Every organism has a different glycosylation profile, and this has a big impact on the therapeutic potential and effectiveness of glycoprotein biologics. In general, the process of glycosylation originates in the endoplasmic reticulum (ER). Many glycosylation stages are completed by Golgi-localized glycosyltransferases that subsequently create mature glycan structures. In order to modify glycosylation and humanize the glycosylation profile of expression hosts, a variety of genetic techniques, including gene knockdown, knockout, and knockin, overexpression and modification, and so on, have been proposed. These techniques alter heterogeneity, sialylation, fucosylation, and branching structures.

Breakthrough
Numerous experimental scenarios have shown that gene knockout is a useful tool in glycan engineering. For instance, highly glycosylated human chorionic gonadotropin (hCG) hormone is expressed by Chinese hamster ovary (CHO) cells that have had GalT4 knockdown and GnT-IV and -V overexpression. RNA interference (RNAi)-mediated knockdown was employed to decrease glycosyltransferase activities that are unique to plants. Additionally, in CHO cells, combined siRNA knockdown of GDP-mannose 4,6-dehydratase (GMD) and α1,6-fucosyltransferase (FUT8) offers a novel approach to producing entirely non-fucosylated therapeutic antibodies with increased ADCC.

Utilizing Precision Genome Editing to Knockout and Knockin
Unwanted enzymes are usually inactivated in glycoengineering methods to maximize activity by preventing the transfer of individual monosaccharides to glycan structures. There have already been reports of knockouts of α1,6-fucosyltransferase (FUT8) in CHO, α1,6-mannosyltransferase (Och1p) in yeast, α1,3-fucosyltransferase (FUT) and β-N-acetylglucosaminidase (β-hex) in insect cells, and β1,2-xylosyltransferase (XylT) in plants. By using gene knockout techniques, the manufacturing of undesirable or host-specific glycol-epitopes could also be stopped. In addition, human-specific glycosyltransferase-based knockin techniques have been developed to humanize plant N-glycosylation. It has been established that the insertion of human glycosylation machinery can be accomplished efficiently by glycoengineering and the combined knockout/knockin strategy of glycosylation-related enzyme genes.