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ISIS Report 09/11/11
Pharm Crops Ignoring Health & Environment
The
promises of pharm crops undermined by threats to
health and environment Prof. Joe Cummins
A fully referenced version of this report is posted on ISIS members website and is otherwise available for download here
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Pharmaceutical
drug production has undergone major changes following the development and
approval of drugs called ‘biologicals’ that are for
the most part proteins produced by genetic engineering. Biologicals make up at
least a quarter of new drug approved, though they are about twice as likely as
chemical drugs to experience regulatory action following approval (see ‘Biologicals’,
Wonder Drugs with Problems, SiS 42). The recombinant protein drugs
are produced using viruses, bacteria, yeast, and cell cultures
from insects, rodents, primates or humans. The use of genetically modified
(GM) crop plants to produce biologics has been an attractive prospect because
the crops are capable of producing vast quantities of recombinant proteins at
low cost. There have been a large number of such transgenic ‘pharm crops’ created
in the laboratory and field trialled, though none have been approved for
commercial drug production. However, some have now progressed to clinical
trials.
The
phases of clinical trials
Clinical
trials are separated into several phases. Phase 0 is
a recent designation for exploratory, first-in-human trials conducted in
accordance with the United States Food and Drug Administration (FDA). Phase 0
trials include the administration of single sub-therapeutic doses of the drug
to a small number of subjects (10 to 15) to gather preliminary data on the
agent's pharmacodynamics (what the drug does to the body) and pharmacokinetics
(what the body does to the drugs). Phase I trials are the first stage of
testing in human subjects involving a small (20-100) group of healthy
volunteers, and designed to assess the safety (pharmacovigilance), tolerability,
pharmacokinetics and pharmacodynamics of a drug. Once the initial safety of the
study drug has been confirmed in Phase I trials, Phase II trials are performed
on larger groups (20-300) to assess the drug’s efficacy, as well as to continue
Phase I safety assessments. When the development process for a new drug fails,
this usually occurs during Phase II trials. Phase III studies are randomized
controlled multicentre trials on large patient groups (300–3 000 or more
depending on the disease/medical condition) and are the definitive assessment
of the drug’s efficacy. When the phase III trial is completed and deemed
satisfactory, the drug may be released for use in treating
the general population.
Pharm
crops in clinical trials
The
first report of a plant-made therapeutic (PMT) protein reaching Phase II human
clinical trials is Locteron of Biolex Therapeutics,
Inc., a controlled-release interferon alfa (IFN-α) for treating chronic
hepatitis C. The current treatment involves weekly administration of an
IFN-α in combination with an antiviral drug ribavarin. In 2005, 32
patients participated in Phase IIa clinical trials of Locteron. The IFN-α
produced in the aquatic plant Lemna was administered fortnightly in
combination with ribavirin in a randomised double blind study. An early 100
percent virological response was achieved in all 16 hepatitis C patients
treated with 480 and 640 μg doses. Early virological response has been
established as a pre-requisite for long-term response in hepatitis C patients.
The first plant-made therapeutic directed for
human use to reach Phase III clinical trials was a carrot cell suspension
Gaucher’s disease therapeutic developed by Protalix BioTherapeutics. Gaucher’s
disease is the most common of the lysosomal storage diseases. It is caused by a
hereditary deficiency of the enzyme glucocerebrosidase (also known as acid
β-glucosidase), which breaks down the glycolipid glucocerebroside,
resulting in its accumulation particularly in white blood cells (mononuclear leukocytes),
and also in the spleen, liver, kidneys, lungs, brain and bone marrow. The
transgenic human glucocerebrosidase (human prGCD) in carrot cells were grown in
a bioreactor system. The purified recombinant protein was tested in Phase I/II
trials in 2006 before entering Phase III trials in 2009. Thirty-one patients
suffering from Gaucher’s disease were tested in a multi-centre, randomised,
double blind trial. The primary endpoint (20 percent average reduction of
spleen volume) was achieved in treated patients after only 6 months, and safety
analysis showed that the treatment was well tolerated with no serious or severe
adverse events reported. Patients who successfully completed this study were
granted expanded access, some for over two
and a half years. On December 1, 2009, Pfizer and Protalix entered into an
agreement to develop and commercialize prGCD for the treatment of Gaucher’s
disease, giving Pfizer exclusive worldwide licensing rights while Protalix
retains commercialization rights in Israel. However, in early 2011, the FDA
declined approval for the drug, asking for additional data from existing
studies, but not requiring any additional trials.
