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ISIS Report 29/04/13
New GM Nightmares with RNA
Small double-stranded RNA (dsRNAs) that aim
to interfere with specific gene expression are increasingly used to create GM
crops; unfortunately they have many off-target effects and can also interfere
with gene expression in all animals exposed to the crops Dr Mae-Wan Ho
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Genetic modification by RNA interference
Most commercially grown genetically modified
(GM) crops are engineered to produce foreign proteins, but new ones are
increasingly engineered to produce RNA of a special kind - double-stranded RNA
(dsRNA) - that aims to interfere with the expression of a specific gene,
usually to silence the gene  (Table 1).
Table 1 GM crops with dsRNA commercially
approved or the approval pipeline
Flav Savr tomato
Withdrawn from market
High oleic acid soybean lines G94-1, G94-19 and G168
approved 2000 Withdrawn from market
New Leaf Y and New Leaf Plus Potato
FSANZ* approved 2001 Withdrawn from market
High oleic acid soybean lind DP-305423-1
FSAMZ* approved 2010
Herbicde tolerant, high oleic acid soybean Line MON87705
Golden mosaic virus resistant pinto bean
Papaya ringspot virus resistant papaya
1996, Canada 2003, Japan
Altered grain starch wheat
Approved for field trials & feeding experiment
*CSIRO Commonwealth Scientific and Industrial Research Organization
*Embrapa Brazilian Agricultural Research Corporation
*FSANZ Food Standards Australia New Zealand
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The ability of
dsRNA to interfere with gene expression was known since the 1980s; and the
biochemistry of the phenomenon - referred to as RNA interference (RNAi) – was
worked out in the roundworm Caenorhabditis elegans in the late 1990s .
The same RNAi pathway has been identified since in practically all plant and
animal kingdoms . DsRNA includes siRNA (short-inhibitory RNA), miRNA (microRNA),
shRNA (short hairpin RNA) etc., all intermediates leading to RNA interference
of protein synthesis. This can happen at transcription, or at translation. Typically,
dsRNA originates from a long RNA molecule with stretches of complementary base
sequences that base pair to form a stem ending in a non-base-paired loop. This
stem-loop structure is then processed into a shorter dsRNA, and one strand, the
guide strand does the job of interfering. It binds to a mRNA (messenger RNA)
molecule in the cytoplasm by complementary base-pairing to prevent the mRNA from
being translated into protein. Alternatively, the guide strand targets and
chemically modifies DNA sequences in the nucleus by adding methyl groups to the
DNA, and cause modification of histone proteins associated with the DNA. The
nuclear pathway is known to inhibit transcription and to seed the formation of
heterochromatin, an inactive, non-transcribed region of chromosomes.
genetic modification has been used before. The first GM crop to be
commercialized, the Flav Savr tomato, created with ‘antisense’ technology to
delay ripening, is now known to act via dsRNA (Table 1).
gene silencing effect of dsRNA can become inherited (either indefinitely, or through
two or more generations) in cells and organisms that are not genetically
modified, but simply exposed to the dsRNA for a period of time. It can happen
via methyl groups added to the DNA, or the modification of histones, without
changing the base sequence of the DNA in the genome [3, 4]. This is another
example of the inheritance of acquired characters now known to occur through
many different mechanisms (see  Epigenetic Inheritance
- What Genes Remember and other articles in the series, SiS 41) that
makes genetic modification all the more hazardous.
Obvious dangers of dsRNA ignored by
DsRNA genetic modification has large
implications on safety based on what is already known (see below): DsRNA is
stable, it resists digestion and may enter the bloodstream; its role in
modifying gene expression is universal and acts across kingdoms; toxicity to
animals have been amply demonstrated and exploited in targeting pests; although
the intended target is a specific gene, many off-target effects have been
identified; finally, plant dsRNA has been found circulating in the human
bloodstream where it can be taken up into cells and tissues to interfere with
the expression of genes. Consequently, animals including human beings eating
the GM food containing dsRNA could well be harmed.
are ignoring and dismissing the findings despite repeated warnings from
scientists. Jack Heinemann at the University of Canterbury, Christchurch, in New
Zealand and his colleagues have had the same experience as independent
scientists everywhere with their national regulators; in Heinemann’s case, the Food
Standards Australia New Zealand (FSANZ). FSANZ has approved for use as human
food at least 5 GM products with modification to produce dsRNA (see Table 1), in
blatant disregard of evidence brought to their notice again and again.
Heinemann and colleagues call it aptly  “regulation by assumption”, and show
how the same applies to regulatory agencies in the US and in Brazil.
DsRNA resists digestion in the gut and
enters the bloodstream
Typically, both DNA and RNA are Generally
Regarded as Safe (GRAS), and assumed to be broken down in the gut when eaten
with GM food and feed. This assumption was already contradicted by experiments
going back to the early 1990s (see my book  Genetic Engineering Dream or Nightmare,
the first edition of which was published in 1998). There have been many
publications documenting the ability of DNA to survive digestion in the gut and
to pass into the bloodstream whenever investigations were carried out with
sufficiently sensitive detection methods (see  DNA in GM Food & Feed (SiS
23). DsRNA in particular, is much more stable than single stranded RNA. DsRNA produced
in GM plants survive intact after passing through the gut of insects and worms
feeding on the plants [8, 9]. Also, oral exposure of insect pests to dsRNA was
effective in inducing RNA interference . Worms can even absorb dsRNA
suspended in liquid through their skin, and when taken in, the dsRNA can
circulate throughout the body and alter gene expression in the animal. In some
cases the dsRNA taken up is further multiplied or induces a secondary reaction
resulting in more and different secondary dsRNA with unpredictable targets (see
 for review).
DsRNA mechanisms is universal to plants
and animals and works across kingdoms
Thus, not only are dsRNA mechanisms
universal to all plants and animals, there is already experimental evidence
that they can act across kingdoms.
Researchers in China have
now shown that miRNA from food can circulate in the human blood stream and may
well turn genes off in the human body  (see  How Food Affects
Genes, SiS 53). They demonstrated that dsRNAs can survive digestion
and be taken up via the gastrointestinal tract. These plant-derived dsRNA
silenced a gene in human tissue culture cells, and in mouse liver, small intestine
and lung. A survey of existing data of small RNA molecules (conducted by
scientists working for Monsanto) from human blood and tissues sources, farm
animals and insects confirmed that regulatory RNAs from plants can be found in
animals including humans . The data also indicated that some dsRNAs from
plants are found more frequently than predicted from their level of expression
in plants; in other words, there may be a selective retention or uptake of some
DsRNA is part of a nucleic acid
intercommunication system throughout the body
The team at several universities in China
has been researching miRNAs for some years, and found them actively secreted
from tissues and cells in the body. They serve as biomarkers for disease, and
could act as signaling molecules in intercellular communication . In fact,
miRNAs and other dsRNAs may be part of a nuclei acid intercommunication system
operating throughout the body (see  Intercommunication
via Circulating Nucleic Acids, SiS 42) that has been coming to light. This not
only lends support to Darwin’s ‘Lamarckian idea’ of the inheritance of acquired
characters and pangenesis  (Darwin’s Pangenesis, the
Hidden History of Genetics, & the Dangers of GMOs, SiS 42), but
also leaves organisms very vulnerable to the ‘unintended side-effects’ of
genetic modification and GM foods.
Toxicities to wild life, domestic
animals and human beings
and reviewed by Heinemann and colleagues , there is evidence that specific siRNAs can be toxic and the toxicity can be
transmitted through food. Thus, GM maize and cotton plants engineered to
express novel dsRNAs intended to be toxic to target insects were transmitted
from plant to insects feeding on the plant, and were further processed in the
animal to a siRNA that silenced one or more genes essential for life, or
essential for detoxifying natural plant toxins (i.e., gossypol in cotton).
Other researchers fed dsRNA directly, or applied dsRNA in liposomes as
insecticides. It has been suggested that one siRNA can
cleave as many as ten target mRNAs.
As mentioned earlier, the effects of gene silencing from RNAi can be
inherited, as is the associated toxicity; it is all part of a spectrum of toxic
effects transmitted across generations, from individuals exposed to the toxin
on to their offspring (see  Epigenetic Toxicology,
SiS 42). Also, GM crops engineered to produce dsRNA may end up producing
additional, unintended secondary dsRNAs, thereby multiplying the toxic effects.
effects of dsRNA known from ‘gene therapy’ experiments since 2003
A worst case
scenario of toxic dsRNA came from a gene ‘therapy’ experiment in mice reported
in 2006, which killed more than 150 animals  Gene Therapy Nightmare for
Mice (SiS 31). The technique – hailed as 2002’s ‘breakthrough of the
year’ in ‘precision’ gene therapy - was found to have many off-target effects
only a year later  Controversy
over gene therapy ‘breakthrough’, SiS 26). In general, researchers
were finding dozens of genes affected by a single siRNA.
main reason for off-target effects affecting other genes and other species is
that the interference depends on complementary base pairing for short sequences
- 21 bases in the case of siRNA, but only 7 for miRNA (and it was the siRNAs
acting as miRNAs that cause many of the unintended effects) - there could well
be similar sequences all over the same genome and in genomes of different
species. In particular, many dsRNAs target regulatory sequences of genes, which
are likely to be common to sets of genes expressed together in certain tissues
and cells. In addition, non-specific effects result from the interferon response
and from response to cationic lipids typically used to deliver the siRNA .
