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Biotechnology and Society---Part
22
Transgenic therapeutics - Medicines in milk
An equation means nothing to me unless it expresses a thought of God. - Srinivasa Ramanujan (1887-1920), Indian mathematician
In a previous article we
described how food crops like corn and cash
crops like tobacco are being
genetically modified to produce human therapeutics through gene transfer. The
same procedure can be adopted to genetically modify animals and produce
therapeutic proteins in their milk. In both these cases, the primary advantage
is the cost of production. Let us examine the process of producing human
proteins in transgenic animals.
Transgenic animal creation: The
first transgenic animal, a lowly mouse, was created in Yale University (New
Haven, CT, USA) in 1980. Since then many other transgenic animals, including
sheep, goats, cows, rabbits, pigs and chicken were created at universities and
corporate research laboratories. After 'Dolly', the first cloned sheep, was
created, the technology and the publicity associated with the prospects and
perils of animal cloning have gained widespread attention.
There
are two methods by which transgenic animals can be produced: pronuclear
microinjection, and nuclear transfer. Pronuclear microinjection is a relatively
simple method involving linking about 200-300 copies of a gene of interest to a
promoter (the forerunner of the gene) of choice and injecting the foreign DNA
through a fine glass needle into fertilised animal eggs which are then implanted
into surrogate mothers to be carried to term and delivered. The success rate of
this method to produce the transgenic animal is between 1 and 5 per cent of
which only a fraction expresses the adventitious gene at a desired high level.
The nuclear transfer method was
used successfully to clone the sheep, Dolly, and later other animal species. The
method involves modification of actively dividing foetal cells with a marker
gene (to identify the modified cells), and the human gene of interest, and
selecting the clones which contain the added genes. The second step is to remove
the unfertilised eggs from the animal chosen, and remove the nucleus from the
egg. The cells selected above are then subjected to electro-fusion (fusion
through application of an electric field) with the enucleated (nucleus removed)
mature oocytes (eggs obtained in the second step above). The fused cells are
then transferred to a recipient animal capable of reproduction. After the normal
gestation period, the surrogate mother will deliver the transgenic animal. The
success rate in this method is better than the microinjection method and is
currently the method of choice to produce transgenic animals.
It is now possible to use
somatic cells (cells other than germ cells) instead of foetal cells for cloning
purposes. Somatic cells can be obtained and cultured in large numbers so they
are amenable to gene manipulation. One of the key advantages of nuclear transfer
over the older microinjection technique is that it saves time and thus reduces
costs. Two features of nuclear transfer are helpful in this: the certainty of
obtaining female founders by using a cell line derived from a female source, and
the ability to store genetically modified cells for use in creating identical
females at any time.
If one were to make therapeutic
proteins in the milk of transgenic animals, the procedures should make sure that
the right control genes are put in besides the gene for the protein in question
and they should be directed to go to the mammary glands of the animal so that
the protein will be expressed in the milk. The success rate then is measured by
the extent of expression of the protein in the milk of the animal.
Production systems and
companies: Most transgenic firms use large farm animals like cows,
sheep, and
goat in order to obtain the economic advantages of obtaining large amounts of
milk from fewer number of herds. GTC Biotherapeutics (Framingham, MA) uses both
goats and cows to produce more than 60 therapeutic proteins, including plasma
proteins, monoclonal antibodies and vaccines. They have both in-house projects
as well as contract projects with other pharmaceutical companies. One product
that is in late stages of testing is recombinant human anti-thrombin III (rhATIII)
(produced in goat milk), an anti-coagulant protein found in blood. Currently,
the product is derived from processing outdated blood but the demand exceeds the
supply.
GTC is also working on another
in-house project to develop a malaria vaccine, MSP-1, under a grant from the
National Institutes of Health, from goat milk. It appears eight goats can
produce enough of this vaccine to inoculate 20 million individuals. A litre of
goat milk can contain up to 9 g/L of the transgenic protein. The cost to produce
a transgenic protein in goat milk can thus be 3 to 30 times cheaper than the
current method using mammalian cell culture. GTC has a 300-acre goat and dairy
farm in Massachusetts that is USDA-certified. Once a transgenic herd is
established then the development costs will be greatly reduced.