SemBioSys has also completed a phase I-II trial
of safflower-produced insulin, and found it to have a similar safety profile to
current recombinant insulin. The insulin is produced in oil bodies allowing for
simplified extraction, and the plants have been grown in open fields.
Pharm crop systems
Production of biological drugs in pharm crops is potentially hazardous,
particularly if it is undertaken in open fields. They should all be done, if at
all, under strictly confined containment. The threat of open field pharm crops ranges
from the contamination of the human food supply to impact of the biological drugs on
wildlife and farm animals. ISIS has written extensively on this issue over the
years (see Pharm crops ,
ISIS reports since 2002). Unfortunately the emphasis has been on the safety of the purified final biological drug and not on the
threat to the human food supply and to wildlife and farm animals. The regulatory
framework envisioned by the advocates of pharm crop drug production is far from
adequate to prevent catastrophic consequences of amplified open-field pharm
crop production.
The main plant-based platforms for production of
biological drugs or vaccines are the production of pharmaceutical proteins and
vaccines in seeds produced in open fields, and/or transient expression systems in
contained environments or bioreactor plant cell cultures. Some examples are given
below.
Seed-based production of biological drugs
Seed-based production of pharmaceutical drugs is achieved by
linking the promoters and signal sequences for seed proteins with the gene for
the pharmaceutical protein. The resulting human or animal pharmaceutical
protein or vaccine is usually stable for several years if maintained in a cool
dry environment. The most commonly employed seed crops include rice, maize and
safflower.
Boothe and colleagues of SemBioSys and Calgary University stated: “Physical containment strategies may be impractical or at least
uneconomical for most seed-based systems.” Biological containment such as male
sterility or “organelle expression” could be used, but for many species, physical
segregation may be the only practical method available. In other words, pharm
crops are to be grown in open fields, but at distances sufficient to isolate
the plants from any food production areas and from any close relatives that
could cross with the pharm crop and spread the gene. They suggested that the
use of largely or completely self-pollinating species could provide an
additional safeguard against transgene escape. And as the most likely cause of
food supply adulteration is through the inadvertent mixing of seed from pharm
crop and food crop of the same species, companies engaged in pharm crop
production must maintain a tight chain-of-custody over their transgenic seed.
For all these reasons, Booth and colleagues think “it is highly unlikely” that pharm
crops will ever be approved for unconfined release.
Nevertheless, SemBioSys has begun the final
stages of a clinical trial for human insulin produced in safflower.
Little or no mention has been made about the impact of
pharm crops on wildlife by those intending grow the crops in open fields. Human
insulin-producing crops were even tested in areas with threatened animal
species (see
GM safflower with
human pro-insulin, SiS 35). DNA
techniques have identified insulin-like peptide genes in invertebrates,
including insects, molluscs and nematodes; these findings clearly establish
that insulin is an evolutionarily ancient hormone present in all higher animals.
Insulin toxic shock may appear in honey bees, birds and other treasured
animals. However, the advocates of open field plant pharmaceutical production
have not been obliged to fully evaluate the impact on wildlife, nor have they
reported comprehensive ecological studies following open field tests.
Transgenic
synthetic approximation of human insulin-like growth
factor was produced at high levels in rice seeds and found to be active in rats.
Insulin-like growth factor has cancer promoting properties making open
field production highly hazardous (see Cancer
promoting transgenic rice, SiS 22).
Another seed-based biological in the pipeline is
for treatment of Alzheimer’s disease (AD). The main signs of AD are cognitive
impairment and plaques composed of amyloid beta (Aβ) protein in patients’
brains. Therefore, therapy for AD focuses on the removal of Aβ. An “edible
vaccine” was developed to boost intestinal immunity. Currently there are no
published reports on the ecological and health impacts of the oral vaccine produced
in open fields.
For the treatment of chronic allergic diseases
such as bronchial asthma and rhinitis, biologicals are being used to induce
oral tolerance to allergens such as those produced by house dust mites (HDM). HDM
allergens are a major cause of such diseases. Oral tolerance is a T
cell-mediated phenomenon involving a specific decrease in immune responses to
antigens previously encountered via the oral route. Oral tolerance has been demonstrated
in numerous species, including humans and mice, and can be achieved using many
different antigens. The dust mite allergens are heat labile, acidic
glycoproteins present in HDM faeces. To further increase the immunogenicity of
this therapy, researchers utilize the native machinery of the host plants. Several
plant seeds contain protein bodies (PBs), which function as sites of protein
storage and are resistant to proteolytic digestion. Bioencapsulation of
allergens into PBs is a strategy to increase the immunogenicity of allergens
for oral immunotherapy. However, the inclusion of an
antigen for which oral tolerance has already been established such as a
previous treatment for rice allergy, may lead to inhibition of responses to
other, unrelated antigens included in the immunization. This phenomenon is
known as ‘bystander suppression’. The induction of bystander suppression
has been demonstrated in multiple allergic diseases, but it has also been shown
to enhance susceptibility to infection by inhibiting antibody production. The
effect of HDM vaccine was antigen-specific because the levels of specific IgE
and IgG in mice secondary immunized with unrelated antigens in transgenic rice
seeds were not affected by oral vaccination with HDM rice. The results demonstrate
the ability of HDM to induce oral tolerance without associated bystander
suppression. The oral rice seed vaccine illustrates several of the problems
involved in production of seeds for oral vaccination including the care with
which oral tolerance must be considered in the event that the human population
is exposed to GM seeds containing antigens which lead to immune suppression. Most dangerous of
all is the use of a staple food crop for so many billions of people around the
world.
Despite concerns of genetic contamination, maize
seeds that produce heat labile E. coli enterotoxin (LT-B) in the seeds
of maize have been approved for testing in the open field in Iowa. The National
Environmental Policy Act evaluation of the field trial failed to take into
account the fact that bees may spread transgenic pollen while birds and small
mammals along with careless seed transportation could contaminate the human
food chain with LT-B and certainly threaten the human food produced in the
state of Iowa. Furthermore, there has been no report on whether or not the LT-B
antigen produced in maize causes induction of bystander suppression frequently
associated with vaccines subject to oral tolerance (see above).
There have been numerous other studies and field
trials on seed based oral vaccines and the studies reported above are typical
of such studies and field trials.
Transient expression systems
Transient expression systems are created in plant viruses with Agrobacterium.
The transient expression methods are capable of producing higher levels of the recombinant
proteins than permanent genetic modifications because many more copies of the
recombinant genes are active in each plant cell. A combination of the transient
expression methods is called magnifection. Magnifection involves using genetically
modified plant virus vectors to transform Agrobacterium strains that are
in turn allowed to infect the crop plant in a mild vacuum. The combined use of Agrobacterium
and plant virus vectors results in a large number of recombinant RNA virus
particles entering the plant cells. These recombinant RNA viruses
epigenetically produce high levels of recombinant proteins in a few days, and
then decline. The crop plants transfected by the modified viruses are used only
once, producing no long term foot print on the environment, it is claimed. The
process involves vacuum infiltration of whole plants with dilute suspensions of
agrobacteria carrying T-DNAs encoding RNA replicons. The bacteria provide
infection and systematic movement throughout the plant while the viral vector
provides short distance spread, amplification and high level expression. The
speed of the process is such that milligrams and grams of recombinant protein
are available in 3 to 4 weeks, and as much as 100 kg within less than a year.
