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Nuclear targeting of plasmids and protein-DNA complexes

My laboratory studies the mechanisms and applications of plasmid and DNA-binding protein nuclear localization. Our long term goals are to develop gene therapy approaches to the treatment of a variety of human diseases by focusing on the development of novel non-viral intra- and extracellular delivery methods. Our main emphasis is in the area of vascular gene delivery and function. Perhaps the major problem hindering gene therapy is the inefficiency of gene transfer to slowly and non-dividing cells. While many aspects of non-viral vector design are being addressed, one critical area that has not received adequate attention is the nuclear import of vector DNA. Clearly, without the translocation of plasmid DNA into the nucleus, no gene expression, or "gene therapy" can take place. My laboratory continues to identify and characterize novel DNA sequences to promote nuclear import of non-viral vectors, both in cultured cells and in vivo.

Recent work from our laboratory has begun to address the nuclear targeting and entry of plasmid DNA. Using cultured cells, we have shown that plasmids are able to enter the nuclei of cells in the absence of cell division and its accompanying nuclear envelope breakdown. Assays used to follow the movement of DNA include in situ hybridization, reporter gene expression, and GFP-, YFP-, and BFP- tagged proteins. As for all other macromolecular exchange between the cytoplasm and nucleus, DNA nuclear entry is mediated by the nuclear pore complex.

Furthermore, we have demonstrated that portions of the 72 bp SV40 enhancer are absolutely necessary for the nuclear entry of plasmid DNA in all eukaryotic cells tested to date; plasmids not containing this sequence remain in the cytoplasm until cell division, whereas plasmids containing the enhancer migrate to the nucleus within several hours. These results demonstrate that transport of DNA into the nucleus is sequence-specific. This 72 bp DNA fragment contains multiple binding sites for various general transcription factors: (click image for larger view)  

Since transcription factors bind to specific DNA sequences and contain nuclear localization signals (NLSs) for their nuclear import, it is likely that these proteins coat the DNA with NLSs, thereby allowing the DNA-protein complex to utilize the NLS-mediated import machinery for nuclear entry:


Interestingly, the SV40 sequence appears to be somewhat unique among viral enhancers: neither the strong promoter/enhancers of CMV or RSV have similar DNA nuclear import activity.

Click left image for larger view

While much of our work is carried out in microinjected and transfected cells, we have also developed novel DNA labeling techniques using triplex-forming peptide nucleic acid (PNA) clamps and have adapted cell-free systems to study the nuclear import of protein and protein-DNA complexes in real time. We have shown using

permeabilized cells that whereas the importin and proteins and RAN are sufficient to drive the nuclear import of an NLS protein, nuclear extracts are also required for sequence-specific plasmid import. Based on these results, we have developed a model for general DNA nuclear import:

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We are focusing on identifying the proteins present in the nuclear extract needed for this activity by subtractive (purification) and additive (using recombinant transcription factors known to bind the SV40 enhancer) approaches. One additional interesting

feature of plasmid nuclear import that we have discovered is that import is inhibited when transcription is arrested. We have demonstrated that this inhibition is not due to lack of transcription of the import substrate (i.e., the plasmid) but manifests its effect at the level of the cell. This is strikingly similar to the transcription mediated nuclear import inhibition seen with certain nuclear shuttling proteins including the mRNA binding protein hnRNP A1. Thus, we are working to understand how transcription regulates nuclear import of DNA and the shuttling of nuclear proteins, especially transcription factors.
  Our model predicts that by using DNA elements containing binding sites for transcription factors expressed in unique cell types, we should be able to create plasmids that target to the nucleus in a cell-specific manner. Using the promoter from the smooth muscle gamma actin (SMGA) gene whose expression is limited to smooth muscle cells, we have created a series of reporter plasmids that are expressed selectively in smooth muscle cells: (click left image for larger view)
Moreover, when injected into the cytoplasm, plasmids containing portions of the SMGA promoter localize to the nucleus of smooth muscle cells, but remain cytoplasmic in fibroblasts and endothelial cells. In contrast, a similar plasmid carrying the SV40 enhancer is transported into the nuclei of all cell types tested. Limited nuclear import of the SMGA promoter-containing plasmids could be achieved when the smooth muscle specific transcription factor SRF was expressed in stably transfected non-smooth muscle cells, supporting our model for the nuclear import of plasmids. Moreover, when binding sites for SRF or another smooth muscle transcription factor, Nkx3, are mutated, import is abolished, further implicating these proteins in nuclear import of the plasmids. In collaboration with Warren Zimmer (Univ. South Alabama) we are continuing to identify the factors that bind to the SMGA promoter to regulate smooth muscle specific transcription and nuclear import using gel shift assays, yeast two-hybrid library screens, transient transfection assays, DNaseI footprinting, and nuclear import assays. Finally, we have demonstrated that these smooth muscle specific nuclear targeting sequences are also able to promote increased gene expression in liposome- and polycation-transfected non-dividing cells in a cell-specific manner, similar to their nuclear import activity. These results provide proof of principle for the development of cell-specific non-viral vectors for any desired cell type. Current studies involve expanding our repertoire of cell-specific DNA nuclear targeting sequences so that we may be able to target genes selectively to any desired cell or tissue type.
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