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Thursday, April 3,2014

Muzzled by Monsanto

Is Big Ag squelching research showing its new RNAi GMOs may be dangerous?

By Caitlin Rockett

After nearly 30 years studying how plants use their genes to defend against viruses, Vicki Vance, a professor at the University of South Carolina, doesn’t see genetically modifying plants as a malevolent or arrogantly God-like endeavor.

“There’s DNA in the world and it gets passed from one organism to another and it’s the natural thing. If that’s the problem you have with transgenic plants, that’s not a good reason to be against them,” Vance says.

She does, however, have a problem with mega corporations allegedly using their money and power to hide the risks of new forms of genetic technology.

“I didn’t use to be an anti-GMO person and I didn’t use to have strong feelings about Monsanto, but …,” she says, her voice trailing off.

But that was before the Chinese research, before the calls from Monsanto, before she couldn’t get funding for work that she feels could change the way we treat cancer and other diseases. Her research put her at odds with one of the most powerful corporations in the world.

Vance isn’t a nobody in the world of RNA research. At a June 2011 conference hosted by the nonprofit International Life Science Institute (ILSI), a group of academics, regulatory professionals from the Environmental Protection Agency, the Department of Agriculture’s Environmental Risk Analysis Program and members of the biotech industry gathered in Washington D.C. They came to evaluate the environmental risks of a promising new technique to protect crops against pestilent insects — a gene-regulating process called RNA interference.

Vance wasn’t just an attendee at the conference; she provided the introduction for the event. She’s studied small interfering ribonucleic acid molecules, siRNA, in plants for most of her career. Her name appears often in academic papers and conference proceedings on the topic of gene silencing, the main function of so-called RNAi technology.

At that time, views of Vance and the other attendees was relatively positive: “No plausible risk hypotheses were identified that can be considered unique to RNAi mechanisms when compared to other genetically engineered plants with similar traits.”

“At the time I was like, ‘Hell yes it’s safe — how is this gonna be dangerous?’” says Vance. “The corn rootworm will take up these siRNAs, which turn off production of essential proteins in pests. Apparently it works really well. Otherwise you’d have to use pesticides, chemicals that are toxic.”

But her stance on RNAi as a pesticide would change shortly after the conference.

Controversial research

RNAi has applications in both the medical world and in agriculture. But these two worlds are not after the same thing when it comes to RNA. While the medical community is trying to perfect processes that will cause the human body to accept modified RNA strands, agriculture corporations working in the GMO field are busy trying to prove that their RNA strands can’t be assimilated by the human body at all.

For example, some microRNAs interfere with cell division and block cancer. These tumor suppressor RNAs are missing in cancer patients. If they can be replaced — an experimental treatment known as microRNA replacement therapy — then doctors could theoretically stop the proliferation of cancer cells. But in agriculture where RNA is being engineered as a pesticide designed to kill insects that feed on crops — such as Monsanto’s RNA efforts aimed at the Western corn rootworm, the most economically destructive pest in corn production — it is paramount that the RNA in and/or on the corn that is later eaten by humans doesn’t subsequently infiltrate our cells causing who knows what kind of unintended consequences.

In short, the medical world needs the genetically modified RNA to be assimilated by our bodies and the agricultural world needs the opposite to occur.

In September of 2011, three months after Vance gave her presentation on RNAi at the ILSI conference, a team lead by Chen-Yu Zhang of Nanjing University in China published a paper in the journal Cell Research claiming that mammals (mice, in the case of their study) take up small RNAs when they eat plants, and those plant RNAs regulate expression of mammalian genes — something the science world refers to as trans-kingdom gene regulation.

The team reported finding small RNA molecules in the bloodstream and tissue of mice and humans. They found that one particular molecule of RNA from rice could inhibit a protein that supports removal of low-density lipoprotein, or “bad” cholesterol from the blood. If such a finding proved to also be true for humans, it would potentially indicate that eating foods contain ing modified RNA could have major implications for heart disease and other health issues tied to cholesterol.

“That had never been reported before. Nobody had thought about that,” says Vance. “What the hell, I mean, you’re eating a plant and taking in plant RNAs and they are regulating the expression of your genes? I think that has to be considered. … There’s been a lot of resistance to that paper. When something really unexpected like that comes up, there’s always a lot of resistance.”

Resistance was apparent even before the Chinese study was published. The team’s manuscript was rejected by wellknown journals Science, Cell and Molecular Cell. Zhang told The Scientist Magazine it was because their discovery was “too extraordinary.”

