Developing a system for PDX in vivo genetic manipulation and selection

项目总结 患者来源的异种移植(PDX)是一种强大的人类癌症模型，其设计的重点是 个性化医疗。它有可能成为癌症患者标准护理的一部分(癌症头像) 以及药物发现、癌症耐药机制研究和筛选的优越平台 肿瘤进展/转移和药物反应的潜在生物标志物。与当前工作相结合 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 井。然而，目前还没有在PDX中进行遗传操作的方法和工具， 这是PDX模型在研究界使用的一个重大障碍。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基因操作 PDX的数量。该方法将允许基于慢病毒的可诱导shRNAs/miRNA的自失活递送， 或cDNAs和荧光素酶直接进入PDX，在小鼠体内生长的同时具有选择感染的能力 肿瘤细胞通过白喉毒素注射。这样的系统将允许强大的肿瘤细胞基因组 体内操作，使PDX模型成为标准的癌症研究平台。这个 目前应用的具体目标是：1)验证我们实验室开发的系统的能力 为了成功感染，选择并在多个不同的癌症PDX中表达所需的cDNAs/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、 基因组/转录组完整性)引入空盒/S并用白喉毒素进行选择 它可能引发的任何潜在变化。这一分析将使我们能够解释任何非特定的影响 方法论可能具有并定义了它的局限性。 为了实现这些目标，我们构建了两个pD-vivos载体(用于表达cdna或sh/miRNA)。 自失活慢病毒的制备及其与抗DPH2和DPH2的shRNA融合的萤火虫荧光素酶的编码 多西环素诱导启动子下插入cDNAs或sh/miRNA的多克隆位点。PDX，成长于 NSG小鼠，将通过瘤内直接注射浓缩病毒股票和通过 腹腔注射白喉毒素。荧光素酶信号将用于控制成功感染 并追踪感染肿瘤细胞的生长/选择情况。由此产生的PD-Vivos修饰的PDX肿瘤将 被收集、冷冻以长期储存，或可直接重新移植到幼小鼠体内以诱导 在多西环素食物/水中表达cDNAshRNA。PD-Vivos-PDX肿瘤将用于基因组和 RNA测序以与亲本PDX进行比较，并确定转基因的影响。 这项工作的合理性是，目前还没有可用于体内遗传操作和 这种系统将极大地扩大PDX的应用范围。

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.