SUMMARYThe nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of non-viral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366 bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, 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 human cytomegalovirus (CMV) immediate early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (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 obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in non-viral vectors may greatly increase their ability to be transported into the nucleus, especially in non-dividing cells.

One factor limiting the success of non-viral gene therapy smooth muscle cells, we have created a series of reporter vectors is the relative inability to target genes specifically plasmids that are expressed selectively in smooth muscle to a desired cell type. To address this limitation, we have cells. Moreover, when injected into the cytoplasm, plasbegun to develop cell-specific vectors whose specificity is mids containing portions of the SMGA promoter localize to at the level of the nuclear import of the plasmid DNA. We the nucleus of smooth muscle cells, but remain cytoplashave recently shown that nuclear import of plasmid DNA mic in fibroblasts and CV1 cells. In contrast, a similar plasis a sequence-specific event, requiring the SV40 enhancer, mid carrying the SV40 enhancer is transported into the a region known to bind to a number of general transcription nuclei of all cell types tested. Nuclear import of the SMGA factors (Dean DA, Exp Cell Res 1997; 230: 293). From promoter-containing plasmids could be achieved when the these studies we developed a model whereby transcription smooth muscle specific transcription factor SRF was factor(s) bind to the DNA in the cytoplasm to create a proexpressed in stably transfected CV1 cells, supporting our tein-DNA complex that can enter the nucleus using the model for the nuclear import of plasmids. Finally, these protein import machinery. Our model predicts that by using nuclear targeting sequences were also able to promote DNA elements containing binding sites for transcription facincreased gene expression in liposome-and polycationtors expressed in unique cell types, we should be able to transfected non-dividing cells in a cell-specific manner, create plasmids that target to the nucleus in a cell-specific similar to their nuclear import activity. These results promanner. Using the promoter from the smooth muscle vide proof of principle for the development of cell-specific gamma actin (SMGA) gene whose expression is limited to non-viral vectors for any desired cell type.

Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluoresceinconjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time-and energydependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin ␣, importin ␤, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import.The nuclear envelope presents an effective barrier between the nuclear and cytoplasmic compartments of the cell. Although it is impermeant to large non-nuclear molecules, a multitude of macromolecules must enter and exit the nucleus across this envelope every second in order for the cell to live. All macromolecular exchange between the nucleus and the cytoplasm studied to date occurs through the nuclear pore complex (NPC), 1 is signal-dependent, and utilizes a series of receptor proteins (for a review see Ref. 1). In the case of proteins destined for the nucleus, the nuclear localization signal (NLS) interacts with one of a growing number of importin family members to target the complex to the NPC. In the classical case of NLS-containing proteins, the protein binds to importin ␣, the NLS "receptor," which in turn interacts with importin ␤. Once at the NPC, the complex interacts with the small GTP-binding protein RAN in its GDP-bound state and its accessory factor NTF2 while being translocated across the NPC. After translocation into the nucleus, the complex disassembles because of the conversion of RAN-GDP to RAN-GTP by exchange or replacement and the importins return to the cytoplasm (2). Similar scenarios of receptor proteins interacting with signals to mediate translocation across the nuclear envelope also occur for the nuclear export of proteins (e.g. exportin and the nuclear export signal) and viral mRNAs (Crm1p, HIV Rev, and the Rev response element), and the nuclear import of small nuclear RNAs that contain both protein-encoded NLSs and the trimethylguanosine cap as import signals (1, 3).We have recently shown that the nuclear import of plasmid DNA (pDNA) also u...

These results demonstrate that primary, non-transformed human corneal epithelial cells and keratocytes display sequence-specific nuclear import of plasmid DNA in the absence of mitosis. The small sequence that mediates nuclear localization of plasmids is active both in microinjected and cationic liposome transfected cells, and leads to increased gene expression. Thus, inclusion of this DNA sequence into non-viral vectors should improve the efficiency of ocular gene transfer in vivo.