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An abstract of the article "Combination of Allogeneic Hematopoietic Stem Cell Transplantation and Allogeneic MHC Gene Transfer Against Solid Cancers," by Kazunori Aoki, Masaki Ohashi, Miwa Kushida, Kimiko Yoshida, Masaki Mandai, Gary J. Nabel and Teruhiko Yoshida is presented.

We have been investigating the efficacy of an intratumoral interferon (IFN)-alpha gene transfer against solid cancers, and found that when the gene is transduced into the subcutaneous tumors, IFN-alpha concentration is markedly increased in the injected tumor but not in the serum. To explain this effective confinement of IFN-alpha to target tissues, we hypothesized that the extracellular matrix in the tumors interacts with IFN-alpha. In this study, a solid-phase-binding assay and immunoprecipitation demonstrated that the IFN-alpha binds directly to matrix proteins. Immunohistochemical staining showed a co-localization of IFN-alpha with pericellular fibronectin. In addition, matrix-bound IFN-alpha protein transduced intracellular signaling and potentiated its cytotoxic activity, suggesting that the retention of IFN-alpha protein on extracellular matrix is likely to play a role in its in vivo biological activity. The data suggest a therapeutic advantage of the intratumoral IFN-alpha gene transfer over the conventional parenteral therapy both in the safety and efficacy.

The interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis and immunomodulation. We previously examined the two antitumor mechanisms, taking advantage of the fact that IFN-alpha did not show cross-species activity in its in vivo effect. In a nude mouse subcutaneous xenograft model using human pancreatic cancer cells, the expression of human IFN-alpha effectively induced cell death of human pancreatic cancer cells, whereas mouse IFN-alpha augmented antitumor immunity by stimulation of natural killer cells. Here, we extended our investigation to a syngeneic pancreatic cancer model, so that the integrated antitumor activity of local IFN-alpha gene therapy, including the antiproliferative, proapoptotic, antiangiogeneic and immunomodulatory effects, can be evaluated rigorously. When a recombinant hamster IFN-alpha adenovirus was injected into syngeneic subcutaneous tumors of hamster pancreatic cancer (PGHAM-1) cells in Syrian hamster, tumor growth was significantly suppressed due to cell death and T cell- and natural killer cell-mediated antitumor immunity. Moreover, in this case, tumor regression was observed not only for the injected subcutaneous tumors but also for the untreated tumors both in the peritoneal cavity and at distant sites. No significant systemic toxicity was observed in the treated hamsters. Moreover, the subcutaneous rechallenge of PGHAM-1 cells was rejected in three of four cured hamsters from the initial tumor challenge. This study further demonstrated that local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its multiple mechanisms of antitumor activity and its lack of significant toxicity.

Interferon alpha (IFN-alpha) is used worldwide for the treatment of a variety of cancers. For pancreatic cancer, recent clinical trials using IFN-alpha in combination with standard chemotherapeutic drugs showed some antitumor activity of the cytokine, but the effect was not significant enough to enlist pancreatic cancer as a clinically effective target of IFN-alpha. In general, an improved therapeutic effect and safety are expected for cytokine therapy when given in a gene therapy context, because the technology would allow increased local concentrations of this cytokine in the target sites. In this study, we first examined the antiproliferative effect of IFN-alpha gene transduction into pancreatic cancer cells. The expression of IFN-alpha effectively induced growth suppression and cell death in pancreatic cancer cells, an effect which appeared to be more prominent when compared with other types of cancers and normal cells. Another strategy we have been developing for pancreatic cancer targets its characteristic genetic aberration, K-ras point mutation, and we reported that the expression of antisense K-ras RNA significantly suppressed the growth of pancreatic cancer cells. When these two gene therapy strategies are combined, the expression of antisense K-ras RNA significantly enhanced IFN-alpha-induced cell death (1.3- to 3.5-fold), and suppressed subcutaneous growth of pancreatic cancer cells in mice. Because the 2',5'-oligoadenylate synthetase/RNase L pathway, which is regulated by IFN and induces apoptosis of cells, is activated by double-strand RNA, it is plausible that the double-strand RNA formed by antisense and endogenous K-ras RNA enhanced the antitumor activity of IFN-alpha. This study suggested that the combination of IFN-alpha and antisense K-ras RNA is a promising gene therapy strategy against pancreatic cancer.

Recently we reported that on-site interferon (IFN)-alpha production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-alpha in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-alpha gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-alpha cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2('),5(')-oligoadenylate synthetase activity, suggesting that IFN-alpha produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-alpha gene therapy for HCV-associated liver diseases.