Development of ESiPSC approach for non-germline GEM modeling

开发用于非种系 GEM 建模的 ESiPSC 方法

• 批准号: 8938101
• 负责人: Terry van Dyke
• 金额: $ 20.24万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8938101
• 关键词: Adenovirus Vector; Adopted; Alleles; Animals; Antineoplastic Agents; Astrocytoma; Biological Assay; Biological Markers; Brain; Breeding; Cancer Model; Cell Line; Cells; Clinical; Collection; Complex; Conscious; Data; Derivation procedure; Development; Disease; Disease Progression; Drug Evaluation; Embryo; Epithelium; Evaluation; Fetal Tissues; Gene Expression; Gene Transfer; Generations; Genetic; Genetic Variation; Genetically Engineered Mouse; Genotype; Goals; Histology; In Vitro; Infection; Intervention; Libraries; Lung; Malignant Neoplasms; Malignant neoplasm of ovary; Messenger RNA; Modeling; Molecular; Molecular Profiling; Mus; Oncogenic; Outcome; Ovarian; Ovary; Pancreatic carcinoma; Pharmaceutical Preparations; Phase; Plasmid Cloning Vector; Population; Preclinical Drug Evaluation; Process; Reagent; Series; Serous; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Ursidae Family; Validation; Variant; animal breeding; base; cancer type; carcinogenesis; clinically relevant; cohort; comparative; cost; design; drug efficacy; efficacy evaluation; genetic analysis; improved; in vivo; induced pluripotent stem cell; melanoma; mouse model; novel strategies; operation; polypeptide; pre-clinical; preclinical study; recombinant virus; research study; response; segregation; tumor; tumor growth; tumor progression; tumorigenesis

A widespread reduction to practice for an approach that employs genetically engineered mouse (GEM) cancer models for more predictive cancer drug efficacy evaluation is largely impeded by significant costs intrinsic to operations involving GEM animals but also due to frequent shortage of appropriate expertise in supporting complex genetics projects. To tangibly streamline the most laborious and costly components of preclinical studies (that, for example, involve manipulations with a sizeable breeding colony of GEM models and frequent genotyping to identify animals with appropriate allelic combinations) we will construct and characterize a library of ES and iPSC lines from a collection of well characterized and proven to be clinically relevant GEM models or carcinogenesis, including those for non-small cell lung, brain, ovarian serous epithelium, and melanoma cancer types. In order to achieve optimal outcomes in the efficiency of iPSC derivation while maintaining sufficient genetic diversity of an input cell population subject to in vitro reprogramming, we will apply an adenoviral vectors gene transfer based technology whereby recombinant viruses transiently express multicistronic self-processing polypeptides upon infection of genetically divergent pools of cells. The latter will in turn be derived from fetal tissues collected from embryos that belong to different litters to assure genetic diversity stipulated by random segregation of alleles in mixed background crosses typical for many multi-allelic GEM models. Resulting iPSC clones (at least five per model) that bear desired set of modified loci will undergo extensive in vitro characterization prior to producing cohorts of chimeric animals, now designated as non-germline GEM (NG-GEM) models, to be used both in preclinical drug evaluation studies and to conduct experiments on basic cancer mechanisms. In the first set of studies, the chimeric animals will be exploited for the purpose of tumorigenesis induction in specific tissues, such as brain or ovaries, and subsequent evaluation of tumors by patho-histology and gene expression techniques vis--vis similarly induced tumors appearing in conventionally bred animals. Selected subsets of chimeric animals will be further subjected to a pharmacological intervention with combinations of therapeutics proven to be efficacious in reverting the tumor growth in de novo cancer mouse models. As above, representative clinical, histological and molecular analyses will be performed to comparatively assess anti-cancer drug response in iPSC-derived chimeric animals employing as baseline available data gathered in a similar set of assays from conventionally bred tumor-bearing mice. Such comprehensive cross-evaluation of two similar though technologically divergent approaches is intended to further validate the robust and cost-conscious strategy of preparing preclinical cohorts of tumor bearing animals based on iPSC-derived non-germline GEM models. To extend the benefits of constructed iPSC library, in another set of in vivo experiments, several cohorts of iPSC derived NG-GEMs developed with different iPSC clones (though bearing an identical combination of inducible oncogenic alleles) will be brought in a comparative juxtaposition to identify variations in carcinogenesis outcomes, putatively driven by strain-specific dissimilarities and/or impact of modifier(s). Direct genetic analyses of iPSC clones will be undertaken to identify such modifier loci.

采用基因工程小鼠 (GEM) 癌症模型进行更具预测性的癌症药物功效评估的方法的广泛减少在很大程度上受到涉及 GEM 动物的操作固有的巨大成本的阻碍，而且还由于支持复杂遗传学项目的适当专业知识经常短缺。为了切实简化临床前研究中最费力和成本最高的部分（例如，涉及对 GEM 模型的相当大的繁殖群体进行操作，以及频繁的基因分型，以识别具有适当等位基因组合的动物），我们将从一系列经过充分表征并被证明与临床相关的 GEM 模型或癌变模型（包括非小细胞肺、脑、卵巢模型）中构建和表征 ES 和 iPSC 系库。 浆液性上皮细胞和黑色素瘤癌症类型。为了在 iPSC 衍生效率方面实现最佳结果，同时保持体外重编程输入细胞群的足够遗传多样性，我们将应用基于腺病毒载体基因转移的技术，通过该技术，重组病毒在感染遗传差异的细胞池时瞬时表达多顺反子自加工多肽。后者将衍生自从属于不同窝的胚胎收集的胎儿组织，以确保在许多多等位基因 GEM 模型中典型的混合背景杂交中等位基因的随机分离所规定的遗传多样性。由此产生的带有所需修饰基因座集的 iPSC 克隆（每个模型至少五个）将在产生嵌合动物群之前进行广泛的体外表征，这些嵌合动物现在被指定为非种系 GEM (NG-GEM) 模型，用于临床前药物评估研究和进行基本癌症机制实验。在第一组研究中，嵌合动物将被用于在特定组织（例如大脑或卵巢）中诱导肿瘤发生，并随后通过病理组织学和基因表达技术对肿瘤进行评估，以对比传统饲养动物中出现的类似诱导肿瘤。选定的嵌合动物子集将进一步接受药理学干预，联合治疗已被证明可有效恢复新生癌症小鼠模型中的肿瘤生长。如上所述，将进行代表性的临床、组织学和分子分析，以比较评估 iPSC 衍生的嵌合动物中的抗癌药物反应，使用从常规饲养的荷瘤小鼠的一组类似测定中收集的可用数据作为基线。对两种相似但技术上不同的方法进行这种全面的交叉评估，旨在进一步验证基于 iPSC 衍生的非种系 GEM 模型制备荷瘤动物临床前队列的稳健且具有成本意识的策略。为了扩展构建的 iPSC 文库的优势，在另一组体内实验中，将使用不同 iPSC 克隆（尽管具有相同的诱导致癌等位基因组合）开发的几组 iPSC 衍生 NG-GEM 进行比较并置，以识别致癌结果的变化，这些变化可能是由菌株特异性差异和/或 修饰符。将对 iPSC 克隆进行直接遗传分析，以确定此类修饰基因座。