ABSTRACT The use of RNA technologies to specifically target genetic alterations in tumor cells has shown great potential of becoming a novel therapy modality for cancer treatment. Nevertheless, systemic delivery of RNA agents such as messenger RNA (mRNA) to tumor cells in vivo commonly faces multiple barriers, including low stability, rapid elimination by renal excretion, insufficient cellular uptake, poor endosomal escape, and transient activities. Our long-term objective is to develop robust nanoparticle (NP) platforms for effective and safe RNA delivery to solid tumors, and along with cancer target validation in vivo, to eventually transition the RNA nanomedicines into clinical development. In the last funding cycle, a lipid-polymer hybrid RNA NP system has been engineered with favorable features, such as small size, high RNA encapsulation, efficient cytosolic translocation, and relatively long blood circulation. We have also pioneered the application of these hybrid NPs for mRNA delivery to restore tumor suppressors (e.g., PTEN) in different cancer types including prostate cancer (PCa) and non-small cell lung cancer, which represents a novel approach to cancer treatment that is independent of oncogene antagonism. In our latest work, we further reveal that PTEN restoration in PTEN-null/mutated murine tumor cell lines can induce immunogenic cell death (ICD). Preliminary in vivo studies show that PTEN mRNA NP treatment triggers cytotoxic T cell responses, modulates the immunosuppressive tumor microenvironment, and improves the responses of immune checkpoint blockade therapy. In this renewal application, we propose to i) address the unique challenge of transient bioactivity in mRNA delivery by developing a new generation of hybrid mRNA NPs, and ii) apply the new hybrid mRNA NPs to explore PTEN restoration-induced ICD and evaluate the anti-tumor efficacy of PTEN restoration along with immune checkpoint blockade. Specifically, the three Aims underlying the proposal are: 1) To optimize the new generation of hybrid NPs and study the NP-mediated long duration of mRNA bioactivity with the goal of achieving prolonged PTEN expression in PCa tumors using as infrequent injections as possible; 2) To apply the optimized mRNA NPs to investigate the mechanisms underlying PTEN-mediated ICD and anti- tumor immune responses and to evaluate the therapeutic effect and safety in subcutaneously grafted, orthotopic, and transgenic models of PCa; and 3) To expand the new hybrid mRNA NPs to systemic co-delivery of PTEN mRNA and CpG oligodeoxynucleotide (a toll-like receptor-9 agonist) for stronger ICD and to test the co-delivery NPs for PCa treatment together with immune checkpoint inhibitors. We expect that successful completion of this project will lead to development of a novel synthetic mRNA nanotherapy that could benefit cancer patients with loss/mutation of PTEN. Moreover, this NP delivery strategy could be readily expanded to other tumor suppressor- encoding mRNA...