“Most of the people [who speculate about our work] just don’t believe it because the concept right now, I have to say, is broken by my results,” Zhang told Boulder Weekly in a recent interview from Nanjing. “They don’t want to believe until I have new data or the other groups reproduce some of our data. And of course some other people, for whatever reason I don’t want to say … I don’t want to even touch … they are just against our discovery no matter what it is.”

The work was so controversial that another preeminent journal, Nature Biotechnology, made a rather unusual move: They published a letter from a team detailing negative findings. In other words, it was a study that presents no new conclusions, only an unsuccessful attempt to recreate Zhang’s findings.

“[T]he new report, resulting from a collaboration between miRagen Therapeutics and Monsanto, clarifies what were controversial findings in [the Zhang study]. The latter study … sparked vigorous debate because it reported the presence of plant microR- NA in human blood plasma and suggested that one in particular, miRNA 168a, from ingested rice could traverse into the circulation of mice resulting in the modulation of miRNA target genes in the animal.”

The editorial goes on to say that the miRagen/ Monsanto study which used three different groups of mice for control and comparison, found no evidence of miRNA 168a in the plasma and liver tissue of mice fed a rice diet, and they attributed altered LDL levels in the animal blood to differences in the nutrition available to mice in different groups.

“One of the issues with eating rice alone without out any sort of protein source is you can get effects on metabolism that really have to do with lack of a balanced diet instead of the transit of microRNA and trans-kingdom gene expression,” says William Marshall, president and chief executive officer for the Boulder-based miRagen (pronounce mir-a-gin) Therapeutics. miRagen is a company researching RNA use in treating cardiovascular disease, metabolic disease and fibrosis.

Marshall says he thinks it’s important that Nature Biotechnology broke tradition and published the miRagen/ Monsanto study.

“One general issue in science publications is that negative studies are not often published. It’s like the finding never gets reported because science journals want to report on innovations and successes,” he says. “But there’s this whole reproducibility initiative that Nature Biotechnology is part of to really attempt to highlight these results, because if you don’t report that, there are questions about the validity of the study, then the study becomes sort of de facto the truth. That’s how scientific literature works. And I think it’s important that we rethink this old system, and it’s becoming a really important theme in science today.

Beyond that, however, Marshall says he was disappointed that the team was unable to recreate Zhang’s results.

“[miRagen] got excited about this from the strict perspective of being able to develop microRNA drugs,” Marshall says. “The unfortunate outcome was that we could not observe that particular microRNA [miRNA 168a] was actually able to transit [from the gut and blood stream into cells]. From our perspective it was disappointing, because we saw this as a real opportunity to expand the horizon for all nucleic acid based drugs. This was going to be ground breaking and so far we’ve not been able to reproduce it. And in the absence of really showing a robust effect here, it’s very difficult contemplating taking it further.”

Monsanto and miRagen weren’t the only team unable to reproduce the results from Zhang’s study. In May of 2012, researchers at Brigham and Women’s Hospital in Boston were unable to detect levels of plant miR- NAs in the blood of healthy athletes who were fed RNA-laden fruit. The team also failed to find RNA traces in mice or bees.

“We conclude that horizontal delivery of microRNAs via typical dietary ingestion is neither a robust nor a frequent mechanism,” wrote the authors.

In June, a research team from Johns Hopkins University published a piece in RNA Biology saying that Zhang’s results were likely a false positive resulting from the technique his group used.

In the Monsanto/miRagen study, the team suggested that Zhang’s results were skewed by environmental contamination of the sequencing equipment they used to detect plant microRNAs in humans.

“That particular plant microR- NA had been used in the past to normalize these studies and so the issue is that it could have been a common contaminate that was used in a lab to do deep sequencing,” says Marshall.

But the fact that miRagen collaborated with Monsanto on this report gives rise to legitimate questions about credibility — the agricultural powerhouse clearly has much to gain from using a genetic technology that will kill the costliest parasite to the world’s most produced grain, but what about miRagen?

What’s at stake?

Some scientists, such as Kevin M. Folta, an associate professor in the horticultural sciences department at the University of Florida, believe miRagen had nothing to gain by discrediting the Zhang study.

“[MiRagen has] a vested interest in identifying mechanisms to orally administer miRNA and detect physiological outcomes,” Folta wrote in a blog piece in November 2013.

“If [miRagen] repeated Zhang et al.’s work it would have been a positive finding for their company, as I’m sure they get plenty of criticism for the viability of their potential therapies,” Folta concluded.

