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.

Conclusion: 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.