Integration of a multimeric STAT consensus site into the adenoviral genome to interfere with IFN signaling represents a novel concept to improve oncolytic virotherapy.

Adenovirus infection triggers an interferon (IFN) response that reduces replication of the viral genome early in the adenoviral life cycle. In oncolytic virus therapy, viral replication is essential for effective cell lysis, which subsequently can activate a systemic immune response against the tumor. We show that different members of the JAK/STAT protein family redundantly contribute to the suppression of adenoviral replication in bladder cancer-derived cells. This mechanism requires the expression of E1A; however, it does not affect expression levels of E1A, but rather its transactivation of E1B55K, and particularly E4. We developed a novel concept to sequester STAT isoforms from host cells by using a multimeric STAT consensus sequence that was introduced into the E3 region of the oncolytic virus XVir-N-31, resulting in XVir-N-14. This virus exhibits a higher potency in replication, cell lysis, and particle formation than XVir-N-31. This approach of sequestering DNA-binding molecules to the viral genome might offer an alternative to combination therapy with JAK/STAT inhibitors, which would not only suppress the desired systemic immune response but also cause serious adverse effects.IMPORTANCESuccessful application of oncolytic virotherapy requires efficient virus replication that can be suppressed by the host's cell antiviral IFN response. We extend in this study the insight into the underlying molecular mechanisms in bladder cancer cells. Suppression of JAK/STAT activity induces an E1A-dependent upregulation of early gene expression, although the E1A expression level is not affected. The effect is not observed when overexpressing E1B55K or E4orf6 in the viral genome, indicating a regulatory role of STAT molecules on the transactivation capacity of E1A. We developed a novel concept to interfere with the JAK/STAT signaling pathway by cloning a multimeric STAT consensus sequence in the E3 region of the oncolytic virus XVir-N-31. The resulting virus XVir-N-14 shows improved replication, cell lysis, and particle formation. Trapping a molecular suppressor to the viral genome supplements previous strategies to modify the host's immune response by cloning shRNAs or molecular inhibitors against the JAK/STAT signaling pathway.