- Azad, Taha;
- Rezaei, Reza;
- Singaravelu, Ragunath;
- Pelin, Adrian;
- Boulton, Stephen;
- Petryk, Julia;
- Onsu, Kemal Alper;
- Martin, Nikolas T;
- Hoskin, Victoria;
- Ghahremani, Mina;
- Marotel, Marie;
- Marius, Ricardo;
- He, Xiaohong;
- Crupi, Mathieu JF;
- Hoang, Huy-Dung;
- Nik-Akhtar, Abolfazl;
- Ahmadi, Mahsa;
- Zamani, Nika Kooshki;
- Golshani, Ashkan;
- Alain, Tommy;
- Greer, Peter;
- Ardolino, Michele;
- Dickinson, Bryan C;
- Tai, Lee-Hwa;
- Ilkow, Carolina S;
- Bell, John C
The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.