Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway

Efficient nuclear delivery of plasmid DNA represents a major barrier in non viral gene transfer. One approach has been to use DNA-binding proteins such as GAL4 from yeast as DNA carriers with nuclear targeting properties. We r ecently showed, however, that GAL4 is inefficient in targeting DNA to the n ucleus because its DNA-binding and nuclear targeting activities are mutuall y exclusive, which relates to the fact that GAL4 nuclear import occurs via a novel pathway. Here, we 'switch' this pathway to a more conventional one by adding a modified poly-lysine to which an optimized nuclear targeting si gnal, based on that of the SV40 large T-antigen, is linked. We also use a c himeric GAL4-alpha -melano-cyte stimulating hormone (MSH) fusion protein to enable gene transfer to cells expressing the MSH receptor. Switching the n uclear import pathway of the transfecting complex significantly enhances re ceptor-mediated gene transfer through enabling interaction with desired com ponents of the cellular nuclear import machinery. The present study represe nts the first demonstration that nuclear targeting signals can enhance rece ptor-mediated gene delivery, the approaches having important relevance to r esearch and clinical applications, such as in generating transgenic or knoc k-out animals, or in gene therapy.