Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies

Abstract

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.

Fig. 1. Integration, functional characterization, and optimization of the split T7 inducible system in vaccinia virus.

a Schematic illustration of the split T7 inducible expression cassette inserted into Thymidine Kinase (TK) open reading frame. The FKBP linked N-terminal portion of the T7 RNAP and the FRB linked C-terminal portion of the T7 RNAP were expressed under continuous vaccinia virus promoters. A Fusion protein consisting of GFP, and firefly luciferase (GFPLuc) was incorporated under T7 promoter and induced by the addition of Rapalogs. mCherry fluorescent protein is expressed from the virus to detect virus infection in cells. b, c Representative fluorescent images and quantitation of luciferase signal (RLU) of the T7 inducible system from U2OS cells 24 h after infection with VV-ST7-iGFPLuc in the presence or absence of 10 nM rapamycin. mCherry indicates virus infection. d Comparison of the luciferase signal from various cell lines 24 h after infection at MOI 0.1 with VV-ST7-iGFPLuc in the presence or absence of 10 nM rapamycin. e Multistep growth curve of VV-ST7-iGFPLuc compared to the control vaccinia virus at different time points from Hela cells infected at MOI 0.01 in the presence or absence of 10 nM rapamycin. f Representative fluorescent images of the T7 inducible GFP system from U2OS cells 24 h after infection with VV-ST7-iGFPLuc at MOI 0.1 in the presence of different Rapalogs at 10 nM concentration. g Relative luminescence emitted after 24 h from U2OS cells infected with VV-ST7-iGFPLuc at MOI 0.1 in the presence of different concentrations of Rapalogs. h Comparison of vaccinia virus growth in the presence of Rapalogs at 10 nM in U2OS and HELA cells 24 h after infection at MOI 0.1. i, j IVIS imaging of HT-29 tumors in CD-1 nude mice. Tumors were injected with VV-ST7-iGFPLuc (1E7 PFU/tumor) when they reached ~150 mm³ in size. Different Rapalogs were administered at 1 mg/kg by their preferred route. After 24 h, luciferase signal was measured, and the control group received rapamycin intraperitoneally. Luciferase signal was measured in all groups again at 72 h. Scale bars = 40 μm in (b, f). Data indicate means ± SD of three (c–e, g, h) to four (j) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.

Fig. 2. Optimization of the doxycycline-inducible system for controlling gene expression in vaccinia virus.

a HeLa cells were infected with vaccinia virus at MOI 10 and RNA was collected at 2, 4, and 8 h post infection. The heatmap, categorized by gene function and reported timing of gene expression, depicts results of the ribosome profiling after cDNA synthesis and sequencing. b Schematic illustration of the tetracycline inducible expression cassette inserted to TK locus. The TetR protein was expressed under a continuous vaccinia virus promoter and various vaccinia virus promoters preceding a TetO element, which binds TetR protein, were incorporated upstream of the GFPLuc fusion protein. The dissociation of the TetR from TetO upon doxycycline (Dox) administration leads to initiation of transcription and gene expression. Blue fluorescence protein (BFP) is continuously expressed from the virus to detect viral infection. c, d Representative fluorescent images and quantitation of luciferase signal (RLU) of U2OS cells 24 h after infection with viruses expressing the GFPLuc fusion protein (VV-TetR-iGFPLuc) at MOI 0.1 under the control of various native and synthetic vaccina promoters. Expression of GFPLuc was induced with 100 ng/ml Dox. BFP images indicate virus infection. e Comparison of luciferase signal (RLU) from different cell lines 24 h after infection at MOI 0.1 with VV-TetR-iGFPLuc in the presence or absence of 100 ng/ml Dox. f Multistep growth curve of VV-TetR-iGFPLuc compared to the control vaccinia virus at different time points using Hela cells infected at MOI 0.01 in the presence or absence of 100 ng/ml Dox. g Quantitation of viral titers within HT-29 tumors from CD-1 nude mice seven days after Dox (625 mg/kg) was introduced into the diet of mice. Tumors were infected with control vaccinia virus (VV) or VV-TetR-iGFPLuc at 1E7 PFU/tumor prior to the introduction of Dox. h IVIS imaging of HT-29 tumors following injection with VV-TetR-iGFPLuc at 1E7 PFU/tumor when tumors reached ~150 mm³ in size. Mice were given Dox (625 mg/kg) in their diet 2 days post viral infection (Group 1) or immediately following viral infection (Group 2) of tumors. For Group 2, Dox was removed from the diet after 2 days. Luciferase signal was monitored at 12 h, 1, 2, 4 and 6 days post viral infection. Scale bars = 40 μm in (c). Data indicate means ± SD of three (e, f), four (d), or ten (g) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.

