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.