Project Summary Atopic dermatitis (AD) is a common, chronic inflammatory skin condition characterized by dry, itchy lesions. Current treatments, including immunomodulators and anti-inflammatory steroids, can sometimes control symptoms but may also have serious side effects and do not offer a long-term cure. These treatments may be insufficient because they do not address the initial role of skin barrier failure in the development of AD, which can lead to a cycle of inflammation, sensitization, and infection. It is believed that the damage to the skin barrier in AD is exacerbated or caused by excessive serine protease activity. Proper proteolytic activity is essential for maintaining the skin barrier and is regulated by proteolysis of the corneodesmosomes, which is predominantly carried out by kallikrein-related peptidases (KLKs). KLK5, 7, and 14 are the core proteolytic enzymes in the epidermis and are inhibited by LEKTI (lympho-epithelial Kazal-type-related inhibitor) fragments. Excessive serine protease activity, particularly from KLK5 and KLK7, is thought to be a primary cause of AD. However, correcting this excess proteolysis in the skin is difficult because many homeostatic pathways in this organ are controlled by proteases, requiring specific modulation with minimal antigenicity. Enzyme replacement using LEKTI has not yet been successful because applied topical peptides are quickly degraded by the many exo- and endo-proteases present in the skin. Therefore, most KLK inhibitor efforts involve modifying natural peptide inhibitors to maintain their activity while increasing their lifetime. These small molecule drug mimics may have significant toxicity concerns due to systemic absorption. We propose that the use of genetically engineered topical bacteria for on-site enzyme replacement could effectively address these challenges. We aim to genetically engineer a harmless bacterium to temporarily populate the skin microbiome and deliver kallikrein inhibitors to treat AD. Our platform for continuous production of peptide drugs on the skin surface has the potential to revolutionize the treatment of atopic dermatitis. In this Phase I SBIR, we will establish the safety of our topical application and demonstrate bioactive secretion of our target inhibitors. This will provide a strong foundation to transition to Phase 2 where we will establish safety and efficacy in murine genetic models.