Transient expression provides recombinant protein at up to 80 percent of the
total cell protein. The process is relatively inexpensive due to the speed of
production and high yield of recombinant protein that simplifies purification.
The whole process may be contained in a greenhouse; a one hectare greenhouse is
capable of producing 500 kg recombinant protein per year. Previous viral
vectors require a thousand times more space. The speed at which large
quantities of recombinant proteins can be produced means that recombinant
proteins can be tailor-made for diseases of individuals (see Magnifection
Safe Pharming or Doomsday Device, SiS 42).
However, it is imperative that the
strictest containment must be applied to the use of these transient systems.
The greenhouse must be closed, not just to insects and rodents, but also
outfitted with air filters that prevent virus particles from escaping.
Otherwise, it could create and spread catastrophic diseases of plants and
animals. Indeed, some of the numerous transient systems being employed include
serious human and animal pathogens. Filoviruses (Ebola and Marburg viruses)
cause severe and often fatal haemorrhagic fever in humans and non-human
primates. The US Centers for Disease Control classifies Ebola and Marburg viruses as ‘category A’ pathogens (defined as posing a risk to national security
as bioterrorism agents). To generate a vaccine against it, a geminiviral
replicon system was used to produce an Ebola immune complex (EIC) in a
tobacco-related species, Nicotiana benthamiana. This EIC induces an
immune response targeting the Ebola glycoprotein GP1 that mediates Ebola viral
infection. The vaccine consists of the Ebola GP1 fused to the heavy chain of humanized
monoclonal antibody that specifically binds to a linear epitope on GP1. This GP1-heavy
chain fusion protein allows the individual
immunoglobulins to bind to each other to form the Ebola immune-complex, which
in-turn induces a high immune response when used as a vaccine. Subcutaneous
immunization of mice with purified EIC resulted in anti-Ebola virus antibody
production at levels comparable to those obtained with a GP1 virus-like
particle. Large quantities of vaccine can be produced in the transient system
in a short time.
To assess the quality of antibodies transiently
expressed to high levels in plants, the human anti-HIV monoclonal antibody,
2G12, was expressed and characterized using both replicating and
non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV)
RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue) of
affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was
used (HT is a general enhancer of protein expression in plants), and the
antibody was retained in the endoplasmic reticulum (ER). The non-replicating
system is based on a disabled cowpea mosaic virus RNA-2, and high-level
expression may be achieved using Agrobacterium-mediated transient
transformation. This non-replicating system contains the virus but does not
prevent the modified Agrobacterium from escaping into the environment. The
HIV antibody produced in plants proved equivalent to the antibody produced in
animal cells. Analysis by mass-spectrometry showed that the glycosylation
pattern of the transgenic protein was determined exclusively by whether the
antibody was retained in the ER and did not depend on whether a replicating or
non-replicating system was used.
To further improve the heterologous protein
yield of tobacco platforms, transient and stable expression of four recombinant
proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas
aeruginosa, and a hyperthermostable a-amylase)
was evaluated in numerous species and cultivars of Nicotiana. Of
the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I
64) produced the highest transient concentrations of recombinant proteins, in
addition to producing a large amount of biomass and a relatively low quantity
of alkaloids, making it the most effective plant host for recombinant protein
production. Tobacco has proven to be the most effective plant for producing transiently
expressed recombinant proteins.
The transient expression systems are proving
attractive for their speed of production, relatively low cost of production and
the absence of adverse environmental impact provided that strict
containment is applied.
Production in Chloroplasts
Expressing transgenes in chloroplasts has caught the attention of
researcher as chloroplasts are claimed to be transmitted by eggs alone and not
spread to wild populations through pollen. Scientists are using this strategy
to develop vaccines for illnesses including malaria and cervical cancer.
