Pharming, recombinant medicines and synthetic biology

Medicines that are proteins

Large or extensively modified therapeutic proteins are usually produced in living cells because correct folding and post-translational processing are difficult to reproduce by chemical synthesis. The required processing determines whether bacterial, yeast or mammalian cells are suitable. Some short peptides are synthesised chemically, and cell-free expression systems exist, so cellular production is a practical requirement for large glycoproteins rather than an absolute rule for all protein drugs.

Before recombinant DNA technology, protein medicines were extracted from an organism that already made them, and the two named examples on the specification show the consequences of that.

Factor VIII is the clotting factor missing in the commonest severe haemophilia. For decades it was concentrated from donated blood plasma, pooled from thousands of donors per batch. In the early 1980s that pooling proved catastrophic. Plasma from a single infected donor contaminated whole batches, and a large fraction of the people treated for haemophilia in that era were infected with HIV, hepatitis C or both. Heat and solvent-detergent treatment of the concentrates, introduced from 1985, stopped transmission of HIV and then hepatitis C.

Recombinant factor VIII went further and removed the donor pool itself. It has been made since the 1990s by cultured mammalian cells carrying the human gene, so no blood-borne pathogen, known or not yet discovered, can reach the product by that route.

Describe the drawing

A graph with the number of donations pooled into one batch on a logarithmic horizontal axis from one to twenty thousand, and the probability that the batch holds at least one infected donation on the vertical axis from nought to one. Three curves are drawn, one for each rate of undetected infection in a single donation: one in a thousand, one in ten thousand and one in a hundred thousand. At the left-hand edge, where the pool is a single donation, the three sit a hundredfold apart and all of them are on the floor of the graph. By the dashed line marking a batch of five thousand donations they have risen to about ninety-nine per cent, thirty-nine per cent and five per cent, and by twenty thousand donations to a hundred, eighty-six and eighteen per cent, so the hundredfold difference between the three has collapsed to less than sixfold.

Adenosine deaminase (ADA) is the enzyme whose absence causes one form of severe combined immunodeficiency, the condition you met in the gene therapy lesson. Before gene therapy, and still alongside it, patients are treated with injections of the enzyme itself, chemically stabilised so it survives in the blood. It was first purified from cattle and since 2018 has been produced recombinantly. The same condition therefore has two recombinant treatments: supplying the protein, or supplying the gene.

Mammalian cells are preferred to a cheaper bacterial fermenter because E. coli transcribes and translates a human gene without carrying out the processing that follows. Factor VIII requires chaperoned folding, disulfide bridges at specific positions, and sugar chains added in a Golgi apparatus. A bacterium cannot supply that processing, so it yields a polypeptide with the correct primary structure and without the required folding or glycosylation. Insulin is small and requires little post-translational modification, so it can be produced in bacteria. The required processing determines the expression host.

Recombinant medicine

A protein drug produced by cells carrying an introduced copy of the human gene, rather than extracted from blood or tissue.

Factor VIII

The clotting protein deficient in haemophilia A, produced recombinantly in mammalian cell culture since the 1990s.

Adenosine deaminase

The enzyme missing in ADA-deficient severe combined immunodeficiency, supplied to patients as a recombinant protein.

Pharming: expression in milk

Pharming uses genetically modified organisms, including plants and animals, to produce pharmaceutical substances. A tissue-specific promoter can direct expression in milk, but yield depends on the construct and species.

Mammalian cell culture is effective but expensive: the cells grow slowly, require a rich sterile medium, and yield from milligrams per litre for complex proteins such as factor VIII to grams per litre for antibodies. A lactating transgenic animal secretes the protein into milk, which is collected by milking and is a simpler starting material to purify than cell culture medium or plasma. Reported yields vary widely between constructs and species, from milligrams to several grams per litre, so quote a figure with its source rather than a general rate.

Describe the drawing

The genetic construct used in pharming, drawn as a length of DNA carrying a promoter taken from a goat milk-protein gene immediately upstream of the human gene for the wanted protein. An arrow leads down through injection into a fertilised egg to a row of four tissue cards for the resulting adult animal: mammary gland, liver, muscle and skin. Every tissue carries the construct, but only the mammary gland card is highlighted as reading it, because the promoter is active nowhere else, so the human protein is secreted into the milk and purified from it.