The Monsanto/miRagen study clearly states that Monsanto asked the Colorado-based therapeutics company to participate in the reproducibility study. Marshall says miRagen was in contact with Monsanto even before the study because of the ag company’s interest in RNAi technology — which included their interest in preventing trans-kingdom gene regulation.

“So this [study] was a win-win scenario for both of us because essentially we would learn why this particular microRNA was able to transit and be orally bioavailable, and at the same time we would work with Monsanto to understand the rules for the ones that don’t,” Marshall adds. “And the idea was if you would want to engineer plants and prohibit any sort of transkingdom gene regulation then we would understand the rules behind that.”

Vance is less convinced in the innocence of the partnership.

“I think Monsanto was trying to get some legitimacy by bringing in these people from [miRagen] because they have some, what I would consider, establishment animal microRNA people — there are some highly thought of people on their scientific advisory boards,” she says.

“But it’s Monsanto who’s spearheading this thing, and they have this company as first author and last author, whereas they’re all in the middle. That’s another thing that’s saying, ‘This isn’t really Monsanto. Pay no attention to the Monsanto people. First author and last author, that’s the important thing,’ and that is the important thing,” Vance says. “First author, that’s typically the one that did the most work. Last author, in [the microbiology] field, is the person usually who is the communicating author, the one who takes responsibility for the work. But is Monsanto driving this from behind?”

Vance believes both companies have a financial interest in discrediting the Chinese paper.

“On the other hand, I think [miRagen] has some interest in discrediting this Chinese paper. They are trying to use microRNAs therapeutically, and it’s hard to get them [through the blood stream to the cell], and so there’s all sorts of things you have to do to make them work and those things are expensive and they have their downsides. And so what this Chinese paper says is, “Well, you don’t have to do any of that stuff, all you have to do is make it in plants and then eat it. All you have to do is ingest it.”

Such a finding would have major implications on drug industry research and development which desires to create expensive, profitable medicines that can be sold as opposed to developing foods that could fight certain diseases simply by being ingested.

While Zhang declined to comment directly on Monsanto steering any research, he did say he felt slighted by Nature Biotechnology.

Zhang published a response to the correspondence from Monsanto/miRagen critiquing Zhang’s study, and while Nature Biotechnology published the response immediately following the critique, Zhang says he’s disappointed that the journal didn’t mention his response in the editorial about the importance of reproducibility.

“They did not mention at all our reply, they just said, ‘Well, somebody reproduced this study and they couldn’t reproduce our data.’ I cannot believe — it’s really unbelievable — that such a decent scientific journal had such unfair and unprofessional behavior,” says Zhang.

“I just want to say,” he adds, “obviously something is going on. It’s not pure science. I just think something is maybe behind them.”

He pauses, then adds quickly, “I don’t want to say anymore.” Zhang seems uncomfortable saying the word Monsanto, often calling it “the company.”

“I don’t want to attach to them,” he says. “[When the paper came out] they contacted me, the Chinese office. I don’t want to have any relationship to them. Even right now I don’t want to say anything about transgenic or GM food.”

Knock, knock? Who’s there? It’s Monsanto. Now show us your research.

After the Zhang paper was published, Vance was so intrigued that she led her lab, without external funding, to design similar but distinctive experiments to test whether they could detect plant small RNA in animals simply by feeding them the plants.

“We designed plants that make a cocktail of three human tumor suppressor RNAs and then fed those plants to mice,” says Vance. “We fed that to the mice once a day for 28 days. The tumor burden was significantly suppressed in the mice. We’re very excited about that. Seems like there’s huge potential here and our work suggests the Chinese paper was right.”

“There’s no toxicity,” she says. “At least in our studies there’s no toxicity, it just has these amazing therapeutic effects.”

But Vance found that scientific journals weren’t as enthusiastic about her research as she is.

“We still can’t get it funded. I don’t know what I’m going to do. Reviewers always say the same things: it stems from work that can’t be replicated, how can you prove the plant small RNAs get in, the Chinese paper is controversial, no one believes it,” she says.

Zhang also dealt with questions about proving that plant RNA was present in human blood and tissue.Skeptical researchers questioned how to differentiate between plant RNA and animal RNA. As miRNA are very short, only about 22 nucleotides long (in comparison to the millions found in DNA), there could be matching sequences between animal and plant.

Despite skepticism from both the public and scientific community, Vance and Zhang continued with their work.

Vance says it was about a year after the ILSI conference when Andrew Roberts, deputy director for ILSI’s Center for Environmental Risk Assessment, called her up to ask if she would be the last author — essentially a point-of-contact for inquiries — on a white paper about the conference’s conclusions.

“That was when I said — the Zhang paper had come out by then — I said I didn’t want my name on the paper,” says Vance. “That’s when Monsanto started to call me.”

A quick fall

Prior to the release of the Zhang paper and Vance’s refusal to be listed on ILSI’s risk assessment paper, Monsanto had invited Vance to give a talk at the International Symposium of Biosafety of GMO Plants, a biennial international meeting organized by the International Society for Biosafety Research.

The meeting was, perhaps oddly enough, held in St. Louis that year, where the agricultural behemoth Monsanto is headquartered. According to Vance, Monsanto was in charge of the session on the safety of RNAi plants.

“They asked me to give the same overview of RNAi that I had given at the [ILSI] meeting. They had already paid my way, made my hotel reservations, I had an abstract, I was listed on the schedule and everything. Then this fuss came up over the [Zhang] paper,” Vance says. “They called me and asked, was I going to talk about [the Zhang paper] at the symposium and I said, ‘Well yeah, that’s part of the story, it has to be discussed.’”

Vance says Monsanto was adamant that she not mention the Zhang paper in her overview. Her insistence on bringing it up only made the situation more complicated.

“I had to participate in a conference call and [Monsanto] had lawyers present. They eventually called me back and uninvited me from the [International Symposium of Biosafety of GMO Plants],” she says.

But the calls didn’t end.

“They kept calling me because I’d said [my lab] had data consistent with the Zhang paper, and they wanted to ‘help me with experiments’ because I had results that were in conflict with their results. They said they wanted to make sure I was doing the right controls on my experiments. I said, ‘I’ve been a scientist for 30 years, I think I know what I’m doing and when I publish the paper you can comment on it.’

According to Vance, Monsanto representatives told her, “We were hoping to get to it before that happens.”

After another series of phone calls in which Monsanto asked if they could send only two scientists instead of a team to Vance’s lab, Vance told them they were simply not invited.

“I was really surprised that Monsanto took the time and effort to try to squash my research because it’s such a contrast — I’m a little old lady running a little lab in South Carolina,” Vance says.

“Maybe I’m being paranoid,” offers Vance, “but I feel there’s an effort from a large company with a lot of money toward discrediting the work of this other group and keeping people from publishing their work.”

She eventually received another call from Andrew Roberts, deputy director of ILSI’s Center for Environmental Research Assessment, who had invited Vance to present at the 2011 conference on RNAi environmental risks. She told Roberts about the recent experience with Monsanto. According to Vance, Roberts called Monsanto and asked them to stop contacting her — and she says the calls did stop.

When BW called Roberts, he declined to comment on Monsanto’s possible interference with Vance’s work. When asked whether he requested that the company back off of the South Carolina researcher he said, “No comment — but I will say this falls in the ‘no good deed goes unpunished’ category.”

More evidence

Vance and Zhang aren’t the only researchers who claim to have promising results that indicate trans-kingdom gene regulation is possible. Eric Lam, a professor in the department of plant biology and pathology at Rutgers University, has been researching the transit of plant small RNA to animal cells for more than a decade.

“The idea is to see if we can actually express small RNAs, which we call silencing RNAs or siRNAs, that target human pathogenic viruses, like the flu virus. Why we have to take flu shots is because viruses such as influenza have a very high neurogenic rate, meaning they can easily evolve new amino acid residues that allow them to escape new vaccines that [are] create[d] every year,” Lam says.

His longest running project produces transgenic tomato plants that express RNA targeted at viruses such as influenza and Hepatitis C.

But reviewers weren’t convinced in the reliability of Lam’s preliminary study, saying it wasn’t clear that the team’s sequencing methods — the process of determining the precise order of nucleotides of the RNA molecules — were actually detecting plant small RNAs in their rabbit test subjects.

“These are small RNAs — they are like 21 base nucleotides long. That’s very small in terms of sequencing. You have to have many copies of those to be such that it’s not just a fluke, just an artifact of the sequencing,” Lam says.

Lam says this is the same argument that the Monsanto/miRagen study made about Zhang’s work.

“The problem that came out, the controversy, is if you only see a couple of variants of one small RNA, how much could be just error of sequencing?” Lam explains. “The argument is whether these are sequencing errors or whether these are bonafide transport of the plant microRNA into the animal system. That’s really the crux of the issue that I saw in [the Monsanto/miRagen studied that was published in] Nature Biotech.”

Lam says his group has been able to produce a tomato plant that contains a 400 base pair fragment of the influenza virus.

“We know which 21 base fragments are present. Now we’re doing the rabbit blood RNA sample to try to find out whether multiple siRNA that are now in food should appear in the blood if this is true. Not a single one, but multiple ones,” Lam says. “Because of this, I believe we have the potential system to resolve this controversy.”

Lam doesn’t feel slighted that his research has come under scrutiny.

“I accept the [preliminary] review — extraordinary claims need extraordinary data to back it up. This is how science is done in the States anyway,” Lam says, no trace of frustration in his voice. “I fully realize there could be a lot of controversy and discussion if this proves to be true, and it could potentially really change the way we do medicine and also understand how we interact with our food. So it is an important finding that needs really tight scrutiny.”

Jonathan Lundgren, a research entomologist at the U.S. Department of Agriculture’s North Central Agriculture Research Laboratory, Lundgren agrees that RNAi needs more scrutiny, especially when used as a pesticide.

“Most of our experiences with RNAi and the risks that it poses have been done in a petri dish or on a sick person. With pesticidal RNA, the scale that we’re talking about, in terms of deployment …” he pauses to collect his thoughts. “Genetically modified crops are planted on 9 percent of the terrestrial land surface of our country. That scale elevates the importance of understanding the risks that are imposed by RNAi technology. It may be safe, it may not, but we should understand that before large deployment is realized,” he says.

Vance still believes in the potential of transgenic plants. Unlike many people, she doesn’t see genetic modification of plants as “playing God,” and in fact sees the process as natural.

“A lot of good things can come from transgenic plants, but I do take objection that [Monsanto] are doing things I can see have a potential risk when they could avoid it. I’m a scientist and I make transgenic plants and I don’t feel like I’m playing God. If it’s a useful thing, we should do it. If a new risk comes up you shouldn’t fight it — if new data shows this is a possible risk, address it.”

A simple step, in Vance’s opinion, would be to engineer corn plants to only express specific RNA in the roots of the plant where the corn rootworm will feed, avoiding consumption by humans.

“Why do they have to express their RNAi in corn seeds? They don’t have to. They could just put it in the roots – it wouldn’t be hard to do. Why don’t they just fix their freaking plants so they won’t be dangerous to people? Even if there’s some small chance it’s dangerous,” Vance pauses as she has many times during conversations about Monsanto, clearly frustrated.

“I just don’t understand the mindset,” she sighs.

Respond: letters@boulderweekly.com


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Thank you, Caitlin Rockett, for this fascinating article.I am grateful that the Boulder Weekly continues its tradition of tough journalism and tackling stories that mainstream corporate media won't go near. 

The GMO industry is an international, pseudo-scientific, profit-driven religion centered in America's heartland which has spawned far more than its share of such cults. The GMO religion is fundamentalist to the core. And like more conventional cults, the GMO religion promises salvation while damning all unbelievers. The GMO religion is a missionary organization which employs corporate technicians (I hesitate to use the word, "scientist"), prosyletizes politicians (they are such easy converts, after all), and hires internet and media shills to promote the new religion. Website tracts (industry-supported propaganda sites) litter the internet even more than Watchtower magazines litter bus stops. 

And this is only the beginning.

 

 

 

 

 

You are confused, CS.

 

Strangely, the author didn't feel the need to note that Lam has a patent on this topic, and a vested interest in it working (or appearing to work). http://www.google.com/patents/WO2012135820A2 Of course, we all know patents aren't peer-reviewed, but some people think that's evidence anyway. So let's be sure to disclose that. Readers might want to know that.

 

Well said Captain Supreme. Te GMO trolls are hitting labeling law states hard. It is the same script depending on the targets. Shooting for anti-government types, hook-em with the "keep government out of our food" rhetoric. Same thing with the scientists, go after their funding, fellowship, and tenure, or blacklist them. They have trolls infiltrating Occupy, homeless camps, and Climate change....They call themselves scientists who are trying to out conspiracy theorists, linking anti-GMO'ers with all deniers which is so ironic since Monsanto's think tank CEOs ARE the denier shills for big oil, tobacco, and big Chem corps...discusting

 

Captain Supreme - I appreciate your eloquent statement of what the GMO industry is about - thank you!

 

REPLY TO THIS COMMENT

Sorry, Pizzle. Perhaps it is you who are confused. 

