High efficiency of gene delivery and long-term expression are key requirements for a good gene therapy vector. Adenoviral vectors come close to meeting this specification — they are among the most efficient delivery vectors, they have a broad range of targets and removing most of the virus's own genes has rendered them non-toxic and mostly non-immunogenic. However, gene expression from the adenoviral genome, which is linear and remains extrachromosomal, is unstable and is almost completely lost within a year. Yant et al. now report a substantial improvement to this gene delivery method — by combining adenovirus-mediated delivery with transposon-mediated integration, they achieve high levels of long-term transgene expression in mice.

In a previous study, the authors showed that the Sleeping Beauty (SB) transposase could cause a plasmid-borne SB transposon to integrate randomly into a mouse chromosome in vivo. Although the integrated transgene was expressed over long periods of time, the applicability of this approach for gene therapy was limited because plasmids, unlike adenoviruses, cannot be delivered efficiently to cells in vivo. Yant et al. therefore engineered a transposon-containing transgene in an adenoviral vector; however, they soon discovered that transposition is efficient only from circular templates — adenoviral DNA is linear. To overcome this problem, the authors introduced an Flp/FRT recombination step into their procedure — Flp recombinase, expressed from a separate vector, was used to mediate recombination between FRT sites that flanked the transposon-containing transgene to circularize it. This circular construct was then a target for SB transposase, which integrates it into the genome.

Once Yant et al. were satisfied that the system worked reliably, they tested it for the persistence of transgene expression by including the lacZ ORF in the SB transposon. The constructs were delivered into the tail vein of immunocompromised mice and their livers were examined five weeks later for β-galactosidase expression. Up to 45% of hepatocytes showed expression. Also, expression was maintained after several rounds of cell division, confirming the stability of the integrated transgene. Because the activity of SB transposase depends on zinc, transgene expression can be turned on or off by regulating zinc levels in the water that the mice drink.

The system was finally tested for expression efficiency by placing a gene for human coagulation factor IX — the protein that is deficient in haemophilia B — in the transposon vector. Even after six months, the level of human coagulation factor IX in transgenic mice was ∼135-fold higher than in controls. Importantly, similar levels had previously been shown to be sufficiently therapeutic in a mouse model of haemophilia B.

The authors are quick to point out the advantages of their system. A major limitation of adenoviral vectors — their instability — has been overcome, but it also turns out that transposon-mediated integration has an important advantage over other types of integration as it doesn't cause chromosomal rearrangements. Far from being ready to rest on their laurels, Yant et al. have already begun working on improving the system by developing a single vector to integrate the properties of the two that were used in this study.