Genetically engineered starch granules for a
malaria vaccine were produced containing plasmodial vaccine candidate antigens in
the unicellular green algae Chlamydomonas reinhardtii. The C-terminal
domains of the apical major antigen (AMA1) from Plasmodium berghei were
fused to the algal granule bound starch synthase (GBSS).The peptides are
efficiently expressed and bound within the chloroplast. Mice were either injected
with the engineered starch particles and Freund adjuvant, or fed with the
engineered particles co-delivered with the mucosal adjuvant, the B-subunit of
heat-labile enterotoxin (LTB) derived from E. coli, and then challenged
intraperitoneally with a lethal inoculum of the malaria parasite, P. Berghei.
Both experimental strategies led to a significantly reduced parasitemia
(parasites circulating in the blood) with an extension of life span including
complete cure for intraperitoneal delivery. In the case of the starch-bound
vaccine, the immune sera or purified immunoglobulin G of mice immunized with
the corresponding starch strongly inhibited in vitro the
intra-erythrocytic asexual development of the most human deadly Plasmodium
species.
Human papillomavirus (HPV) causes cervical
cancer in women worldwide, which is currently prevented by vaccines based on
virus-like particles (VLPs). However, these vaccines have certain limitations
in their availability to developing countries, largely due to elevated costs. There
are also serious doubts over efficacy and safety (The HPV Vaccine
Controversy, SiS 41) yet to be addressed. As an alternative to VLPs, capsomeres have been shown to be
highly immunogenic and can be used as a vaccine candidate. Capsomeres form the
outer surface of a viral particle. Furthermore, coupling of an adjuvant-like Escherichia
coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its
immunogenicity and reduce the costs related to separate co-administration of
adjuvants. Two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB
as a fusion protein were expressed in tobacco chloroplasts. Western blot
analysis showed that the LTB-L1 fusion protein was properly expressed in the
plastids and the recombinant protein was estimated to accumulate up to 2 percent
of total soluble protein. However, all transplastomic lines showed chlorosis,
male sterility and growth retardation, which persisted in the ensuing four
generations studied. Nevertheless, the plants reached maturity and produced
seeds by pollination with wild-type plants. Production of pharmaceuticals in
chloroplasts has been promoted as a way to limit the spread of transgenic pharm
crops, however. As this study shows, the transformation of the chloroplast may
create toxic side effects on the transformed plant. In spite of the toxic side effects,
the tobacco plants produced significant quantities of vaccine peptides.
We have refuted the dogma that
proclaims chloroplast genetic modification offers fool proof containment of
transgenes. Many plant species transmit chloroplasts through pollen alone while
others transmit chloroplasts through both pollen and egg. Even strictly
maternal chloroplast transmitters will transmit the chloroplast through pollen
under stressful conditions (see Molecular Pharming by Chloroplast
Transformation, SiS 27).
To Conclude
Between the years 1991 and 2011 there have been 101 open field
trials of crops modified with genes for pharmaceutical proteins in the United States alone. Modified maize was released in 51 of the trials, modified rice in 21
of the trials, modified tobacco in 18 trials, modified safflower in 3 trials, tomato
was modified in 1 trial, rapeseed was modified in 1 trial, alfalfa in 1 trial
and the Tobacco mosaic virus (TMV) in 6 trials while the Tobacco etch virus (TEV)
was modified in 1 trial. The management of the environmental impact of the most
of the modified crops cannot be independently evaluated in most releases
because the information is withheld as trade secrets. However, in the case of those
crops with which environmental impact studies were reported,
the safety requirements employed appeared to be very primitive and in most
cases ineffective. Immediate efforts should be made to evaluate the level of
contamination of the United States maize and rice crops with the genes and
proteins from the carelessly managed field trials.
The production of pharmaceuticals appears to be
most safely done using transient expression systems contained in greenhouses. But
they need to be virus proofed, as well as insects and rodent-proof. The seed
based production of pharmaceuticals can be safely undertaken in greenhouses
which prevent release of both pollen and seeds (and hence must be rodent and
insect proof), but not at all in the open fields. The use of primary food and
feed crops such as maize and rice in open fields is pure folly. Furthermore,
the horizontal transfer of the genes for pharmaceutical products deserves much
full attention (GM DNA Does Jump
Species, SiS 47).
