PROJECT SUMMARY Approximately 50 million people suffer from one or more autoimmune diseases in the US alone. Over the last decades, the discovery of biologics has considerably improved the care for patients with autoimmune diseases. However, current treatments require weekly or more frequent injections and this can lead to missed doses and to poor patient compliance, especially in the younger population, resulting is inconsistent or sub- therapeutic drug levels. Since many of the therapeutics on the market or being developed to treat rheumatoid arthritis (RA), multiple sclerosis, and other T lymphocyte-mediated chronic autoimmune diseases are biologics, we propose a novel bioengineering approach for the non-invasive delivery of recombinant peptides in autoimmune diseases. More specifically, we propose to produce an immunomodulatory peptide by a probiotic for oral administration to treat RA. As our immunomodulator, we have chosen a recombinant analog of ShK, a small venom peptide that blocks Kv1.3 channels and thus inhibits the activation of human and rat effector memory T lymphocytes. As a probiotic, we have selected Lactobacillus reuteri LJ01, a well-characterized probiotic that survives transit through the gastrointestinal tract. In preliminary work, we have introduced an ShK analog in L. reuteri to generate LrS235 that secretes a functionally active peptide upon induction, resulting in the block of Kv1.3 channels and the inhibition of human TEM cell proliferation in vitro and in a reduction of clinical severity in a small pilot trial using the collagen-induced arthritis rat model of RA. Through this project, we will define the pharmacokinetics, biodistribution, and efficacy of ShK-235 produced by LrS235 in preventing and treating rat models of RA (Specific Aim 1) and we will define and optimize the efficacy and safety of our probiotic based gut delivery (Specific Aim 2). At the end of this project, we will have established proof-of-concept for a new therapeutic strategy for the treatment of RA and other autoimmune diseases mediates by effector memory T lymphocytes through the oral delivery of a probiotic engineered to secrete an immunomodulatory Kv1.3 blocking venom peptide.