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Cellular delivery of a replication-conditional herpes simplex virus type 1 (HSV-1) vector provides a means for gene therapy of invasive tumor cells. LacZ -bearing neural precursor cells, which can migrate and differentiate in the brain, were infected with a ribonucleotide reductase-deficient HSV-1 mutant virus (rRp450) that replicates only in dividing cells. Replication of rRp450 in neural precursor cells was blocked prior to implantation into the tumor by growth arrest in late G 1 phase through treatment with mimosine. Viral titers in the medium of mimosine-treated, rRp450-infected neural precursor cells were below detection levels 3 days after infection. In culture, after removal of mimosine and passaging, cells resumed growth and replication of rRp450 so that, 7 days later, virus was present in the medium and cell death was evident. Mimosine-treated neural precursor cells injected into established intracerebral CNS-1 gliomas in nude mice migrated extensively throughout the tumor and into the surrounding parenchyma beyond the tumor over 3 days. Mimosine-treated neural precursor cells, infected with rRp450 and injected into intracerebral CNS-1 tumors, also migrated within the tumor with the appearance of foci of HSV–thymidine kinase-positive (TK + ) cells, presumably including tumor cells, distributed throughout the tumor and in the surrounding parenchyma over a similar period. This migratory cell delivery method has the potential to expand the range of delivery of HSV-1 vectors to tumor cells in the brain.

Background This study investigates elements of herpes simplex virus type 1 (HSV-1) which influence transgene expression in tetracycline-regulated expression systems. Methods Different HSV-1 mutants were used to infect Vero cells that had been transfected with plasmids containing the luciferase gene under the control of tet-off or tet-on tetracycline-regulation systems. Results The baseline level of luciferase expression was elevated after infection with HSV-1 mutants lacking one or more immediate early genes encoding transactivating factors: ICP27, ICP4 and ICP0. With the tet-off system, not only was baseline expression elevated, but there was a complete loss of induction upon removal of tet when this regulatory system was brought into the cell by infection with helper virus-free amplicon vectors. Elevation of luciferase expression was also observed upon infection with the same HSV-1 mutants following transfection with a plasmid containing only a CMV minimal promoter driving luciferase (pUHC13-3). Only one HSV mutant (14HD3), which bears a disruption in the transactivation domain of VP16 and is deleted for both ICP4 genes, did not increase baseline luciferase expression after transfection of pUHC13-3. The disregulating effects were dependent on virus dose and were not influenced by treatment with interferon (IFN)-alpha, which suppresses viral gene expression. Additional assays involving cotransfection of pUHC13-3 with a plasmid encoding of the HSV-1 transactivating factor ICP4 revealed that ICP4 was the most potent inducer of gene expression from the tetO/CMV minimal promoter. Conclusion These results indicate that proteins encoded in the HSV-1 genome, especially the transactivating immediate early gene products (ICP4, ICP27 and ICP0) and the VP16 tegument protein can activate the tetO/ minimal CMV promoter and thereby interfere with the integrity of tetracycline-regulated transgene expression. Copyright # 2000 John Wiley & Sons, Ltd.

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 107 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. Copyright © 1999 John Wiley & Sons, Ltd.

Publisher Summary There is a rich array of possible intervention points and times in stimulation of central nervous system (CNS) repair using genetic engineering techniques. These can be broken down into three cell categories: neurons, pathways, and innervation targets—and three time intervals—immediately after injury, during neurite regrowth, upon target recognition, and upon reinnervation. At the time of injury it should be possible to inject vectors and cells directly into the damaged site. The most nontoxic, yet efficient direct gene delivery vectors in the current formulations would be adenovirus and adenoassociated virus (AAV), gutless adenovirus, helper virus-free herpes simplex virus (HSV) amplicons, and lentivirus. Genes that might be helpful for neurons in the injury period include those coding for anti-apoptotic proteins or protective proteins for free radicals. Grafted cells could be preprogrammed genetically with a pro-drug-activation suicide gene, so that systemic application of the pro-drug would lead to cell death. An ideal system would be activation of a cell death geneunder the control of a tetracycline inducible promoter.