PROJECT SUMMARY: Cancer immunotherapy is based on the premise of immune-recognition and targeted killing of tumor cells, thus possesses the promising power to eradicate aggressive disease. Notably to date, immunotherapy with immune checkpoint inhibitors significantly prolongs the survival of patients. Unfortunately, recurrent and metastatic disease occurs to a significant portion of patients within 5 years largely due to resistance to the immune checkpoint inhibitor. This largely stems from the local immunosuppressive tumor microenvironment (TME), which is enriched with immunosuppressive myeloid cells. To reprogram the TME into a “hot” environment enriched with functional myeloid cells, we designed a dual action immunostimulatory nanoparticle (dual-NP) that triggers a highly potent activation of the dysfunctional tumor-resident myeloid cells. The premise of our strategy is based on three key components: (1) Our previous studies showed that V domain Immunoglobulin Suppressor of T cell Activation (VISTA) is a novel myeloid cell-intrinsic immune checkpoint protein, which controls antitumor immunity. We showed VISTA is highly expressed on immunosuppressive myeloid cells and blocking VISTA can synergize with TLR9 agonist to reprogram the immunosuppressive myeloid cells to boost antitumor immunity. (2) The dual-NP is co-loaded with a VISTA siRNA and the TLR9 agonist CpG. Upon intratumoral administration, the dual-NP ensures the simultaneous uptake of its synergistic cargoes by the same tumor-resident myeloid cells and proficient intracellular delivery of each cargo, thus achieving optimal effects to activate these cells. Our recent studies show that simultaneous silencing of the VISTA gene and stimulation of TLR9 leads to a synergistic T cell-mediated tumor clearance and curative responses with protective immunological memory against tumor recurrence. (3) We developed a simple and controllable method to generate ionizable lipid dual-NPs of different sizes (i.e., 30, 40 or 60 nm) with high degree of uniformity and consistency. Our studies show that small dual-NPs achieve widespread distribution and predominant uptake by myeloid cells throughout the tumor volume upon intratumoral administration. Innovation: To our knowledge, this is the first effort to combine advanced nanoparticle design, simultaneous delivery of siRNA and a TLR agonist, and silencing of a gene related to an immune checkpoint protein specific to myeloid cells. AIM 1: Optimize the design of the dual-NP and test the ex vivo and in vivo efficacy in reprograming tumorassociated myeloid cells. AIM 2: Evaluate the short and long-term safety profile of the dual-NP and characterize the mechanism of antitumor immune responses associated with dosage and frequency of dual-NP administration. AIM 3: Evaluate the therapeutic efficacy of the dual-NP as a monotherapy and in combination with standard immune checkpoint inhibitors in murine models of advanced melanoma and metastatic breast cancer.