Figure 3.

Construction of a dual-module adenovirus expressing mouse telomerase reverse transcriptase-targeting ribozyme (mTERT-TR)-regulated HSVtk and sPD1-Ig. (a) Ad5mTR.sPD1 encodes mTERT-TR-HSVtk under the control of the CMV promoter in the E1 region and sPD1-Ig under control of the EF1α promoter in the E3 region. Details of the construction are described in Material and Methods section. (b) HEK293 cells (4 × 10⁵) were infected with the indicated adenovirus as an multiplicity of infection of 10 for 24 hours, followed by lysis in RIPA buffer and immunoblot analysis. sPD1-Ig expression by Ad5mTR.sPD1 (total of 80 µg protein per lane) was confirmed using an anti-PD1 antibody. (c) The enzymatic activity of HSVtk in phosphorylating ganciclovir was evaluated in CT26 cells by measuring the accumulation of radio-labeled [H³]PCV (1 µCi/ml) as described in Materials and Methods section. Data represent the mean ± SD of triplicate assays. (d) Culture supernatant harvested from cells infected with adenovirus (5 or 10 MOI) was mixed with cocultures of ovalbumin (OVA)-specific CD8 T cells (1 × 10⁶) purified from OT-1 mice and MC38/OVA cells (1 × 10⁴) in 6-well plates. After 72 hours, culture supernatants were collected and interferon-γ levels were measured. Data represent the mean ± SD. P, unpaired t-test. ψ, packaging signal; δE1 and δE3, deletion of E1 and E3; CMV, cytomegalovirus promoter; EF1α, elongation factor 1α promoter; HSVtk, herpes simplex virus thymidine kinase; ITR, inverted terminal repeats; MOI, multiplicity of infection; Rz, mouse TERT targeting trans-splicing ribozyme; pA, poly A; PCV, penciclovir.