Development of ESiPSC approach for non-germline GEM modelling

使用基因工程小鼠(GEM)癌症模型进行更具预测性的癌症药物疗效评估的方法被广泛减少到实践中，这在很大程度上受到涉及GEM动物的手术固有的巨大成本的阻碍，也因为在支持复杂的遗传学项目方面经常缺乏适当的专业知识。为了切实简化临床前研究中最费力和最昂贵的部分(例如，涉及大量GEM模型的繁育群体的操作和频繁的基因分型，以识别具有适当等位基因组合的动物)，我们将从一系列具有良好特征并被证明与临床相关的GEM模型或癌变，包括非小细胞肺癌、脑、卵巢浆液上皮和黑色素瘤癌症类型的集合中构建和表征ES和IPSC系的文库。为了实现IPSC衍生效率的最佳结果，同时保持经过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的基因转移技术，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处理多肽。后者将从从属于不同胎次的胚胎收集的胎儿组织中获得，以确保在许多多等位基因宝石模型中典型的混合背景杂交中由等位基因随机分离所规定的遗传多样性。产生的带有所需的一组修饰基因的IPSC克隆(每个模型至少五个)将经过广泛的体外鉴定，然后产生嵌合动物队列，现在被称为非生殖系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的实验。在第一组研究中，嵌合动物将被用于在特定组织(如大脑或卵巢)中诱导肿瘤形成的目的，以及随后通过病理组织学和基因表达技术与传统饲养动物中出现的类似诱导肿瘤相比对肿瘤进行评估的目的。选定的嵌合动物亚组将进一步接受药物干预，治疗方法的组合被证明在逆转新生癌症小鼠模型的肿瘤生长方面有效。如上所述，将进行具有代表性的临床、组织学和分子分析，以比较评估IPSC来源的嵌合动物的抗癌药物反应，使用从传统培育的荷瘤小鼠的类似测试中收集的可用数据作为基线。这种对两种相似但技术上不同的方法的全面交叉评估旨在进一步验证基于IPSC衍生的非生殖系GEM模型准备肿瘤动物临床前队列的稳健和成本意识策略。为了扩大构建的IPSC文库的好处，在另一组体内实验中，用不同的IPSC克隆开发的几组IPSC衍生的NG-gem(尽管携带相同的可诱导致癌等位基因组合)将被引入比较并列，以确定致癌结果的差异，可能是由菌株特异性差异和/或修饰物的影响(S)驱动的。将对IPSC克隆进行直接遗传分析，以确定这些修饰基因座。

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.