Treatment of Hepatocellular Carcinoma with Adeno-Associated Virus Encoding Interleukin-15 Superagonist Chia-Ming Chang, 1,2 Chia-Hui Lo, 1,2 Yao-Ming Shih, 3 Yin Chen, 2 Ping-Yi Wu, 2 Koichi Tsuneyama, 4 Steve R. Roffler, 2 and Mi-Hua Tao 1,2 Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, but effective therapies are still needed. The liver has been identified as an important immune organ and is heavily populated with various lymphocyte subsets known to play important roles in cancer immunosurveillance. We hypothesized that acti- vation of hepatic lymphocytes by interleukin (IL)-15, a cytokine known for its ability to trigger proliferation and activation of natural killer (NK) cells, natural killer T cells, and memory CD8 þ T cells, might offer an alternative therapy for HCC. We employed hepatotropic adeno-associated virus serotype 8 (AAV8) to deliver an IL-15 superagonist (IL-15-IL-15RaS), consisting of IL-15 covalently linked to the N-terminal sushi domain of the IL-15 receptor a chain, to achieve local sustained cytokine expression in the liver environment. We observed that a single injection of AAV8 expressing IL-15-IL-15RaS, but not IL-15 alone, greatly expanded the number of hepatic mononuclear cells, mainly NK cells, for at least 21 days. AAV8=IL-15-IL-15RaS treatment generated potent antitumor activity in a liver metastatic murine HCC model (BNL cells), and significantly prolonged the survival time of treated animals. The antitumor effect depended mainly on NK cells, not on CD8 þ and CD4 þ T cells, because AAV8=IL-15-IL-15RaS treatment greatly enhanced the cytolytic activity of hepatic NK cells and de- pletion of NK cells abrogated the therapeutic effect. Importantly, no apparent liver toxicity was observed during AAV8=IL-15-IL-15RaS treatment. Together, our data demonstrate that AAV8-delivered IL-15-IL-15RaS provides an effective and safe therapy against metastatic HCC. Introduction H epatocellular carcinoma (HCC), the fifth most common cancer worldwide, is one of the most difficult cancers to treat (Avila et al., 2006; El-Serag et al., 2008; Llovet and Bruix, 2008). Surgical resection and liver transplantation are considered curative therapies, but are feasible for only a small number of patients. Other treatment options, such as percutaneous ethanol injection, arterial chemoembolization, and radiofrequency ablation, are only partially effective in controlling localized tumors, and are usually accompanied by a high rate of metastatic recurrence after treatment. Therefore, there is an urgent need to develop more effective therapeutic strategies to treat HCC. CD8 þ T cells and natural killer (NK) cells play important roles in cancer immunosurveillance by directly killing ma- lignant cells (Street et al., 2004; Zitvogel et al., 2006; Wald- hauer and Steinle, 2008). Many studies demonstrate that in- terleukin (IL)-15 is a powerful cytokine for triggering the proliferation and activation of NK, natural killer T (NKT), and memory CD8 þ T cells (Fehniger et al., 2002; Budagian et al., 2006; Waldmann, 2006), suggesting that IL-15 might be a potential candidate for cancer immunotherapy. Indeed, IL-15 could increase the antitumor activity of adoptively transferred CD8 þ T cells or the chemotherapeutic agent cy- clophosphamide through NK cell- and T cell-dependent mechanisms, but when administered alone IL-15 showed only marginal antitumor effects (Evans et al., 1997; Chapoval et al., 1998; Klebanoff et al., 2004; Teague et al., 2006). The failure of IL-15 therapy is likely due to the limiting avail- ability of in vivo IL-15 receptor a chain (IL-15Ra), which is present on antigen-presenting cells (dendritic cells and 1 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan. 2 Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan. 3 Graduate Institute of Microbiology, National Taiwan University, Taipei 10051, Taiwan. 4 Department of Diagnostic Pathology, Graduate School of Medicine and Pharmaceutical Science, University of Toyama, Toyama 930–0194, Japan. HUMAN GENE THERAPY 21:611–621 (May 2010) ª Mary Ann Liebert, Inc. DOI: 10.1089=hum.2009.187 611