Antiviral Applications of RNA Interference
5-10 April, 2008
St Feliu de Guixols, Spain

Organisers:

Jens Kurreck, Free University of Berlin, Germany
Ben Berkhout, University of Amsterdam, Netherlands

Draft Report

Introduction

Pandemic virus infections are a major threat for humans. In recent years, the prevalence of infections with viruses such as the human immunodeficiency virus (HIV) and hepatitis B and C viruses (HBV/HCV) has steadily been increasing and new viruses like the coronavirus causing the severe acute respiratory syndrome (SARS) have emerged. Adaptation of the avian flu to humans is expected to result in a severe pandemic with millions of deaths.

RNA interference (RNAi) is a cellular mechanism, which has been discovered less than 10
year ago, that allows sequence-specific inhibition of gene expression. Double stranded
RNA molecules induce degradation of a homologous RNA. RNAi is considered to be an
evolutionary conserved natural antiviral defense mechanism of cells. In plants, RNAi is the basis for systemic immunity after local infection with a virus. In contrast, mammals have developed a sophisticated immune system. However, the RNAi machinery is still
considered to be helpful to treat virus infections in humans. In recent years, two clinical
trials have been initiated to treat viral infections with RNAi-based strategies. In one of the trials, short double-stranded RNA molecules, known as small interfering RNAs (siRNAs), are employed to treat infections with the respiratory syncytial virus (RSV). For the second trial blood stem cells of AIDS patients are treated ex vivo (outside the body) and cells protected against the human immunodeficiency virus (HIV) are then re-infused into the patient.

A great advantage of RNAi as compared to other antiviral approaches is the possibility to rapidly adopt the technology to new genetic variants of viruses or even to completely new types of viruses. In principle, the development of RNAi therapeutics can begin once the genome sequence of an emerging viral pathogen is known whereas the development of small molecule antiviral requires the prior elucidation of much biological and functional detail.

In addition to the use of small RNAs as antiviral agents, micro RNAs (miRNAs) have come
into the focus of recent research. These molecules as endogenously expressed in cells
and are now considered to be major regulators of gene expression at the post-transcriptional level. Recently, miRNAs have been found to play an important role in the
interplay between viruses and their hosts. Several viruses encode their own miRNAs, eg
Herpesviruses use them to control the state of latency. Other viruses like the hepatitis C virus rely on cellular miRNAs for their replication. In addition, some cellular miRNAs seem to protect cells from infections and as a countermeasure several viruses inhibit the cellular miRNA machinery.

Scientific Content
The opening lecture by Mark Kay dealt with viral vector mediated delivery of siRNAs against the hepatitis B virus. He addressed two important topics: the delivery issue and specificity of RNAi-treatments. Various talks throughout the meeting dealt with either non- viral of viral delivery of siRNAs. For the first strategy, cationic lipids are usually employed, but one speaker also reported on the use of chitosan as a carrier for siRNAs. For the latter approach, different types of viral vectors can be employed that were elaborated on by various speakers throughout the meeting. The most widely used viral vectors are derived from lentiviruses, adenoviruses and adeno-associated viruses. As Mark Kay further pointed out, it is important to dose viral vectors or siRNAs with great care, in order not to saturate the endogenous miRNA pathway or to induce other types of unspecific effects. Further talks in the first session dealt with chemical synthesis of siRNAs and methods to evaluate the outcome of an RNAi application.

The second session focused on RNAi in plants. An important feature, in which plants differ from mammals, is the systemic spread of RNAi: After virus infection of a certain tissue of the plant, double-stranded RNAs spread throughout the plant and confer systemic resistance against the virus. Another topic that became a major issue throughout the conference was also introduced in this session: The interplay between viruses and the host RNAi-pathway. Several plant viruses encode suppressors of the host RNAi machinery. This interaction was also an important topic in the next session on RNAi in the fruitfly Drosophila melanogaster. In these insects RNAi constitutes the antiviral immune system. It is an interesting focus of current research to understand the similarities and differences of RNAi in various classes of organisms like insects and mammals.

Sessions 4 to 8 of the conference mostly dealt with viruses pathogenic to humans. For several mammalian viruses, it is well accepted that they encode and express miRNAs, which are important to regulate their life cycle. For HIV-1, however, it became a controversial debate, whether this virus encodes miRNAs or regulates cellular miRNAs. Mammals developed two lines of defense against viruses, the innate immunity including interferon responses to infections and the adaptive immunity (antibodies). The question therefore arises, whether RNAi is still part of the antiviral defense in mammals.

Another focus of the conference was the use of RNAi as a new therapeutic approach against HIV-1. With more than 30 million people infected with the virus worldwide, there is a great need to develop new antivirals to treat infections with HIV-1. A major drawback of this approach, however, is the development of escape mutants upon prolonged virus inhibition. An approach to extend the inhibitory activity of an RNAi application is to combine several siRNAs in analogy to the combination of antiviral drugs in conventional virus therapy. RNAi might also become a weapon against HIV-1 variants being resistant against low molecular weight drugs by specifically targeting the mutated sequences. Furthermore, cellular targets can be silenced to block viral spread and to prevent viral escape.

In the session on hepatitis B and C viruses the concept of combination of RNAi approaches with interferon was discussed. Interferons can either be supplied separately or induced by immuno-stimulatory motifs of the siRNAs. This combination has improved antiviral activity as compared to a single treatment.

Other viruses were dealt with in a separate session. RNAi has been employed by various groups against enteroviruses or their receptor. In addition, RNAi has been used as a research tool to decipher the detailed mechanism of virus entry into the cells. Large scale or even genome-wide screens with siRNAs targeting thousands of human genes have been employed to identify host factors required by the virus for entry into the cells and replication.

The last session dealt with the clinical application of RNAi. Although not directly related to the virological topic, one speaker was invited to report on the experiences during clinical development of RNAi as an anti-cancer agent. The company Silence Therapeutics intends to bring an siRNA into the clinic in 2008 that helps to prevent the emergence of metastasis of tumors.

Assessment of the results & their potential impact on future research or applications
It has become possible to design efficient siRNAs against any given virus and some progress has been made in recent years with respect to the delivery of siRNAs in animals. New deep sequencing technologies also allow obtaining comprehensive pictures of miRNAs involved in cellular processes.

Several topics were heavily under debate during the meeting. One of the most controversial points was whether RNAi plays an important role as a defense mechanism
against viruses in mammalian cells. Furthermore, there was no consent on whether HIV-1 encodes relevant miRNAs. These topics will definitively have to be addressed in future
research.

An important question that cannot be answered today is whether experimental RNAi approaches can be translated into therapeutics. As outlined above, important issues will
have to be solved including the delivery of siRNAs and specificity of RNAi treatments.
Various trials with RNAi against viruses have commenced only recently and the outcome of these trials will be important for the further development of the field. It will be necessary to see, whether RNAi-based approaches will be suitable to cope with the problem of viral escape and if they might be even suitable in cases in which viruses have developed resistance against the antiviral agents existing to date.