PROJECT ABSTRACT Osteoarthritis (OA) affects the entire joint. There is growing evidence that cartilage catabolism and synovial inflammation are interdependent and act as primary drivers of OA. In this competitive renewal, we build upon our findings in the first funding period, and explore the central hypothesis that cartilage and synovium are key therapeutic targets for the protective effect of 4-aminobutyrate aminotransferase (ABAT) loss-of-function (LOF) against OA. Important discoveries made during the first funding period defined completely novel targets in the pathogenesis of OA and significantly advanced the field. We discovered that DNA methyltransferase 3b (Dnmt3b) expression is reduced in murine and human OA chondrocytes, and drives the disease process. In rigorous genetic mouse models, we established that Dnmt3b LOF (loss-of function) is catabolic in chondrocytes and accelerates OA, whereas Dnmt3b GOF (gain of function) is anabolic and protects against OA. Integrated analysis of DNA methyl-seq and RNA-Seq identified Abat as a key downstream target of Dnmt3b. Dnmt3b was found to methylate the Abat promoter and suppress its expression, while suppression of Dnmt3b reduced promoter methylation and increased Abat expression, thereby establishing a reciprocal Dnmtb3b-Abat axis in articular chondrocytes. Using lentiviral approaches, we showed that Abat over-expression is catabolic and accelerates OA, while Abat suppression is anabolic and protects against OA. Vigabatrin, a small-molecule inhibitor of Abat, also blocked the development of OA and synovial inflammation in mice with knee injury. To i) definitively establish the Dnmt3b-Abat axis in vivo in cartilage; and ii) determine if Abat LOF protects against OA by acting on cartilage and/or synovium, we created innovative conditional Abat GOF and LOF mice. Specific Aim 1 will use conditional Abat GOF/LOF in combination with Dnmt3b GOF/LOF mouse models to determine whether Abat is the key downstream mediator of Dnmt3b in regulating cartilage homeostasis. Specific Aim 2 will use tissue specific conditional gene deletions in cartilage (Aim 2A), in synovial fibroblasts (Aim 2B), and in synovial myeloid cells (Aim 2C) to determine if the protective effect of Abat LOF against OA is dependent on both cartilage and synovium. Specific Aim 3 will determine whether siAbat gene therapy using a peptide nanoparticle delivery platform confers protection against OA in mice. These findings will definitively show that Abat is the key signal downstream of Dnmt3b, define the relative role of Abat on cartilage and synovium in protection against OA and pain, and establish delivery of siAbat nanoparticles as a novel translational approach for the treatment of OA and joint pain.