Developing a system for PDX in vivo genetic manipulation and selection

开发 PDX 体内遗传操作和选择系统

• 批准号: 9756342
• 负责人: Elena Nikolaevna Pugacheva
• 金额: $ 7.5万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-06 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9756342
• 关键词: 3-Dimensional; Academia; Adverse effects; Bacterial Toxins; Binding; Biology; Breast Cancer Patient; Cancer Biology; Cancer Model; Cancer Patient; Cancer cell line; Cell Culture Techniques; Cell Proliferation; Cell Survival; Cells; Clinical Trials; Cloning; Code; Collaborations; Collection; Communities; Complementary DNA; Cooperative Human Tissue Network; Core Facility; Corynebacterium; DTR gene; Detection; Diphtheria; Diphtheria Toxin; Doxycycline; EWS/FLI 1 Type 2 gene; Environment; Enzymes; Epidermal Growth Factor Receptor; Evaluation; Exotoxins; Firefly Luciferases; Food; Freezing; Gene Expression Profile; Generations; Genomics; Glioblastoma; Goals; Growth; Human; Imagery; Immune system; In Vitro; Industry; Infection; Injections; Intraperitoneal Injections; Knowledge; Laboratories; Luciferases; Malignant Neoplasms; Malignant neoplasm of brain; Metabolism; Methodology; MicroRNAs; Modeling; Modification; Morphology; Mus; Neoplasm Metastasis; Outcome; Pathology; Patients; Peptide Elongation Factor 2; Pharmaceutical Preparations; Pre-Clinical Model; Production; Protein Biosynthesis; Protocols documentation; Reporting; Reproducibility; Research; Research Personnel; Resistance; Signal Transduction; Site; Subfamily lentivirinae; System; Targeted Toxins; Technology; Therapeutic; Transgenes; Transplantation; Validation; Variant; Viral; Water; Work; Xenograft Model; Xenograft procedure; anticancer research; base; cancer biomarkers; cancer cell; cancer drug resistance; clinically relevant; design; drug discovery; efficacy testing; follow-up; genetic approach; genetic manipulation; genetic selection; genomic RNA; human model; humanized mouse; in vivo; malignant breast neoplasm; minimally invasive; neoplastic cell; new therapeutic target; novel; novel anticancer drug; performance tests; personalized medicine; potential biomarker; profiles in patients; promoter; research and development; response; screening; small hairpin RNA; standard care; tool; transcriptome; transcriptome sequencing; tumor; tumor heterogeneity; tumor progression; tumor xenograft; vector

PROJECT SUMMARY The patient-derived xenograft (PDX) is a powerful model of human cancer designed with the focus on personalized medicine. It has the potential to become part of a cancer patient’s standard care (cancer avatars) and a superior platform for drug discovery, mechanistic studies on cancer drug resistance, and screening of the potential biomarkers of cancer progression/metastasis and drug response. In combination with current work on developing humanized mice, it will be able to explain interactions within the patient’s immune system as well. However, the methodology and tools for genetic manipulations in PDX are currently unavailable, representing a significant barrier for usage of PDX models in the research community. Our objective is to validate developed in our laboratory PD-VivoS toolkit and methodology to enable genetic manipulation of PDXs. This methodology will allow for self-inactivating lentivirus-based delivery of inducible shRNAs/miRNA, or cDNA and luciferase directly into the PDX growing in a mouse along with the ability to select for the infected tumor cells via diphtheria toxin administration. Such a system will allow for the robust tumor cell genomic manipulation in vivo, enabling the inclusion of PDX models as a standard cancer research platform. The specific aims of the current application are: 1) to validate the system developed in our laboratory for the ability to successfully infect, select and express the desired cDNA/or shRNA in multiple different cancer PDXs available via our PDX core facility; 2) to systematically examine the PDX biology (growth, pathology, genomic/transcriptome integrity) upon introduction of the empty cassette/s and selection with diphtheria toxin for any potential changes it might induce. This analysis will allow us to account for any non-specific effects this methodology might possess and define its limitations. To achieve these goals, two PD-VivoS vectors (to express cDNA or sh/miRNA) were generated for production of self-inactivating lentivirus, and coding for firefly luciferase fused with shRNA against DPH2 and multiple cloning site for cDNA or sh/miRNA insertion under doxycycline inducible promoter. PDXs, grown in NSG mice, will be infected through direct intratumoral injections of concentrated viral stock and selected via intraperitoneal injections of diphtheria toxin. The luciferase signal will be used to control for successful infection and to follow up the growth/selection of infected tumor cells. The resultant PD-VivoS modified PDX tumor will be collected, frozen for longer term storage or could be directly re-transplanted into naïve mice to induce cDNA/shRNA expression with doxycycline food/water. The PD-VivoS-PDX tumor will be used for genomic and RNA sequencing to compare to parental PDX and define the effects of transgene transduction. The rational for this work is that there is no system currently available for in vivo genetic manipulation and selection, and that such a system will significantly increase the range of application for PDX.

项目摘要 患者来源的异种移植物（PDX）是一种强大的人类癌症模型，其设计重点是 个性化医疗它有可能成为癌症患者标准护理的一部分（癌症化身） 为药物发现、肿瘤耐药机制研究和肿瘤细胞筛选提供了一个上级平台。 癌症进展/转移和药物反应的潜在生物标志物。结合当前工作 在开发人源化小鼠时，它将能够解释患者免疫系统内的相互作用， 好.然而，目前还没有PDX基因操作的方法和工具， 这是研究界使用PDX模型的一个重大障碍。我们的目标是 验证我们实验室开发的PD-VivoS工具包和方法，以实现遗传操作 的PDX。该方法将允许基于自失活慢病毒的诱导型shRNA/miRNA的递送， 或者cDNA和荧光素酶直接导入在小鼠中生长的PDX中，该PDX沿着具有选择感染的 通过白喉毒素给药治疗肿瘤细胞。这样的系统将允许稳健的肿瘤细胞基因组 在体内操作，使PDX模型作为标准的癌症研究平台。的 本申请的具体目标是：1）验证我们实验室开发的系统的能力 为了成功感染、选择并在多种不同的癌症PDX中表达所需的cDNA/或shRNA， 可通过我们的PDX核心设施获得; 2）系统地检查PDX生物学（生长，病理学， 基因组/转录组完整性），并用白喉毒素进行选择 任何可能的变化这种分析将使我们能够解释任何非特异性的影响， 方法论可能拥有并定义其局限性。 为了实现这些目标，产生了两种PD-VivoS载体（以表达cDNA或sh/miRNA），用于 产生自失活慢病毒，并编码与针对DPH 2的shRNA融合的萤火虫荧光素酶， 多西环素诱导型启动子下的cDNA或sh/miRNA插入的多克隆位点。PDX，生长在 NSG小鼠将通过直接瘤内注射浓缩的病毒储备液进行感染，并通过 腹腔注射白喉毒素。荧光素酶信号将用于控制成功感染 并跟踪受感染肿瘤细胞的生长/选择。所得到的PD-VivoS修饰的PDX肿瘤将 收集、冷冻以长期储存或可直接重新移植到幼稚小鼠中以诱导 用多西环素食物/水进行cDNA/shRNA表达。PD-VivoS-PDX肿瘤将用于基因组和 RNA测序以与亲本PDX进行比较并确定转基因转导的影响。 这项工作的理由是，目前还没有可用于体内遗传操作的系统， 选择，这样的系统将大大增加PDX的应用范围。