Modulating expression of candidate genes to improve lentiviral vector production in stable cell line

调节候选基因的表达以提高稳定细胞系中慢病毒载体的产量

• 批准号: 2752732
• 金额: --
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2752732
• 关键词: Modulating; expression; candidate; genes; improve

The use of gene therapy products for the treatment of a large range of indications and as potential vaccines in the clinic shows great promise but producing high titre viral vectors and manufacturing at scale remains a challenge and contributes to the cost of these therapies. Further, recent advances in the use of mRNAs as treatments and preventative vaccines also requires further development of their design and manufacturing to reduce cost and improve the potency of such therapies.At the University of Kent we have used HET293 cells to produce viral vectors, particularly adenovirus associated virus (AAV), for the delivery of gene therapies and through our studies and the literature identified cellular targets that are increased or decreased in expression when comparing conditions leading to lower or amounts of correctly assembled and packaged AAVs. We have also developed approaches to design mRNAs that when produce by in vitro transcription (IVTmRNAs) give higher protein expression for use as therapies or vaccine candidates. The proposed PhD project will use a combination of genome editing, siRNA knockdown and over-expression studies to determine the impact of manipulation of individual and multiple targets on AAV production from HEK293 cells, thus furthering our understanding of the basic cell biology that underpins AAV production from HEK293 cells and creating novel, industrially relevant HEK293 cell lines for enhanced AAV production. We will also work with the researchers at Southampton to determine if enzymes capable of mRNA editing impact the function of IVTmRNAs. The programme of research is; Phase 1: Knockdown by siRNA or over-express 10-20 candidate genes in HEK293 cell lines and determine the impact on AAV production and genome packaging. From the resulting data, select the top 5 candidate genes with the greatest impact on AAV production. Phase 2: Combine top 2 targets by performing double knockdown, double over-expression or knockdown + over-expression in HEK293 expressing cell lines, assess impact on AAV production. Select top combination(s). Phase 3: Assess impact of IVTmRNA sequence on RNA editing and protein expression from these IVTmRNAs in HEK293 cells. Phase4: Assess the impact of top combination(s) form Phase 2 either alone or in combination on AAV vector production from HEK293 cells.

基因治疗产品用于治疗大范围适应症和作为临床潜在疫苗显示出巨大的希望，但生产高滴度病毒载体和大规模生产仍然是一项挑战，并增加了这些疗法的成本。此外，在使用mrna作为治疗和预防性疫苗方面取得的最新进展也需要进一步发展其设计和制造，以降低成本并提高此类疗法的效力。在肯特大学，我们使用HET293细胞生产病毒载体，特别是腺病毒相关病毒（AAV），用于基因治疗的递送。通过我们的研究和文献，在比较导致正确组装和包装AAV的降低或数量的条件时，确定了表达增加或减少的细胞靶标。我们还开发了设计mrna的方法，当通过体外转录（ivtmrna）产生时，可以提供更高的蛋白表达，用于治疗或候选疫苗。拟议的博士项目将使用基因组编辑、siRNA敲低和过表达研究的组合来确定单个和多个靶点的操作对HEK293细胞产生AAV的影响，从而进一步了解HEK293细胞产生AAV的基础细胞生物学，并创建新的、工业相关的HEK293细胞系，以增强AAV的产生。我们还将与南安普顿的研究人员合作，确定能够编辑mRNA的酶是否会影响ivtmrna的功能。研究方案是；第1阶段：在HEK293细胞系中通过siRNA敲低或过表达10-20个候选基因，并确定对AAV产生和基因组包装的影响。从得到的数据中选出对AAV产量影响最大的前5个候选基因。第二阶段：在HEK293表达细胞系中，通过双敲低、双过表达或敲低+过表达，将前2个靶点组合在一起，评估对AAV产生的影响。选择最优组合。第三阶段：评估IVTmRNA序列对HEK293细胞中这些IVTmRNA的RNA编辑和蛋白质表达的影响。第4阶段：评估第2阶段的顶部组合单独或联合对HEK293细胞AAV载体生产的影响。