These problems are well-recognized among researchers using RNAi to study gene
function, or in potential gene therapy. So there is no excuse for regulators of
GMOs to ignore this glaring evidence.
identified between target sequences in GM crops and human genes
Matches between the dsRNA sequences from difference species
are already known. Heinemann and colleagues describe the range of matches as
sequence matches of approximately 21 nucleotides long
or exceeding 95 % identity over a stretch of 40 nucleotide
(7 or more) contiguous identical matches in the 3’ untranslated region of
mRNA (gene regulatory region), which can be more determinative than the
number of nucleotide matches overall
To counter the regulators’ assumption that as
plants and humans and other animals have very different genomes their DNA/RNA
sequences would also be very different, Heinemann conducted a first simple comparison
in August 2012 between the DNA sequence of the human genome and a DNA sequence
from the wheat SBE1 gene provided to the database Genbank by CSIRO. The actual
sequences used by CSIRO to construct the dsRNA were not made known to Heinemann
at the time. Later, he found out from another source that these appeared in a
publication 5 years before. Based on this information, Heinemann reconstructed
some of the intended novel DNA sequences used to create the GM wheat, and
looked again for matches in the human genome and selected parts of the human
genome. He came up with similar results both times .
There were four perfect matches of 21
nucleotides and another 13 nucleotide stretch match, within a wheat gene
sequence of just 536 nucleotides. And this does not include comparisons of
secondary unintended dsRNAs that may be induced in the GM plant, as indeed, in
any GMO, including those not explicitly engineered to create dsRNA.
Unanticipated off-target adverse effects can be difficult to detect and they are impossible to
reliably predict using bioinformatics techniques such as sequence matching, as
Heinemann points out .
technology to silence genes based on specific sequence matching has numerous
unintended off-target effects, and is no improvement over the conventional hit
and miss GM technology that has already proven every bit as hazardous as some
of us have predicted (see ISIS’ recent reviews ( Bt Crops Failures
and Hazards, SiS 53,  Why
Glyphosate Should Be Banned, ). We have been calling for a global ban on GM
crops and a shift to sustainable non-GM agriculture since 2003  The Case
for A GM-Free Sustainable World (Independent Science Panel Report, ISIS
publication). The case is stronger than ever now.
Heinemann JA. Agapito-Tenfen SZ and
Carman JA. A comparative evaluation of the regulation of GM crops or
products containing dsRNA and suggested improvements to risk assessment.
Environment International 2013, 55, 43-55, http://bit.ly/14i7pyG
Fire A, Xu S, Montgomery MK, Kostas SA,
Diver SE and Mello CC. Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 1998,
Cogoni C and Macino G.
Post-transcriptional gene silencing across kingdoms. Curr Opinion Genet
Dev 2000, 10, 638-43.
Hawkins PG, Sharon Santoso S, Adams C,
Anest V and Morris KV. Promoter targeted small RNAs induce long-term
transcriptional gene silencing in human cells. Nucleic Acids Research
2009, 37, published online 20 March 2009, doi:10.1093/nar/gkp127.
Ho MW. Genetic
Engineering Dream of Nightmare? The Brave New World of Bad Science and Big
Business, Third World Network, Gateway Books, MacMillan, Continuum,
Penang, Malaysia, Bath, UK, Dublin, Ireland, New York, USA, 1998, 1999,
2007 (reprint with extended Introduction).
Heinemann JA. Update on “Evaluation of
risks from creation of novel RNA molecules in genetically engineered wheat
plants and recommendations for risk assessment”. 21 March 2013 email@example.com
Rory Short Comment left 29th April 2013 17:05:47 The GM nightmare only seems to get worse. I personally feel that the GM nightmare is but a manifestation of a more fundamental problem. This problem arises from how, certainly the people with money power, view their relationship with the rest of life. They view themselves as apart from life not as just a part of life. This misunderstanding will eventually see us off the planet.
sharlene neal Comment left 30th April 2013 09:09:13 Wonderful research! We need more of this to show the public. Many of us already see the genetic changes expressing themselves in our bodies including plant materials growing within. Biopsies revealed this in my tissues.We have all become unwilling participants in a huge genetic experiment. This is a nighmare! Help us find a way out. Thank you so much for all the hard work!!! I am grateful!