A simeprevir-inducible molecular switch for the control of cell and gene therapies

Abstract

Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.

Fig. 1. Identification and characterization of HCV NS3/4A PR:simeprevir (PRSIM)-based chemical inducer of dimerization (CID) modules.

a Schematic of PRSIM modules. b The crystal structure of the HCV PR:simeprevir complex (PBD code: 3KEE) shows that simeprevir binds in a shallow surface groove of HCV PR with a large surface area of simeprevir remaining solvent exposed. The structure is visualized using The PyMOL Molecular Graphics System Version 2.1.0 (Schrödinger). c PRSIM_23 binds to HCV PR in complex with simeprevir (blue closed circles) but not to HCV alone (blue open circles) in a homogenous time-resolved fluorescence (HTRF) assay. The observed assay signal decrease at high PRSIM_23 concentrations is likely due to depletion of detection reagent. No binding of a control Tn3 (black circles) to HCV PR is observed. Each data point represents the mean of two independent replicates. d HCV PR binds PRSIM_23 in the presence of 10 nM simeprevir with an apparent affinity of 6.1 nM, in a Surface Plasmon Resonance (SPR) assay. No binding of HCV PR alone is seen at the same HCV PR concentration. e HCV PR shows low-affinity binding (6.2 µM) to PRSIM_23 in the absence of simeprevir. f Dose-dependent heterodimerization of HCV PR and PRSIM_23 was induced by simeprevir in an SPR assay with an EC50 of 4.0 nM. Each data point represents the mean ± s.d. of three independent replicates. The dose–response curve was fit to the data using 3 parameter nonlinear regression. g PRSIM_23 binds to HCV PR in complex with simeprevir (blue) but not to HCV PR in complex with other HCV protease inhibitors in an HTRF assay. Each data point represents the mean of two independent replicates. The dose–response curve was fit to the data using 4 parameter nonlinear regression excluding the data points at the two highest concentrations of simeprevir where detection reagent depletion is observed. Source data for c–g are provided as a Source Data file.

Fig. 2. The PRSIM-based CID module can regulate expression of exogenous and endogenous genes.

a Schematic of the simeprevir-inducible split transcription factor (STF) luciferase reporter assay. AD is activation domain and DBD is DNA-binding domain. b Left: the PRSIM_23-based STF system (blue) achieves a greater fold-induction of luciferase expression compared to the rapalog-induced iDimerize system (green). Right: A 3-fold lower level of baseline luciferase expression is observed from the PRSIM_23-based system (blue) than from the iDimerize system (green). RLU is relative luminescence units. c One (triangles) or two (squares) additional copies of PRSIM_23 fused to the AD lead to greater fold-induction of luciferase gene expression compared to a single copy (circles). d Schematic of AAV delivery of inducible IL-2 to cells. The PRSIM_23 CID STF and an inducible IL-2 transgene (iIL-2) are packaged into a single AAV particle. e Simeprevir-induced dose-dependent expression of IL-2 (squares) after transduction. Open squares indicate untransduced cells. f Levels of IL-2 expression induced by the PRSIM_23-based CID module (blue) were comparable to IL-2 levels from an AAV containing a constitutively active IL-2 transgene (orange). g Schematic of AAV vectors for delivery of the PRSIM_23 CID module-based STF and inducible luciferase transgene to cells in trans. h Simeprevir dose-dependent luciferase expression was observed after co-transduction (blue) but not after transduction of either AAV individually (green, orange) or in untransduced cells (black). i Schematic of the PRSIM_23 CID-based CRISPRa system used to regulate endogenous IL-2 expression. j Cells were co-transfected with an IL-2-targeting gRNA plasmid and a plasmid encoding the PRSIM_23 CID module-based STF. Expression of endogenous IL-2 was induced with 300 nM simeprevir (blue) or vehicle (grey) and either a single gRNA or two gRNAs in combination. For b, c, e and h, dose–response curves were fit to the data using 4-parameter nonlinear regression. Each data point represents the mean of two (e), three (b), (f; inducible IL-2), (h), (j), or four (c), (f; constitutive IL-2) independent replicates. Error bars in b, c, f, h, and j are s.e.m. Source data for b, c, e, f, h, and j are provided as a Source Data file.

Fig. 3. The PRSIM-based CID module can regulate a Caspase 9-based kill switch in vitro.

a Schematic of the Caspase 9-based kill switch. The PRSIM_23-based CID protein components are fused to a dimerization-deficient Caspase 9 activation domain (AD). b Phase contrast images of HEK293 (left), HCT116 (middle), and HT29 (right) cells stably transduced with the wild-type kill switch show rapid cell death upon treatment with 10 nM simeprevir (bottom) but not in untreated cells (top). Images were acquired after 60 min (HEK293, HCT116) or 90 min (HT29). c Caspase 3 activity is induced in wild-type kill switch-transduced HEK293 cells after treatment with 10 nM simeprevir (SIM; blue) relative to untransduced untreated HEK293 cells (grey). Each data point represents the mean ± s.d. of four independent experiments. d Caspase 3 activity is induced in wild-type kill switch-transduced HCT116 (orange) or HT29 cells (blue) after treatment with 10 nM simeprevir relative to untransduced, untreated parental cells. Each data point represents the mean ± s.d. of four independent experiments. e Simeprevir-dose-dependent cell viability 4 hrs (left) and 48 hrs (right) post simeprevir dosing in HCT116 (closed orange circles) or HT29 cells (closed blue circles) transduced with the kill switch. No cell killing is observed in untransduced parental cells (open circles). Each data point represents the mean of three independent replicates ± s.d. Data were fit to a dose-response curve using 4-parameter nonlinear regression. f Phase contrast images of HEK293 cells stably transduced with the kill switch S196A mutant show rapid cell death upon treatment with 10 nM simeprevir (bottom) but not in untreated cells (top). g Caspase 3 activity is induced by 10 nM simeprevir to comparable levels in the wild-type (WT; blue) and S196A mutant (purple) kill switch relative to treated untransduced HEK293 cells. Each data point represents the mean of five independent experiments ± s.d. For c, d, and g, data was analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons. For b, e, and f, a representative of n = 4 independent experiments is shown. Source data for c–e, and g are provided as a Source Data file.

Fig. 4. The PRSIM-based CID module can be used to regulate a Caspase 9-based kill switch in therapeutically relevant cells.

a Simeprevir (SIM)-induced dose-dependent cell killing up to 4 hrs post simeprevir dosing in kill switch-transduced Sa121 hESCs (blue circles). No killing is observed in parental Sa121 hESCs (orange circles) or without simeprevir stimulation. Each data point represents the mean of three independent replicates ± s.d. b Functionality of the kill switch engineered into primary T cells was determined by flow cytometry, measuring the disappearance of live kill switch+ (CD19+) cells following 10 nM (blue) or 10 µM simeprevir (orange) treatment for 7, 14, or 21 days. Untreated cells are in black. Data is presented for n of 3 human donors with percentage killing denoted above each bar. Source data are provided as a Source Data file.

Fig. 5. The PRSIM-based CID can be used to regulate a Caspase 9-based kill switch in vivo.

a Schematic of mouse model. b Complete tumor regression is induced by a single 200 mg/kg dose of simeprevir in a kill switch-transduced HT29 xenograft mouse model. The dashed line represents the day of simeprevir dosing. Each line represents an individual mouse. Black circles are mice treated with vehicle only, blue circles are mice treated with simeprevir after the average group tumor size reached 250 mm³, and orange circles are mice treated with simeprevir after the average group tumor size reached 500 mm³. Source data for b are provided as a Source Data file.