Ulrich Herrlinger

Pre-existing herpes simplex virus 1 (HSV-1) immunity decreases, but does not abolish, gene transfer to experimental brain tumors by a HSV-1 vector.

The influence of pre-existing anti-herpes simplex type 1 (HSV-1) immunity on HSV-1 vector-mediated gene transfer to glioma cells was analyzed in this gene marking study using intracranial D74 gliomas in syngeneic Fischer rats. The HSV-1 mutant virus used, hrR3, is defective in ribonucleotide reductase and bears the marker genes E. coli lacZ and HSV-1 thymidine kinase (HSVtk). Initial marker gene expression in tumors 12 h after direct virus injection was reduced in immunized animals to about 15% of that in nonimmunized animals. Marker gene expression in both sets stayed at initial levels for 2 days after intratumoral injection and declined markedly on day 5. Inflammatory infiltrates in the tumor were more prominent in HSV-1-immunized, as compared with nonimmunized animals, at 12 and 24 h, but appeared similar at 2–5 days after injection. By day 10, the immune reaction had subsided in immunized animals and macrophages remained only in nonimmunized animals. In conclusion, gene transfer to brain tumors using a HSV-1 vector was greatly reduced, but not competely abolished, under pre-immunization conditions. Pre-existing antibodies to HSV-1 may also serve a positive role in providing an increased margin of safety in intracranial application of HSV-1 vectors by limiting spread of the virus within the brain and to other tissues.

Intraarterial Delivery of Adenovirus Vectors and Liposome± DNA Complexes to Experimental Brain Neoplasms

This study investigated the intraarterial delivery of genetically engineered replication-deficient adenovirus vectors (AVs) and cationic liposome-plasmid DNA complexes (lipoDNA) to experimental brain tumors. Adenovirus or lipoDNA was injected into the internal carotid artery (ICA) of F344 rats harboring intracerebral 9L gliosarcomas, using bradykinin (BK) to selectively permeabilize the blood-tumor barrier (BTB). Brain and internal organs of the animals were collected 48 hr after vector injection and stained for expression of the marker gene product, beta-galactosidase (beta-Gal). Intracarotid delivery of AV to 9L rat gliosarcoma without BTB disruption resulted in transgene expression in 3-10% of tumor cells distributed throughout the tumor. Virus-mediated expression of beta-gal gene products in this tumor model was particularly high in small foci (< or = 0.5 mm), which had invaded the normal brain tissue surrounding the main tumor mass. In these foci more than 50% of tumor cells were transduced. BK infusion increased the amount of transgene-expressing cells in larger tumor foci to 15-30%. In the brain parenchyma only a few endothelial cells expressed beta-gal owing to AV-mediated gene transfer. Intracarotid delivery of lipoDNA bearing a cytoplasmic expression cassette rendered more than 30% of the tumor cells positive for the marker gene without BTB disruption. The pattern of distribution was in general homogeneous throughout the tumor. BK infusion was able to increase further the number of transduced tumor cells to more than 50%. Although lipoDNA-mediated gene transfer showed increased efficacy as compared with AV-mediated gene transfer, it had less specificity since a larger number of endothelial and glial cells also expressed the transgene. AV and lipoDNA injections, in the absence and presence of BK, also resulted in transduction of peripheral organs. AV showed its known predilection for liver and lung. In the case of lipoDNA, parenchymal organs such as liver, lung, testes, lymphatic nodes, and especially spleen, were transduced. These findings indicate that intracarotid application of AV and lipoDNA vectors can effectively transduce tumor cells in the brain, and that BTB modulation by BK infusion can further increase the number of transgene-expressing tumor cells.

Helper virus-free herpes simplex virus type 1 amplicon vectors for granulocyte-macrophage colony-stimulating factor-enhanced vaccination therapy for experimental glioma.

