Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents.

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

MDM2 oncogene as a target for cancer therapy: An antisense approach.

The MDM2 oncogene is amplified or overexpressed in human cancers. It has also been suggested that MDM2 levels are associated with poor prognosis of several human cancers. The MDM2 oncoprotein binds to the p53 tumor suppressor protein and serves as a negative regulator of p53. The p53 tumor suppressor also has an important role in cancer therapy, with p53-mediated apoptosis being a major mechanism of action for many clinically used cancer chemotherapeutic agents and radiation therapy. Therefore, the negative regulation of p53 by MDM2 may limit the magnitude of p53 activation by DNA damaging agents, thereby limiting their therapeutic effectiveness. The investigators hypothesize that, by inhibiting MDM2 expression, the MDM2 oncoprotein level will be reduced and the MDM2 negative feedback inhibition of p53 will be diminished, resulting in a significant increase of functional p53 levels that will modulate p53-mediated therapeutic effects. The overall objective of the present study was to investigate the functions of MDM2 oncogene in tumor growth and the potential value of MDM2 as a drug target for cancer therapy. The role of MDM2 in tumor growth is determined by inhibiting MDM2 expression in in vivo models of human cancers. The in vivo synergistically therapeutic effects of MDM2 inhibition and DNA damaging agents were also evaluated. Significant in vitro antitumor activities were found in cell lines, human osteosarcoma SJSA and choriocarcinoma JAR, in a time-, concentration-, and sequence-dependent manner. Following i.p. administration of anti-MDM2 antisense oligonucleotides, in vivo antitumor activity was observed in nude mice bearing SJSA and JAR xenografts in a dose-dependent manner. Moreover, in vivo synergistically therapeutic effects of MDM2 inhibition and DNA damaging agents adriamycin and 10-hydroxycamptothecin were observed. This study should provide the basis for future development of anti-MDM2 antisense oligonucleotides as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.

Infectivity enhancement for adenoviral transduction of canine osteosarcoma cells.

The full realization of conditionally replicative adenoviruses (CRAds) for cancer therapy has been hampered by the limited knowledge of CRAd function in vivo and particularly in an immunocompetent host. To address this issue, we previously proposed a canine adenovirus type 2 (CAV2)-based CRAd for clinical evaluation in canine patients with osteosarcoma (OS). In this study, we evaluated infectivity-enhancement strategies to establish the foundation for designing a potent CAV2 CRAd with effective transduction capacity in dog osteosarcoma cells. The results indicate that the native CAV2 fiber-knob can mediate increased binding, and consequently gene transfer, in both canine osteosarcoma immortalized and primary cell lines relative to previously reported Ad5 infectivity-enhancement strategies. Gene delivery was further enhanced by incorporating a polylysine polypeptide onto the carboxy terminus of the CAV2 knob. This vector demonstrated improved gene delivery in osteosarcoma xenograft tumors. These data provide the rationale for generation of infectivity-enhanced syngeneic CAV2 CRAds for clinical evaluation in a dog osteosarcoma model.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy.

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on 'xenotype' knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.