The therapeutic application of biotechnological products, including proteins and DNA, is limited by the lack of an efficient delivery system to the target cells. Recent findings on peptide transduction domains (PTDs) have raised the hope that these bioactive macromolecules can be transported directly into the cytoplasmic compartment of mammalian cells. Our long-term goal is to develop an efficient delivery system that, by virtue of the membrane transduction activity of PTDs, can be used for protein and gene therapy. Currently, the development of this novel delivery system is hindered by the lack of quantitative measurements of peptide transduction. In this application, a recently developed method that is capable of determining peptide transduction into cytoplasma without the interference from concurrent endocytosis will be used to study this novel membrane transport process. The separation of the cytoplasmic fraction from the endocytotic vesicles in cell homogenates will be achieved by using gel filtration chromatography. FITC-dextran will be included in the cell uptake assay to serve as an internal standard for measuring vesicle-rupture during the homogenization, and cells will be washed with heparin-buffer to eliminate membrane-bound PTD-peptides before the homogenization.
The specific aims of this project are (1) to characterize cationic oligopepetides in membrane transduction, (2) to investigate the application of PTD-carriers for cytoplasmic delivery of bioactive macromolecules including anti-proliferative polypeptides and antisense oligonucleotides, (3) to demonstrate the intracellular processing of PTD-peptides, and (4) to elucidate the mechanism of PTD-mediated membrane transduction. Studies will be focused on (a) the identification of PTD-peptides with high transduction efficiency as drug carriers regarding the size, charge, and structure, (b) the criteria for selecting polypeptides and polynucleotides to be transported via PTD-mediated transduction, (c) the chemical linkages between PTD-peptide and proteins for maintaining biological activity in cytoplasma, (d) the determination of the effect of intracellular processing on the bioactivity of the macromolecules and (e) the identification of specific binding sites on cell membrane, such as heparan sulfate proteoglycans, that mediate the membrane transduction activity. This proposal will provide timely and essential information for developing an effective delivery system that can be used to transport genetically engineered proteins or genes into cytoplasma of target cells for the treatment of various human diseases.
