Fig. 1: Principle of homologous recombination (HR). Flanking of the ingoing sequence (red) by surrounding wild type sequences of the targeted locus (green) allows for the precise exchange of a defined stretch of genomic information. The technique enables knock-out and knock-in as well as any punctual or small-stretch modifications.
Genetic manipulation of producer-organisms becomes crucial, when the quality of the produced pharmaceutical protein fails to meet the required standards. Such quality determinants could be e.g. a specific structure of N-glycans but as well post-translational trimming by a specific human protease, which is not present in the producing organism.
In contrast to systems based on mammalian, insect or other plant-cells, Genome modification is straightforward and effective in Physcomitrella. Timelines and effort for such modifications are in line with those of a yeast system.
Like in the latter, genome engineering in the moss is based on a mechanism known as homologous recombination (HR; Fig. 1).
Physcomitrella is unique amongst higher eukaryotes in the occurring rate of HR, which routinely reaches values of about 50% among transgenics. This high rate opens the opportunity to use HR as a technological tool for engineering the mosses genome.
Further prerequisites for such efficient engineering approaches come from two sides:
The first is the sequenced genome, which is highly annotated and therefore supplies the essential basis for planning any engineering approach.
The second is the haploid nature of the predominant stages within the Physcomitrella life cycle. Haploid cells harbour only one copy of their genome. As a consequence, genome modifications directly take effect and cannot be compensated by a second, unmodified copy as in all diploid systems. Basically, the laborious steps of sexual crossing and Mendelian selection are gratuitous in the moss, ensuring extremely short timescales.
Efficient HR, together with genomic data and the haploid nature of the moss, enable us to customise the genomic background (Fig. 2) of our production system for every specific product.
Fig. 2 : Genome customisation.
Greenovation has used genome customising extensively to optimise the N-Glycan structures of produced proteins.
Fig. 3: Glyco-Engineering. Removal of plant-specific, potentially immunogenic glycan-residues.
Plant N-Glycans carry two specific carbohydrate-residues lacking in other organisms. Those are an α-1,3-linked fucose and a β-1,2-linked xylose (Fig. 3), which are discussed to bear immunogenic potential when present on recombinant, pharmaceutical proteins.
Fig. 4: Glyco-peptide_MS.
Greenovation managed to remove those structures quantitatively from the mosses N-Glycans. We therefore engineered the genome by making use of the HR-technique [link HR] and knocked out the loci of two Glycosyl-Transferases, responsible for the attachment of xylose and fucose to the N-glycan-structures.
N-glycan structures on proteins produced in this double-knockout strain are 100% free of plant-specific xylose and fucose residues. Their typyical structure is bisected, ending with an N-acetyl-Glucosamin (GlcNAc)-residue at both ends (Fig. 4 ).