Fig. 3. Cumate-inducible system controls viral gene expression in vitro and in vivo.

a Schematic illustration of the cumate-inducible expression cassette inserted into TK locus. The CymR protein was expressed under a continuous vaccinia virus promoter and a GFPLuc fusion protein incorporated under various vaccinia virus promoters preceding a CuO element which binds CymR protein. The dissociation of the CymR from CuO upon cumate administration leads to initiation of transcription and gene expression. Blue fluorescence protein is expressed from the virus to monitor its presence in infected cells. b, c Representative fluorescent images and quantitation of luciferase signal (RLU) from U2OS cells 24 h after infection at MOI 0.1 with viruses expressing the GFPLuc fusion protein (VV-CymR-iGFPLuc) under the control of various native and synthetic vaccinia promoters. Expression of GFPLuc was induced with 100 µg/ml cumate. d Quantitation of viral titers within HT-29 tumors from CD-1 nude mice seven days following infection with VV-CymR-iGFPLuc (1E7 PFU/tumor) and treatment with varying amounts of cumate. e, f C57BL/6 mice were fed with varying amounts of cumate in their diet for 25 days and weighed at regular intervals for up to 55 days to measure cumate toxicity. Serum was collected at days 25 and 55 to measure different toxicity indicators. g–j HT-29 tumors from CD-1 nude mice were infected with VV-CymR-iGFPLuc (1E7 PFU/ml) and mice fed cumate diets according to the schedule and amounts shown. Bioluminescence images were taken, and luciferase activity quantified 1 and 2 days following initial virus treatment. After day 2, cumate diets were switched to a normal rodent diet and the control group received a diet containing 6000 mg/kg of cumate. Additional images were acquired following the diet switch, at day 4 post virus infection. Bar graphs show the total flux signal measured in the tumor area at the indicated days. Scale bars = 40 μm in (b). Data indicate means ± SD of three (c), twenty (d), and five (e–j) five biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.

Fig. 4. Double inducible chemogenetic switches overcome major challenges with single molecule systems.

