PROJECT SUMMARY The process of receptor-mediated transcytosis is a critical, yet highly selective, mechanism for the transport of proteins from the intestinal lumen into systemic circulation. Despite the fundamental importance of this transport mechanism and the potential to exploit it to revolutionize oral delivery of protein therapeutics, our understanding of intestinal transcytosis has been limited by both the lack of a more accurate, scalable in vitro model of the human intestinal epithelium and the difficulty in identifying the full repertoire of receptors that can actively and selectively transport cargo from the luminal side to the basolateral side of this physical barrier. In this R01 proposal, we will synergistically integrate stem cell engineering, directed protein evolution, and synthetic biology approaches to first uncover new mechanisms of receptor-mediated transcytosis and to then apply these findings to create a novel platform for oral delivery of insulin. More generally, our approaches and results should have broad utility in understanding the formation of epithelial barriers and in efficiently delivering drugs across them in a highly selective manner.