Development of ESiPSC approach for non-germline GEM modeling

一种采用基因工程小鼠（GEM）癌症模型进行更具预测性的癌症药物疗效评估的方法的广泛实践受到涉及GEM动物的操作固有的显着成本的阻碍，但也由于经常缺乏支持复杂遗传学项目的适当专业知识。切实简化临床前研究中最费力、最昂贵的部分（例如，涉及对GEM模型的相当大的繁殖群体的操作和频繁的基因分型以鉴定具有适当等位基因组合的动物），我们将从充分表征并证明是临床相关的GEM模型或致癌作用的集合构建和表征ES和iPSC系的文库，包括非小细胞肺癌、脑癌、卵巢浆液性上皮癌和黑色素瘤癌症类型。为了在iPSC衍生的效率方面实现最佳结果，同时维持经受体外重编程的输入细胞群体的足够遗传多样性，我们将应用基于腺病毒载体基因转移的技术，由此重组病毒在感染遗传上不同的细胞池时瞬时表达多顺反子自加工多肽。后者将反过来来源于从属于不同窝的胚胎收集的胎儿组织，以确保由许多多等位基因GEM模型典型的混合背景杂交中等位基因的随机分离规定的遗传多样性。产生的带有所需的一组修饰基因座的iPSC克隆（每个模型至少五个）将在产生嵌合动物的群组之前进行广泛的体外表征，嵌合动物现在被指定为非种系GEM（NG-GEM）模型，用于临床前药物评价研究和进行关于基本癌症机制的实验。在第一组研究中，嵌合体动物将用于在特定组织如脑或卵巢中诱导肿瘤发生的目的，随后通过病理组织学和基因表达技术维斯维斯在常规饲养的动物中出现的类似诱导的肿瘤评价肿瘤。所选的嵌合动物亚组将进一步接受药理学干预，其中治疗剂的组合被证明在从头癌症小鼠模型中有效逆转肿瘤生长。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，所述嵌合动物采用在来自常规繁殖的荷瘤小鼠的一组类似测定中收集的基线可用数据。这种对两种相似但技术上不同的方法的全面交叉评价旨在进一步验证基于iPSC衍生的非生殖系GEM模型制备携带肿瘤动物的临床前队列的稳健和成本意识策略。为了扩展构建的iPSC文库的益处，在另一组体内实验中，将用不同iPSC克隆（尽管携带诱导型致癌等位基因的相同组合）开发的iPSC衍生的NG-GEM的几个群组进行比较并置，以鉴定致癌结果的变化，这是由菌株特异性差异和/或修饰剂的影响驱动的。将进行iPSC克隆的直接遗传分析以鉴定此类修饰基因座。

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.