Abstract Pediatric brain tumors are the leading cause of death in children with cancer in the U.S. Among them, pediatric high-grade gliomas (pHGGs) are one of the most common and aggressive forms of brain cancer, with a median survival of 9-15 months.1-3 One of the prominent subgroups of pHGG that arises in cerebral hemispheres encodes for G34R/V substitutions in the histone H3F3A, along with ATRX and TP53 inactivating mutations. The current standard of care, consisting of tumor resection followed by radiation and chemotherapy,1-4 only leads to a modest increase in median survival. One of the reasons for the limited therapeutic outcomes is tumor recurrence, caused by the spread of pHGG cells that infiltrate the brain.1-4 Treatment effectiveness for pHGG has also been limited due to the blood-brain barrier (BBB),5 which precludes the efficient delivery of chemotherapeutic compounds to the tumor mass. Therapeutic strategies involving local delivery of chemotherapeutic agents to the tumor are emerging as attractive approaches. To explore novel therapeutic modalities for the G34R/V pHGG subtype, we developed a de novo mouse model harboring the genetic lesions using the Sleeping Beauty (SB) transposase-mediated system.6-8 Our preliminary data demonstrate that the H3.3G34R mutation reduces the expression of genes involved with DNA repair, rendering the cells more susceptible to ionizing radiation in vivo and to DNA damage sensitizers such as Olaparib, a PARP inhibitor. In this application, we propose to deliver Olaparib into the TME using high-density lipoprotein (HDL)-mimicking nanodiscs (NDs) that can be specifically internalized into tumor cells via scavenger receptor class B-1 (SR- B1) and caveolae lipid rafts endocytosis.9 We observed that SR-B1 is expressed in H3.3G34R pHGG neurospheres (NS) derived from the SB model, as well as in H3.3G34R pHGG patient-derived cells. In this study, we will develop chemo-immunotherapy delivery vehicles based on sHDL NDs loaded with CpG, a Toll- like receptor 9 (TLR9) agonist, together with Olaparib, a chemotherapeutic agent, for targeting H3.3G34R pHGG. We demonstrated that local delivery of sHDL NDs loaded with chemo-immunotherapeutics, in an intracranial syngeneic mouse glioma model, elicited tumor regression and anti-tumor CD8+ T cell responses in the brain tumor microenvironment (TME) without overt off-target effects.10 These data indicate that sHDL NDs are an attractive drug delivery platform for pHGG, which we hypothesize will result in tumor regression and long-term survival. The proposed delivery system has significant potential for clinical translation.