Karen M. Johnston

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oris, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep¯) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep¯ hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells. Herpes simplex virus/adeno-associated virus (HSV/AAV) hybrid vectors combine the advantages of two gene delivery vehicles, namely HSV-1 amplicons and recombinant (r) AAV vectors. Hybrid vectors bear the oris and pac features of HSV-1 amplicons and a lacZ transgene cassette flanked by AAV inverted terminal repeat (ITR) sequences, with or without the AAV rep gene outside the cassette. Dividing U87 cells infected with the hybrid vectors retained transgene expression longer than those infected with a conventional HSV-1 amplicon vector, presumably through on site amplification and/or genomic integration of the ITR-flanked transgene cassette. The presence of the rep gene appeared to increase the stability of expression in this system. By using the helper virus-free packaging system, cytotoxic effects were virtually eliminated. This vector has the potential to achieve stable transgene delivery to both dividing and non-dividing cells through “episomal” amplification and/or chromosomal integration of the transgene cassette.

Brain tumors have an immunoprivileged status which contributes to their refractoriness to treatment. In this study, immune rejection of GL261 glioma tumors in the mouse brain was achieved by subcutaneous vaccination with GM-CSF-transduced glioma cells. Cultured GL261 cells were transduced to secrete murine GM-CSF using a retrovirus vector, then irradiated, and injected subcutaneously into H-2 matched C57BL/6 mice. In prevaccination studies, the median survival time (MST) of animals vaccinated with 5 x 10(4) or 5 x 10(5) GM-CSF-transduced cells 7 days prior to intracranial injection of 10(6) nontransduced, nonirradiated GL261 cells was significantly prolonged by 45-50% compared with animals vaccinated in parallel with nontransduced, irradiated glioma cells. In treatment of established gliomas, the MST of animals, which were treated subcutaneously with 5 X 10(6) irradiated GM-CSF-transduced cells 3 days after intracranial injection of 2 x 10(4) nontransduced cells, was prolonged significantly by 36% compared with animals treated with the same number of nontransduced, irradiated cells or to sham-treated animals. In prevaccination studies, histology of brain tumors 4 days after intracranial tumor cell injection revealed infiltrates of CD8+ lymphocytes and eosinophils, the latter exclusively in animals vaccinated with GM-CSF-transduced cells, Thus, subcutaneous injection of irradiated GM-CSF-transduced glioma cells can induce a potent immune response to intracranial gliomas both as a vaccination against subsequent intracranial glioma cell implantation and for treatment of established intracranial glioma.