Cell-specific nuclear import of plasmid DNA

One factor limiting the success of non-viral gene therapy vectors is the re lative inability to target genes specifically to a desired cell type. To ad dress this limitation, we have begun to develop cell-specific vectors whose specificity is at the level of the nuclear import of the plasmid DNA. We h ave recently shown that nuclear import of plasmid DNA is a sequence-specifi c event, requiring the SV40 enhancer, a region known to bind to a number of general transcription factors (Dean DA, Exp Cell Res 1997; 230: 293). From these studies we developed a model whereby transcription factor(s) bind to the DNA in the cytoplasm to create a protein-DNA complex that can enter th e nucleus using the protein import machinery. Our model predicts that by us ing DNA elements containing binding sites for transcription factors express ed in unique cell types, we should be able to create plasmids that target t o the nucleus in a cell-specific manner. Using the promoter from the smooth muscle gamma actin (SMGA) gene whose expression is limited to smooth muscl e cells, we have created a series of reporter plasmids that are expressed s electively in smooth muscle cells. Moreover, when injected into the cytopla sm, plasmids containing portions of the SMGA promoter localize to the nucle us of smooth muscle cells, but remain cytoplasmic in fibroblasts and CVI ce lls. In contrast, a similar plasmid carrying the SV40 enhancer is transport ed into the nuclei of all cell types tested. Nuclear import of the SMGA pro moter-containing plasmids could be achieved when the smooth muscle specific transcription factor SRF was expressed in stably transfected CV1 cells, su pporting our model for the nuclear import of plasmids. Finally, these nucle ar targeting sequences were also able to promote increased gene expression in liposome- and polycation-transfected non-dividing cells in a cell-specif ic manner, similar to their nuclear import activity. These results provide proof of principle for the development of cell-specific non-viral vectors f or any desired cell type.