Biomolecules that represent important therapeutic leads are often impractical because they cannot reach their targets, most commonly because of failure to cross cell membranes and reach appropriate subcellular destinations. Cell-penetrating peptides (CPPs) hold great promise for overcoming these failures. CPPs are capable of mediating penetration of the plasma membrane by molecules to which they are coupled, allowing delivery of ?cargos? to cell interiors, a potentially transformative platform technology that can enable an array of specific applications. Nevertheless, development of CPP therapeutics has been disappointing because traditional CPP-cargo molecules largely remain trapped in endosomes rather than reach the cytoplasm. The largest technical hurdle to development of CPP therapeutics is failure to escape from endosomes ? our technology solves this problem. Our innovative approach is the use of high affinity but reversible noncovalent coupling to attach cargos to CPPs. Our prototype CPP-adaptor fusion protein, TAT-Calmodulin (TAT-CaM), consists of the cell penetrating moiety from HIV transactivator of transcription and human calmodulin. TAT-CaM binds CaM binding-site (CBS) containing cargos with nM affinity in the presence of calcium but negligibly in its absence. Because mammalian cells typically maintain low resting concentrations of calcium, cargos dissociate from the CPP-adaptor once inside the cell, releasing cargo to the cytoplasm or other subcellular destination. This R15 AREA renewal application describes efforts to elucidate the mechanisms, kinetics and other basic issues of CPP biology and develop methods to use our CPP-adaptors to deliver nucleic acids such as siRNA for research and therapeutic purposes. We will also utilize them to deliver cargo molecules that will demonstrate the broad utility of our system and its advantages over transfection, namely efficiency, speed and tunability. Success in these endeavors will validate that our strategy is an adaptable tool for delivery of a wide array of macromolecules including nucleic acids, potentially enabling the development of a new generation of therapeutics and research tools.
Cell-penetrating peptides offer the tantalizing prospect of exogenously delivering user-defined macromolecular cargos through the membranes of eukaryotic cells. Our innovative core technology is based on cell-penetrating peptide-coupled adaptor proteins that allow the spontaneous loading of almost any desired biomolecular cargo for rapid delivery into cells. We will gain understanding of the mode of entry and subcellular trafficking of CPPs and cargos and develop innovative assays for an array of research questions.
