Rapid Synthetic Viral Vectorology for Future Pandemic Response

Characterizing viral biothreats and predicting potential evolutionary pathways is crucial to mitigate future pandemics. Approaches utilizing in silico modeling are ultimately limited by the lack of empirical data and the immense scale of the theoretical evolutionary landscape. Conversely, laboratory experiments which aim to study viral biothreats are effective but must be performed at high biosafety levels or chimeric viruses. To overcome these challenges, we have employed a multidisciplinary approach combining viral vectorology and basic virological study to establish RAVEN (Rapid Attenuated Virus EvolutioN). In the same way that viral vector gene therapy approaches replace viral genes with a genetic “payload,” RAVEN similarly replaces critical replication genes with suitable reporter genes. The excised viral genes are then expressed in trans, permitting viral vector assembly. The resulting viral particles are infectious and essentially identical to wild type but cannot replicate. For this reason, viruses requiring high biocontainment can be propagated in a Biosafety Level-2 environment. While the aim of RAVEN is to establish a broad toolkit applicable to a wide variety of viruses, our current work has focused on the establishment of a vector model of SARS-CoV-2. We have produced a synthetic SARS-CoV-2 vector which lacks key replication genes. These open reading frames have been replaced with reporter genes which were stitched into the polyprotein sequence. All homology between the transgenes and vector was removed to prevent recombination that could restore a replication-competent virus. Resulting vector particles are infectious and are capable of GFP transduction of cells. Critically, these vectors are not replication competent in the absence of transgene expression and the absence of transgene RNA in viral vector preparations has been confirmed. We believe this approach supports safer, faster characterization of existing and future viral threats and will serve as a critical tool to reduce the impacts of future viral pandemics.