a Schematic illustration of the ST7-Tet double inducible expression cassette inserted into TK locus. The TetR and split T7 fragment proteins were expressed under continuous vaccinia virus promoters and a GFPLuc fusion protein was incorporated under T7 promoter preceding a TetO element which binds TetR protein. The binding of split T7 fragments in the presence of Rapalogs and dissociation of the TetR from TetO upon Dox administration leads to initiation of transcription and gene expression. b, c Representative fluorescent images and quantitation of luciferase activity from U2OS cells 24 h following infection with VV-ST7-TetR-iGFPLuc in the presence or absence of Dox and rapamycin. mCherry signal indicates virally infected cells. d Schematic illustration of the ST7-Cumate double inducible expression cassette inserted into the same locus as described in A. The CymR and split T7 fragment proteins were expressed under continuous vaccinia virus promoters and GFPLuc was incorporated under T7 promoter preceding a CuO element which binds CymR protein. The binding of split T7 fragments in the presence of Rapalogs and dissociation of the CymR from CuO upon cumate administration leads to initiation of transcription and gene expression. e, f Representative fluorescent images and quantitation of luciferase activity from U2OS cells 24 h following infection with VV-ST7-CymR-iGFPLuc in the presence or absence of cumate and rapamycin. g Schematic illustration of the Cu-Tet double inducible expression cassette inserted into the same locus as described in A. The CymR and TetR proteins were expressed under continuous vaccinia virus promoters and GFPLuc was incorporated under different vaccinia virus promoters preceding a CuO element which binds CymR protein, and a TetO element which binds TetR protein. Dissociation of the CymR from CuO and TetR from TetO upon cumate and Dox administration leads to initiation of transcription and gene expression. h Luciferase signal (in RLU) was quantified 24 h after infection of U2OS cells with viruses expressing GFPLuc under the control of CuTet and various native and synthetic vaccinia promoters in the presence or absence of 100 µg/ml cumate and 100 ng/ml Dox. i, j Representative fluorescent images and quantitation of luciferase activity from HEK293T, Hela, Vero, HT-29, 786-O cells infected with VV-CymR-TetR-iGFPLuc in the presence or absence of cumate and Dox after 24 h. IRF signal indicates virally infected cells. k Schematic illustration of the CuTet double inducible expression cassette inserted in the intergenic location between UL26 and UL27 open reading frames of the HSV-1 genome. The CymR-T2A-rtTA3 fusion protein was expressed under the UBC promoter. For expression of the GFPLuc fusion protein, TetO followed by a minimal CMV promoter and CuO was used. In the presence of Dox, rtTA3 binds to the TetO and enhances CMV promoter activity which will be at maximum activity if CymR is also dissociated from CuO upon cumate administration. IRF is continuously expressed from the virus, to monitor viral infection. l, m Representative fluorescent images and quantitation of luciferase activity from U2OS cells infected with VV-CymR-TetR-iGFPLuc in the presence or absence of cumate and Dox after 24 h. IRF signal indicates virally infected cells. Scale bars = 40 μm in (c, f, i, m). Data indicate means ± SD of three (b, h, j, l) or four (e) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.

Fig. 5. Application of the tetracycline inducible system as a safety switch to generate a conditionally replicating vaccinia virus.

a Schematic illustration of the tetracycline inducible system controlling the expression of the D13L vaccinia virus gene which leads to the conditional growth of the vaccinia virus in the presence of Dox. b Representative images of U2OS cells 48 h after infection with VV-TetR-iD13 (MOI 0.01) in the presence or absence of 100 ng/ml Dox. c, d A549, Hela, HT-29, and SKOV3 cells were infected with control vaccinia virus or VV-TetR-iD13 at MOI 0.01 in the absence or presence of Dox at 100 ng/ml. After 48 h, cells were stained with crystal violet, and cell viability assessed by measuring absorbance at 570 nm using resazurin. e, f Multistep growth curves of VV-TetR-iD13 compared to control vaccinia virus at different time points from U2OS and Hela cells when infected at MOI 0.01 in the presence or absence of 100 ng/ml Dox. g, h IVIS imaging of HT-29 tumors in CD-1 nude mice following injection with VV-TetR-iD13 (1E7 PFU/tumor) when tumors reached 150 mm³. Mice were given a Dox diet either 2 days post viral infection (Group 1, blue bars) or immediately following viral infection (Group 2, brown bars). For Group 2, Dox was removed from the diet after 2 days. Luciferase signal was measured at 12 h, 1, 2, 4 and 6 days after virus and drug administration. Bar graphs show the average total luciferase signal emitted in the tumor area. Scale bars = 200 μm in (b). Data indicate means ± SD of three (c, e, f) or four (h) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.

