Diffuse midline glioma (DMG) is a lethal pediatric brain tumor without a cure and with a poor prognosis. The H3K27M mutation is present in 80% of DMG patients and is the primary driver of the disease. Therefore, targeting this oncohistone using mRNA-based gene therapies may represent a promising treatment option. However, mRNA as a drug needs to overcome the blood-brain and blood-tumor barriers to reach the tumor site, which is difficult due to the location of DMG in the brain stem. During my postdoctoral fellowship, I have developed non- toxic lipid nanoparticles (LNPs) that can effectively deliver mRNA to DMG tumors in vivo. This proposal aims to further optimize and evaluate the efficacy of these LNPs in delivering mRNA, explore the mechanism behind their delivery, and develop a new therapeutic approach targeting the H3K27M gene in DMG. In Aim 1, I will test the delivery of mRNA with different bases modifications and LNP chemistries to maximize LNP transfection and protein translation in DMG mouse models. In Aim 2, I will test the hypothesis that microglia take up LNPs and repackage them in extracellular vesicles that are subsequently delivered to DMG cells ‘in relay’. In the independent phase (Aim 3), I will explore LNP-mRNA-based gene therapy approach to target oncohistone H3K27M in DMG. Altogether this proposal aims to advance our understanding of DMG and establish a reliable and effective nucleic acid delivery approach for DMG. Completing the proposed project will allow me to build a strong scientific foundation under the mentorship of Profs. Kathryn A. Whitehead (lipid nanoparticle expert), Ian F. Pollack (pediatric brain tumor expert) and Drew Weissman (mRNA expert). An interdisciplinary advisory team has carefully been assembled, consisting of Prof. Robert S. Langer for translational drug delivery expertise, Profs. Maria G. Castro, Carl Koschmann and Sameer Agnihotri for DMG biology and mouse model expertise, Prof. Xiaoming Hu for microglia expertise and Prof. Samira Kiani for gene therapy expertise. Together, this research proposal, mentorship team, advisory committee and institutional support from Carnegie Mellon University will lay the scientific groundwork and provide the necessary training to reach my ultimate goal of successfully starting my independent academic career.