient in a mouse influenza model. In order to identify host factors on which the virus depends large scale screens were performed and a large number of factors from the spliceosome were among the hits.
Jörg Kaufmann (Silence Therapeutics AG, Berlin) presented data on Atu027, an anti-cancer siRNA delivered systemically for the treatment of gastrointestinal cancer. Although not an antiviral RNAi approach, this presentation nicely listed the challenges of the clinical development of RNAi therapeutics. First of all, a formulation was developed, Atuplex, which consists of liposomes of ~120 nm containing a cationic lipid and a helper-lipid PEG-lipid, in which the siRNA is incorporated, a blunt ended 23-mer with 2'-O-methyl modification for stabilisation. The complex could be lyophilized and stored long-term at 4°C without significant loss of efficacy, an important requirement for clinical development. Furthermore, biodistribution, toxicology and efficacy studies were conducted in various animal models. The siRNA was found mostly in the endothelial cells of the lung but did not penetrate the tumor. No cytokines were induced, indicating that siRNA administration is safe. Furthermore, metastasis was reduced in a prostate cancer model. Combined, the data showed that Atu027 is effective and safe. Currently, Silence Therapeutics is preparing for a phase I clinical trial that is expected to start this year.
In light of the new data presented at this meeting it is clearly too early to close the door on an antiviral function of RNAi in mammals. Instead, data in favour of an antiviral role of RNAi in mammals are accumulating. In addition, viruses and the cellular RNAi machinery interact in multiple different ways. This meeting has shown that both fundamental research on RNAi and viruses and the applications of RNAi technology are developing fast. An important discussion point during the meeting was about the future for RNAi therapeutics . RNAi can be very potent and specific, underscoring the great potential of this mechanism. However, increasing concerns about toxicity and off-target effects have tempered these initial expectation for a rapid introduction of RNAi-based drugs in the clinic. Despite these concerns, pharmaceutic companies are investing in the further development of RNAi-based therapeutics. Currently, it is safe to say that we have only a limited understanding of the RNAi pathway and its functions. A more thorough understanding will contribute to the fine-tuning of RNAi-based drugs such that safe and effective RNAi based therapeutics can be developed.
We thank Y.P. Liu for her advice and suggestions during preparation of the manuscript. The meeting was made possible by support of the ESF in partnership with the European Molecular Biology Organisation (EMBO). RNAi research in the Berkhout lab is sponsored by ZonMw (Vici grant and Translational Gene Therapy program), NWO-CW (Top grant), the European Union (LSHP-CT-2006-037301) and the Technology Foundation STW (grant AGT.7708).
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