Mechanism of Nuclear Import of a Smooth Muscle Cell-Specific Plasmid Vector for Gene TherapyPublic Deposited
Cardiovascular disease is a leading cause of morbidity and mortality worldwide. While many therapies have been used to treat vascular disease symptomatically, gene therapy offers a means of treating the molecular dysfunctions driving disease pathogenesis. Unfortunately, gene therapy has achieved only modest therapeutic success in clinical trials due to a lack of tissue-specific targeting of vectors and low levels of gene transfer. We have begun to address these limitations by designing plasmid vectors that achieve efficient gene expression by targeting constructs to the nuclear compartment of specific cell types. Nuclear import of plasmid DNA (pDNA) is essential for successful gene therapy, since the nucleus is the site of transcription. Recently, the smooth muscle gamma actin (SMGA) promoter was demonstrated to mediate nuclear import of pDNA specifically in smooth muscle cells (SMCs) both in vitro and in vivo. Constructs lacking this import sequence, referred to as a DNA nuclear targeting sequence (DTS), remain confined to the cytoplasm until cell division or degradation. Since the SMGA promoter contains multiple binding sites for SMC-specific transcription factors, including serum response factor (SRF) and NK3, I hypothesized that these factors bind to the DTS in the cytoplasm of SMCs and coat the plasmid with nuclear localization signals (NLSs) that interact with karyopherins (importins) to facilitate plasmid nuclear import. In this thesis, I demonstrate that SRF and NK3 transcription factors are necessary and sufficient to drive plasmid import in SMCs and that the SRF NLS is required. In addition, I use plasmid affinity chromatography, mass spectrometry, and live-cell pulldowns of plasmid-protein complexes to identify import proteins (including Ran, importin β1 and importin 7) that exhibit DTS-specific interactions with DTS plasmid constructs both in vitro and in vivo. Finally, I show that importin β1 is required for plasmid nuclear import of DTS constructs. A better understanding of this route of gene delivery will facilitate the development of SMC-specific gene therapy vectors for therapeutic use.
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