At present, there are few methods to selectively transfer genes to non-dividing vascular smooth muscle cells. This is a major problem in the development of gene therapy approaches to treat vascular proliferative diseases such as atherosclerosis and restenosis, which affect up to 5% of the US population. We have developed a way to overcome this problem. While many aspects of vector design have focused on targeting DNA to and into cells, the fate of the DNA once inside the cytoplasm has not been addressed. Without the translocation of exogenous DNA into the nucleus, no gene expression or """"""""gene therapy"""""""" can take place. We have identified a DNA sequence from the smooth muscle gamma actin promoter that increases nuclear localization and subsequent gene expression uniquely in smooth muscle cells, a critical target in vascular gene therapy. We have shown that the transcription factors SRF and Nkx3.1/3.2 bind to the SMGA promoter and mediate transcriptional control of this promoter. Incorporation into an expression plasmid of a 176 bp fragment of the SMGA promoter containing binding sites for SRF and Nkx3.1/3.2 causes nuclear import and subsequent high level gene expression solely in cultured smooth muscle cells, but not other cell types. Mutation of binding sites for SRF and/or Nkx3 in this sequence abolishes nuclear import activity. Moreover, the SMGA promoter also acts in the intact vasculature of rats to target genes to the smooth muscle layer, but not other vascular cells. We hypothesize that the cell-selective nuclear import of the SMGA promoter is mediated by the transcription factors SRF and Nkx3.1/3.2 that are co-expressed in smooth muscle cells but not other cells of the vasculature. With our development of a new method for highly efficient gene delivery to the vasculature of living animals, we are in a unique position to test the effects of this cell-selective nuclear import sequence on smooth muscle transfection in animal models for intimal hyperplasia. This proposal is designed to test these hypotheses and will lead to the creation of new vascular gene therapy vectors that are both cell-specific and capable of greater gene transfer efficiencies. Finally, we will use the information gained to test the efficacy of these vectors in several complementary models of neoinitmal formation using the cdk inhibitors p21 and p27.
The specific aims of this proposal are: (1) characterize the mechanisms of SRF- and Nkx3.1/3.2-mediated smooth muscle cell specific DNA nuclear import. (2) Determine the efficacy of the cell selective SMGA plasmid nuclear targeting sequences for driving cell-specific gene transfer in vivo. (3) Test whether cytostatic gene therapy with smooth muscle-specific nuclear targeting constructs inhibits neointimal hyperplasia in vivo.
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