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Safety requirements for gene therapy currently focus on preventing acute patient toxicity caused by the introduction of exogenous DNA. This project aimed to investigate the possibility that more subtle changes in cell regulation may occur as a result of transfection or transgene expression. Mouse B16.F1 melanoma and CMT93 colorectal cell lines transfected with a plasmid (pNASS) or retroviral (pBabeNeo) construct were used as a model. Expression of the transgene was driven by a 2540bp (plasmid) or 769bp (retroviral) fragment of the mouse tyrosinase promoter. Results have shown that transfection per se can cause small changes in the proliferation rate of B16.F1 and CMT93 cells, but transgene expression did not produce this effect. However, expression of an IL-2 transgene in B16.F1 cells did decrease the rate of substrate attachment via an intracellular mechanics. These data suggest that cell regulation can be disrupted by transfection and transgene expression. Despite these changes, DDRT-PCR analysis of approximately 25% of the mRNA population indicated few changes in gene expression between parental and transfected cells. Loss of transgene expression has proved an obstacle to success in clinical trials and was observed in both cell lines. In B16.F1 cells containing pNASS/IL-2, levels of IL-2 protein were reduced by more than 90% after 15 weeks in culture and this was mirrored by a decrease in IL-2 mRNA levels. The transgene was intact in high passage cells and restriction digest analysis did not indicate changes in methylation status. The continued presence of the construct was confirmed by treatment with 10-6 M melanocyte stimulating hormone, which induced IL-2 trnasgene mRNA and protein expression in cells that had lost expression from pNASS/IL-2. Induction was not observed in cells with the shorter promoter fragment in pBabeNeo/IL-2, indicating that the mechanism of downregulation can be overridden if the promoter contains suitable response elements. |