GTC is also in collaboration
with various companies such as Abbott, Centocor (a subsidiary of Johnson &
Johnson), Alexion Pharmaceuticals, Immunogen, BASF, Bristol Myers Squibb and
Eli
Lilly among others.
Some other companies such as
Pharming, NV (Leiden, The Netherlands) and PPL Therapeutics (Edinburgh, UK, and
Blacksburg, VA) have either severely curtailed their operations or suspended
their programmes completely due to monetary constraints or withdrawal of
collaborative partners. Pharming, NV developed the first transgenic bull
(Herman) in the late 1980s and developed a line of transgenic cows to produce
several proteins including human lactoferrin and alpha glucosidase. PPL was
working with rabbits and sheep to produce alpha-1-antitrypsin, fibrinogen, and a
lipase to treat pancreatic insufficiency in digesting dietary lipids.
Departing from the trend line
of using large farm animals, Bio-Protein Technologies (Paris) specialises in
producing therapeutic proteins in rabbit milk. The main advantage of using
rabbits is the shorter development time and time-to-market. The gestation time
for rabbits is only one month (as opposed to 9 months for goats and cows) and
the female rabbits mature sexually in just four months. Rabbits are also
prolific breeders. These two features will more than compensate for the low milk
production from each rabbit. A cow can produce 20 litres of milk per day
compared to 0.25 litres per day for the rabbit. Bio-Protein’s potential
products include antibodies, plasma proteins, and hormones.
Purification: Expressing a
human protein in animal milk is only the first step. The level of the
therapeutic protein is hardly 1 per cent of the total proteins in the milk. The
total proteins represent only 4 per cent of the milk by weight. Hence, the
challenge will be to purify the foreign protein first by isolating it from the
rest of the constituents and then purifying it to exacting standards. One
important point to remember is to get rid of the last traces of milk proteins
from the therapeutic protein since a small but significant portion of the
population is allergic to milk proteins. The purification costs for processing
transgenic milk will not be very different from those incurred in cell culture.
Safety issues: While the
traditional biotechnology companies making therapeutics via fermentation and
cell culture have addressed the safety concerns very well, the transgenic
companies face some new safety concerns such as difficult-to-detect pathogens
such as prions and the unknown impact of animal viruses on humans. The incidence
of bovine spongiform encephalopathy (BSE), and transmissible spongiform
encephalopathy (TSE) known as 'Mad cow disease' (in cows) and 'scrapies' (in
sheep) prevalent in the beef and meat industry is also a red flag.
The transgenic firms counter
that charge by pointing out that milk-producing animals do not typically harbour
viral pathogens that plague humans. Besides, animal lineage can be traced
rigorously. Once a pedigree is deemed clear of prions, the controlled
environment in which the animals are reared guarantees continued safety. The
regulatory agencies in the US and Europe are not overly concerned about the
safety issues although they have set up appropriate guidelines.
Another
issue that might come up in the future concerns the possibility of the
transgenic animals being slaughtered and brought into the food chain.
The transgenic animals which express foreign protein in milk will also
express it in other tissues at very low levels. Other concerns involve
animal welfare. Calves and lambs produced through cloning often have
higher birth weights and longer gestation times than regular ones. As
a result, births are often difficult and require a Caesarean delivery.
The Center for Science in the
Public Interest, a watchdog organisation in the US, emphasies that it is
imperative that transgenic animals are not released into the environment or
allowed to enter the food supply without a thorough assessment by the
government.
Ultimately, it is not the
actual safety issues or the declaration of safety by government agencies that
will determine the success of the transgenic animal therapeutics but the market
economics and the public perception of the safety issues, as is the current
situation with genetically modified crops.
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