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

基因治疗产品用于治疗大范围的适应症和作为临床中的潜在疫苗显示出巨大的前景，但生产高滴度病毒载体和大规模生产仍然是一个挑战，并导致这些治疗的成本。此外，mRNA用作治疗和预防疫苗的最新进展也需要进一步开发其设计和制造，以降低成本并提高此类疗法的效力。在肯特大学，我们使用HET 293细胞来生产病毒载体，特别是腺病毒相关病毒（AV），对于基因治疗的递送，并且通过我们的研究和文献，当比较导致较低的正确组装的或量的细胞靶时，包装的AAVs。我们还开发了设计mRNA的方法，当通过体外转录（IVTmRNA）产生时，所述mRNA提供更高的蛋白质表达以用作疗法或疫苗候选物。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，以确定单个和多个靶点对HEK 293细胞AAV生产的影响，从而进一步加深我们对HEK 293细胞AAV生产基础细胞生物学的理解，并创建新的，工业相关的HEK 293细胞系以增强AAV生产。我们还将与南安普顿的研究人员合作，以确定能够编辑mRNA的酶是否会影响IVTmRNAs的功能。研究计划是;第一阶段：在HEK 293细胞系中通过SiRNA敲减或过表达10-20个候选基因，并确定对腺相关病毒生产和基因组包装的影响。从所得数据中，选择对AAV生产影响最大的前5个候选基因。第二阶段：通过在表达HEK 293的细胞系中进行双敲低、双过表达或敲低+过表达来组合联合收割机前2个靶标，评估对AAV生产的影响。选择最佳组合。第3阶段：评估IVTmRNA序列对HEK 293细胞中这些IVTmRNA的RNA编辑和蛋白表达的影响。第4阶段：评估第2阶段的最佳组合单独或组合对HEK 293细胞的AAV载体生产的影响。

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.