Incorporation of Novel MADR-GESTALT Technology into UCLA SPORE in Brain Cancer

摘要（总体） 胶质母细胞瘤是成人中最常见的原发性脑肿瘤，是所有人类癌症中最致命的一种。 这种疾病需要创新的方法和更有效的治疗。耐药性表现为 基本屏障，并考虑到恶性胶质瘤的异质性， 为了实现这一目标，我们需要更好地了解基因， 疾病的多样性和异质性，以及更好的临床前动物模型， 胶质瘤异质性一种最先进的方法学-双重组酶介导的镶嵌分析 用于谱系追踪的合成靶阵列的基因组编辑（GESTALT）- 满足了这一需求。MADR为单拷贝体细胞转基因（或突变）提供了一种灵活的手段， CRISPR/Cas9）。MADR-GESTALT提供了一套工具来研究癌症中出现的异质性 通过提供一个一致的，单拷贝的，遗传框架的表达， “个性化”患者驱动基因。围产期电穿孔用于将基因递送到脑干， 祖细胞，并避免多拷贝插入，一种用于体内单拷贝转基因插入的新技术 开发了使用双重组酶的镶嵌分析（MADR）采用双重组酶介导的嵌合分析。 用于将转基因高效插入单个遗传基因座的盒交换。模拟功能丧失 在MADR-GESTALT系统中，CRISPR/Cas9介导的基因编辑也被纳入MADR-GESTALT系统。几 MADR的领域可以扩展和利用，以加强加州大学洛杉矶分校脑癌的具体项目 此外，它还将独立验证MADR-GESTALT在更广泛的癌症中的效用。 研究社区。该提案利用IMAT资助的技术来增强三个正在进行的项目 在我们的SPORE计划中：1）通过DC疫苗接种和PD-1/CSF-1 R靶向免疫治疗抗性 抑制作用2)克服胶质母细胞瘤中药物诱导的内在凋亡抗性。MADR将用于 开发具有神经胶质瘤的同基因免疫活性小鼠，GESTALT将用于评价条形码 有和没有治疗的多样性，以进一步了解免疫和小 分子抑制剂压力。3)针对儿童H3.3G34突变的适应性免疫治疗 胶质母细胞瘤MADR-GESTALT模型将用于研究特定组蛋白 突变影响肿瘤发生和G34 R突变胶质母细胞瘤的免疫治疗。

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.