Project Summary In osteoarthritis (OA), intra-articular inflammation is a key mediator of joint destruction and chronic joint pain. Unfortunately, current strategies to control joint inflammation have largely failed. To address this challenge, our team is developing an innovative metabolic reprogramming strategy for the treatment of knee OA. In our strategy, indoleamine 2,3-dioxygenase (IDO), an immunosupressive enzyme, will be intra-articularly delivered to catabolize tryptophan into kynurenines. Based on IDO’s effect in other tissues, this redirection of tryptophan metabolism will likely drive the polarization of joint-level immune cells toward an anti-inflammatory state. Importantly, our strategy differs from other intra-articular delivery strategies for protein and synthetic drugs, as our enzyme will continuously produce anti-inflammatory metabolites in the OA-affected joint and thereby create prolonged anti-inflammatory effects that potentially reset immune homeostasis in the joint. However, while IDO can continuously produce anti-inflammatory metabolites, free IDO is subject to joint clearance. To address this challenge, we will also fuse IDO to a carbohydrate-binding protein, thereby extending IDO’s joint residence time via a novel tissue anchoring approach. Morever, because tissue-anchored IDO does not need to release to generate anti-inflammatory signals, the anchored IDO will continue to produce anti-inflammatory kyneurenines without the need for our ‘drug’ (IDO) to release and bind a specific target. Our preliminary data demonstrate that tryptophan metabolism is altered in both human OA and rodent models, our tissue anchoring strategy can extend the residence time of an enzyme from a few days to over 4 weeks, and that intra-articular delivery of an IDO fusion protein can shift tryptophan metabolism, reduce inflammation, and reverse pain-related behaviors in a rat knee OA model. As such, this R01 proposal seeks to evaluate intra-articular delivery of an I