Defining ubiquitous chromatin opening element (UCOE) molecular mechanisms of action to expedite their biotechnological applications

该项目的主题福尔斯属于生物安全研究中心的优先领域“工业生物技术”，目的是开发生物资源，用于生产和加工材料和化学品，包括生物制药。该项目也有合成生物学的元素，这是BBSRC的另一个优先领域。普遍存在的染色质开放元件（UCOE）由一个或多个具有来自管家基因的相关无甲基化CpG岛的启动子组成（Neville JJ等人，生物技术高级，2017年）。UCOE具有显著的染色质重塑能力，赋予稳定和增强的转基因表达。用于蛋白质治疗性生物制造的基于UCOEs的表达平台已由Merck MilliporeSigma提供多年，并且已经取得了相当大的商业成功（例如参见https：//tinyurl.com/yyfwfhxp; Neville等人，2017年）。此外，UCOE提供了从慢病毒载体表达的前所未有的稳定性（Neville等人，2017).在即将结束的BBSRC iCASE奖下，申请人和Merck MilliporeSigma通过优化UCOE-病毒启动子组合和包含提高前mRNA加工效率的元件，改进了基于UCOE的质粒载体设计，用于生物制造。然而，这些质粒载体的大小仍然很大，这可能妨碍它们的操作容易性和一旦引入细胞的完整性。我们建议进行以下调查，以克服这些限制。UCOE的功能机制在很大程度上仍然未知。介导UCOE活性的关键转录因子（TF）尚未鉴定。这种缺乏基本的UCOE分子生物学知识，阻碍了它们的完善和进一步应用。因此，本申请的目的是使用跨学科的计算机模拟和功能性基因表达分析来定义UCOE作用机制，特别是关于鉴定介导其活性的TF，这反过来将导致设计合成的、紧凑的、更通用的UCOE，产生新的IP并进一步加速其生物技术应用。为了实现这一目标，将进行以下调查：

The theme of this project falls under the BBSRC priority area "industrial biotechnology" with the objective of developing biological resources for producing and processing materials and chemicals including biopharmaceuticals. This project also has elements of synthetic biology, another BBSRC priority area. Ubiquitous chromatin opening elements (UCOE) consist of one or more promoters with associated methylation-free CpG island from housekeeping genes (Neville JJ et al., Biotech Adv., 2017). UCOE possess dominant chromatin remodelling capabilities confering stable and enhnaced transgene expression. UCOEs-based expression platforms for protein therapeutic biomanufacturing has been offered by Merck MilliporeSigma for many years and has seen considerable commercial success (eg see https://tinyurl.com/yyfwfhxp; Neville et al., 2017). In addition, UCOEs provide unprecedented stability of expression from lentiviral vectors (Neville et al., 2017).Under a BBSRC iCASE award, which is about to end the applicant and Merck MilliporeSigma have advanced UCOE-based plasmid vector designs for biomanufacturing by optimising UCOE-viral promoter combinations and inclusion of elements to enhance efficiency of pre-mRNA processing. However, the size of the these plasmid vectors remains large, which can be a hinderance to their ease of manipulation and intactness of once introduced into cells. We propose the following investigation to overcome these limitations. UCOE mechanisms of function remain largely unknown. Crucially transcription factors (TFs) that mediate UCOE activity are unidentified. This lack of basic UCOE molecular biology knowledge has held up their refinement and further application. Thus, the aim of this application is to use interdisciplinary in silico and functional gene expression analyses to define UCOE mechanisms of action, especially with respect to identifying the TFs that mediate their activity, which will in turn lead to the design of synthetic, compact, more versatile UCOEs generating new IP and further expediting their biotechnological application. In order to achieve this aim the following lines of investigation will be undertaken: