PROJECT SUMMARY Each year, 15,000 infants die in the U.S. from diseases for which there are no effective therapies, and many more suffer through invasive procedures to address the diseases that are treatable. Here, we propose a non-invasive therapy based on the genetic engineering of breast milk cells to enable unprecedented production and delivery of therapeutics in infants. The approach is similar to the recently FDA-approved CAR-T therapy, except simpler, less invasive, and with broader potential use. Ultimately, we envision that cells will be isolated from freshly expressed milk, sorted by type, and transfected with RNA or DNA. Cells will then be reincorporated into milk for consumption. Each breast milk cell type has the potential for unique therapeutic functions: epithelial cells can operate as protein replacement “factories”, immune cells can produce peptide antigens for vaccination or immunotherapy, and stem cells can be programmed through the expression of transcription factors for cell therapy. This proposal will take the first, most important steps in achieving our long-term therapeutic vision. First, we will establish a basic understanding of breast milk cell subsets, their biodistribution in mouse neonates, and their transport mechanisms. We will determine if breast milk cell uptake is facilitated by cell attributes, other milk components, and/or the nature of the neonatal gut. We will also establish breast milk cell persistence times in the neonate as a function of location. Several transfection methods, including viral, non-viral, and microfluidic systems will be tested for their ability to maximize protein output without compromising cell transport. Finally, we will pursue therapeutic applications informed by our fundamental studies, including protein replacement, vaccination, immunotherapy, and stem cell therapy. Initial successes will set the stage for follow-up studies in animal models of neonatal disease and the eventual non-invasive treatment of infants for whom there are no currently available therapies.