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Feasibility of gene therapy for hepatoma based upon hepatoma-cell specific expression of the herpes simplex virus thymidine kinase (HSV-tk) gene was investigated. Alb e/p-pNT 230 retroviruses, which contain the HSV-tk gene under the transcriptional control of the murine albumin gene enhancer and promoter, were produced from ecotropic Psi 2 retroviral packaging cells. Infection of Alb e/p-pNT 230 retroviruses to hepatoma cells did not affect the cell proliferation at all. The retroviral-infected hepatoma cells, however, were susceptible to ganciclovir and acyclovir toxicity, while uninfected parental hepatoma cells were not. Ganciclovir was proven to exhibit much stroner cytotoxicity upon the retroviral-infected hepatoma cells than acyclovir. On the other hand, Alb e/p-pNT 230 retroviral-infected non-hepatoma cells were resistant to ganciclovir, and the sensitivity was more than 100-fold different as compared with the same retroviral-infected hepatoma cells, indicating the potential of hepatoma cell-specific elimination without affecting any other tissues by use of Alb e/p-pNT 230 retroviruses. Furthermore, when mice bearing a bulky established tumor consisting of Alb e/p-pNT 230 retroviral-infected hepatoma cells were treated with ganciclovir, complete tumor regression was observed in 11 of 14 mice. These results indicate the feasibility of gene therapy for heptoma using the HSV-tk and ganciclovir system. Finally, 3ganciclovir-resistant clones were established from 3tumors that were not abrogated by ganciclovir treatment. These clones were proven not to lose the HSV-tk gene transferred by Alb e/p-pNT 230 retroviruses. The gene was, however, heavily methylated, and 5-azacytidine treatment partially restored the sensitivity of these 3clones to ganciclovir, indicating that methylation plays an essential role in expression of the retrovirally transferred gene.

Recent remarkable developments in molecular biology and in vitro culture techniques have provided a promising new treatment, gene therapy, for cancer and congenital genetic diseases. For in vivo gene therapy toward cancer, it is indispensable to direct the expression of exogenous genes exclusively to cancer cells. I have constructed a recombinant retroviral vector in which a tissue-specific regulatory element is used as an internal promoter. It contains the murine albumin enhancer and promoter as an internal promoter and the lacZ gene coding bacterial β-galactosidase as a reporter between the two Moloney murine leukemia virus long terminal repeats. This vector was introduced into ecotropic retroviral ψ2 packaging cells to produce recombinant retroviral particles. Various cell lines were infected with the recombinant retrovirus to assess its tissue specificity in vitro. Expression of the lacZ gene was detected solely in hepatoma cell lines but not in fibroblasts nor in B lymphoma cells. In vivo infection was carried out to evaluate tissue specificity of this system. The recombinant retrovirus was injected into a murine subcutaneous hepatoma and expression of the lacZ gene was observed in hepatoma cells but not in surrounding connective tissues. Then the retrovirus was injected into murine livers and no expression was detected in normal hepatocytes. The retrovirus was again injected into the livers of partially hepatectomized mice, resulting in the gene expression in only a few regenerating hepatocytes. Ratio of expression of the exogenous gene was, however, much lower than that in subcutaneous hepatoma. Finally, the gene expression of higher efficiency was confirmed as the retroviral infections were repeated. These results indicate that repeated gene transfer by means of the recombinant retrovirus which contains a tissue-specific regulatory element as an internal promoter should possess high potential for selective elimination of hepatoma cells in vivo.