Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells

Abstract

Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat the most common liver cancer-hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells, leaky anticancer gene expression and possible toxicity. To improve the safety, we employed synthetic biology to engineer BV for transgene expression regulation. We first uncovered that miR-196a and miR-126 are exclusively expressed in HCC and normal cells, respectively, which allowed us to engineer a sensor based on distinct miRNA expression signature. We next assembled a synthetic switch by coupling the miRNA sensor and RNA binding protein L7Ae for translational repression, and incorporated the entire device into a single BV. The recombinant BV efficiently entered HCC and normal cells and enabled cis-acting transgene expression control, by turning OFF transgene expression in normal cells while switching ON transgene expression in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in separate culture and mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and non-HCC normal cells for selective transgene expression control and killing of HCC cells.

Figure 1.

Involvement of miR-196a in HCC tumorigenicity. (A) MiR-196a expression in different HCC cells. (B) Recombinant BVs that expressed scramble sponge (Bac-Scramble), miR-196a sponge (Bac-s196) and pre-miR-196a (Bac-m196). (C–E) miR-196a levels in HepG2 (C), PLC (D) and Huh-7 (E) cells after BV transduction. (F–G) Spheroid formation in PLC (F) and Huh-7 (G) cells after BV transduction. The miR-196a levels in various HCC and normal cells were quantified by TaqMan^® RT-qPCR and normalized to that in human hepatocytes. HCC cells were mock-transduced or transduced with recombinant BVs at MOI 200 and the miR-196a levels were analyzed by RT-qPCR at 1 dpt. The data represent means±SD of triplicated culture experiments.

Figure 2.

Identification of OncoMiR and NormalMiR. (A) Expression of miR-126, miR-134, miR-155, miR-218 and miR-196a in PLC, HUVEC, human hepatocyte and liver tissue. (B–D) miR-196a (B), miR-126 (C) and miR-218 (D) levels in mouse livers and PLC cells. (E) miR-126 levels in BV-transduced PLC cells. The miRNA levels were analyzed by RT-qPCR and normalized to those in PLC. The data represent means ± SD of at least three independent experiments.

Figure 3.

Recombinant BVs carrying the synthetic switch for transgene expression regulation. (A) Schematic illustration of recombinant BVs LKE and CirCE. CMV, cytomegalovirus immediate-early promoter. WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. PA, poly A signal. (B) Schematic illustration of transgene expression induction (ON) and repression (OFF) in HCC and normal cells. CirCE transduction of PLC cells (high miR-196a, low miR-126) would turn ON EBFP expression due to the negligible miR-126 levels and high miR-196a levels that could turn OFF L7Ae translation. In contrast, CirCE transduction of HUVEC cells (low miR-196a, high miR-126) would turn ON L7Ae/EYFP expression due to the lack of miR-196a-mediated inhibition and turn OFF EBFP due to the miR-126 and L7Ae binding to the EBFP mRNA.

Figure 4.

Selective transgene (EBFP) expression in HCC cells. (A) Fluorescence micrographs of cells after BV transduction. (B) Percentage of EYFP⁺ cells. (C) Mean EYFP intensity (×10³ a.u.). (D) Percentage of EBFP⁺ cells. (E) Mean EBFP intensity (×10³ a.u.). (F) L7Ae levels. (G) miR-196a levels. The HUVEC and PLC cells were transduced with LKE or CirCE at MOI 200 and examined by fluorescence microscopy or flow cytometry at 1 dpt. The L7Ae and miR-196a levels were analyzed by RT-qPCR at 1 dpt. The data represent means±SD of triplicated culture experiments.

Figure 5.

Selective killing of HCC cells in separate culture by the synthetic switch-based BV. (A) Schematic illustration of recombinant BV CirCB. CirCB was similar to CirCE except that the transgene was proapoptic hBax. (B) Cell morphology. (C) Viable cell density. (D) Viability. (E) Apoptosis induction. The HCC (PLC) and normal (HUVEC) cells were separately seeded to 6-well plates (3 × 10⁵ cells/well) and were mock-transduced or transduced with CirCB. At 1 dpt, the cell morphology was observed under the microscope. All cells were then harvested and stained by trypan blue, followed by viable cell density and viability analyses. The cells were also subjected to luminescence-based Caspase 3,7 assay to quantify apoptosis. Higher relative luminescence units (RLU) indicate higher degrees of apoptosis. The data represent means±SD of triplicated culture experiments.

Figure 6.

Selective transgene expression and killing of HCC cells in PLC/HUVEC co-culture. (A) Fluorescence micrographs of co-cultured PLC and HUVEC cells. HUVEC and PLC cells were co-cultured at a seeding density ratio of 1:1 in 6-well plates (1.5 × 10⁵ cells/well for each cell) and transduced with LKE or CirCE at MOI 200. The co-cultured cells were immunolabeled with anti-CD31 antibody and observed with a fluorescence microscope. (B–D) Immunofluorescence double labeling and flow cytometry analysis of BV-transduced co-cultured cells. HUVEC and PLC cells were co-cultured and transduced as in (A), immunostained with anti-EGFR and anti-CD31 antibodies, followed by flow cytometry analysis. The CD31⁻/EGFR⁺ PLC and CD31⁺/EGFR⁻ HUVEC cells were gated (B) and the EBFP expression in PLC (c) and HUVEC (D) was elucidated. (E) Selective killing of HCC cells in co-culture by CirCB. HUVEC and PLC cells were co-cultured as in (A), mock-transduced or transduced with CirCB, immunostained with anti-EGFR antibody and analyzed by flow cytometry. The data are representative of at least 3 independent culture experiments.