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

基因治疗产品用于治疗多种适应症并在临床上作为潜在的疫苗显示出巨大的前景，但生产高滴度病毒载体和规模化生产仍然是一个挑战，并增加了这些治疗的成本。此外，在使用mRNAs作为治疗和预防性疫苗方面的最新进展也需要进一步开发它们的设计和制造，以降低成本和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，并通过我们的研究和文献确定了在比较导致较低或正确组装和包装的AAVs的条件时，表达增加或减少的细胞靶点。我们还开发了设计mRNAs的方法，当通过体外转录(IVTmRNAs)产生时，这些mRNAs可以提供更高的蛋白质表达，用于治疗或候选疫苗。拟议的PHD项目将结合基因组编辑、siRNA敲除和过度表达研究来确定单个和多个靶点的操纵对HEK293细胞产生AAV的影响，从而加深我们对支撑HEK293细胞产生AAV的基本细胞生物学的了解，并创建新的、具有工业相关性的HEK293细胞系，用于增强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IVTmRNAs的功能。研究计划是；第一阶段：在HEK293细胞系中用siRNA敲除或过表达10-20个候选基因，并确定对AAV生产和基因组包装的影响。从得到的数据中，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HEK293的细胞系中进行双重敲除、双重过度表达或敲除+过度表达，结合前2个靶点，评估对AAV产生的影响。选择TOP组合(S)。第三阶段：评估IVTmRNA序列对HEK293细胞中这些IVTmRNAs的RNA编辑和蛋白质表达的影响。阶段4：评估TOP结合(S)形成的阶段2单独或联合对HEK293细胞产生AAV载体的影响。

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.