Fig. 6. Conditionally replicating vaccinia virus as a safe alternative for vaccine development.

a–e Evaluation of the safety of the conditionally replicating virus. Mice were injected intravenously with either control vaccinia virus at 1E6 pfu or VV-TetR-iD13 at 1E7 pfu. Formation of pox lesions on mice feet and tails was monitored (a) and quantified (d) 5 days post i.v. injection. IVIS imaging was performed on days 5 and 7 post virus injection (b) and body weight were measured at consistent intervals (c). Organs were harvested at day 10 and tissues processed for quantifying viral titer (e). f, g Mice were intraperitoneally injected with 1E6 pfu of control and vaccine viruses with serum collected at days 7, 14, and 21. Subsequently, a RBD ELISA assay was performed and endpoint titer for all the samples and days were quantified (f). A SARS-CoV-2 pseudotyped neutralization assay was performed on day 14 samples (g). h, i Mice were intraperitoneally injected with 1E6 pfu of the VV-S-Hexapro-TetR-iD13 in the presence or absence of 625 mg/kg Dox in the diet for two days. Serum was collected at day 14 and was used for an RBD ELISA to quantify the normalized ELISA absorbance versus dilution factor in different conditions (h). Serum was also used for a SARS-CoV-2 pseudotyped neutralization assay. j, k Mice were intraperitoneally injected with 1E6 pfu of the VV-S-HexaPro-TetR-iD13 or MVA-S-HexaPro. Serum was collected at day 14 and was used for a RBD ELISA to quantify the normalized ELISA absorbance versus dilution factor in different conditions (j). Serum was also used for a SARS-CoV-2 pseudotyped neutralization assay (k). S and S-HexaPro represent SARS-CoV-2 wild-type spike protein and proline stabilized version of the SARS-CoV-2 spike respectively. Data indicate means ± SD of five (d, f–j) or ten (e) biological replicates. Data indicate means ± SEM of five (c) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.

Fig. 7. Doxycycline-inducible system as an effective strategy to regulate expression of a toxic fusogenic protein.

a Schematic illustration and representative images of GFP expressing cells transfected with Arenavirus glycoproteins including Isfahan virus (ISFV), Machupo virus (MACV), Sabiá virus (SABV), Guanarito virus (GTOV), Ippy virus (IPPYV), Lassa virus (LASV), Pichinde virus (PICV), Ekpoma virus-1 (EKV-1), Ekpoma virus-2 (EKV-2), Latino virus (LATV), Junín virus (JUNV), Tamiami virus (TAMV), Paraná virus (PRAV), lymphocytic choriomeningitis virus (LCMV) and Reovirus P14, P15, P22 FAST proteins. Six hours post transfection of GFP expressing cells, cells were mixed with a separate population of mCherry expressing cells. After 24 h, cells were monitored for signs of cell-cell fusion. b HEK293, U2OS, Vero, A549, and Hela cells were transfected with TAMV-GP (Tamiami virus glycoprotein), P14, and P15 FAST proteins. Twenty-four hours after transfection, an Alamar blue viability assay was performed. c HEK293, A549, Hela, MiaPaCa2, and Vero cells were infected with either VV-TetR-iP14 or VV-TetR-iTAMV-GP (MOI 0.1) in the presence or absence of 100 ng/ml Dox or ST-246, an anti-poxvirus drug. Representative images were taken 24 h post infection. d, e Multistep growth curves of Hela and U2O2 cells infected with VV-TetR-iTAMV-GP (MOI 0.01), in the presence or absence of 100 ng/ml Dox added at the same time, or 48 h after virus infection. f–h A549 tumors were injected with either PBS or VV-TetR-iTAMV-GP (1E7 PFU/tumor) at a size of ~150 mm³. Dox was given in the diet of mice (625 mg/kg) as indicated two days after virus injection. Tumors were measured at regular intervals (f). The image shown compares tumor size across all treatment groups and mice in the study (g). Tumor weight was also measured (h). i A549 tumors were injected with VV-TetR-iTAMV-GP (1E7 PFU/tumor) at a size of ~150 mm³ in size and depending on the treatment group received 625 mg/kg of Dox in their diet two days after virus injection. Tumors were harvested at day 4 and day 12 post virus injection and processed for immunohistochemistry staining using vaccinia virus, HA and caspase 3/7 antibodies. Scale bars = 40 μm in (a, c). Data indicate means ± SD of three (b, d, e) or ten (f, h) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.

