Control of alphavirus-based gene expression using engineered riboswitches

•Alphavirus-based replicons containing riboswitches were generated.•This allowed conditional control of transgene expression via input ligand.•Regulation of both DNA-launched and VRP-packaged replicon expression was achieved.•The type I IFN response to these replicons was controlled by riboswitch modulation.•Riboswitch-mediated control of TC-83 virus replication was also achieved (1160-fold).

Alphavirus-based replicons are a promising nucleic acid vaccine platform characterized by robust gene expression and immune responses. To further explore their use in vaccination, replicons were engineered to allow conditional control over their gene expression. Riboswitches, comprising a ribozyme actuator and RNA aptamer sensor, were engineered into the replicon 3′ UTR. Binding of ligand to aptamer modulates ribozyme activity and, therefore, gene expression. Expression from DNA-launched and VRP-packaged replicons containing riboswitches was successfully regulated, achieving a 47-fold change in expression and modulation of the resulting type I interferon response. Moreover, we developed a novel control architecture where riboswitches were integrated into the 3′ and 5′ UTR of the subgenomic RNA region of the TC-83 virus, leading to an 1160-fold regulation of viral replication. Our studies demonstrate that the use of riboswitches for control of RNA replicon expression and viral replication holds promise for development of novel and safer vaccination strategies.