AUTONOMOUS PARVOVIRUS TRANSDUCTION OF A GENE UNDER CONTROL OF TISSUE-SPECIFIC OR INDUCIBLE PROMOTERS

Several classes of viruses are in use, or are being developed as gene therapy vectors. Viruses with small genomes containing few essential g enes have the advantage of requiring only simple complementation syste ms to allow packaging of foreign DNA, substituted for the entire viral coding sequences. Retroviruses and the dependent parvovirus AAV (aden o-associated virus) have been used in this way, and both possess an ef ficient integration mechanism which should allow long-term expression of transduced genes. in some situations, however, long-term persistenc e may be undesirable and there is a need for small, non-integrating vi ral vectors. Autonomous parvoviruses, such as LuIII, have potential as such vectors for short-term expression of therapeutic genes. We previ ously described recombinants of LuIII that transduced reporter genes, expressed using the viral constitutive promoter, P4. We have now gener ated several recombinants containing regulated promoters. A Virus incl uding a liver-specific enhancer directed 10- to 20-fold preferential e xpression of the luciferase reporter in transduced human hepatoma (Hep G2) versus HeLa cells. In additional LuIII recombinants, the luciferas e reporter was linked with chimeric promoters containing binding seque nces for either the yeast GAL4 protein or the bacterial tetracycline r epressor. Luciferase expression was strongly activated when these viru ses were used to infect cells containing a cognate trans-activator (GA L4 or tTA, a tetracycline repressor fusion with VP16 of herpes simplex ), introduced by transfection. The response to tTA could be abolished, or reduced in a graded manner, by exposure of the infected cells to t etracycline. Further results suggested that an increase in basal expre ssion, apparently mediated by the viral left terminal inverted repeat, could be minimized by interposing polyadenylation signals between thi s sequence and the promoter. These results confirm that appropriate tr anscriptional regulation can be achieved for genes transduced by an au tonomous parvovirus vector. Such vectors therefore show promise for th e delivery of therapeutic genes in situations requiring cell-specific, short-term expression, eg in targeting suicide genes for ablation of cancer cells.