Fig. 8. Applications of the cumate “safety switch” in regulating expression of potentially toxic cytokines.

a IL-2, IL-12 and IL-18 concentrations measured by ELISA from supernatants of U2OS cells 6 h after infection with VV-CymR-iIL12-2-18 (MOI 1) and treated with 100 µg/ml cumate or PBS. b IL-2, IL-12 and IL-18 concentrations measured by ELISA from supernatants of U2OS cells 6 h after infection with VV-CymR-iIL12-2-18 (MOI 1) and varying concentrations of cumate. c, d IFN-gamma and TNF-alpha levels measured by ELISA. Supernatants were taken from mouse splenocytes cultured ex vivo with conditioned media from U2OS cells treated with 100 µg/ml cumate or PBS following infection with VV-CymR-iIL12-2-18 (MOI 1). Neutralizing antibodies against IL-2, IL-12 and IL-18 were added to conditioned media for 1 h prior to transferring them to splenocytes. e–h Treatment-related mortality and toxicity were assessed 5 days after intratumorally injection of HT-29 tumors with control vaccinia virus, VV-IL12-2-18 or VV-CymR-iIL12-2-18 (1E7 PFU/tumor). Tumors were ~150 mm³ in size at the time of injection. Mice treated with VV-CymR-iIL12-2-18 were given a cumate (6000 mg/kg) or regular diet. Water in the lungs (an indication of pulmonary edema) (g) and alkaline phosphatase activity (h) in serum were measured as indicators of toxicity. i–m Treatment-related mortality and toxicity were assessed 5 days after intratumoral injection of HT-29 tumors with control vaccinia virus, VV-IL12-2-18 or VV-CymR-iIL12-2-18 (1E7 PFU/tumor). Tumors were ~150 mm³ in size at the time of injection. Mice treated with VV-CymR-iIL12-2-18 were given a regular diet, or a diet with varying amounts of cumate (1000, 2000 or 6000 mg/kg; j). Water in the lungs (k), and serum levels of alkaline phosphatase (l) and aspartate aminotransferase activity (m) were assessed. n Survival analysis of C57BL/6 mice injected intraperitoneally with 5E5 MC38 cells and treated with either PBS, control vaccinia virus, or VV-CymR-iIL12-2-18 at 3E7 pfu/mouse. Treatments were given at days 6, 8, and 10 post cell injection. A cumate diet (1000 mg/kg) was given to the VV-CymR-iIL12-2-18 treatment groups the same day, or 5 days after virus injection as indicated. o Crystal violet staining of U2OS and Vero cells 48 h after infection with VV-CymR-iIL12-2-18/TetR-iD13 at different MOIs in the presence of 100 µg/ml cumate (+Cu), 100 ng/ml doxycycline (+Dox), or both (+Dox +Cu). p IL-12, IL-2, and IL18 concentrations measured by ELISA in supernatants of U2OS cells 6 h following infection with VV-CymR-iIL12-2-18/TetR-iD13 (MOI 1) in the presence or absence of 100 ng/ml Dox, 100 µg/ml of cumate or both. q Survival analysis of C57BL/6 mice injected intraperitoneally with 5E5 MC38-WT cells and treated with either PBS, control vaccinia virus, or VV-CymR-iIL12-2-18/TetR-iD13 at 3E7 pfu/mouse. viruses were injected at days 6, 8, and 10 post cell injection, with mice receiving either Dox (625 mg/kg), cumate (1000 mg/kg) diets, or both for 5 days. Data indicate means ± SD of three (a–d, p) or five (e–n, q) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.