A novel approach using transcomplementing adenoviral vectors for gene therapy of adrenocortical cancer.

Current therapies for adrenocortical carcinomas do not improve the life expectancy of patients. In this study, we tested whether a gene-transfer therapy based upon a suicide gene/prodrug system would be effective in an animal model of the disease. We employed E4- and E1A/B-depleted, herpes simplex virus-thymidine kinase-expressing adenoviral mutants that transcomplement each other within tumor cells, hereby improving transgene delivery and efficacy by viral replication in situ. Transcomplementation of vectors increased the fraction of transduced of tumor cells. This increase was accompanied by greater tumor volume reduction compared to non-transcomplementing approaches. Survival time improved with non-replicating vectors plus GCV compared to controls. However, transcomplementation/replication of vectors led to a further significant increment in anti-tumor activity and survival time (p < 0.02). In treated animals, we observed a high number of apoptotic nuclei both adjacent to and distant from injection sites and sites of viral oncolysis. Ultrastructural analyses exhibited nuclear inclusion bodies characteristic of virus production in situ, and provided further evidence that this therapy induced apoptotic cell death within tumor cells. We conclude that the efficacy of suicide gene therapy is significantly amplified by viral replication and, in combination with GCV, significantly reduces tumor burden and increases survival time.

AV.TK-mediated killing of subcutaneous tumors in situ results in effective immunization against established secondary intracranial tumor deposits.

Gene transfer vectors expressing herpes simplex thymidine kinase (HSVtk), in addition to direct killing of tumor cells, often have an associated local "bystander effect" mediated by metabolic coupling of tumor cells. A systemic antitumor effect mediated by the immune system, termed the distant bystander effect, has also been reported. We have observed the development of cytotoxic T-lymphocyte (CTL) populations and long-lasting antitumor immunity following treatment of subcutaneous tumors with an adenoviral vector expressing HSVtk (AV.TK) and ganciclovir (GCV) in rat glioma model. This vaccination effect seen with AV.TK/GCV treatment of subcutaneous tumor could even abrogate or retard growth of previously established secondary intracranial tumors.

Adeno-retroviral chimeric viruses as in vivo transducing agents.

Several hybrid viral gene transfer systems have been described that exploit the favorable features of the two parent viral species. We have developed a hybrid adeno-retroviral vector system to generate a retroviral vector in situ. The system consists of adenoviruses encoding MoMLV gag.pol (Axtet.gag.pol), the VSV-G viral envelope (Axtet.VSV-G), the retroviral vector LXSN expressing the neomycin phosphotransferase gene (AV-LXSN) and a transcriptional regulator to control expression of gag.pol and envelope (AV-rtTA). In vitro, biologically active retroviral vector preparations were generated following adeno-retroviral transduction of 9L rat glioma cells. In vivo the transcomplementing adeno-retroviruses were co-administered intratumorally into subcutaneous 9L glioma tumors in rats and human A375 melanoma xenografts in nude mice. In the 9L rat model, G418 cell cultures were only obtained when 9L cells were harvested from tumors injected with all four transcomplementing adeno-retroviruses. Molecular analysis of DNA extracted from 9L G418 populations derived both in vitro and in vivo showed appropriate integration of the LXSN proviral sequence. Tumor cells were harvested 1, 3 and 4 weeks after adeno-retrovirus administration to the human A375 xenografts. The percentage of G418 colonies recovered from tumors transduced with all of the transcomplementing adeno-retroviruses increased with time, whereas no increase was observed in tumors transduced with AV-LXSN alone. DNA extracted from G418 A375 cell populations showed the presence of integrated proviral sequences only in animals that received the full complement of adeno-retroviruses. These results demonstrate that adenoviral vectors expressing transcomplementing genes for retroviral proteins and retroviral vector RNAs can be used for in situ transduction of target cells.

Adenovirus vectors as transcomplementing templates for the production of replication defective retroviral vectors.

We have generated an adenovirus containing a retroviral vector sequence encoding the neomycin phosphotransferase (neo) gene (AV-LXSN). AV-LXSN transduction of retroviral packaging cell lines led to production of LXSN retroviral vector with alternative viral envelopes; exposure of target cells to retroviral containing supernatants confirmed envelope specific tropism. Retroviral titers (G418 cfu/ml) were comparable to those produced by standard techniques. Retrovirus could be detected in supernatants within 24 hours of AV-LXSN transduction and persisted as long as 120 hours. Southern blot analysis of DNA purified from populations of G418 cells showed the presence of a single neo containing restriction fragment of the appropriate size that could only be generated by reverse transcription of LXSN to produce LXSN provirus. This adeno-retroviral chimeric vector system could simplify the generation and testing of different retroviral vectors, particularly where assessment of vectors with alternative envelopes carrying novel targeting ligands is required.

