A NOVEL PIGGYBACK PACKAGING SYSTEM FOR HERPES-SIMPLEX VIRUS AMPLICON VECTORS

Recombinant and amplicon vectors derived from herpes simplex virus typ e 1 (HSV-1) have proven to be an efficient means of gene delivery to c ells in culture and in vivo, In this study, a system was developed to make propagation of the amplicon vector and helper virus mutually depe ndent on each other, in a 'piggyback' fashion, This combined system su pports maintenance and enrichment of the amplicon vector when propagat ing stocks, while allowing the helper virus to serve as a recombinant vector in its own right, Amplicons bearing a gene essential for HSV-1 replication, IE3, as well as the Escherichia coil lacZ marker gene, we re propagated using a mutant virus (d120) deleted in the same essentia l gene, Vector stocks could be propagated in Vero cells and other cult ured cells not transfected with the IE3 gene with markedly delayed cyt opathic effects, as compared to wild-type virus. Relatively high titer s of amplicon vectors (6 x 10(7) infectious units/ml) were achieved wi th this piggyback system in Vero cells, with an apparent ratio of ampl icon vector: helper virus of up of 5:1 under some conditions; however, recombinant wild-type virus was also generated, Injection of these st ocks into experimental gliomas in rodent brain revealed gene delivery to tumor cells mediated by both amplicon vectors (lacZ) and helper vir us (HSV-thymidine kinase), with no apparent neuropathology of normal b rain, This basic piggyback vector model is amenable to modifications t o promote conditional propagation of vectors in vivo and to allow inco rporation of multiple transgene elements into both the amplicon and re combinant helper virus vectors.