ABSTRACT
Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.
Subject(s)
Deoxycytidine/analogs & derivatives , Genetic Therapy/methods , Melanoma/therapy , Promoter Regions, Genetic , Proteins/genetics , Animals , Antiviral Agents/pharmacology , Bromodeoxycytidine/analogs & derivatives , Cell Line, Tumor , Cell Separation , Cloning, Molecular , Deoxycytidine/pharmacology , Dependovirus/genetics , Enhancer Elements, Genetic , Extracellular Matrix Proteins , Female , Flow Cytometry , Ganciclovir/pharmacology , Gene Transfer Techniques , Genes, Reporter , Humans , Immunosuppressive Agents/pharmacology , Melanoma/genetics , Mice , Mice, Nude , Models, Genetic , Monophenol Monooxygenase/genetics , Neoplasm Proteins , Neoplasm Transplantation , Plasmids/metabolism , Simplexvirus/genetics , Thymidine Kinase/genetics , Time Factors , Tissue DistributionABSTRACT
Selective killing of tumors can be achieved by targeting the transcription of suicide genes via specific DNA control elements to malignant cells. Three different enhancer-promoter systems were constructed and evaluated for their capability to direct gene expression to melanoma. Two tissue-specific (tyrosine and MIA) promoters and one weak viral promoter were fused to multiple tandem copies of a melanocyte-specific enhancer element. Reporter gene assays revealed a maximum increase in transcription by combining each promoter with 3-4 copies of the enhancer and demonstrated that all enhancer-promoter combinations exhibited tissue-specific activity. Though this activity was still significantly less than that of the strong but unspecific cytomegalovirus (CMV) promoter. In contrast, when those combinations were employed to drive the expression of two suicide genes, encoding the diptheria toxin A chain (DT-A) and the prodrug-activating herpes simplex virus thymidine kinase (TK), respectively, only those constructs in which transcription was under control of tissue-specific promoter elements mediated selective killing of melanoma cells. This killing was in the range of cell death induced by CMV promoter activity. Our data indicate that the enhancer/tyrosinase and enhancer/MIA promoter constructs but not the viral promoter constructs can provide a valuable tool for selective suicide gene expression in melanoma.
