Utilizing tumor hypoxia to enhance oncolytic viral therapy in colorectal metastases.

Hypoxia, a reduction in normal tissue oxygen levels, is a common condition of solid tumors. Rapidly proliferating tumors outstrip their vascular supply resulting in hypoxic regions. These areas are reported to have oxygen concentrations less than 2%.1 Oxygen-deficient cancer cells exhibit an increased rate of genetic mutation, local invasion, and metastatic spread.2,3 Similarly, low tumor oxygen tension has been correlated with increased metastases and poor survival in various cancers.4 In addition to the aggressive nature of these tumors, hypoxia confers resistance to standard radiation therapy and chemotherapy.5,6 Hypoxic tumors are thus associated with a poor outcome regardless of treatment modality.3 The development of new therapeutic strategies is necessary. Lower tumor oxygen levels compared with surrounding normal tissues may be used to selectively target cancer cells in gene therapy.7 Hypoxia is a potent signal that induces the production of proteins, which allow cells to adapt to a low oxygen environment. Hypoxia-inducible factor 1 (HIF-1) is a key transcriptional regulator controlling the expression of diverse hypoxia-inducible genes related to angiogenesis, oxygen transport, cellular adhesion, and glycolysis.8–10 Although involved in adaptive physiologic processes, HIF-1 is also overexpressed in diverse cancers and is associated with tumor progression and metastases.11,12 HIF-1 consists of an oxygen-regulated α subunit (HIF-1α) and a constitutive β subunit (HIF-1β). In hypoxia, HIF-1α translocates to the nucleus where it dimerizes with HIF-1β. This heterodimer binds to the hypoxia-responsive element (HRE) of target genes to stimulate transcription.13 The HRE consensus sequence has been characterized in the 5′ untranslated region of the vascular endothelial growth factor (VEGF) gene.14 Replication-competent, multimutated herpes simplex viruses (HSV) have been successful in the treatment of a broad spectrum of malignancies in animal models, including brain, hepatic, gastric, prostate, head and neck, pancreatic, and colorectal.15–20 Genetically engineered oncolytic viruses have been attenuated to have reduced toxicity to normal tissues, while maintaining their inherent ability to kill tumor cells. G207 is a second-generation HSV type-1, containing multiple mutations, which significantly minimize reversion to a pathogenic phenotype. G207 has deletions in both copies of the neurovirulence gene γ134.5, which is responsible for viral spread and replication within the central nervous system. This virus also contains an insertional inactivation of UL39, the gene encoding the large subunit of ribonucleotide reductase (RR).16 RR is the main rate-limiting enzyme for viral DNA synthesis and replication, controlling the nucleotide substrate pool by regulating the conversion of ribonucleotides to deoxyribonucleotides.21 Viral mutants with a defective UL39 gene exclusively replicate in and lyse rapidly dividing cancer cells, which provide sufficient levels of RR.22 Increasing tumor RR levels may potentially enhance viral replication and tumor cell lysis. This study sets out to determine whether replacement of UL39 under control of a hypoxia-responsive enhancer would promote more vigorous killing of cancer cells by an oncolytic herpes virus and increase tumor specificity through hypoxia.