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最常见的原发脑瘤，也是人类所有癌症中最致命的癌症之一。 这种疾病需要创新的方法和更有效的治疗方法。治疗抗性呈现出一种 基本障碍，并鉴于恶性胶质瘤的异质性，结合了多种 可能需要一些方法来克服它。要做到这一点，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 胶质瘤的异质性。一种最新的方法学--双重重组酶介导的镶嵌分析 盒式磁带交换(MADR)和用于谱系追踪的合成目标阵列的基因组编辑(格式塔)- 满足了这一需求。MADR为单拷贝体细胞转基因(或突变)提供了灵活的方法 CRISPR/CAS9)。MADR-格式塔提供了一套工具来研究癌症中的紧急异质性 通过为多个基因的表达提供一致的、单拷贝的遗传框架来形成和复发 “个性化”的患者驾驶基因。围产期电穿孔被用来将基因输送到脑干和 为避免多拷贝插入，体内单拷贝转基因插入的新技术 是发展起来的。双重组酶介导的马赛克分析 用于高效地将转基因插入到单个遗传基因座的盒式交换。建立功能丧失模型 突变，CRISPR/Cas9介导的基因编辑也被整合到MADR-格式塔系统中。几个 MADR的领域可以扩大和利用，以加强加州大学洛杉矶分校脑癌的具体项目 孢子，此外，它还将独立验证MADR-格式塔对更广泛的癌症的效用 研究社区。该提案利用了IMAT资助的一项技术，增强了三个正在进行的项目 在我们的孢子计划内：1)靶向免疫治疗-DC疫苗和PD-1/CSF-1R的耐药性 抑制力。2)克服药物诱导的胶质母细胞瘤对内源性细胞凋亡的抵抗。MADR将习惯于 开发具有神经胶质瘤和格式塔的同基因免疫活性小鼠将用于评估条形码 有无治疗的多样性，以进一步了解免疫治疗和小肿瘤的演变 分子抑制压力。3)针对儿童H3.3G34突变的适应性免疫治疗 胶质母细胞瘤。MADR-格式塔模型将被用来研究特定组蛋白 突变影响G34R突变的胶质母细胞瘤的肿瘤发生和免疫治疗。

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.