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

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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: