Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene d elivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin o f replication and early promoter. In this report, we show that, although fr agments throughout this region can support varying degrees of nuclear impor t, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the h uman cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma vi rus LTR, were unable to direct nuclear localization of plasmids. The inabil ity of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent prot ein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obt ained with more than 1000 copies per cell of plasmids lacking the SV40 sequ ence. However, the levels of expression were the same for both plasmids aft er cell division in cytoplasmically injected cells and at all times in nucl ear injected cells. Thus, the inclusion this SV40 sequence in nonviral vect ors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells. (C) 1999 Academic Press.