Osteoarthritis (OA) is a chronic, debilitating musculoskeletal disease characterized by progressive loss of joint function, leading to pain and functional limitation. It is the leading cause of chronic disability in the US, affecting 40% of adults over the age of 70. This disease lacks effective diagnostics, prognostic biomarkers and treatments, due mainly to a poor understanding of its etiology. Insights into the molecular mechanisms of OA, and the contributions of extra-articular tissues, are key to the development of desperately needed clinical biomarkers and novel therapeutics. Because aging is the greatest risk factor for developing OA, understanding the mechanism of how aging leads to OA is a potential key to unraveling pathogenesis. Our preliminary data in human samples has shown evidence for epigenetic dysregulation in articular cartilage, but how individual risk factors influence the epigenome during OA development remains unknown. In this project, we hypothesize that age-related dysregulation of DNA methylation at specific sites leads to changes in the expression of genes that are involved in osteoarthritis development and progression. To test this, we will, for the first time, investigate epigenetic patterns in murine models of OA. We will first identify age- related changes in DNA methylation and gene expression in articular cartilage that occur with the development of OA under the influence of three major OA risk factors: age, trauma (using the DMM model) and obesity (using the high fat diet model). Second, we will then identify the changes in DNA methylation/gene expression that are prevented/reduced in articular cartilage of the same mouse models of OA by rapamycin treatment, which reduces aging and induces autophagy in articular cartilage. Third, we will determine the effects of targeted manipulation of DNA methylation in a key age-associated OA autophagy gene (PPARG) and other genes we identify in the first two aims as shared among various risk factors in human OA chondrocytes in vitro using cutting-edge Cas9- based epigenetic editing. Discoveries achieved by these aims will form the preliminary data for strong R01 applications to confirm novel diagnostic OA associations and develop new therapeutic strategies to treat this devastating disease. This project will provide the first data epigenetic patterns in murine OA, and the first direct evidence regarding the functional consequences of epigenetic editing in human chondrocytes.