ABSTRACT (Overall) Glioblastoma, the most common primary brain tumor in adults, is one of the most lethal of all human cancers. This disease requires innovative approaches and more effective treatments. Treatment resistance presents a fundamental barrier, and given the heterogeneous nature of malignant gliomas, a combination of diverse approaches will likely be needed to overcome it. To achieve this, we need a better understanding of the genetic diversity and heterogeneity of the disease, as well as better pre-clinical animal models that recapitulate human glioma heterogeneity. A state-of-the-art methodology—mosaic analysis with dual recombinase-mediated cassette exchange (MADR) and genome editing of synthetic target arrays for lineage tracing (GESTALT)— addresses this need. MADR provides a flexible means for single-copy somatic transgenesis (or mutation with CRISPR/Cas9). MADR-GESTALT provides a suite of tools to study the emergent heterogeneity in cancer formation and recurrence by providing a consistent, single-copy, genetic framework for the expression of multiple “personalized” patient driver genes. Perinatal electroporation is used to deliver genes to brain stem and progenitor cells, and to avoid multiple copy insertion, a new technique for single-copy transgene insertion in vivo was developed. Mosaic Analysis with Dual Recombinases (MADR) employs dual recombinase-mediated cassette exchange for high efficiency insertion of transgenes to a single genetic locus. To model loss-of-function mutations, CRISPR/Cas9-mediated gene editing is also incorporated into the MADR-GESTALT system. Several areas of MADR can be expanded and leveraged to enhance specific projects within the UCLA Brain Cancer SPORE, which, in addition, will independently validate the utility of MADR-GESTALT for the wider cancer research community. This proposal utilizes an IMAT-funded technology that enhances three ongoing projects within our SPORE program: 1) Targeting immunotherapy-resistance with DC vaccination and PD-1/CSF-1R inhibition. 2) Overcoming drug-induced resistance to intrinsic apoptosis in glioblastoma. MADR will be used to develop syngeneic immunocompetent mice with gliomas and GESTALT will be used to evaluate barcode diversity with and without treatment to further understand tumor evolution under immunotherapeutic and small molecule inhibitor pressure. 3) Adaptive immunotherapy to target the H3.3G34 mutation in pediatric glioblastoma. MADR-GESTALT models will be used to examine mechanisms by which particular histone mutations affect oncogenesis and immunotherapy for G34R mutant glioblastoma.