The construct is the part to describe precisely. The human gene is joined downstream of the promoter of a milk protein gene, such as β-lactoglobulin. The transcription factors that switch that promoter on exist only in mammary gland cells during lactation. The construct is microinjected into the nucleus of a fertilised egg, or introduced into cultured cells from which an embryo is then cloned by nuclear transfer, and the embryo is implanted into a surrogate.

Microinjection can produce mosaic founders, in which only some cells carry the insert, so animals must be screened and a confirmed line established before production begins. A female of that established line carries the gene in every cell and expresses it in one tissue. Her milk carries the human protein, her blood and other tissues carry very little, and purification starts from milk.

Antithrombin is the licensed example: an anticoagulant protein produced in the milk of a herd of transgenic goats. It was approved as a drug in Europe in 2006, the first medicine from a transgenic animal to be licensed. A herd of goats can supply quantities that would otherwise require very large numbers of blood donations.

Pharming

The use of genetically modified organisms, including plants and animals, to produce pharmaceutical substances; in livestock, classically in milk.

Mammary-specific promoter

The promoter of a milk protein gene, placed in front of an introduced gene so that the gene is transcribed only in mammary gland cells during lactation.

Plants as factories for medicines

Pearson Edexcel Biology A names three kinds of genetically modified organism used to make drugs: microorganisms, animals and plants. Microorganisms make human insulin in fermenters, and animals are covered above. Plants are the third.

The human gene is joined to a promoter that plant cells recognise and introduced with Agrobacterium tumefaciens or a gene gun. Two approaches are used. In a stable transgenic plant the gene is inserted into a chromosome and inherited through seed, so the crop can be grown year after year. A seed-specific promoter can confine the protein to the seeds, where it keeps well in storage, much as a milk-protein promoter confines a goat's product to her milk.

In transient expression, the leaves of Nicotiana benthamiana, a fast-growing relative of tobacco, are flooded with agrobacteria carrying the gene. The leaf cells make the protein for a few days without the gene entering their chromosomes, and the leaves are then harvested and the protein extracted. Transient expression takes weeks, where establishing a transgenic line takes years.

Three examples show the range. Taliglucerase alfa is an enzyme given to people with Gaucher disease, whose cells lack it, and is made by genetically modified carrot cells grown in culture vessels. In 2012 it became the first plant-made medicine approved for human use in the United States. ZMapp, a mixture of three monoclonal antibodies against Ebola virus, was made by transient expression in Nicotiana benthamiana and given to some patients in the 2014 West African outbreak. The trial that followed was too small to show conclusively whether it worked. A COVID-19 vaccine made of virus-like particles produced in the same plant was approved in Canada in 2022, though the company that made it closed the following year.

Containment has failed before. In 2002 maize plants engineered to make a vaccine protein for pigs sprang up from fallen seed in a Nebraska field that had been replanted with soya beans. The soya harvested there had to be destroyed. Most plant-made medicines are now produced in non-food plants such as Nicotiana benthamiana, in greenhouses, or in plant cell culture in closed vessels. That removes the escape route at the cost of some of the cheapness.

Plant-made pharmaceutical

A medicine, usually a protein, produced by genetically modified plants or plant cells carrying the gene for it.

Transient expression

Production of a protein from a gene delivered into cells for a few days without being inserted into their chromosomes.

Welfare and regulatory considerations

The arguments about pharming are set out below on each side, with the evidence for each.

Welfare, against. Making a founder animal is inefficient. Microinjection succeeds in a small percentage of eggs, so producing one transgenic goat means many surrogate pregnancies, failed implantations and discarded embryos. Where cloning is used it brings its own losses, since a large fraction of cloned pregnancies fail late or produce unhealthy offspring. Critics add a deeper objection: that redesigning an animal's genome to serve as production equipment treats a sentient creature as an instrument, whatever its day-to-day conditions.

Welfare, for. Once the founder exists, the producing herd is bred normally and kept as a dairy herd. Milking does not harm the animal, and the commercial requirement for clean, consistent milk means the animals are housed and fed to a high standard. Supporters also compare the alternatives: the protein would otherwise come from pooled human plasma, with the safety record described above, or from cell culture at a cost some health systems will not meet.