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There are 3 comments on this article so far. Add your comment
| joe cummins Comment left 9th November 2011 21:09:15
A late breaking development: Human blood serum in GM rice
Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. The production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds bearing a synthetic approximation of the human albumin gene. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA).The recombinant rice is planned to be produced in open fields according to the guidelines laid out by the United States USDA (1).
The pollution of rice food crops with GM rice making human serum albumin poses a significant threat to the human food chain and that threat is very real based on the widespread contamination of US rice crops with a gene for herbicide tolerance which originated from GM rice being tested in small open field plots.
Human serum albumin is the most abundant protein in human blood plasma. It is produced in the liver. Albumin transports hormones, fatty acids, and other compounds, buffers pH, and maintains osmotic pressure, among other functions. Human albumin is often used to replace lost fluid and help restore blood volume in trauma, burns and surgery patients. Serum albumin may undergo a slow non-enzymatic glycation. Glycation can result in the formation of Advanced Glycosylation End-Products (AGE), which result in abnormal biological effects. Accumulation of AGEs leads to tissue damage via alteration of the structures and functions of tissue proteins, stimulation of cellular responses, through receptors specific for AGE-proteins, and generation of reactive oxygen intermediates. AGEs also react with DNA, thus causing mutations and DNA transposition. Thermal processing (cooking) of proteins and carbohydrates brings major changes. People suffering from chronic inflammation, severe infection, etc. may show high albumin levels in serum (hyperalbuminemia). High blood albumin may also mean, a person may be suffering from an abnormal swelling, immune system disorder, cancer, malnutrition, liver disorder and kidney disorder. The point here is that pollution of the food chain with a gene for human albumin may lead to untoward consequences. For example AGES has been implicated in the gene damage associated with kidney disease observed in type 2 diabetes which is also associated with high cancer incidence. The consequences of consuming a diet based on GM rice heavily polluted with human serum albumin must be brought to light.
1.He Y, Ning T, Xie T, Qiu Q, Zhang L, Sun Y, Jiang D, Fu K, Yin F, Zhang W, Shen L, Wang H, Li J, Lin Q, Sun Y, Li H, Zhu Y, Yang D GM Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Natl Acad Sci U S A. 2011 Oct 31. [Epub ahead of print]
| Todd Millions Comment left 20th November 2011 08:08:15
And as we all know-there is never any 'accidental'escapes of GM untested unapproved modifided varities.And if the ever were,they would be of no human health effect(or more importantly liability)being as they are based on already approved mods in other crops.So says the holy dogma of the changeling and his sainted predeccesors.Any evidence contrary?-"First door too the left one cross each."
this is all sadly pedictable by the history of biowarfare dating back too the great war.Thanxs for outlineing so well new variants of a predictable story. | Todd Millions Comment left 1st December 2011 08:08:35
Prof Cummins-
Having printed off and considered this post properly(particularly your timely and important comment)-Any info on when these rice albumen tests began?
Rice is a vital 'fuel'staple too me.When US supplied rice began too make me ill-I first did isolate feed sessions too confirm it was the rice.The determining symptoms were;
1-organ inflamation and upset.
2-joint pain(sharp and dull)with loss of fine motor control.
I then did two feed trials of 100 grams and 200 grams-the 300,I didn't do since severity was much greater on the 200.So toxic rather than allergenic is indicated,but not confirmed.
I have set asside sample of the rice used.So it may be possible too determine if this is new herbiside,or mod escape.Or -'A beautiful synergy'.
I of course didn't inform anyone in health or ag canaduh-so the PR denial 'science' is unprimed and still leashed.This will also protect and screen anyone who would check this from-night of long knife attack.Also a vital precaution in -volenteer too be 3rd world shithole frosty bannana republics,with'report too supervisor'whistle blower'protection'-as it was under the despised gliberals(see;Starlink).
So-can you supply reliable dates for when these feild trials of the blood albuenem rice started?I will have the date that I noticed these symptoms begin on a post office stamped letter.If these trials began after this date-We would have a causal link.There is more-I'll put it in the letter. |
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