VASCULAR SMOOTH MUSCLE SPECIFIC GENE THERAPY VECTORS

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DESCRIPTION (Adapted from Investigator's Abstract): It is presently quite difficult to transfer genes to non-dividing vascular smooth muscle cells (SMC) and this has limited the development of gene therapy approaches for the treatment of human vascular diseases such as atherosclerosis and restonosis. The applicant has developed a novel strategy that may overcome this problem and has focused upon mechanisms of DNA fate in cytosol. They have demonstrated that plasmid DNA nuclear import in non-dividing eukaryotic cells occurs through the nuclear pore complex and this DNA nuclear traffick is sequence specific. Moreover, they have identified a DNA sequence that mediates nuclear import of plasmid DNA in only smooth muscle cells. The DNA is the proximal portion of the smooth muscle H-actin (SMGA) promoter which contains binding sites for several SMC-specific transcription of this promoter and it is hypothesized that they also mediate the nuclear import of DNA. The hypothesis predicts that transfactors containing nuclear localization signals (NLS) for their nuclear import bind to specific SMGA DNA sequences thereby coating the DNA with NLSs and allowing the DNA to utilize the NLS-mediated import machinery for nuclear entry. If the transfactors are expressed uniquely in SMCs, import should occur only in those cells. The experiments in this application will utilize four specific aims to examine this hypothesis. The specific aims are: 1) To identify DNA sequences in the SMGA promoter needed for smooth muscle cell-specific DNA import. This will be done by studying plasmid containing portions of this promoter, using microinjection and in situ hybridization to identify sequences that mediate import in SMCs only. To define the transcription factors which bind to imported SMGA sequences and potentially mediate cell selective import. This will employ electrophoretic mobility shift assays to identify the respective binding proteins interacting with the sequences identified in Aim 1. Their role will be tested by transfecting their genes into non- muscle cells to reconstitute cell-specific nuclear import. 3) To identify the cellular factors involved in DNA nuclear import. Here they will test the hypothesis that transcription factors identified in Aim 2 and which are unique to smooth muscle cells can mediate SMGA DNA nuclear import using a permeabilized cell assay. The smooth muscle specificity will be confirmed by also studying non-muscle cells as well. 4) To test the efficacy and cell-specificity of SMGA promoter constructs in an in vitro vascular smooth muscle cell proliferation model. Here they will test the ability of the imported sequences to increase cell-specific gene expression by transfection, and apply this to an in vitro model and prevent SMC proliferation.