Viral Replicon Systems and Their Biosafety Aspects

Viral RNA replicons are self-amplifying RNA molecules generated by deleting genetic information of one or multiple structural proteins of wild-type viruses. Remaining viral RNA is used as such (naked replicon) or packaged into a viral replicon particle (VRP), whereby missing genes or proteins are supplied via production cells. Since replicons mostly originate from pathogenic wild-type viruses, careful risk consideration is crucial.A literature review was performed compiling information on potential biosafety risks of replicons originating from positive- and negative-sense single-stranded RNA viruses (except retroviruses).For naked replicons, risk considerations included genome integration, persistence in host cells, generation of virus-like vesicles, and off-target effects. For VRP, the main risk consideration was formation of primary replication competent virus (RCV) as a result of recombination or complementation. To limit the risks, mostly measures aiming at reducing the likelihood of RCV formation have been described. Also, modifying viral proteins in such a way that they do not exhibit hazardous characteristics in the unlikely event of RCV formation has been reported.Despite multiple approaches developed to reduce the likelihood of RCV formation, scientific uncertainty remains on the actual contribution of the measures and on limitations to test their effectiveness. In contrast, even though effectiveness of each individual measure is unclear, using multiple measures on different aspects of the system may create a solid barrier. Risk considerations identified in the current study can also be used to support risk group assignment of replicon constructs based on a purely synthetic design.