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

通过使用普遍存在的染色质开放元件 (UCOE) 改善多能干细胞及其分化后代中的转基因表达

• 批准号: 244040345
• 负责人: Professor Dr. Thomas Moritz
• 金额: --
• 依托单位: Institut Experimentelle Hämatologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/244040345?language=en
• 关键词: Improved; Transgene; Expression; Pluripotent; Stem

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.

对于多能细胞如胚胎干（ES）和诱导多能干（iPS）细胞及其分化后代的有效遗传修饰，转基因沉默是主要障碍之一。在最近的一项原理验证研究中，我们的团队证明，小鼠模型中普遍存在的染色质开放元件 (UCOE)，特别是源自人类 HNRPA2B1/CBX3 位点 (A2UCOE) 的 1.5 kb 遗传元件，可以通过稳定转基因来克服这个问题在非分化 ES/iPS 细胞及其造血后代中的表达。利用确定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（造血和心脏）以及替代小鼠（神经元、肝脏和心脏）分化途径。此外，我们打算在应用四倍体互补技术的已建立的小鼠胚胎发生模型中评估 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之外，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 A2UCOE 的确定的缺失和取代突变体进行结构功能研究。此外，我们将分析特定病毒整合位点及其表观遗传环境对 A2UCOE 功效的作用。为此，将对一组具有单次半随机整合的 ESC/iPSC 克隆进行克隆分析，并结合对在同一指定位点包含或不包含 A2UCOE 的载体的克隆进行研究。这些克隆将通过翻转重组酶介导的盒交换（FRMCE）产生，旨在研究转录沉默异染色质中以及活跃转录基因转录起始位点附近的两种整合。因此，我们项目的成功完成将建立一种通用方法来规避多能干细胞及其衍生后代中的转基因沉默，从而对从多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际意义。此外，这里建议的研究旨在增强我们对分子，特别是对 UCOE 功效负责的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转基因沉默的分子和表观遗传机制。