PROJECT SUMMARY/ABSTRACT Compared to other types of cancer that have benefited from considerable advances in therapy, therapeutic outcomes in glioblastoma (GBM) patients still remains dismal due to its resistance to conventional therapies. Such poor response is often related to aberrant dysregulation of gene expressions in tumor cell and tumor- supportive tumor microenvironment (TME) that is educated by the tumor cell through immune editing, indicating that the tumor is not primed for the treatment due to the unfavorable gene expressions. In order to prime the tumor cells and the immunosuppressive TME to improve the clinical outcomes in GBM patients from current treatment options or even new therapeutics, both tumor cells and tumor-associated macrophages (TAMs) need to be reprogrammed by reversing the aberrantly dysregulated gene expressions across various signaling pathways. Tumor suppressive microRNAs (miRNAs) have been proposed as the most suitable therapeutic target to perform the tumor reprogramming role. We recently found that a putative tumor suppressive miRNA, miR-138, has a potential to reprogram GBM tumor cells through down-regulation of major oncogenic pathways CD44 and EGFR, which governs cell proliferation and immune evasion through PD-L1 and cytokine expressions. However, little progress has been made in miRNA-based therapy mainly due to the lack of efficient and safe targeted delivery method into tumor cells across the blood brain tumor barrier (BBTB). In addition, dual targeting of both tumor cells and TAMs to delivery such therapeutically useful tumor suppressive miRNAs has never been attempted. Previously, we have shown that RNA nanovector (RNV) platform based on the Three-Way-Junction (3WJ) motif of packaging RNA (3WJ-pRNA) in the DNA packaging motor of bacteriophage phi29 with conjugation of folate (FA) (FA-RNV) has a potential to selectively target folate receptor (FR) positive GBM cells and deliver a small RNA with favorable bio-distribution and pharmacokinetic profiles in mice model. We hypothesize that targeted delivery of tumor suppressive miR-138 into GBM tumor cells and TAMs by our ligand-conjugated RNV will reprogram both GBM tumor and tumor- associated innate immunity to result better tumor regression through direct tumor cell killing and susceptibility to conventional immunotherapy. Here, we propose to optimze the RNV derived from the three-way junction (3WJ) motif to achieve this challenging task by testing different cell-specific ligand or RNA aptamers (Aim 1). In addition, we will test the preclinical impact of simultaneous targeted delivery of miR-138 into tumor cells and TAMs by cell-specific RNVs (Aim 2). To our knowledge, this is the first preclinical study attempting to simultaneously modulate tumor cells and innate immunity by delivering a tumor suppressive miRNA using the RNV delivery system for GBM treatment. Our study results can be rapidly translated into human clinical trials in a combination with curre...