PROJECT SUMMARY Biomolecules that represent important therapeutic leads often fail because they cannot reach their targets, commonly because of inability to cross cell membranes and reach appropriate subcellular destinations. Cell- penetrating peptides (CPPs) have long held great promise for overcoming these failures. They 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, engineer next-generation improvements in adaptors and cargos and develop methods to use them to efficiently deliver cargos for research and therapeutic purposes. 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 innovative therapeutics and research tools.