Adenovirus hexon protein enhances nuclear delivery and increases transgeneexpression of polyethylenimine/plasmid DNA vectors

Inefficient nuclear delivery restricts transgene expression using polyelect rolyte DNA vectors. To increase transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexon protein to polyethylenimine (PE I, 800 kDa). Activity of the conjugate was compared with PEI and PEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expr ession in HepG2 cells than PEI/DNA or complexes containing albumin, without increasing cell uptake. Following cytoplasmic injection into Xenopus laevi s oocytes, hexon-containing complexes showed reporter gene expression to be elevated by 10-fold compared with PEI/DNA. The ability of hexon to promote nuclear delivery of PEI/DNA nanoparticles was compared with that of classi cal nuclear localization sequences (NLS) by measuring transgene expression following intracytoplasmic microinjection of hexon-PEI/DNA complexes and NL S-albumin-PEI/DNA complexes in rat-1 fibroblasts. The resulting nuclear tra nsfer efficiency was in the following order: hexon-PEI/DNA > NLS-albumin-PE I/DNA > PEI/DNA > DNA alone > albumin-PEI/DNA. The activities of both NLS-a lbumin-PEI and hexon-PEI were abolished by co-injection of wheat germ agglu tinin, suggesting that both act by means of the nuclear pore complex (NPC); in contrast, excess free NLS-albumin abolished transgene expression with N LS-albumin-PEI/DNA, but only partially inhibited hexon-PEI/DNA. Nuclear tra nsfer efficiency following cytoplasmic injection was dependent on DNA conce ntration for ail materials, although hexon conjugates showed much better ac tivity than NLS-albumin at low DNA doses (500-1000 plasmids/cell). Our data are consistent with hexon mediating nuclear delivery of plasmid complexes by means of the NPC, using mechanisms that are only partially dependent on the classical NLS import pathway. The hexon-mediated mechanism of nuclear i mport enables substantially better transgene expression, particularly when DNA concentrations in the cytoplasm are limiting.