title: A novel 'piggyback' packaging system for herpes simplex virus amplicon vectors.
abstract: Recombinant and amplicon vectors derived from herpes simplex virus type 1 (HSV-1) have proven to be an efficient means of gene delivery to cells in culture and in vivo. In this study, a system was developed to make propagation of the amplicon vector and helper virus mutually dependent on each other, in a "piggyback' fashion. This combined system supports maintenance and enrichment of the amplicon vector when propagating 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 coli lacZ marker gene, were propagated using a mutant virus (d120) deleted in the same essential gene. Vector stocks could be propagated in Vero cells and other cultured cells not transfected with the IE3 gene with markedly delayed cytopathic effects, as compared to wild-type virus. Relatively high titers of amplicon vectors (6 x 10(7) infectious units/ml) were achieved with this piggyback system in Vero cells, with an apparent ratio of amplicon vector: helper virus of up of 5:1 under some conditions; however, recombinant wild-type virus was also generated. Injection of these stocks into experimental gliomas in rodent brain revealed gene delivery to tumor cells mediated by both amplicon vectors (lacZ) and helper virus (HSV-thymidine kinase), with no apparent neuropathology of normal brain. This basic piggyback vector model is amenable to modifications to promote conditional propagation of vectors in vivo and to allow incorporation of multiple transgene elements into both the amplicon and recombinant helper virus vectors.
authors.affiliation: Department of Neurology, Massachusetts General Hospital East, Boston, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Pechan
authors.forename: M
authors.initials: M
authors.lastname: Fotaki
authors.forename: R L
authors.initials: RL
authors.lastname: Thompson
authors.forename: R
authors.initials: R
authors.lastname: Dunn
authors.forename: M
authors.initials: M
authors.lastname: Chase
authors.forename: E A
authors.initials: EA
authors.lastname: Chiocca
authors.forename: X O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002522:Chlorocebus aethiops; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D014709:Vero Cells