Advancing the UCOE-based gene expression platform fo r improved bioma nufacturing and gene therapy applications.

背景普遍存在的染色质开放元件（UCOE）由至少一个启动子区域和来自管家基因的相关的无甲基化CpG岛组成，并赋予稳定的转基因表达。基于UCOE的载体在生物制造中取得了商业成功，并为基因治疗应用提供了前所未有的慢病毒载体（LV）表达稳定性。然而，对转录和转录后控制机制的理解的进展意味着基于UCOE的载体的设计可以改进。该项目旨在改进基于UCOE的载体，用于（i）生物制造和（ii）基因治疗。该工作计划将使用来自人HNRPA 2BI-CBX 3（A2 UCOE）和鼠Rps 3基因座的原型UCOE。对可重复和稳定的普遍存在或组织特异性功能的需求是研究基因和蛋白质功能、有效基因治疗和工业蛋白质生物制造过程的基础研究的核心。因此，鉴于本项目中计划的基于UCOE的病媒开发的一般性质，这项工作的影响将在学术和工业部门产生深远的影响。强大的和多功能的下一代基于UCOE的载体的开发将大大加快学术界研究人员的工作，生物制造。通过提供基因表达领域最先进的技术，为临床和基因治疗提供帮助。研究方法。候选人将获得基础分子生物学的广泛知识，并获得广泛的哺乳动物细胞组织培养和分子生物学（例如重组DNA）方面的培训。细胞转染、慢病毒载体、DNA/RNA分析、流式细胞术）研究技术。重要的是，该项目将提供将基础科学调查纳入商业规模使用考虑的第一手经验。获得的技能将用于解决以下项目目标：目标1。提高生物制造中质粒载体的效率。前体mRNA剪接、3 '末端形成和转录终止是基因表达中的限速步骤。因此，内含子、多聚腺苷酸化和转录终止元件将被添加到现有的EMD Millipore A2和Rps 3 UCOE质粒载体中，以有效地提供所有这些转录后事件。生成具有增强安全性特征的基于UCOE的LV在学术带头人的实验室中调整现有LV以生成通用的最小化A2 UCOE盒，用于插入任何感兴趣的基因。LV设计旨在最小化A2 UCOE遗传毒性，其可以通过异常剪接和通读A2 UCOE转录物发生，将通过包括适当定位的miRNA靶位点来解决。基于UCOE的睡美人（SB）转座子载体的产生：尽管UCOE在基因治疗应用中越来越有吸引力，但还没有在SB载体中进行测试。因此，A2 UCOE为基础的盒，在LV（目标2）将在SB载体进行测试。目标4探索UCOE的作用机制，为未来的发展。进行生物信息学调查，以突出占据的转录因子（TF）结合位点共同UCOE，可能是其功能的基础。通过突变分析和细胞表达研究，对鉴定的TF结合位点进行功能性检测。

Background. Ubiquitous chromatin opening elements (UCOEs) consist of at least one promoter region with associated methylation-free CpG island from housekeeping genes and confer stable transgene expression. UCOE-based vectors have seen commercial success in biomanufacturing and provide unprecedented stability of expression from lentiviral vectors (LVs) for gene therapy applications. However, advances in understanding of transcriptional and post-transcriptional control mechanisms imply design of UCOE-based vectors can be improved. This project aims to improve UCOE-based vectors for (i) biomanufacturing and (ii) gene therapy. The workplan will use the prototypical UCOE from the human HNRPA2BI-CBX3 (A2UCOE) and the murine Rps3 loci.Importance of project. The need for reproducible and stable ubiquitous or tissue specific function is at the core of basic research investigating gene and protein function, effective gene therapy and industrial protein biomanufacturing processes. Therefore given the generic nature of the UCOE-based vector developments planned in this project, the impact from this work will be far reaching in both academic and industrial sectors. The development of robust and versatile next generation UCOE-based vectors to be developed will greatly expedite the work of researchers within academia, biomanufacturing. and gene therapy settings by providing access to state-of-the-art technology in the gene expression field.Research methods.The candidate will acquire a broad knowledge of basic molecular biology and gain training in a wide range of mammalian cell tissue culture and molecular biology (e.g. recombinant DNA. cell transfection, lentiviral vectors, DNA/RNA analysis, flow cytometry) research techniques. Importantly, the project will provide first-hand experience of taking a basic science investigation through to consideration for commercial scale use. The skills acquired will be used to address the following project aims:Aim 1. Improving the efficiency of plasmid vectors in biomanufacturing.Pre-mRNA splicing, 3'end formation and transcription termination are coupled, rate limiting steps in gene expression. Thus intronic, polyadenylation & transcription termination elements will be added to existing EMD Millipore A2 and Rps3 UCOE plasmid vectors to effectively provide all of these post-transcriptional events.NOTE: promising designs to be tested at 5L bioreactor scale-up during industrial placement.Aim 2. Generation of UCOE-based L Vs with enhanced safety features.Adapt existing LVs in academic lead 's laboratory to generate generic minimised A2UCOE cassettes for the insertion of any gene of interest. LV designs aimed at minimising A2UCOE genotoxicity, which can occur via aberrant splicing and read through transcripts from the A2UCOE, will be addressed by inclusion of appropriately located miRNA target sites.Aim 3. Generation ofUCOE-based Sleeping Beauty (SB) transposon vectors.Although increasingly attractive for gene therapy applications, UCOEs have yet to be tested within SB vectors. Thus A2UCOE based cassettes as those in LVs (Aim 2) will be tested in SB vectors.Aim 4 Explore UCOE mechanisms of action for future advances.Conduct a bioinfofmatics investigation to highlight occupied transcription factor (TF) binding sites common to UCOEs that may underlie their function. Functionally test activity of identified TF binding sites by mutational analysis and cell expression studies.