Project Summary/Abstract Patients with lethal castration-resistant prostate cancer (CRPC) are currently treated with agents targeting androgen receptor (AR) signaling. However, AR inhibition has not dramatically improved CRPC patient survival, underscoring the need to discover novel oncogenic mechanisms in CRPC and develop new therapies targeting these mechanisms. Cancer cells are addicted to aberrant RNA polymerase II (Pol II) transcription, which includes initiation, elongation, and termination phases, as well as a recycling step critical to repeated Pol II transcription of the same gene after the initial transcription cycle. While studies have already indicated that uncontrolled transcriptional initiation and elongation have oncogenic roles, it is unknown whether other Pol II transcription processes contribute to cancer-relevant transcriptional outcomes and cancer growth. In preliminary studies, our newly developed in vitro and cell-based transcription recycling assays have found that Pol II recycling is a key yet overlooked transcription process with relevance to prostate cancer. We have found that Mediator complex subunit 31 (MED31) drives Pol II recycling in CRPC cells, enhancing mRNA output during the recycling process. Importantly, high expression of MED31 is both sufficient and necessary for prostate cancer castration-resistant growth and is associated with poor prognosis of CRPC patients. While these findings identify the oncogenic MED31 as a new therapeutic target for CRPC, transcription regulators such as MED31 are generally considered untargetable by traditional, small molecule-based drug design. We have developed a safe lipid nanoparticle (LNP) system for targeted delivery of the CRISPR/Cas13d system to efficiently and specifically knock down oncogenic transcription regulators at the mRNA level. In preliminary studies, we have demonstrated that the LNP-Cas13d system effectively and safely knocks down MED31 mRNA and decreases CRPC cell growth in vivo, establishing the proof of the concept that the therapeutic window exists for targeting MED31 in CRPC. Together, our preliminary findings support the hypothesis that MED31-governed transcription recycling is a novel oncogenic driver for CRPC progression and that an LNP-Cas13d-based RNA targeting system can counteract oncogenic transcription driven by MED31 in CRPC with safety, specificity, and efficacy. In Aim 1, we will delineate the molecular mechanism, biological function, and clinical relevance of MED31-mediated transcription recycling. In Aim 2, we will target MED31-mediated transcription recycling using a CRISPR/Cas13d-based nanoparticle system. The successful completion of these aims will significantly elucidate the critical role of Pol II recycling in lethal prostate cancer and will provide an experimental basis for future clinical trials testing the utility of an LNP- Cas13d RNA targeting system to target this novel oncogenic mechanism in CRPC patients.