PROJECT SUMMARY/ABSTRACT RNA interference (RNAi) using short interfering RNA (siRNA) is a promising strategy to block disease- causing targets that are difficult to selectively and/or potently block with conventional small molecule drugs. Five siRNA drugs are now in clinical use, all gaining FDA approval since 2018. The first siRNA drug was a lipid nanoparticle, but subsequent drugs are “carrier-free” molecular conjugates enabled by chemical modifications to the RNA that provide nuclease stability. Carrier-free technologies are of great interest due to manufacturing simplicity and reduced risk of carrier (cationic polymer or lipid) associated side effects and toxicities. siRNAs can be readily designed and optimized for hitting any gene in the body and, therefore, have tremendous therapeutic potential. However, all siRNA drugs currently approved by the FDA aim to silence gene expression in the liver, and no new siRNA drugs have been approved for treatment of diseases localized to or emanating from other sites in the body. We are optimizing siRNA conjugates for treatment of osteoarthritis (OA), a degenerative joint disease that is a leading cause of physical disability in the United States. Current OA treatments, such as steroids, reduce pain but can cause side effects and do not halt or slow progression of the disease. siRNA therapeutics offer the option to block the genetic underpinnings of the disease and have the potential to yield the first disease modifying osteoarthritis drugs (DMOADs). The proposed project aims build from our recent work developing siRNA molecules end-modified through a PEG linker with a diacyl lipid (siRNA-L2), which spontaneously forms a molecular complex with albumin (alb/siRNA-L2) in situ following intravenous injection. We have shown this albumin “hitchhiking” siRNA-L2 to be safe and to have a high level of accumulation and on-target gene silencing activity in injured joints. Cells residing in OA-afflicted joints produce inflammatory cytokines and matrix metalloproteinases (MMPs) that initiate and propagate the joint degenerative process. The overall goal of this proposal is to implement siRNA-L2 to systemically treat and block progression of PTOA and to build from promising data showing that silencing of MMP13 has therapeutic benefit in post-traumatic OA. Specifically, this project will (1) elucidate the mechanism of action of siRNA-L2 against MMP13 in OA, (2) test new chemical modifications for improving joint retention and activity of siRNA-L2, (3) test for efficacy of siRNA-L2 targeting MMP13 in a larger (non-rodent) animal model, and (4) establish the platform nature of siRNA-L2 against other gene targets associated with pathogenesis of OA. The investigative team for this interdisciplinary project spans RNA chemistry/therapeutics (Duvall), OA biology and single cell RNA sequencing (Maerz), and OA clinical care (Crofford). These successful scientists cover the areas of expertise required for the proposed work...