Cell-specific nuclear import of plasmid DNA

Abstract

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.

Sequence Requirements for Plasmid Nuclear Import

“…Comparison of the transcription factor consensus binding sequences in these two promoters (20), and the sites identified experimentally (21), reveals that the binding sites for several transcription factors, including AP2 and AP3, are not present. To test the hypothesis that AP2 alone mediates the nuclear import of SV40 DNA, we tested the smooth muscle gamma actin promoter, which contains an AP2 site approximately 200 bp upstream of the transcriptional start site (22). Plasmids containing fragments of this promoter, ranging from 400 bp to 2.2 kbp, were unable to mediate nuclear import of the plasmids in TC7 and CV1 cells as analyzed by in situ hybridization (22).…”

“…To test the hypothesis that AP2 alone mediates the nuclear import of SV40 DNA, we tested the smooth muscle gamma actin promoter, which contains an AP2 site approximately 200 bp upstream of the transcriptional start site (22). Plasmids containing fragments of this promoter, ranging from 400 bp to 2.2 kbp, were unable to mediate nuclear import of the plasmids in TC7 and CV1 cells as analyzed by in situ hybridization (22). Most likely, AP2 is only one of a larger complex of proteins that forms on the SV40 promoter/enhancer region.…”

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

“…In addition, inclusion of the SMGA DTS in a luciferase construct increased the expression by 40-fold in arrested cells. 10 On the basis of the presence of binding sites for several SMC-specific transcription factors in the SMGA DTS, we hypothesize that SMC-specific factors bind the SMGA DTS to mediate cell-specific nuclear import. Indeed, overexpression of SRF in non-SMCs increased import, supporting this model.…”

“…[3][4][5][6][7][8] Our laboratory has begun to design plasmid vectors that achieve efficient gene expression by targeting constructs to the nuclear compartment of specific cell types. [9][10][11][12][13] Initially, we demonstrated that inclusion of as little as 72 bp of the simian virus 40 (SV40) promoter/ enhancer in plasmid vectors facilitates nuclear import in all cell types tested. 12 Due to the ability of this sequence to target DNA to the nucleus, we refer to it as a DNA nuclear targeting sequence (DTS).…”

“…[19][20][21] We have also developed a tissue-specific DTS, utilizing the smooth muscle g-actin (SMGA) promoter, that mediates nuclear import of pDNA specifically in smooth muscle cells (SMCs). 10 The SMGA DTS was shown to drive SMCspecific plasmid nuclear import in rat mesenteric vessels following electroporation into living animals, proving its viability for in vivo gene transfer. In addition, inclusion of the SMGA DTS in a luciferase construct increased the expression by 40-fold in arrested cells.…”