title: Combined HSV-1 recombinant and amplicon piggyback vectors: replication-competent and defective forms, and therapeutic efficacy for experimental gliomas.
abstract: BACKGROUND
  The versatility of HSV-1 vectors includes large transgene capacity, selective replication of mutants in dividing cells, and availability of recombinant virus (RV) and plasmid-derived (amplicon) vectors, which can be propagated in a co-dependent, 'piggyback', manner.
  
  
  METHODS
  A replication-defective piggyback vector system was generated in which the amplicon carries either of two genes essential for virus replication, IE2 (ICP27) or IE3 (ICP4), as well as lacZ; the RV is deleted in both these genes, and vector stocks are propagated in cells transfected with one of the complementary genes. In the replication-competent system, the amplicon carries the IE2 and lacZ; the RV had a large deletion in the IE2; and stocks are propagated in untransfected cells. Titers over successive passages, recombination between amplicon and RV, and the structural integrity of vector genomes were evaluated. The replication-competent system was tested for therapeutic efficacy in subcutaneous 9L gliosarcoma tumors in nude mice with activation of ganciclovir via the viral HSV-thymidine kinase gene.
  
  
  RESULTS
  Both systems generated high titer amplicon vectors (about 10(7) tu/ml) and amplicon:RV ratios (0.6-3.0). No replication-competent RV was generated in either system. The replication-defective system showed low toxicity and increased packaging efficiency of amplicon vectors, as compared to single mutant RV helper virus. The replication-competent system allowed co-propagation of amplicon and RV; injection into tumors followed by ganciclovir treatment inhibited tumor growth without systemic toxicity.
  
  
  CONCLUSIONS
  New replication-defective and replication-competent piggyback HSV, vector systems allow gene delivery via amplicon vectors with reduced toxicity and co-propagation of both RV and amplicon vectors in target cells, with effective tumor therapy via focal virus replication and pro-drug activation.
authors.affiliation: Department of Neurology, Massachusetts General Hospital East, Harvard Medical School Neuroscience Program, Boston 02129, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Pechan
authors.forename: U
authors.initials: U
authors.lastname: Herrlinger
authors.forename: M
authors.initials: M
authors.lastname: Aghi
authors.forename: A
authors.initials: A
authors.lastname: Jacobs
authors.forename: X O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003673:Defective Viruses / Q000235:genetics / Q000502:physiology; D017781:Genes, Immediate-Early; D015316:Genetic Therapy; D005822:Genetic Vectors; D018316:Gliosarcoma / Q000628:therapy*; D006378:Helper Viruses / Q000235:genetics / Q000502:physiology; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D007763:Lac Operon; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011995:Recombination, Genetic; D014709:Vero Cells; D014779:Virus Replication