Biopharmaceuticals are nowadays
still mostly produced either in bacteria or in mammalian cells.
The great disadvantage of bacterial systems is that proper
protein folding and modification like glycosylation (addition of
sugar moieties) is not assured. Mammalian systems on the other
hand are very cost intensive and have the risk of carrying
viruses that are able to infect humans. That is why many
researchers aim at using plants as inexpensive and safe
bioreactors.
The drawback of many of these
attempts is very low protein yield which makes purification
still too expensive. But recently researchers have discovered
that seeds are a promising alternative, as transgenic proteins
seem to accumulate at a higher level. A second advantage is that
seeds can be stored for weeks and even months without the
product being degraded or destroyed, which would make
manufacturing and transportation much easier. In addition there
already exist well-established seed fractionation procedures for
the major crops.
However public concern about
possible outcrossing of transgenes is high, which is why
self-containment has to be ensured. “As rice is primarily a
self-pollinated crop, it offers particular attraction in terms
of containment of transgenes, in addition to providing obvious
advantages associated with producing protein-based medicines in
seeds for direct administration to human patients (novel drug
delivery).” explained Dr. Ravinder Sardana from the Department
of Cellular and Molecular Medicine at the
University of Ottawa.
An additional advantage is that
rice is edible, which could in certain cases eliminate the need
for purification of the product. This is why Dr. Sardana´s
research group chose rice seeds to produce a widely used and so
far very expensive drug, the cytokine Granulocyte
Macrophage-Colony Stimulating Factor (GM-CSF). Cytokines are
molecular messages that organisms use to communicate. For
instance in humans, cytokines can be released to let the immune
system know there is an infection.
This cytokine is essential in
fighting infections by regulating production and function of
granulocytes and macrophages. Abnormally low numbers of these
immune cells, a state called neutropenia, is a common result of
cancer chemotherapy. However, neutropenia can also result from
other causes such as hereditary diseases or viral infection.
Recombinant GM-CSF is clinically used also after bone marrow
transplantation to accelerate immune system recovery and has a
number of other possible applications, such as in HIV patients.
But the high costs of producing it in yeast and E.coli so far
limits its widespread use.
In their work, which was
published in a recent issue of
Transgenic
Research, the authors introduced the full coding sequence of
the human GM-CSF gene attached to a special rice promoter into
rice cells by the help of Agrobacterium.
After breeding a couple of
plants, they confirmed the presence of their target by several
methods. An important outcome was that the cytokine accounted
for 1.3% of total protein in the rice varieties, which is much
higher than in the attempts tried before with tobacco and
sugarcane plants. In addition this is enough to imagine
reasonable low production costs for commercial use.
The second fundamental question
was of course, if the purified cytokine is active and usable.
Therefore the researchers tested a human cell line which is
known to grow only in the presence of GM-CSF. They showed that
cells indeed grew when adding rice seed extract and when they
compared cell numbers with a sample where they used commercially
available GM-CSF, they found it to be similar. This was an
important finding because it showed that rice-produced GM-CSF
can be used as a biopharmaceutical.
As previously mentioned,
modification of a protein, in this case glycosylation, is an
important issue. The authors show in their publication, that the
protein is indeed glycosylated, although they want to spend more
time to find out, if the glycosylation pattern of this
plant-derived protein is the same as in human. Previous research
on GM-CSF showed that modification is not essential for its
activity, but it could well influence activity levels and immune
system answers.
To further improve their
findings, Dr. Sardana´s group is considering coupling the gene
to another promoter, which already showed to augment protein
yields to 4% of total protein. This would decrease production
costs further.
So there is some fine-tuning,
which can be done, but as the authors explain in their
publication in Transgenic Research, “This is the first report
where an additional protein, biologically active human
recombinant protein GM-CSF, has been produced in the seeds of
transgenic rice plants.” The method was already patented by the
group, which is the first step to go for commercial production
of GM-CSF in rice.
Frauke Focke is a Science
Writer for Checkbiotech in Basel, Switzerland.
Publication:
Ravinder Sardana et. al.
Biologically active human GM-CSF produced in the seeds of
transgenic rice plants.
Transgenic
Research 2007 Dec;16(6):713-21.
CONTACT:
Dr. Anil Dudani
Department of Cellular and Molecular Medicine,
Faculty of Medicine,
University of Ottawa
451 Smyth Road
Ottawa, ON, K1H 8M5
Canada
Patent:
http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=7214862.PN.&OS=PN/7214862&RS=PN/7214862
