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

推进基于 UCOE 的基因表达平台，以改善生物制造和基因治疗应用。

• 批准号: 1668272
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1668272
• 关键词: Advancing; UCOE; based; gene; expression

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.

背景资料。无处不在的染色质开放元件(UCOE)由至少一个启动子区域和管家基因的CpG岛组成，提供稳定的转基因表达。基于UCOE的载体在生物制造方面取得了商业上的成功，并为基因治疗应用提供了前所未有的慢病毒载体表达的稳定性。然而，对转录和转录后调控机制的理解的进步意味着基于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类HNRPA2BI-CBX3(A2UCOE)的原型UCOE和小鼠RPS3定位的UCOE。对可重复和稳定的泛在或组织特异性功能的需求是研究基因和蛋白质功能、有效的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，鉴于该项目中计划的基于UCOE的病媒开发的一般性，这项工作将对学术界和工业部门产生深远的影响。即将开发的强大和通用的下一代基于UCOE的载体的开发将极大地加快学术界和生物制造领域的研究人员的工作。研究方法。应聘者将获得广泛的基础分子生物学知识，并接受广泛的哺乳动物细胞组织培养和分子生物学(如重组DNA)方面的培训。细胞转染法、慢病毒载体、DNA/RNA分析、流式细胞术)研究技术。重要的是，该项目将提供从进行基础科学调查到考虑商业规模使用的第一手经验。所获得的技能将用于解决以下项目目标：目的1.提高载体在生物制造中的效率。前-mRNA剪接、3‘端形成和转录终止是耦合的，基因表达中的速率限制步骤。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微孔A2和RPS3 UCOE质粒载体中，以有效地提供所有这些转录后事件。注意：有望在5L生物反应器中进行工业放大测试的设计。目的2.生成具有增强安全特性的基于UCOE的L VS。在学术领导的S实验室中适配现有的LV，以生成用于插入任何感兴趣基因的通用最小化A2UCOE盒。旨在最小化A2UCOE遗传毒性的LV设计，可以通过异常剪接和阅读A2UCOE的转录本来实现，将通过包含适当定位的miRNA靶点来解决。Aim 3.基于UCOE的睡美人(SB)转座子载体的生成。尽管UCOE越来越吸引基因治疗应用，但UCOE尚未在SB载体中进行测试。因此，基于A2UCOE的盒式磁带将在SB载体上进行测试。目的4探索UCOE的作用机制。进行生物信息学研究，以突出UCOE共同占据的转录因子(TF)结合部位，这些结合部位可能是UCOE的功能基础。通过突变分析和细胞表达研究，对确定的转铁蛋白结合位点进行功能测试。