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--用双重组酶（MADR）进行镶嵌分析， 用于谱系追踪的合成靶阵列的基因组编辑（GESTALT）解决了这一需求。MADR提供 用于单拷贝体细胞转基因（或用CRISPR/Cas9突变）的灵活手段。MADR-GESTALT 提供了一套工具来研究癌症形成和复发的紧急异质性， 一致的、单拷贝的、用于表达多个“个性化”患者驱动基因的遗传框架。 围产期电穿孔用于将基因递送到脑干和祖细胞，并避免多拷贝 插入，开发了一种用于体内单拷贝转基因插入的新技术。使用Dual进行镶嵌分析 重组酶（MADR）采用双重重组酶介导的盒交换，用于高效插入 转基因到一个基因位点。为了模拟功能丧失突变，CRISPR/Cas9介导的基因编辑 也被纳入MADR-GESTALT系统。MADR的几个领域可以扩展和利用 加强加州大学洛杉矶分校脑癌孢子内的具体项目，其中，此外，将独立验证 MADR-GESTALT在更广泛的癌症研究领域的应用。该提案利用了IMAT资助的 这项技术增强了我们SPORE计划中的三个正在进行的项目：1）主动免疫疗法 结合检查点调节治疗胶质母细胞瘤。MADR将用于开发同基因 将使用具有神经胶质瘤和GESTALT的免疫活性小鼠来评估条形码多样性， 治疗，以进一步了解肿瘤在免疫压力下的演变。2)遗传易感性 小儿神经胶质瘤的发展。MADR-GESTALT将用于建立一个中等通量，高保真， 患者特异性体内建模平台，以了解新型生殖系变体的致病性，它们对 基因表达，以及它们对儿童高级别胶质瘤易感性的贡献。3)适应性免疫治疗 针对胶质母细胞瘤中的H3.3G34突变。MADR-GESTALT模型将用于研究机制 特定组蛋白突变通过其影响肿瘤发生和G34 R突变型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 recombinases (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) Active immunotherapy combined with checkpoint modulation for 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 pressure. 2) Genetic susceptibility in pediatric glioma development. MADR-GESTALT will be used to establish a moderate-throughput, high-fidelity, patient-specific in vivo modeling platform to understand pathogenicity of novel germline variants, their effects on gene expression, and their contribution to pediatric high-grade glioma susceptibility. 3) Adaptive immunotherapy to target the H3.3G34 mutation in glioblastoma. MADR-GESTALT models will be used to examine mechanisms by which particular histone mutations affect oncogenesis and immunotherapy for G34R mutant glioblastoma.