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Unlike the development of drugs based on small chemical entities there are no conventional regulatory toxicity studies established for DNA-based products. As the potential for insertional mutagenesis is of particular concern for gene therapy, we have investigated the mutagenicity of model non-viral DNA-based products at the HPRT locus in Chinese hamster V79 cells. Cultures were transfected with a pL3112BSKS plasmid in combination with a number of non-viral transfection facilitators: Effectene, Lipofectamine 2000 and ExGen 500. The plasmid contains a green fluorescent protein gene, which was used as a reporter of transfection efficiency. Flow cytometry was used to analyse large numbers of cells. Small scale transient transfection efficiencies (7-90%) were obtained at low cytotoxicity, however, scaling up the process led to decreased transfection and increased cytotoxicity. Stable transfection (chromosomal) was observed, but only at very low levels (&lt;1.5%). Two of the non-viral delivery facilitators (Effectene and ExGen 500) themselves induced mutation at the HPRT locus, although they were considerably less potent than the positive control ethyl methanesulphonate. In transfection experiments, neither of the non-viral delivery facilitators (Effectene and Lipofectamine 2000) had a mutagenic effect, whereas with ExGen 500 there was evidence of a mutagenic effect, consistent with the mutagenicity observed with the non-viral transfection facilitator alone. Moreover, treatment with the plasmid pL3112BSKS itself was able to induce a 3- to 7-fold increase in the mutation frequency at the HPRT locus. Our studies highlight some of the problems associated with using exisiting genetic toxicology testing procedures for the assessment of DNA-based products used in novel gene therapy approaches. However, given the limited level of sophistication of the current approach, the data suggest that non-viral gene therapy may present a detectable mutation risk and more appropriate testing strategies are needed to evaluate the nature of the risk.

Evaluating the genetic toxicology of DNA-based products using existing genetic toxicology assays