What if our pharmaceuticals could be grown in plants? It might sound like science fiction, but two VIB/UGent research groups are working hard to scrap the “fiction” part. In a recent paper published in Nature Biotechnology, Prof. Nico Callewaert and Prof. Ann Depicker describe how they addressed pharming’s most pressing problem: plant glycosylation.
“Pharming,” or the use of plants as production systems for therapeutic proteins, has been problematic for a long time. The biggest hurdle lies in the process of protein glycosylation: animal and plant cells show important differences in sugar residues present on the proteins. Because of this, plant-produced therapeutic proteins that are injected into mammals or humans can cause severe allergic and immunogenic reactions, since the plant glycans are recognized as foreign substances.
Shearing off the sugars
Through a close collaboration, the VIB/UGent research groups of Prof. Nico Callewaert and Prof. Ann Depicker are now well on their way to solving this longstanding problem.
“Different strategies are possible for this problem,” Callewaert says. “You could shut off the genes coding for the enzymes that carry out glycan synthesis, for instance. This is not as evident as it sounds, as there are many genes involved in glycosylation. Generating many knock-out lines and breeding them into one stable line would be a very time-consuming project. Although such an approach is possible with model plants, it wouldn’t be feasible with true crops, which have long generation times. Many crops are also polyploid, further complicating the matter.”
“We opted to use a different strategy, applying our so-called GlycoDelete technology. By combining a single knock-out with one overexpression, we were able to completely remove the sugar residues from the proteins, and for many protein types this is a robust solution.”
The GlycoDelete technology was developed in Callewaert’s lab, originally for use in mammalian cells to circumvent glycan heterogeneity and simplify protein purification. GlycoDelete encompasses two interventions: knocking out the gene encoding the GnT I enzyme (N-acetylglucosaminyltransferase I) and overexpressing the endoT enzyme (endo-N-acetyl-β-D-glucosaminidase) present in the fungus Hypocrea jecorina. In doing so, Callewaert’s team created an efficient tool for cleaving glycans at a molecular level. Combined with the transgene of a therapeutic protein of interest, plants can produce glycan-free pharmaceuticals that won’t elicit allergic reactions.
GlycoDelete: no downsides
Of course, glycosylation happens for a reason. Doesn’t cutting these sugar chains impair protein functionality? Not according to Callewaert:
“In our earlier studies with GlycoDelete, we thoroughly checked for function loss in a number of protein categories, but we found none. We also witnessed no phenotypical changes in the mammalian GlycoDelete cell lines, and the GlycoDelete plants developed in a perfectly normal fashion.”
“We mainly focus on monoclonal antibodies, since these are such an important part of today’s pipeline of medicines. Antibodies are used for various purposes: some are meant to kill tumor cells, while others are designed simply to neutralize certain factors in the body. Our GlycoDelete antibodies mainly belong to the second group. By using GlycoDelete, we do not make proteins lose their function — we modulate their function. Of course, this technique isn’t the ultimate answer to every protein production problem in plants, but it still offers a good solution for many of them.”
Beans as pills
Plants offer one very big plus in terms of protein production: storage. Plant seeds can contain a large amount of proteins and store them for extended periods of time. Beans and peas, for instance, can be preserved for years in a dried state without losing their germination potential — all essential components remain present within the seeds. This would be extremely useful in situations such as pathogen outbreaks, where vast amounts of therapeutic proteins would be needed quickly. The possibility to store therapeutic proteins would decouple production from downstream processing for the first time, enabling a more efficient and flexible workflow.
But that’s not the only advantage of seeds as protein storage vehicles:
“Also, the administration of therapeutic proteins to the large intestine is facilitated by seed storage,” Callewaert explains. “Seeds containing therapeutics can simply be eaten. The seed matrix also acts as a protective capsule for the protein as it passes through the harsh environments of the stomach and small intestine. Seeds are very well suited for those applications.”
The applications for GlycoDelete plants are numerous, but, as always, a lot of work still needs to be done. “Our publication deals with a proof of concept study in Arabidopsis,” Callewaert says. “The next step will be implementing the GlycoDelete technology in crops with protein-rich seeds.”
We surely look forward to hearing more about this exciting technology!
Piron, Robin, et al. "Using GlycoDelete to produce proteins lacking plant-specific N-glycan modification in seeds." Nature biotechnology 33.11 (2015): 1135-1137.
Meuris, Leander, et al. "GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins." Nature biotechnology 32.5 (2014): 485-489.
Photo courtesey © VIB