Exploring the utility of a stable synthetic “nano”chromosome in a biotech-friendly yeast for production of humanized glycoproteins

We propose a new approach to genetically engineering yeast cells so that they make therapeutic molecules more reliably, quickly, and cheaply. This involves providing yeast cells with a completely new, synthetic, extra chromosome into which multiple human genes can be inserted and then edited with precision. An increasing number of new drugs are proteins rather than small organic molecules. Virtually all therapeutic proteins have chains of sugars (or “glycans”) that are attached to them and hence they are called “glycoproteins”. Examples include monoclonal antibodies, clotting factors, cytokines and enzymes. More and more glycoprotein therapeutics are receiving approval for treating conditions from Alzheimer’s disease to arthritis, asthma, heart disease and cancer. They cannot be synthesized in the chemistry laboratory but must be “bio”manufactured, i.e. produced biologically, using genetically modified cells. Today, most glycoproteins are biomanufactured using engineered mammalian cells. Our study is a partnership with Fujifilm Diosynth Biotechnologies, the world’s second largest biopharmaceutical producer. The biomanufacture of therapeutic glycoproteins is a multibillion-pound industry and the cost and complexity of production in mammalian cells contributes to the expense of rolling out potentially breakthrough therapies. This delays approvals, or makes them barely affordable, exacerbating health inequalities across the globe. As an alternative to relying on mammalian cells cultured in expensive media and elaborate facilities, worldwide interest has focussed on “microbial cell factories” based on engineered bacteria or yeast that, unlike mammalian cells, grow quickly and cheaply to high densities in simple facilities. But a problem with using microbes is the need for the aforementioned glycans that are chemically complicated and must be attached at precisely defined locations on the protein. While mammalian cells decorate proteins with glycans that closely resemble human ones, this key capability is beyond most microbes. Bacteria do not add sugars at all while yeast produces proteins bearing chemically simple glycans consisting mainly of multiple mannose units, unsuitable for humans. Although high-mannose glycans can be removed, “deglycosylated” proteins are not as stable, long-lived in plasma, or functionally active as glycoproteins bearing human-like glycans. We propose to reprogramme the biochemical pathways that construct glycans in yeasts. Using the “biotechnology-friendly” yeast Pichia pastoris as host cells, we will insert a tiny but stable synthetic chromosome (a “nanochromosome”) of our own design, which has dedicated "landing pads" for new genes. We'll use these landing pads to add at least ten genes required to synthesise human-like glycans. Our engineered yeast cells will still attach high-mannose sugars to their own glycoproteins, a capability that can be repressed when the expression of human genes from the nanochromosome is induced. This will allow cells to grow healthily up to the point when they are instructed to start making the target humanised glycoprotein. The extensive genetic manipulations needed for this approach cannot be performed on the cell’s natural chromosomes without causing damage that multiplies with the number of gene insertions, since each insertion requires cleavage and repair of native DNA. Conversely, insertions of genes into our nanochromosome is, by design, more precise and reproducible, making it easier to use automation in order to systematically optimise the process, and to edit the inserted genes. By providing a dedicated vehicle, the nanochromosome, for carrying new genes, our strategy will yield robust yeast strains with pristine native genomes, suitable for commercial biomanufacture of human-like therapeutic glycoproteins or, indeed, other high-value products.