Improved Transgene Expression in Pluripotent Stem Cells and their Differentiated Progeny by the Use of Ubiquitous Chromatin Opening Elements (UCOE)

对于胚胎干细胞(ES)和诱导多能干细胞(IPS)及其分化后代等多能细胞的高效遗传修饰，沉默转基因是主要障碍之一。在最近的一项原则证明研究中，我们的团队证明了在小鼠模型中，普遍存在的染色质开放元件(UCOE)，特别是来自人类HNRPA2B1/CBX3基因座的1.5kb遗传元件(A2UCOE)可以通过稳定未分化的ES/iPS-细胞及其造血祖细胞中的转基因表达来克服这一问题。利用已定义的小鼠和人类分化模型，我们现在想要研究，这一概念是否也可以转移到人类间充质细胞(造血和心脏)以及可供选择的小鼠(神经、肝脏和心脏)分化途径。此外，我们打算利用四倍体互补技术在建立的小鼠胚胎发生模型中评价A2UCOE在ESC/IPSC体内分化过程中的转基因促进活性。除了这些更多的翻译方面，我们还想探索UCOE转基因稳定作用的分子基础。在这里，我们将利用已定义的A2UCOE的缺失和替换突变体进行结构功能研究。此外，我们将分析特定的病毒整合位点及其表观遗传环境对A2UCOE疗效的作用。为此，对一组具有单一半随机整合的ESC/IPSC克隆的克隆分析将与在同一定义位点上含有或不含有A2UCOE载体的克隆的研究相结合。这些克隆将通过翻转重组酶介导盒交换(FRMCE)产生，旨在研究转录沉默的异染色质中的两个整合以及活跃转录基因的转录起始点附近的整合。因此，我们的项目的成功完成将建立一种通用的方法来避免多潜能干细胞及其衍生后代中的转基因沉默，从而对从多潜能细胞来源产生转基因细胞治疗产品的整个领域具有直接的实用意义。此外，这里建议的研究旨在增强我们对UCOE疗效的分子，特别是表观遗传决定因素的了解。因此，从长远来看，该项目还将有助于更好地理解逆转录病毒基因转移后控制转基因沉默的分子和表观遗传机制。

For the efficient genetic modification of pluripotent cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells and their differentiated progeny silencing of transgene represents one of the main obstacles. In a recent proof-of-principle study, our group demonstrated that ubiquitous chromatin opening elements (UCOE) and specifically a 1.5 kb genetic element derived from the human HNRPA2B1/CBX3-locus (A2UCOE) in murine models can overcome this problem by stabilizing transgene expression in non-differentiated ES/iPS-cells as well as their hematopoietic progeny. Employing defined murine and human differentiation models, we now want to investigate, whether this concept can be transferred also to human mesenchymal (hematopoietic and cardiac) as well as to alternative murine (neuronal, hepatic, and cardiac) differentiation pathways. Furthermore we intend to evaluate the transgene promoting activity of the A2UCOE during the in vivo-differentiation of ESC/iPSC in an established murine embryogenesis model applying tetraploid complementation technology. In addition to these more translational aspects we also want to explore the molecular basis underlying the transgene stabilizing effect of UCOEs. Here, we will perform structure-function studies utilizing defined deletion and substitution mutants of the A2UCOE. Furthermore we will analyse the role of specific viral integration sites and their epigenetic environment on the efficacy of the A2UCOE. To this end, a clonal analysis of a panel of ESC/iPSC clones with single semi-random integration will be combined with the study of clones harbouring vectors containing or not containing the A2UCOE at the same defined site. These clones will be generated by flip recombinase-mediated cassette exchange (FRMCE) and it is intended to study two integrations each in transcriptionally silent heterochromatin as well as near to the transcriptional start site of actively transcribed genes. Successful completion of our project thus would establish a universal approach to circumvent transgene silencing in pluripotent stem cells and thereof derived progeny, and thereby would be of immediate practical relevance for the whole field of generating transgenic cell therapy products from pluripotent cell sources. In addition, the studies suggested here are designed to enhance our knowledge about the molecular and specifically the epigenetic determinants responsible for the efficacy of UCOEs. Hence, in the long run this project also will contribute to a better understanding of the molecular and epigenetic mechanisms governing the silencing of transgenes following retroviral gene transfer.