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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 [1] (Table 1).

Table 1   GM crops with dsRNA commercially approved or the approval pipeline

ProductCompanyStatus
Flav Savr tomatoMonsantoWithdrawn from market
High oleic acid soybean lines G94-1, G94-19 and G168MonsantoFSANZ* approved 2000
Withdrawn from market
New Leaf Y and New Leaf Plus PotatoDupont PioneerFSANZ* approved 2001
Withdrawn from market
High oleic acid soybean lind DP-305423-1Dupont PioneerFSAMZ* approved 2010
Herbicde tolerant, high oleic acid soybean Line MON87705Monsantoapproved 2011
Golden mosaic virus resistant pinto beanEmbrapa*Brazil approved 2011
Papaya ringspot virus resistant papayaHawaii UniversityUSA 1996, Canada 2003, Japan 2011
Altered grain starch wheatCSIRO*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 [2]. The same RNAi pathway has been identified since in practically all plant and animal kingdoms [3]. 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.  

Actually, dsRNA 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).

Interestingly, the 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 [5] 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 regulators

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.  

However, regulators 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 [1] “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 [6] 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 [7] 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 [10]. 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 [1] 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 [11] (see [12] 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 [13].  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 miRNA molecules.

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 [14]. In fact, miRNAs and other dsRNAs may be part of a nuclei acid intercommunication system operating throughout the body (see [15] 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 [16] (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

As highlighted and reviewed by Heinemann and colleagues [1], 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 [17] Epigenetic Toxicology, SiS 42). Also, GM crops engineered to produce dsRNA may end up producing additional, unintended secondary dsRNAs, thereby multiplying the toxic effects.

Off-target 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  [18] 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 [19] Controversy over gene therapy ‘breakthrough’, SiS 26). In general, researchers were finding dozens of genes affected by a single siRNA.

One 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 [20]. 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.

Matches 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 follows:

  • Perfect sequence matches of approximately 21 nucleotides long
  • Approaching or exceeding 95 % identity over a stretch of 40 nucleotide
  • Short (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 [21].

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 [21].

To conclude

DsRNA 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 ([22] Bt Crops Failures and Hazards, SiS 53, [23] 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 [24] The Case for A GM-Free Sustainable World (Independent Science Panel Report, ISIS publication). The case is stronger than ever now.

References

  1. 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
  2. Fire A, Xu S, Montgomery MK, Kostas SA, Diver SE and Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegansNature 1998, 391, 806-11.
  3. Cogoni C and Macino G. Post-transcriptional gene silencing across kingdoms. Curr Opinion Genet Dev 2000, 10, 638-43.
  4. 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.
  5. Ho MW. Epigenetic inheritance, what genes remember. Science in Society 41, 4-5, 2009.
  6. 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).
  7. Ho MW. DNA in food and feed. Science in Society 23, 34-36, 2004.
  8. Gordon KHJ and Waterhouse PM. RNAi for insect-proof plants. Nat Biotech 2007, 23, 1231-2.
  9. Mao Y-B, Cai W-J, Wang J-W, et al. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotech 2007, 25, 1307-13.
  10. Chen J, Zhang D, Yao Q, et al. Feeding-based RNA interference of a trehalose phosphate synthase gene in the brown plant hopper, Nilaparvata lugens. Insect Mol Biol 2010, 19, 777-86.
  11. Zhang L, Hou D, Chen X. et al. Exogenous plant MiR168a specifically targets mammalian LDIRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res 2012a, 22, 107-26.
  12. Ho MW. How food affects genes. Science in Society 53, 12-13, 2-12.
  13. Zhang Y. Wiggins E. Lawrence C, Petrick J, Ivashuta S and Heck G. Analysis of plant-derived miRNAs in animal small RNA datasets. BCM Genomics 2012, 13.
  14. Zhang Y, Liu D, Chen X. et al. Secreted monocytic miR-150 enhances targeted endothelial cell migration. Molecular Cell 2010, 39m 133-44.
  15. Ho MW. Intercommunication via circulating nucleic acids. Science in Society 42, 46-48, 2009.
  16. Ho MW. Darwin’s pangenesis, the hidden history of genetics & the dangers of GMOs. Science in Society 42, 42-45, 2009.
  17. Ho MW. Epigenetic toxicology. Science in Society 42, 12-15, 2009.
  18. Ho MW. Gene therapy nightmare for mice could human be next? Science in Society 31, 25, 2006.
  19. Ho MW. Controversy over gene therapy breakthrough. Science in Society 26, 38, 2005.
  20. Thermo Scientific Tech Support. Off-target effects: disturbing the silence of RNA interference (RNAi). Tech Review. 2010, accessed 17 April 2013, http://www.thermoscientificbio.com/uploadedFiles/Resources/off-target-tech-review.pdf
  21. 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 jack.heinemann@canterbury.ac.nz
  22. Sirinathsinghji E. Bt crops failures and hazards. Science in Society 53, 14-15, 2012.
  23. Sirnathsinghji E and Ho MW. Why glyphosate should be banned. Science in Society 56, 21-32, 2012.
  24. Ho MW, Lim LC, et al. The Case for A GM Free Sustainable World, Independent Science Panel Report, ISIS/TWN, London/Penang, 2003. http://www.i-sis.org.uk/TheCaseforAGM-FreeSustainableWorld.php
There are 2 comments on this article so far. Add your comment
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!

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