Hypoxia- and radiation-activated Cre/LoxP molecular switch vectors for gene therapy of solid tumors

Rationale: Tumor hypoxia is a well-known negative prognostic factor for most solid tumors. Nevertheless, hypoxia represents a physiological difference that can be targeted for selective cancer gene therapy. In this study, hypoxia-mediated gene therapy was combined with ionizing radiation. We have shown that gene promoters containing hypoxia regulatory elements (HREs) from the Erythropoietin (Epo) gene and CArG elements from the Early Growth Response 1 (Egr1) gene are activated by hypoxia and/or radiation, ensuring therapeutic gene expression within the tumor mass. To achieve tight and specific gene regulation and produce high and sustained levels of the therapeutic gene herpes simplex virus thymidine kinase (HSVtk), an amplification system based on Cre/loxP recombination was devised. In this ‘molecular switch’, control of transgene expression is transferred from the inducible promoter to the strong constitutive cytomegalovirus immediate early (CMV IE) promoter.

Methods: Plasmid constructs were delivered to human tumor cells using DNA-lipid complexes. Cre-mediated excision of the Stop cassette was demonstrated by PCR assay. HSVtk was revealed by immunostaining. For in vitro studies, cell monolayers were transiently transfected with the hypoxia- and radiation-responsive switch vectors, and cell kill was measured after promoter activation and incubation with the prodrug ganciclovir (converted by HSVtk into a cytotoxin). For in vivo studies, vector-modified cell lines were established and grown as xenografts in nude mice. Tumor growth delay was measured after radiation and ganciclovir delivery. Hypoxia was detected by pimonidazole staining.

Results: In U87MG, U373MG glioblastoma and MCF-7 breast adenocarcinoma cells, the HRE/CArG promoters induced robust and selective gene induction after clinically significant doses of radiation (2-3 Gy) and at a range of physiological oxygen concentrations (0-1%). In the molecular switch context, the HRE/CArG promoter activated Cre expression within 2 h of hypoxic incubation, and Cre production was further induced after 2 Gy X-rays. Cre was localized in the nucleus and its activity was preserved in the intracellular environment under oxic as well as hypoxic conditions. Excision of the Stop cassette led to high and selective HSVtk synthesis that was sustained for over 48 h after hypoxia and radiation. The efficacy of the ‘molecular switch’ vectors for cancer gene therapy was tested in vitro and in vivo. After hypoxia and/or radiation, higher and more selective GCV-mediated cell kill was achieved with the switch vectors, when compared with the inducible promoters directly driving HSVtk expression. As expected, no toxicity was observed after sham-treatment. In animal models, the HRE/CArG-switch vector was almost as efficient as the strong CMV IE construct, inducing a significant growth delay, and, in some cases, tumor eradication. Effectiveness of the HRE/CArG-switch vector correlated with exposure to radiation and intratumoral hypoxia.

Conclusions: Hypoxia- and radiation -activated ‘molecular switch’ vectors represent a novel system for both targeted and robust gene therapy of solid tumors, especially where areas of hypoxia are a major hindrance to treatment