Manipulation and engineering of lipid metabolic pathways in CHO cells to enhance processability of cell culture supernantants

CHO 细胞中脂质代谢途径的操作和工程，以增强细胞培养上清液的可加工性

• 批准号: 2873335
• 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: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2873335
• 关键词: Manipulation; engineering; lipid; metabolic; pathways

The CHO cell is the current industrial system of choice for the expression of complex, post-translationally modified recombinant biopharmaceutical proteins Current CHO host cells have been developed for the commercial manufacture of monoclonal antibodies in particular, however many new protein biologics are difficult to express and/or cellular components can present problematic downstream processing issues. One area which has received little attention in CHO cell biology is lipid biosynthesis and the impact of lipids on cell phenotype (cell growth, product yields). Lipids are the major component of cellular membranes, are integral to energy metabolism, cell signalling, cell growth and survival, organelle formation, and transport/secretion via vesicle formation and trafficking. At Kent we have shown that manipulation of lipid biosynthesis can result in increased secreted recombinant protein production and changes in the morphology and nature of vesicles released from CHO cells. The proposed project will elucidate key genes and pathways in lipid metabolism in CHO DG44 and CHOK1 host cell lines and then manipulate these, determining the subsequent impact on culture viability, cell growth, yield and quality of secreted biotherapeutic protein, vesicle (specifically exosome) production and on primary downstream processing events. Specifically the programme of research will involve; Work package (WP) 1: Perform siRNA and CRISPR knockdown/out experiments of key lipid metabolism targets in the CHO host and recombinant biotherapeutic producing cell lines and confirm knockdown/out by qRT-PCR, genome sequencing and western blotting. WP2: Assess the impact of knockdown/out of targets confirmed in WP1 on cell phenotype (growth, recombinant protein yield and quality), lipid metabolism, vesicle/exosome production and primary recovery steps from cell culture supernatants. WP3: Generate novel, engineered CHO host cell lines with target genes either knocked down or over expressed based on WP2 results (select top 2-5 targets) and assess these for recombinant protein production, exosome production and primary recovery.

CHO细胞是目前表达复杂的、翻译后修饰的重组生物制药蛋白的工业系统选择。目前的CHO宿主细胞已被开发用于单克隆抗体的商业化生产，然而许多新的蛋白质生物制品难以表达和/或细胞成分可能会带来有问题的下游处理问题。 CHO 细胞生物学中很少受到关注的一个领域是脂质生物合成以及脂质对细胞表型（细胞生长、产物产量）的影响。脂质是细胞膜的主要成分，是能量代谢、细胞信号传导、细胞生长和存活、细胞器形成以及通过囊泡形成和运输进行运输/分泌的组成部分。在肯特大学，我们已经证明，脂质生物合成的操作可以导致分泌型重组蛋白产量的增加以及 CHO 细胞释放的囊泡的形态和性质的变化。拟议的项目将阐明 CHO DG44 和 CHOK1 宿主细胞系脂质代谢的关键基因和途径，然后对其进行操作，确定随后对培养物活力、细胞生长、分泌生物治疗蛋白的产量和质量、囊泡（特别是外泌体）产生的影响以及主要下游加工事件。具体来说，研究计划将涉及：工作包 (WP) 1：对 CHO 宿主和重组生物治疗药物生产细胞系中的关键脂质代谢靶点进行 siRNA 和 CRISPR 敲除/敲除实验，并通过 qRT-PCR、基因组测序和蛋白质印迹确认敲除/敲除。 WP2：评估 WP1 中确认的靶点敲除/敲除对细胞表型（生长、重组蛋白产量和质量）、脂质代谢、囊泡/外泌体产生以及细胞培养上清液初级回收步骤的影响。 WP3：根据 WP2 结果（选择前 2-5 个靶标）生成新的、工程化的 CHO 宿主细胞系，其中靶基因被敲低或过度表达（选择前 2-5 个靶标），并评估这些细胞系的重组蛋白生产、外泌体生产和初级回收。