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ABSTRACTBrain tumors that have disseminated into cerebrospinal fluid (CSF) pathways are an unresolved therapeutic problem, especially in pediatric neurooncology. Here a gene therapy approach using the herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) paradigm was tested using an HSV vector in a rodent model of disseminated central nervous system tumors. 9L-gliosarcoma cells were implanted simultaneously into the brain and the CSF of syngeneic rats. Five days later, resulting intracerebral and leptomeningeal tumors were treated by intrathecal injection of a replication-conditional HSV vector. This vector was defective for the ribonucleotide reductase gene, but contained an intact HSV-tkgene. Systemic GCV treatment was started 2 days after vector application and continued for 14 days. Tumor-free, long-term survival (LTS) was achieved in 90% of the animals treated with this combined therapeutic approach, whereas only 30% LTS was found in animals that had received the vector alone and 10% LTS in untreated animals. This therapeutic response probably involves oncolytic, on-site replication of the vector, activation of GCV by a HSV-TK, and a strong immune response both to the vector and to 9L cells. Apparent vector-related mortality was observed in 20% of animals without subsequent GCV therapy, but no vector-related mortality was found when the animals were treated with GCV after vector application. Given the successful outcome of this experimental treatment and the apparent potential of GCV to control HSV-related toxicity, intrathecal application of HSV vectors combined with GCV treatment may be a promising approach for treatment of disseminated brain tumors.

ABSTRACTRecombinant and amplicon vectors derived from herpes simplex virus type 1 (HSV-1) have proven to be an efficient means of gene delivery to cells in culture and in vivo. In this study, a system was developed to make propagation of the amplicon vector and helper virus mutually dependent on each other, in a piggyback fashion. This combined system supports maintenance and enrichment of the amplicon vector when propagating stocks, while allowing the helper virus to serve as a recombinant vector in its own right. Amplicons bearing a gene essential for HSV-1 replication, IE3, as well as the Escherichia coli lacZmarker gene, were propagated using a mutant virus (d120)deleted in the same essential gene. Vector stocks could be propagated in Vero cells and other cultured cells not transfected with the IE3gene with markedly delayed cytopathic effects, as compared to wild-type virus. Relatively high titers of amplicon vectors (6 107infectious units/ml) were achieved with this piggyback system in Vero cells, with an apparent ratio of amplicon vector: helper virus of up of 5:1 under some conditions; however, recombinant wild-type virus was also generated. Injection of these stocks into experimental gliomas in rodent brain revealed gene delivery to tumor cells mediated by both amplicon vectors (lacZ) and helper virus (HSV-thymidine kinase), with no apparent neuropathology of normal brain. This basic piggyback vector model is amenable to modifications to promote conditional propagation of vectors in vivoand to allow incorporation of multiple transgene elements into both the amplicon and recombinant helper virus vectors.

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 ( 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.

ABSTRACTA second-generation replication-conditional herpes simplex virus type 1 (HSV) vector defective for both ribonucleotide reductase (RR) and the neurovirulence factor 34.5 was generated and tested for therapeutic safety and efficiency in two different experimental brain tumor models. In culture, cytotoxic activity of this double mutant HSV vector, MGH-1, for 9L gliosarcoma cells was similar to that of the HSV mutant, R3616, which is defective only for 34.5, but was significantly weaker than that of the HSV mutant hrR3, which is defective only for RR. The diminished tumoricidal effect of the 34.5 mutants could be accounted for by their reduced ability to replicate in 9L cells. The MGH-1 vector did not achieve significant prolongation of survival in vivoin the syngeneic 9L rat gliosarcoma model for either single brain tumor focus or multiple intracerebral and leptomeningeal tumors, when the vector was applied intratumorally or intrathecally, respectively, and with or without subsequent ganciclovir (GCV) treatment. In identical 9L brain tumor models with single and multiple foci, application of hrR3 with or without GCV was previously shown to result in marked long-term survival. Contrary to the findings with intrathecal injection of hrR3, no vector-related mortality was observed in any animals treated with MGH-1. Thus, in these rat brain tumor models, the double mutant, replication-conditional HSV vector MGH-1 showed a higher therapeutic safety than the RR-minus vector, hrR3, but had clearly decreased therapeutic efficiency compared to hrR3. The development of new HSV vectors for brain tumor gene therapy will require a balance between maximizing therapeutic efficacy and minimizing toxicity to the brain. Standardized application in brain tumor models as presented here will help to screen new HSV vectors for these requirements.

ABSTRACTNovel 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 lacZreporter 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 repgene (repand 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 repas 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.

ABSTRACTThe therapeutic use of neurotrophic factors for neurodegenerative diseases is promising, however, optimal methods for continuous delivery of these substances to the human central nervous system (CNS) remains problematic. One approach would be to graft genetically engineered human cells that continuously secrete high levels of a biologically produced and processed neurotrophic factor. This ex vivogene therapy approach has worked well in animal models of neurodegenerative diseases using a variety of nonneuronal cell types to deliver the transgene. In our studies, we have been investigating the potential of astrocytes, a cell type normally present in the CNS, as a vehicle for ex vivogene therapy. Here, we demonstrate that astrocytes in the human fetal cortex can be isolated and efficiently infected with an amphotropic retrovirus harboring mouse -nerve growth factor (NGF). These transduced astrocytes express high levels of NGF mRNA and secrete bioactive NGF protein as demonstrated by stimulation of neurite outgrowth from adrenal chromaffin cells. NGF ELISA showed that these astrocytes secrete NGF protein at a rate of 41 ng/day per 105cells after 2 weeks in vitro, whereas NGF is undetectable in medium conditioned by normal astrocytes. These data suggest that human fetal astrocytes can be used for delivering biologically produced neurotrophic factors to the human CNS.