Transfection of glycoprotein encoding mRNA for swift evaluation of N-glycan engineering strategies

Bydlinski Nina; Coats Michael T.; Maresch Daniel; Strasser Richard; Borth Nicole

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Transfection of glycoprotein encoding mRNA for swift evaluation of N-glycan engineering strategies

机译：糖蛋白的转染编码mRNA进行迅速评估N-Glycan工程策略

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N-glycosylation is defined as a key quality attribute for the majority of complex biological therapeutics. Despite many N-glycan engineering efforts, the demand to generate desired N-glycan profiles that may vary for different proteins in a reproducible manner is still difficult to fulfill in many cases. Stable production of homogenous structures with a more demanding level of processing, for instance high degrees of branching and terminal sialylation, is particularly challenging. Among many other influential factors, the level of productivity can steer N-glycosylation towards less mature N-glycan structures. Recently, we introduced an mRNA transfection system capable of elucidating bottlenecks in the secretory pathway by stepwise increase of intracellular model protein mRNA load. Here, this system was applied to evaluate engineering strategies for enhanced N-glycan processing. The tool proves to indeed be valuable for a quick assessment of engineering approaches on the cellular N-glycosylation capacity at high productivity. The gene editing approaches tested include overexpression of key Golgi-resident glycosyltransferases, partially coupled with multiple gene deletions. Changes in galactosylation, sialylation, and branching potential as well as N-acetyllactosamine formation were evaluated.

机译：N-糖基化被定义为大多数复杂生物治疗剂的关键质量属性。尽管许多N-Glycan工程努力，但在许多情况下仍然难以满足不同蛋白质可能改变不同蛋白质的所需的N-Glycan曲线的需求。稳定地生产具有更苛刻的加工水平的均匀结构，例如高度的分支和末端唾液化，特别具有挑战性。在许多其他影响因素中，生产力水平可以使N-糖基化朝向更少成熟的N-聚糖结构转向。最近，我们介绍了一种MRNA转染系统，其能够通过细胞内模型蛋白mRNA载荷的逐步增加阐明分泌途径中的瓶颈。这里，应用该系统以评估增强的N-Glycan处理的工程策略。该工具证明确实是有价值的，以便在高生产率下对工程方法的快速评估进行快速评估工程方法。测试的基因编辑方法包括关键的高压血糖转移酶的过表达，部分地偶联与多基因缺失。评价半乳糖基化，唾液酸化和支链潜力以及N-乙酰氢胺形成的变化。

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《Biotechnology Progress》 |2020年第4期|共6页
作者
Bydlinski Nina; Coats Michael T.; Maresch Daniel; Strasser Richard; Borth Nicole;
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作者单位

BOKU Univ Nat Resources &

Life Sci Dept Biotechnol Muthgasse 18 A-1190 Vienna Austria;

BOKU Univ Nat Resources &

Life Sci Dept Biotechnol Muthgasse 18 A-1190 Vienna Austria;

BOKU Univ Nat Resources &

Life Sci Dept Chem Vienna Austria;

BOKU Univ Nat Resources &

Life Sci Dept Appl Genet &

Cell Biol Vienna Austria;

BOKU Univ Nat Resources &

Life Sci Dept Biotechnol Muthgasse 18 A-1190 Vienna Austria;

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正文语种 eng
中图分类生物科学;
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机译：正常，压力，禁食和复苏大鼠的α亚1酸糖蛋白和血清白蛋白Ib肝脏的mRNa定量

Transfection of glycoprotein encoding mRNA for swift evaluation of N-glycan engineering strategies

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