Bramsen, Jesper Bertram

Kjems, Jørgen

Lorenzen, Niels

Schyth, Brian Dall

Wengel, Jesper

Small interfering RNAs (siRNAs) are regarded as promising new active compounds in gene medicine. They are small 21-22bp long double stranded RNAs which act by targeting and inhibiting expression of specific mRNAs through base complementarity to one of their strands. But one serious problem with siRNA based treatment is the non-specific activities of double stranded RNAs when formulated in some effective delivery reagents. Cellular reactions upon double stranded RNAs include those of the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in innate antiviral defence mechanisms. Following injection of formulated siRNAs we have shown that we are able to detect the effect of such defence mechanisms as lowered mortality of rainbow trout infected with the fish pathogenic virus Viral Haemorrhagic Septicaemia Virus (VHSV).We used the trout and VHSV to screen siRNAs containing various chemical modifications of the RNA backbone and found that was possible to modify the backbone so as to reduce the antiviral effect of the RNA. Antiviral protection was also dependent upon localisation of the modified nucleotide residues in the RNA strands and we found some evidence of an effect of both base composition and thermal stability of the double strands.We conclude that our model is a potent tool for gaining insight into the triggering of antiviral cellular reactions towards RNAs in living fish. The overall perspective is to learn how to avoid nonspecific antiviral responses of RNA-based gene medicine, but the knowledge gained also has a potential for use in the design of adjuvants (although adjuvance effect has not been tested for any of our siRNAs yet). The model can also be used for screening various commercial and noncommercial delivery reagents with the same perspective

Chemical modification of RNA-based medicine can be used to reduce its induction of the innate immune response

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