Assessment of the correlation between nitrite concentration and listericidal activity in cultures of resident and elicited murine macrophages.

Reactive nitrogen intermediates (RNI) derived from L-arginine have been implicated as important anti-bacterial agents in the control of Listeria monocytogenes by murine macrophages. However, not all evidence is consistent with this conclusion. In the present study, this issue was examined using a simple experimental system to assess the correlation between macrophages Listericidal activity and production of nitrite (a stable end product of RNI) in culture. Various levels of nitrite production were achieved by activating macrophages with interferon-gamma (IFN-gamma) (20 or 500 U/ml) with or without lipopolysaccharide (LPS) (10 ng/ml) for 20 h before the Listericidal assay, and by using normal and arginine-free culture medium during the Listericidal assay. Nitrite concentration was measured for the same wells used to assess Listericidal activity. There was essentially no correlation between initial or final nitrite concentration and Listericidal activity in resident peritoneal macrophages. Significant correlations were noted between initial and final nitrite concentration and Listericidal activity in proteose peptone-elicited peritoneal macrophages. However, the correlation coefficients (0.34 and 0.52) suggested marginal biological relevance. In addition, no correlation was noted when LPS-activated macrophages were omitted from analysis. A previous study suggested that the enhanced Listericidal activity of LPS-treated macrophages could be accounted for by an enhanced rate of phagocytosis during the initial phase of the assay. These results suggest RNI are probably not the predominant bactericidal agents used by macrophages from female CD-1 mice to kill L. monocytogenes. However, it remains possible that RNI are important anti-bacterial agents in highly activated (LPS-treated) macrophages, and that there are other mechanisms whereby RNI contribute to host resistance to L. monocytogenes.

Effect of macrophage activation on killing of Listeria monocytogenes. Roles of reactive oxygen or nitrogen intermediates, rate of phagocytosis, and retention of bacteria in endosomes.

The role of macrophage activation in the killing of L. monocytogenes is unclear. Some studies suggest that activation for enhanced production of reactive oxygen and nitrogen intermediates may not be of central importance. Recent data have indicated an important role for interferon-gamma (IFN-gamma) induced retention of L. monocytogenes in endosomes. Data from the present study indicate that proteose peptone-elicited macrophages from DBA2/J, CD-1, and C3H/HeN mice are listericidal. Activation of these cells in vitro for 20 h by IFN-gamma (20 or 500 U/ml) increased H2O2 or nitrite production, but did not increase the number of L. monocytogenes killed during a subsequent 6-h or 7-h culture. Incubation of macrophages with IFN-gamma plus lipopolysaccharide (LPS) caused greater activation and increased the number of Listeria killed during a 6-h or 7-h culture. However, this seems primarily attributable to enhanced phagocytosis. Proteose peptone-elicited macrophages were significantly more effective than resident macrophages in preventing the escape of L. monocytogenes from endosomes into the cytoplasm. This capability was not significantly enhanced by IFN-gamma in vitro, but was enhanced by IFN-gamma plus LPS. This correlates well with the effects of these activation stimuli on killing of L. monocytogenes by proteose peptone-elicited macrophages. These results indicate that enhanced retention of L. monocytogenes in endosomes is induced by proteose peptone elicitation and that further macrophage activation in vitro by IFN-gamma does not improve listericidal activity.

Quantitative aspects of the feeder cell phenomenon: direct assessment of enhanced cystine uptake by lymphocytes.

It has been suggested that feeder cells and 2-mercaptoethanol enhance the survival and growth of murine lymphocytes in culture by increasing cysteine availability. We previously reported that although feeder cells produce thiols, they support lymphocyte growth at densities too low for measurable thiol production. This suggested that increasing the availability of cysteine might not be the major mechanism of feeder cell action. In the present study, [35S] cystine was used to directly monitor cyst(e)ine uptake in lymphocyte-feeder cell co-cultures. The results demonstrate that feeder cells substantially increase cyst(e)ine uptake by lymphocytes, even in the absence of detectable free thiols. Data are presented which suggest an explanation for this unexpected observation.