Mutations from in Vitro Evolution Affect Existence of Replicon RNA and Transcription of Subgenomic

Replicon RNA that self-replicates in cell is promising to be used for vaccination and cancer therapy. However, it’s still challenge to find a replicon construct expressing stronger and longer both in vitro and in vivo. Here, we evolved the replicon constructs encoding with mCherry in Jurkat cells with the selection pressure of sorting the higher mCherry expression cells. Five mutations were enriched and identified after ~2⁶⁰ generations with 5 times selection. One of the mutations, localizing at nsP2, is a synonymous mutation. Another four mutations, in nsP2 or nsP3, resulted in amino acid changes from Gly to Arg, Lys to Glu, Ser to Gly, and Glu to Asp/Val to Met, and denoted by mutation D, C, F, and G, respectively. Seventeen replicon constructs were derived from the five mutations and validated. We found the mutations D and C played critical roles in replicon existence in cells. In contrast, the mutations of F and G played important roles of subgenomic transcription. Interestingly, F or G could suppress the phenotypes of C mutation, but not the D mutation. The synonymous mutation, however, played less role in either replicon existence in cells or subgenomic transcription. Furthermore, the mutations of C and D showed less interferon responses. Consistent to the in vitro data, the replicon constructs with any of the mutations D, C, F, and G could stay longer in B16F10 melanoma. In conclusion, we developed a method that evolves replicon constructs in vitro to get better constructs and identified 4 mutations plays critical roles in existence or subgenomic transcription of replicon RNA. These findings shed light on how to find better constructs, not limited to replicon, by in vitro evolution and pave the road for replicon RNA applications in vaccination and cancer therapy.