Inhibitor-Mediated Programming of Glycoforms

Our long-term vision is to routinely produce recombinant proteins as single defined glycoforms without the need for genetic manipulation. This will functionally be the equivalent for glycans as site-directed mutagenesis. We will showcase this by the production of therapeutically relevant proteins with homogenous pre-defined N-linked glycoforms. Our approach is a radically new approach to the manipulation of cell function; we will use the delivery of computationally defined mixtures of enzyme-specific inhibitors to tune the activity of glycosyltransferases and thereby the glycans displayed on secreted proteins such as antibodies, hormones and other therapeutic modalities. The untemplated nature of glycan synthesis leads to inherent heterogeneity in these glycans. This variation can compromise the behaviour of these complex molecules which are increasingly important in modern disease treatments. The complexity of glycan biosynthesis is staggering; in mammals there are up to 200 different glycosyltransferases (GTs) that compete for substrates within the Golgi, the organelle in which most glycans are synthesized. Generation of a given glycan structure depends on the activities and levels of a suite of glycosylation enzymes in each of the Golgi’s sub-compartments (cisternae). In order to manipulate glycans, we will build an understanding of how the arrangement of key biosynthetic enzymes, their activities and their locations specify glycan structures. Our approach, termed Inhibitor-Mediated Programming of Glycoforms (IMProGlyco) will provide an effective strategy to manipulate these enzymes and thereby deliver defined mixtures of glycosylated proteins. This strategy will be adaptable and expandable into other cell types and organisms and thereby enable scalable production of highly specified recombinant biomolecules. Supply of these will enable both production of high performance biotherapeutics and modulation of cell function together with fundamental studies of the consequence of variation in glycosylation patterns in laboratory studies.