414. A Novel System to Systematically Analyze Diverse Types of Conditionally Replicating Adenoviruses Expressing Immunostimulatory Genes in Syngenic Hamster Models
Authors: Nobuhiro Ijichi, Hirotaka Kawakami, Yuki Obama, Ken-ichiro Kosai
Journal: Molecular Therapy (2016)

We developed a method to efficiently construct diverse "conditionally replicating adenoviruses regulated with multiple cancer-specific factors" (m-CRAs) for an ideal gene-viro-therapy (Gene Ther 2005), after we had developed the immunological gene therapy, including granulocyte macrophage colony-stimulating factor (GM-CSF) (PNAS 1995, 1996, Cancer Res 1996). We then identified by systematic m-CRA analyses that survivin-responsive m-CRA (Surv.m-CRA), in which E1A is regulated by survivin promoter, could achieve stronger and more cancer-selective potentials than competing m-CRAs (Cancer Res 2005). The new Surv.m-CRA, of which an altered E1B promoter is regulated with another cancer-specific promoter (CS-Pr), further increased cancer specificity without reduced anticancer effects (Cancer Gene Ther 2011). Moreover, the unique feature of the increased effectiveness against cancer stem cells suggests that Surv.m-CRA is promising anticancer agent (J Trans Med 2014). In addition, we have developed a novel m-CRA strategy that specifically eliminates undifferentiated cells in pluripotent stem cell-based regenerative medicine (Mol Ther Methods Clin Dev 2015). Taken altogether, we are about to start its clinical trial in Japan. As the first-in-class oncolytic virus expressing GM-CSF was recently approved, m-CRAs expressing immunostimulatory genes should be promising. In general, the development of oncolytic adenoviruses with cytokines has been hampered by two technical limitations in the vector construction and the viral replication-permissive animals. Here we demonstrate a novel system to efficiently generate diverse types of m-CRAs expressing candidate immunostimulatory genes under candidate promoters, and systematically analyze their potentials in syngenic hamster models. We tested the feasibility of this methodology, and succeeded in the rapid generation of the new Surv.m-CRAs comprising 18 different therapeutic units, including a mouse or human cytokine gene downstream to a ubiquitously active promoter (UA-Pr) or CS-Pr. To test the utility of our experimental system, we first analyzed in vivo antitumor efficiencies and toxicities of Surv.m-CRA expressing GM-CSF under a strong UA-Pr in xenograft syngeneic tumor of syrian hamster. An intratumoral injection of the control Surv.m-CRA without GM-CSF inhibited a tumor growth and extended a survival to some degrees. Whereas Surv.m-CRAs expressing mouse GM-CSF more drastically reduced tumor growth, some of these animals died of excessive GM-CSF expressions (showing the remarkable splenomegaly) within 3 days. The results importantly suggest the necessity of systematical analyses to identify the best combination of cytokine genes and promoters to achieve the optimal cytokine levels (Int J Oncol 2004), and we are now analyzing 18 different Surv.m-CRAs, including those expressing GM-CSF at a moderate level and/or in the cancer-specific manner. In conclusion, we have developed a novel system to efficiently generate the candidate m-CRAs expressing diverse immunostimulatory genes under several promoters and to systematically analyze them in syngenic hamster models.