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

CHO细胞是目前用于表达复杂的、翻译后修饰的重组生物制药蛋白的工业系统的选择。目前，CHO宿主细胞已被开发用于商业生产单抗，然而许多新的蛋白质生物制品难以表达和/或细胞成分可能存在问题的下游加工问题。在CHO细胞生物学中，一个很少受到关注的领域是脂类的生物合成以及脂类对细胞表型(细胞生长、产物产量)的影响。脂类是细胞膜的主要成分，是能量代谢、细胞信号传递、细胞生长和存活、细胞器形成以及通过囊泡形成和运输运输/分泌所必需的。在肯特，我们已经证明，操纵脂质生物合成可以导致分泌的重组蛋白产量增加，并改变CHO细胞释放的囊泡的形态和性质。这项拟议的项目将阐明CHO DG44和CHOK1宿主细胞系中脂类代谢的关键基因和途径，然后对这些关键基因和途径进行操作，确定随后对培养活力、细胞生长、分泌生物治疗蛋白、囊泡(特别是外切体)生产的产量和质量以及对初级下游加工事件的影响。具体来说，研究计划将包括：工作包(WP)1：对CHO宿主和重组生物治疗生产细胞系中的关键脂类代谢目标进行siRNA和CRISPR基因敲除/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中确认的靶基因敲除/敲除对细胞表型(生长、重组蛋白产量和质量)、脂肪代谢、囊泡/外切体产生以及从细胞培养上清液中初步回收步骤的影响。WP3：根据WP2的结果(选择前2-5个目标)，建立目标基因被击倒或过度表达的新型工程CHO宿主细胞系，并评估这些细胞系用于重组蛋白生产、外切体生产和初步回收。

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.