Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

Abstract

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1a) that interacts with a 'hypoxia-responsive element' (HRE), the consensus sequence of which ( A / G CGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.The parvovirus Minute virus of mice, MVM(p), replicates preferentially in oncogenic-transformed cells, where it completes a lytic life cycle (Rommelaere & Cornelis, 1991). This property is exploited in gene-therapy vectors derived from autonomous parvoviruses (Russell et al., 1992;Cornelis et al., 2004). The MVM(p) genome is composed of two transcription units, encoding two sets of viral proteins, NS (non-structural) and VP (capsid), under the control of the P4 and P38 promoters, respectively (Cotmore et al., 1983). The non-structural protein NS1 plays an important role in viral DNA replication (Nüesch et al., 1998), is toxic in transformed cells (Caillet-Fauquet et al., 1990) and is required for transactivation of the P38 promoter, which is otherwise only expressed at a very low basal level (Rhode, 1985).The mechanisms underlying the preferential expression of MVM(p) in transformed cells are only poorly understood, but it is at least in part linked to the upregulation of the P4 promoter in these cells compared with normal cells. We therefore wanted to determine whether P4 activity could be further enhanced and whether this could improve vector production. We have explored the possibility of producing MVM vectors under low oxygen tension (hypoxia). Gene expression and production of the human erythrovirus B19 virus are increased under these conditions (Caillet-Fauquet et al., 2004;Pillet et al., 2004). Regulation of promoter activity in hypoxia occurs through the interaction of the transcriptional regulator hypoxia-inducible factor (HIF-1) with a 'hypoxia-responsive element' (HRE) (Semenza, 2001). The MVM(p) promoter contains four putative HREs (consensus sequence A / G CGTG), two in each of the sense and antisense orientations.The presence of the HRE in the promoter region is necessary, but not sufficient, for promoter regulation. Other cellular elements are probably...

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