verbatim): The circulatory system, via the microcirculation, accesses every cell in the body. Exploitation of this to deliver therapeutic genes and other molecules to target organs is limited by endothelial barrier function and selective permeability. The mechanisms regulating selective endothelial permeability are poorly defined, and a better understanding may lead to enhanced methods for delivering genes and macromolecules to tissues and organs. We hypothesize that specific peptide sequence motifs are associated with targeting macromolecular translocation across continuous endothelia, such as that lining the coronary microvasculature. We further hypothesize that specific peptide motifs are also involved in intracellular targeting and trafficking of macromolecules. We contend that definition of these pathways could lead to viable strategies to efficiently deliver therapeutic genes and molecules to the heart. We will use the power of peptide phage display in a model of endothelial barrier function to identify peptide motifs associated with translocation of macromolecules across the endothelium. We will also use phage display to investigate the role such peptide motifs play in endocytosis of macromolecules and their subsequent intracellular trafficking. We will use a complimentary combination of biochemical, immunohistochemical, molecular and ultrastructural approaches, to investigate these events. Finally, we will engineer targeted AAV and adenovirus vectors to investigate the ability of these defined peptide motifs to direct macromolecule translocation and intracellular trafficking in the context of these gene therapy vehicles. We will fulfill the following specific aims: 1) To identify peptide sequences capable of overcoming endothelial barrier function and facilitating macromolecular translocation across continuous endothelia; and to investigate the mechanisms by which this translocation occurs; 2) To identify peptide sequences capable of binding and internalization into cardiac. myocytes, investigate the internalization pathways targeted by these peptide motifs, and determine the role of caveolin 3 in cardiomyocyte endocytosis. 3) To investigate the ability of specific peptide targeting motifs to direct intracellular trafficking and facilitate macromolecular translocation across the continuous endothelia in the context of targeted AAV and adenovirus vectors.
