TARGETING OF CYTOKINE GENE-EXPRESSION TO MALIGNANT-MELANOMA CELLS USING TISSUE-SPECIFIC PROMOTER SEQUENCES

Background: Transduction of tumour cells in vitro with cDNAs encoding various cytokines and/or immune accessory molecules has been shown to diminish or eliminate tumorigenicity when such cells are returned in v ivo to syngeneic animals. One method being explored for in situ gene t herapy is to use tissue-specific promoters to direct expression of the therapeutic genes to the tumour cells. Design: This study used the 5' flanking region of the murine tyrosinase gene to direct expression of three different cytokine genes [murine interleukin 2 (IL-2), IL-4 and macrophage colony-stimulating factor (M-CSF)] specifically to murine melanoma cells. Results: Expression of the IL-2 gene, from 2.5 kbp of the 5' flanking region of the murine tyrosinase gene, was detected in 11 out of 55 puromycinresistant B16 clones isolated after transfection . The highest producing clone secreted 2000 pg/Ml/10(6) cells/48 hours as determined by enzyme-linked immunosorbent assay. The IL-2 was test ed for biological activity by its ability to stimulate proliferation o f the IL-2 dependent CTLL cell line. No detectable level of IL-2 expre ssion occurred in 58 clones of drug-resistant NIH 3T3 cells derived af ter transfection with the same construct. Similar results were obtaine d following transfection of these two cell lines with the tyrosinase-I L-4 minigene construct. Expression of IL-2 in the murine melanoma cell s completely abrogated their tumorigenicity in syngeneic mice. However , progressively growing tumours were produced from clones in which the IL-2 gene was no longer expressed (as determined by reverse transcrip tase polymerase chain reaction). Direct injection of DNA encoding cyto kine genes, expressed from the tyrosinase promoter, into established B 16 melanomas in syngeneic mice resulted in gene expression within the tumour mass. While no change in tumour growth was observed following s uch treatment, the results demonstrate that direct injection of naked DNA into a neoplasm can result in uptake and expression of cytokine ge nes up to 16 days post-injection. Conclusion: The use of tissue-specif ic promoters can limit expression to the required target cell, while t he choice of appropriate gene should result in an alteration in tumour burden.