ABSTRACT Heterotopic ossification (HO) is a condition involving pathologic bone formation in extra-skeletal soft tissues (e.g. tendons, ligaments, muscle) which can occur following orthopaedic surgeries or injuries such as high impact trauma and burns. There is an unmet clinical need to develop effective therapies to treat HO, particularly at early stages post-injury. In this proposed project, our objective is to explore small RNA targeting approaches (microRNA, siRNA) in a clinically relevant mouse model of trauma-induced HO to: i) develop and test new nanoparticle-based strategies to treat this debilitating condition, and ii) advance our understanding of the mechanisms driving HO and identify new potential therapeutic targets. From an unbiased microarray study to identify microRNA (miRNA) expression signatures in developing long bones, we identified miR-138 as being differentially-expressed in distinct regions of the growth plate. We subsequently showed that miR-138 has bone inhibitory function, including the ability to suppress HO in an established trauma-induced HO mouse model. Additionally, we demonstrated that a major mechanism by which miR-138 inhibits osteogenesis is by targeting and suppressing the cytoskeletal regulator, RhoC. We subsequently showed that direct inhibition of RhoC could also inhibit osteogenesis. Whether or not knockdown of RhoC alone can also dampen trauma-induced HO in vivo has yet to be investigated. Inflammation also plays a critical role in HO formation. Pilot data from our group show suppression of IL- 1-induced catabolic genes by either miR-138 over-expression or RhoC inhibition. In addition, p65 (a transcription factor component of the NF-kB complex and an important regulator of inflammation) is another reported target of miR-138, the function of which has not yet been examined in the context of HO. Taken together, we hypothesize that strategies to modulate miR-138 activity (and its downstream targets) will result in suppression of trauma-induced HO, including associated inflammation and pain, and also inform new mechanisms regulating this pathologic process. Two specific aims are proposed to address our hypothesis. Specific Aim 1 is focused on developing a small RNA targeting approach to suppress HO. The effects of over-expressing miR-138 or siRNAs targeting RhoC and p65 will be tested in the trauma-induced HO mouse model. Nanoparticle-based technology will be utilized to enhance translational RNA-targeting capabilities. In Specific Aim 2, a multi-omics approach will be used to determine the effects of nanoparticle treatments and how genetic-based over-expression of miR-138 in specific cell types can suppress HO. These studies will advance our knowledge on cellular processes driving HO, and provide evidence to support new therapeutic approaches, involving small RNA nanoparticle targeting, as a promising treatment for HO.