Combined HSV-1 recombinant and amplicon piggyback vectors: Replication-competent and defective forms, and therapeutic efficacy for experimental gliomas

Background The versatility of HSV-1 vectors includes large transgene capaci ty, selective replication of mutants in dividing cells, and availability of recombinant virus (RV) and plasmid-derived (amplicon) vectors, which can b e propagated in a co-dependent, 'piggyback', manner. Methods A replication-defective piggyback vector system was generated in wh ich the amplicon carries either of two genes essential for virus replicatio n, IE2 (ICP27) or IE3 (ICP4), as well as lacZ; the RV is deleted in both th ese genes, and vector stocks are propagated in cells transfected with one o f the complementary genes. In the replication-competent system, the amplico n carries the IE2 and lacZ; the RV had a large deletion in the IE2; and sto cks are propagated in untransfected cells. Titers over successive passages, recombination between amplicon and RV, and the structural integrity of vec tor genomes were evaluated. The replication-competent system was tested for therapeutic efficacy in subcutaneous 9L gliosarcoma tumors in nude mice wi th 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 gener ated in either system. The replication-defective system showed low toxicity and increased packaging efficiency of amplicon vectors, as compared to sin gle mutant RV helper virus. The replication-competent system allowed co-pro pagation 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 HS V, vector systems allow gene delivery via amplicon vectors with reduced tox icity and co-propagation of both RV and amplicon vectors in target cells, w ith effective tumor therapy via focal virus replication and pro-drug activa tion. Copyright (C) 1999 John Wiley & Sons, Ltd.