Department of Biotechnology and Biomedicine, Section for Medical Biotechnology, Biotherapeutic Glycoengineering and Immunology, Technical University of Denmark, Kongens Lyngby, Denmark.
Department of Biotechnology and Biomedicine, Section for Medical Biotechnology, Biotherapeutic Glycoengineering and Immunology, Technical University of Denmark, Kongens Lyngby, Denmark.
N Biotechnol. 2024 Nov 25;83:101-109. doi: 10.1016/j.nbt.2024.07.004. Epub 2024 Jul 28.
Engineering of extracellular vesicles (EVs) towards more efficient targeting and uptake to specific cells has large potentials for their application as therapeutics. Carbohydrates play key roles in various biological interactions and are essential for EV biology. The extent to which glycan modification of EVs can be achieved through genetic glycoengineering of their parental cells has not been explored yet. Here we introduce targeted glycan modification of EVs through cell-based glycoengineering via modification of various enzymes in the glycosylation machinery. In a "simple cell" strategy, we modified major glycosylation pathways by knocking-out (KO) essential genes for N-glycosylation (MGAT1), O-GalNAc glycosylation (C1GALT1C1), glycosphingolipids (B4GALT5/6), glycosaminoglycans (B4GALT7) and sialylation (GNE) involved in the elongation or biosynthesis of the glycans in HEK293F cells. The gene editing led to corresponding glycan changes on the cells as demonstrated by differential lectin staining. Small EVs (sEVs) isolated from the cells showed overall corresponding glycan changes, but also some unexpected differences to their parental cell including enrichment preference for certain glycan structures and absence of other glycan types. The genetic glycoengineering did not significantly impact sEVs production, size distribution, or syntenin-1 biomarker expression, while a clonal influence on sEVs production yields was observed. Our findings demonstrate the successful implementation of sEVs glycoengineering via genetic modification of the parental cell and a stable source for generation of glycoengineered sEVs. The utilization of glycoengineered sEVs offers a promising opportunity to study the role of glycosylation in EV biology, as well as to facilitate the optimization of sEVs for therapeutic purposes.
通过对其亲本细胞进行遗传糖基工程化，实现细胞外囊泡 (EVs) 的工程改造，以实现更有效的靶向和特定细胞摄取，从而为其作为治疗剂的应用带来了巨大的潜力。碳水化合物在各种生物相互作用中发挥着关键作用，是 EV 生物学的基础。目前尚未探索通过对其亲本细胞的遗传糖基工程化，EV 上糖基化修饰的程度能够达到何种程度。在这里，我们通过基于细胞的糖基工程化，通过修饰糖基化机制中的各种酶，引入了 EV 的靶向糖基化修饰。在“简单细胞”策略中，我们通过敲除（KO）与 N-糖基化（MGAT1）、O-半乳糖胺糖基化（C1GALT1C1）、糖脂（B4GALT5/6）、糖胺聚糖（B4GALT7）和唾液酸化（GNE）相关的必需基因，修饰了主要的糖基化途径，这些基因涉及糖链的延伸或生物合成。基因编辑导致细胞表面相应的聚糖发生变化，这可以通过差异凝集素染色来证明。从细胞中分离出的小细胞外囊泡 (sEVs) 显示出整体相应的聚糖变化，但与亲本细胞也存在一些意想不到的差异，包括对某些聚糖结构的富集偏好和缺乏其他聚糖类型。遗传糖基工程化对 sEVs 的产生、大小分布或 syntenin-1 生物标志物的表达没有显著影响，而对 sEVs 产生产量的克隆影响则观察到。我们的研究结果表明，通过对亲本细胞进行遗传修饰，可以成功实现 sEVs 的糖基工程化，并为生成糖基工程化 sEVs 提供稳定的来源。糖基工程化 sEVs 的利用为研究糖基化在 EV 生物学中的作用以及为治疗目的优化 sEVs 提供了一个有前途的机会。