Regulation. A drug from a living animal poses questions a steel fermenter does not. Milk composition varies with the animal, the season and the feed, so every batch must be purified and tested to prove it identical to the last. The herd must be certified free of infections, and of prion disease in particular, that could pass into the product. The animals themselves must be contained, because a transgenic goat that bred into an ordinary herd would spread a pharmaceutical gene that could never be recalled. Regulators answered with closed, certified herds, animals identified and tracked individually, and approval processes that took years. The animals are also patented, one instance of the ownership question treated below.

Synthetic biology: parts, genomes and pathways

Recombinant DNA technology introduces one gene at a time into an organism that is otherwise unchanged. Synthetic biology assembles characterised genetic components and pathways for defined functions, treating DNA as an engineering material that is specified, synthesised to order, and assembled into circuits and pathways not found in any organism.

It has three levels. Standard parts: promoters, ribosome binding sites, genes and terminators catalogued with defined, measured behaviour, designed so that any part joins to any other, the way electronic components share standard connections. An engineer picks parts from the registry and assembles a device instead of hunting each sequence from scratch. Genome synthesis: whole genomes built chemically from their sequence data.

A bacterial genome of about a million base pairs was synthesised and installed in a recipient cell in 2010, and the cell ran on it. Stripped-down versions since have asked what the minimum set of genes for life actually is. Engineered pathways: several genes, from several organisms, assembled so that a cell runs a metabolic production line it never had.

Describe the drawing

Three tiers showing the synthetic biology method. At the top, five separated standard parts sit in a row: a promoter, three genes and a terminator, each drawn as a labelled block. An arrow carries them to the middle tier where the same blocks are joined end to end with no gaps, promoter first and terminator last. A second arrow drops the assembly into a large rounded outline standing for a yeast cell, inside which a short pathway runs from sugar through an intermediate to artemisinic acid, with each arrow between them labelled as an enzyme made from one of the added genes, and a final line notes that a few chemical steps in a factory finish the antimalarial artemisinin.

The named pathway example is artemisinin, the antimalarial used in combination therapy worldwide. It is extracted from sweet wormwood, a crop whose yield and price varied substantially between years. A pathway of enzymes, some from the plant and some derived from yeast's own metabolism, was assembled in brewer's yeast. The yeast converts sugar into artemisinic acid, which industrial chemical steps then convert into artemisinin. Production began at scale in 2013 as a supply that does not depend solely on the harvest, but it was suspended within about two years when plant-derived artemisinin became cheaper.

Synthetic biology

The design and construction of new biological parts, devices and pathways, and the redesign of existing organisms, treating DNA as a specifiable engineering material.

Standard part

A DNA sequence with a defined function, catalogued and designed to be assembled with any other standard part.

Biosecurity and ownership

The same capability, DNA synthesised to order, underlies both the applications and the risks of synthetic biology. The specifications require the arguments on each side, which are paired below.

Biosecurity. Poliovirus was assembled from mail-ordered DNA fragments and published in 2002, and an extinct relative of smallpox was synthesised in 2017 for around one hundred thousand dollars. The barrier to recreating a dangerous pathogen is therefore money and published sequence, not rare skill. Against that: the same openness lets any public-health laboratory synthesise a new pathogen's genes within days of its sequence appearing, and vaccine development now starts that way.

The response to the risk has been screening, not secrecy. Synthesis companies check orders against databases of dangerous sequences and flag the customers who place them. Screening is voluntary and coverage differs between suppliers, and the point at issue is whether voluntary screening suffices. Date any claim about screening practice or regulation, since both change.

Ownership. Engineered organisms and standard parts can be patented, and the case for is the usual case for patents. Development costs run to hundreds of millions, and no company recovers that without a period of exclusivity, so patents pay for the goats, the yeast and the trials. The case against has two layers. One is access: a patented medicine is priced by its owner, and the diseases synthetic biology could treat most cheaply are concentrated where patients are poorest.

The other is displacement, and artemisinin is the worked case. Fermenter production stabilises the drug supply and also competes with the smallholder farmers who grow wormwood, so the same process cuts supply risk for health systems and cuts income for those growers. A boundary question is also unresolved in law. An engineered gene sequence can be patented, while a naturally occurring human gene has not been patentable in the United States since 2013. Whether the design of an entire organism can be owned is not settled.