Efficient small nucleic acids to combat human respiratory viruses

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Antisense procedures have long been in development, for therapeutic use in humans, and also for crop protection. They aim to inactivate (‘silence’) cellular or pathogenic target RNAs or to modulate their function. Central functional components are small nucleic acids (NAs) such as small interfering RNAs (siRNAs) or antisense DNA-oligonucleotides (ASO), which mostly direct endonucleases to the targets. After considerable progress in research and (pre)clinical trials, the concept now experiences a revival. First approved therapies impressively demonstrate that siRNA and ASO pharmacology is safe and commercially viable. Formulations are being developed, which improve the efficacy, safety and pharmacology of small NA.Nevertheless, antisense procedures still encounter considerable technical limitations. Due to their high structural complexity, it was yet impossible to reliably identify regions in target RNAs that are accessible for NAs and are referred to as ‘accessible sites’ or a-sites in the following. Consequently, the design of siRNA or ASO agents relied on uncertain in silico predictions or empirical tests. There is an urgent need to solve this problem. Our laboratory characterizes RNA-binding proteins (Rbps) and RNA motifs involved in the replication of human, animal and plant pathogenic RNA viruses. In plant, we study the antiviral immune response, which is centrally based on RNA silencing. Here, we developed an experimental method, the ‘eNA screen’, which reliably identifies in vitro siRNAs and ASOs that are capable to associate to a-sites of complex structured RNA molecules such as mRNAs or genomic RNAs of plant viruses. Both in vitro and in planta, protein expression and/or replication of these target RNAs can be efficiently inhibited with eNAs identified in this way (e thus stands for efficient). For the first time, siRNAs and ASOs can be identified in the ‘test-tube’ and then be used in vivo with high specificity and efficiency. The eNA screen technology thus has the potential to significantly increase the potency and safety of antisense procedures.In the proposed project, we want to investigate how structures of complex RNA molecules are formed under eNA screen conditions and define the characteristics of a-sites. Moreover, we will apply the screen to two examples of human respiratory viruses, influenza A and respiratory syncytial virus. For antiviral applications, we consider it particularly important to use multivalent combinations of eNAs targeting many a-sites in viral RNAs. Thus, fast-replicating viruses and also variants (quasispecies) could be fought and escape prevented. By exclusively using highly efficient eNAs, our long-term goal is to develop well-tolerated oral/nasal treatments against viruses for which little or no antiviral substances and/or vaccines are available. These could help to reduce the viral load in early stages of infection, inhibit viral spread and reduce the risk of severe disease progression and pandemics.