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Oncolytic conditionally replicating adenoviruses (CRAd) can exclusively replicate in and lyse tumor cells and are therefore promising tools in cancer therapy. In this study, we combined the oncolytic potential of a CRAd with its ability to deliver a suicide gene (herpes simplex virus thymidine kinase suicide gene, HSVtk) in order to further enhance tumor cell killing in a human retinoblastoma (RB) mouse model. We could demonstrate that CRAd driven by the human telomerase reverse transcriptase (hTERT) promoter and armed with the HSV thymidine kinase suicide gene/ganciclovir (HSVtk/GCV) could very effectively reduce growth of human RB in an orthotopic nude mouse model. These findings suggest that hTERT promoter-driven CRAd in combination with HSVtk/GCV gene therapy could be a promising new approach for the treatment of RB. In addition, we found that hTERT promoter-driven CRAd replication occurred exclusively in human RB cells but not in primary human retinal pigment epithelial cells (hRPE), indicating that application of hTERT promoter-driven CRAd for the treatment of RB would be safe.