2039 Background: Immune checkpoint inhibition has not improved outcomes for glioblastoma patients, and single- cell approaches reveal the glioblastoma microenvironment is largely comprised of immunosuppressive cells. We hypothesized intratumor convection enhanced delivery (CED) of adenoviral gene therapy could recruit and activate anti-tumor immune cells in the glioblastoma microenvironment. Methods: Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma (3 x 104) cells were implanted into the frontal lobe of immunocompetent C57BL/6J mice (18 mice/arm). Intracranial bioluminescence (BLI) and body weight (BW) measurements were used to assess glioblastoma growth and treatment toxicity, respectively. After tumor engraftment, glioblastomas were treated with conformal ionizing radiation mimicking stereotactic radiosurgery (SRS) in human patients as a positive control for tumor inhibition (18Gy/1Fx). Attenuated adeno-associated virus 9 (AAV9) vectors encoding Gfp as a negative control, or encoding experimental cytokines driving recruitment and activation of anti-tumor immune cells in other intracranial tumors ( Ccl4, Il1b, or Apoa1) were delivered using CED (2x1011 vg/mouse). Glioblastomas were collected for histologic, single-cell, and molecular analyses 5 days after treatment (6 mice/arm) and at endpoints after monitoring for survival (12 mice/arm). CED targeting was validated using AAV9-GFP and confocal microscopy. Treatment responses were assessed using H&E, IHC, multiplexed cytokine assays, and single cell mass cytometry (CyTOF) to define immune cell types in the glioblastoma microenvironment. Results: Histologic analyses revealed AAV9-CCL4, AAV9-IL1B, or SRS induced glioblastoma macrophage infiltration, and AAV9-IL1B, AAV9-APOA1, or SRS induced glioblastoma T cell infiltration. AAV9-APOA1 (18.5 days versus 15 days, p < 0.001) or AAV9-IL1B (16.5 days versus 15 days, p = 0.001) CED treatments prolonged median survival from SB28 allografts. Glioblastoma cytokine analysis revealed inhibition of pro-tumor cytokines (IL6, LIF) after experimental CED treatments. Systemic cytokines were minimally changed by CED treatments. CyTOF showed decreased immunosuppressive macrophage infiltration and increased CD8+ T cell or microglia infiltration of the glioblastoma microenvironment after experimental CED treatments. There was no evidence of systemic or central toxicity in any treatment condition. Conclusions: Convection-enhanced delivered of adenoviral gene therapy reprograms the glioblastoma immune microenvironment and improves survival in an immunologically “cold” syngeneic glioblastoma model.