Subcutaneous vaccination therapy with glioma cells, which are retrovirally transduced to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), has previously proven effective in C57BL/6 mice harboring intracerebral GL261 gliomas. However, clinical ex vivo gene therapy for human gliomas would be difficult, as transgene delivery via retroviral vectors occurs only in dividing cells and ex vivo glioma cells have a low growth fraction. To circumvent this problem, a helper virus-free herpes simplex virus type 1 (HSV-1) amplicon vector was used. When primary cultures of human glioblastoma cells were infected with HSV-1 amplicon vectors at an MOI of 1, more than 90% of both dividing and nondividing cells were transduced. When cells were infected with an amplicon vector, HSVGM, bearing the GM-CSF cDNA in the presence of Polybrene, GM-CSF secretion into the medium during the first 24 hr after infection was 1026 ng/10(6) cells, whereas mock-infected cells did not secrete detectable GM-CSF. Subcutaneous vaccination of C57BL/6 mice with 5 x 10(5) irradiated HSVGM-transduced GL261 cells 7 days prior to intracerebral implantation of 10(6) wild-type GL261 cells yielded 60% long-term survivors (>80 days), similar to the 50% long-term survivors obtained by vaccination with retrovirally GM-CSF-transduced GL261 cells. In contrast, animals vaccinated with the same number of nontranduced GL261 cells or with GL261 cells infected with helper virus-free packaged HSV-1 amplicon vectors carrying no transgene showed only 10% long-term survivors. In conclusion, helper virus-free HSV-1 amplicon vectors appear to be effective for cytokine-enhanced vaccination therapy of glioma, with the advantages that both dividing and nondividing tumor cells can be infected, no viral proteins are expressed, and these vectors are safe and compatible with clinical use.

Targeting gene therapy vectors to CNS malignancies

Gene therapy offers significant advantages to the field of oncology with the addition of specifically and uniquely engineered mechanisms of halting malignant proliferation through cytotoxicity or reproductive arrest. To confer a true benefit to the therapeutic ratio (the relative toxicity to tumor compared to normal tissue) a vector or the transgene it carries must selectively affect or access tumor cells. Beyond the selective toxicities of many transgene products, which frequently parallel that of contemporary chemotherapeutic agents, lies the potential utility of targeting the vector. This review presents an overview of current and potential methods for designing vectors targeted to CNS malignancies through selective delivery, cell entry, transport or transcriptional regulation. The topic of delivery encompasses physical and pharmaceutic means of increasing the relative exposure of tumors to vector. Cell entry based methodologies are founded on increasing relative uptake of vector through the chemical or recombinant addition of ligand and antibody domains which selectively bind receptors expressed on target cells. Targeted transport involves the potential for using cells to selectively carry vectors or transgenes into tumors. Finally, promoter and enhancer systems are discussed which have potential for selectivity activating transcription to produce targeted transgene expression or vector propagation.

HSV‐1 infected cell proteins influence tetracycline‐regulated transgene expression

This study investigates elements of herpes simplex virus type 1 (HSV‐1) which influence transgene expression in tetracycline‐regulated expression systems.

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

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.

HSV-1 Vectors for Gene Therapy of Experimental CNS Tumors.

Gliomas account for about 60% of all primary CNS tumors; two-thirds of all gliomas comprise the most malignant form, glioblastoma multiforme, or glioma grade IV. Although much progress has been achieved in the treatment of other solid tumors over the last few decades, the median survival of patients with glioblastoma remains at around 12 mo after standard treatment, which includes bulk resection and irradiation, as well as chemotherapy in some cases (1). Essentially, no patient can expect to survive 5 yr. New treatment modalities like immunotherapy have been applied so far with only limited success (2). With the improvement of methods for in vivo and ex vivo gene delivery, gene therapy became a new, promising approach to glioma therapy. Gliomas appear to be a particularly good target for a gene therapy approach using locally applied vectors, as the growth of gliomas is restricted to the brain. Clinical trials are under way using retrovirus and adenovirus vectors which carry the herpes simplex virus type-1 (HSV-1) thymidine kinase gene (HSV-tk). This gene encodes a prodrug-activating enzyme, which in infected cells converts the nontoxic prodrug, ganciclovir (GCV), to its cytotoxic phosphorylated form (3-5). There is an ever-increasing list of other prodrug-activation systems that showed efficacy in culture and in preclinical studies using rodent glioma models. These include, for example, cytosine deaminase converting 5-fluorocytosine to 5-fluoro-uracil (6), cytochrome P450-2B1 converting cyclophosphamide to phosphoramide mustard (7), deoxycytidine kinase phosphorylating cytosine arabinoside (8), and the Escherichia coli guanine phosphoribosyl transferase (gpt) metabolizing 6-thioxanthine and 6-thioguanine to toxic nucleoside analogs (9). Moreover, gene therapy approaches to brain tumors include the viral transfer of immune-enhancing cytokines, particularly granulocyte/macrophage colony-stimulating factor (10), or antisense to TGF-β to glioma cells (11) used for vaccination purposes. Other approaches use the transfer of genes that modulate angiogenesis (12,13) or are involved in apoptosis like p53 (14). All aforementioned gene-transfer methods use nonreplicative viral vectors.