title: Control of alphavirus-based gene expression using engineered riboswitches.
abstract: 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.
authors.affiliation: Novartis Vaccines, Inc., Cambridge, MA, USA.
authors.forename: Christie L
authors.initials: CL
authors.lastname: Bell
authors.affiliation: Novartis Vaccines, Inc., Cambridge, MA, USA. Electronic address: dong.x.yu@gsk.com.
authors.forename: Dong
authors.initials: D
authors.lastname: Yu
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA, USA.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Smolke
authors.affiliation: Novartis Vaccines, Inc., Cambridge, MA, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Geall
authors.affiliation: Novartis Vaccines, Inc., Cambridge, MA, USA.
authors.forename: Clayton W
authors.initials: CW
authors.lastname: Beard
authors.affiliation: Novartis Vaccines, Inc., Cambridge, MA, USA. Electronic address: peter.mason@regeneron.com.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Mason
mesh_terms: D000524:Alphavirus / Q000235:genetics* / Q000502:physiology; D052157:Aptamers, Nucleotide / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D005818:Genetic Engineering / Q000379:methods; D008967:Molecular Biology / Q000379:methods*; D058928:Riboswitch / Q000187:drug effects*; D014773:Virology / Q000379:methods*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D052157:Aptamers, Nucleotide; D058928:Riboswitch
keywords: Alphavirus; Aptamer; Replicon; Riboswitch; Venezuelan equine encephalitis virus