Comment on the use of the cre/loxP recombinase system for gene therapy vectors

In the recent editorial ‘Radiation to control gene expression’, Stackhouse and Buchsbaum1 gave an excellent summary of the published research on the use of radiation to target gene therapy for cancer. They also comment on several issues that will have to be addressed before this approach can have a practical application. In the article, they outline our own work2 on a ‘molecular switch’ to enhance therapeutic gene expression following a comparatively low induction dose of radiation. Commenting on the use of the cre/loxP recombination system, they state: ‘The hypothesis that once cre recombinase is produced it will continue to create a functional therapeutic transcript has not been tested. The durability of expression of the cre gene and the stability of the cre enzyme will be critical determinants for the continued expression of the therapeutic gene’. In fact, the basis of the system we describe obviates the need for the continued expression of cre via the radiation-responsive promoter. Consequently, a brief review of the mechanism we have utilised is appropriate to clarify the situation.

The cre enzyme catalyzes the site-specific recombination of DNA between 34bp loxP sites.3 The result of recombination is governed by the relative orientation of the loxP sites.4 A segment of ‘spacer’ DNA flanked by loxP sites (‘floxed’) which are unidirectionally orientated, can then be excised by intramolecular loxP recombination via the action of the cre protein.

In our vector the spacer DNA is a transcriptional ‘stop cassette’, positioned between the strong, constitutive CMV IE promoter and the therapeutic gene, preventing its expression. However, radiation-induced expression of cre from a synthetic Egr1 promoter5 results in excision of the stop cassette by recombination, permitted the CMV promoter to control therapeutic transcript production. Thus, following the initial cre-mediated recombination event and deletion of the stop cassette, cre is no longer needed for the continued expression of the therapeutic gene. Indeed the efficacy of the molecular switch is evidenced by the data in Scott et al (Figure?2a) showing that the therapeutic gene is activated as effectively via the switch as when it is regulated directly by the CMV promoter.

However, as the editorial suggests, a number of issues need to be addressed before this vector system can be of practical use. These include the optimisation of the radio-induction response of the promoters for doses used in radiotherapy, the identification of the best prodrug activating gene/radiosensitiser combination (with particular regard to the bystander effect) and the replacement of CMV promoter with tumour-specific promoters producing prolonged, high-level expression of therapeutic genes. The method of delivery will also of course be vital to success.