The GMO industry is driven by profit and market control; hard science is a secondary or tertiary consideration. Propaganda and indoctrination are critical to its financial success. Such is business as usual in today's Corporate America. Where have you been? 

 

 

 

 

Your original comment is full of crazy. "The GMO industry is an international, pseudo-scientific, profit-driven religion..." Let's take this point-by-point. the GMO industry is: 1)International- true 2)psuedo-scientific- false 3)profit driven- true 4)a religion- insane Therein lies my confusion. Your response to my comment, on the other hand, are actually cogent. I can argue with your points on your second comment.... I can't argue with crazy.

 

REPLY TO THIS COMMENT

I want to thank Caitlin for writing about my story.  The article is well researched and well written - and it presents a balanced viewpoint.  She is my idea of a real journalist.  I think such dedicated people are few and far between. So...thanks to Caitlin and thanks to Boulder Weekly!

 

I applaud your bravery ! Don't pay any attention to the goons at Monsanto. I too, have been attacked for my work investigating their products, but am not deterred, our research continues investigating both parts of the their equation, herbicides and GM plants. Its laughable that Monsanto resorts to the opinion of food and wine critics and invited internet trolls to attack scientists. After my first paper was published on their flagship chemical glyphosate, I sent a shoutout to Hugh Grant president of Monsanto in a syndicated radio interview, giving him stock advice to sell his stock while there is still time. Shortly after, CEO Hugh Grant sold off 40,000 shares at $97.74, and both Janet Holloway and Gerald Steiner - both high-level Monsanto executives ditched more than 10,000 shares each. Tom Hartley also bailed on another 6,000 shares at $100.15," Learn more: http://www.naturalnews.com/041853_hedge_funds_Monsanto_shares_stock_market.html##ixzz2y1EUfarP

 

Vicki--can you tell me more about the sequence match that concerns you from the RNA that targets the rootwoom? I looked at the sequence and I wasn't able to see what might be "dangerous to people". Please be specific, with either sequence IDs and coordinates or the actual sequence. I can take it.

 

In answer to Mary Mangan: Sequence predictions are incapable of ruling out harm. This has been shown numerous times, eg, from the EPA white paper (http://www.epa.gov/oppfead1/cb/csb_page/updates/2014/rnai-whitepaper.html): "The specificity of the Dicer enzyme in the generation of siRNA species is not clear so that the need for a 21nt stepwise or other modified bioinformatic analysis to predict off-target effects is still unsubstantiated." Or this paper http://www.epa.gov/oppfead1/cb/csb_page/updates/2014/rnai-whitepaper.html "OTEs [off target effects] were not predicted by current bioinformatic algorithms, nor by the total number of OTEs induced. Instead, pathway analysis was required for them to be identified."

 

Dr.Vance, can you tell us more about the mouse study? It says that you fed them "tumor suppressor RNA". Is the hypothesis that these are being converted to active small RNAs, or are they being taken up and translated, etc as they are? I know there's no room for data here, but just curious. I hope you push that work to publication. Find some good reviewers ahead of time for your grant proposals too... I get frustrated at the lack of vision and risk they want to take on something really edgy. This is a very interesting story and I'm excited to watch it develop. I've been intrigued with the Zhang paper ever since it first emerged. It would be really great finding in many ways.

 

@Mary Mangan The RNAi plants express a double stranded RNA molecule that is rapidly processed into small RNA duplexes (21-22 nucleotides, both strands because they derive from the original double stranded trigger). There is a population of such molecules and for each duplex, one strand incorporates into a protein complex called RISC, where it acts as a guide to find target messenger RNAs (by the base-pairing rules). The other strand is rapidly degraded. A number of investigators have shown that the population of small RNAs is not equally distributed - some are much more abundant than others. At least in some cases that has to do with the purine content of the particular small RNA, but this has not been investigated in detail in many cases. So what I am saying is that these transgenic plants contain a large and diverse population of small RNAs that represent both strands of the original targeted sequence. These small RNAs do not need to be completely complementary to a messenger RNA in order to target that message. So it becomes a very difficult problem to predict the off target effects of a particular RNAi gene. I should point out here that plants naturally produce many many small RNAs and that the pathways for the biogenesis of these RNAs are ancient. RNAi constructs such as the one Monsanto has produced in maize to combat corn rootworm are of particular concern, however. That is because these plants are making small RNAs that do not exist in nature, so we have not co-evolved with these RNAs. If it is true that plant-produced small RNAs are taken up by mammals (including humans) when we eat the plant and that they are functional in the mammal, then that is a safety issue that should be addressed. Perhaps it is not true, but that is a matter that is not yet settled. research to determine if the Zhang paper is correct should be encouraged. As things currently stand, Monsanto (a company with deep pockets and a strong financial interest in discrediting the Zhang result)has bombarded the literature with their negative results. Other investigators, on the other hand, are finding that it is very difficult indeed to get funding or published in this atmosphere. That's not how it should be in my opinion.

 

@Vicki Vance: Yeah, I'm down with the biology. But I see now, you are saying you don't have specific hazards in mind about the rootworm sequence. You have generic fears about this ancient mechanism. Got it. I thought based on your statement you knew of some specific danger. But I don't see any reason that we can't address any concerns with current technologies. And this has the capacity to be so much more precise than previous pesticide mechanisms that it's really valuable to investigate. I'd really like to see less chemical pesticide use for this problem.

I'm sorry that funding has been a challenge for you. I know that's true all around science--not everybody gets to blame Monsanto for that though! I firmly advocate for more public dollars for science--especially plant science.

I'm sure you are aware of the mechanism for pre-publication. I doubt Monsanto could stop you from publishing to arXiv or BiorXiv. In fact, you should see this really interesting paper about sequence contamination on arXiv: http://arxiv.org/abs/1401.7975 , it's relevant to the Zhang work. Might be worth a try for your papers, you can get credit for it and get public feedback.

 

Not crazy. CS has used a good metaphor.

 

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This RNAi technology has been experimentally demonstrated to kill or harm many different species when expressed from transgenic plants. There seems no fundamental reason to treat the Zhang research as an "extraordinary result". It is an "extraordinary result" largely only in that threatens a multi-million dollar (potential) product/technology. Which is why this standard is not scientific, in this case and many other.

Also I was at the previous at the previous ISBGMO conference (two years previously in Buenos Aires, Arg.) where I was given exactly 5 minutes to explain why I thought the existing scientific consensus on the safety of RNAi was wrong. That consensus has/had far too many flaws to explain in fifty five minutes (let alone 5), but the main argument used against what I was proposing was that RNAs would be digested in the gut. Prior to the Zhang paper I didnt have anything other than a cautionary answer to that since there was no data. But that is why the Zhang paper is extraordinarily important to Monsanto and Co. At that meeting it was clear that RNAi safety was a charged subject (my five minutes ended in have fingers pointed at me). The main invited speaker, however, had few qualifications (e.g. no publications on RNAi safety) and only the blandest and most superficial of opinions about the subject. Even published researchers from the EPA weren't invited, though I believe they applied to speak (I invited myself).

 

Thanks Jonathan, this whole thing is intriguing. What I'm trying to understand is why Zhang was the last paper, two years ago. Certainly it is a breeze to sequence small RNAs these days from any isolated source. If plant miRNAs were getting into the human body and eliciting physiological effects, do you think there needs to be specific transport, sequestration and amplification steps? If I do the math, seems like there would have to be. Maybe most importantly, if we really care about safety, shouldn't every animal or plant we consume be tested for miRNAs that can have human consequences? Did we breed them out because these were the plants that didn't kill us? I'm being really honest and just am trying to wrap my head around this whole thing. To me, Zhang et al is still an 'extraordinary result', and someone needs to publish something (especially Zhang et al) confirming or expanding the original work.

 

@Kevin Folta (question #1) You question why no one has published a result confirming or expanding the original Zhang report two years ago. If fact several publications have come out reporting that they see no evidence of plant small RNAs being taken up by mammals. Monsanto has spearheaded this explosion of negative results. They almost immediately published that the Zhang findings were due to "contamination" of the sequencing reactions with plant RNA. Most recently, they were able to publish a correspondence in Nature Biotechnology claiming that they tried to replicate the Zhang study but could not detect plant small RNAs in mouse tissues. However, they were also unable to detect abundant plant small RNAs in the plant tissues they fed the mouse!! This was one of multiple serious flaws in the Nature Biotechnology correspondence, which was none the less published and touted as discrediting the Zhang study. I will simply say (and this is from personal experience) that it is very difficult to get funding to do experiments in the onslaught of negative press provided by Monsanto. Reviewers take this immediate challenge to mean that the work is not true. The negative results are not held to any standard at all, but any data consistent with the original paper is held to extraordinary standards.

 

@Kevin Folta question #2. You asked me to explain my preliminary mouse experiments a bit more. Here's what we did. When the Zhang paper came out saying that plant-made small RNAs are taken up by mammals (including humans)when eating and that those plant small RNAs were able to regulate expression of mammalian genes, we had an idea. We thought this process could be a way to engineer edible plants to deliver beneficial small RNAs. In particular, it is known that certain small RNAs called microRNAs (miRNAs)are lost during many disease states in humans. This is best studied in cancer, where there are a set of miRNAs called "tumor suppressor miRNAs" that are lost at various stages of oncogenesis. In animal models, if these tumor suppressor miRNAs can be replaced, it blocks the cancer. So we designed an experiment to determine if plant-produced tumor suppressor miRNAs could prove to be an effective delivery system to block colon cancer (we used a mouse model for colon cancer for this experiment - apc min mice - these poor little mice are fine when they are young, but they ALL develop many intestinal tumors as they develop). Our results were very promising and highly statistically significant: apc min mice that ingested the tumor suppressor miRNAs showed a dramatic decrease in tumor burden. Furthermore, the daily dose of plant RNAs over the period of a month that produced this therapeutic result, showed no toxicity whatsoever - all the mice grew and gained weight and appeared as healthy as the control mice. This result is consistent with the Zhang paper showing that plant miRNAs are taken up during digestion and can function in the animal. However, we have not been able to get funding to pursue this line of work in spite of our preliminary results. Reviewers demand that we prove that the bioassay results (reduced tumor burden) is actually due to uptake of plant RNA (they ignore the bioassay results). Unfortunately, direct physical detection of plant small RNAs is technically difficult. This is because plant small RNAs are methylated on the 2' position of the 3' nucleotide (animal small RNAs are not methylated). The methylation interferes with techniques for high throughput sequencing of small RNAs or quantitative PCR methods that are required to detect small RNAs that are present at low levels. This set of circumstances means that it may not be possible to definitively demonstrate that the plant miRNAs are present in mouse tissues. This is because the small RNA we feed the mice is identical to the endogenous mouse miRNA except for the methylation at the 3' end of the plant small RNA. So we are left in this unenviable position. We think we have an effective, nontoxic and affordable chemopreventive and we cannot get funding to bring this remarkable technology to the world! How can this possibly be?? Well, it's in part because Monsanto is using the same technology to kill corn rootworms. They express their lethal small RNAs in the whole plant (not just the roots) and, if those small RNAs might also be taken up by humans and be dangerous, then they would have to invest in work to address this concern. They prefer to discredit the work. By doing so, they do a disservice to all of society.

 

Thank you Vicki, for your work and persistence. Stories of professional bullying about GMOs, including behind the scenes attempts by the industry and its friends to suppress contrary results and suppress debate, have unfortunately happened many times (for example, see several stories in Nature and Nature Biotechnology by Emily Waltz from several years ago: http://www.nature.com/news/2009/090902/full/461027a.html and http://www.emilywaltz.com/Biotech_crop_research_restrictions_Oct_2009.pdf). I hope you are spared more of this and continue with your important work. In addition to the current uncertainties about possible harm to humans and other mammals (and other vertebrates) from off-target effects, there is a need to better understand possible harm to many other organisms important to agroecosystems, e.g. beneficial invertebrates. These organisms are critical to the proper functioning farming, such as pollinating crops and killing harmful pests which reduces the need for harmful pesticides. For those organisms, we already know that small regulatory RNAs can be absorbed and have big effects. And of course, many important beneficial species are much more closely related to targeted pests than we are, so not only off-target, but also on-target (or "near-target”) effects could be important. For those who are not biologists, we have learned a lot over the past two decades that many genes are “conserved” (very similar) between many species (not usually the exact DNA sequence, but often without huge changes). On average, the closer the species are to each other, the more similar their genes. But even organisms distantly related can have stretches of sequences in common for some genes, and their function may be affected by on-target or off target effects from regulatory RNAs. There remain many important unanswered questions. But not unexpectedly, small RNA technology is already being touted as the next big thing, and as a bludgeon aimed at those who raise critical questions (this just occurred in a comment by a scientist to an article that I was asked to write for a technology journal). Your experience continues to raise important questions not only about the technology, but even more so about the process of science.

 

Dr. Vance and Dr. Latham, Thank you both for your scientific contributions to food safety and food sovereignty. As a Native American scientist I have great concerns for my people who evolved with corn and the risk they take for eating foods that are genetically modified, especially corn. The fact that many Americans have not co-evolved with the GE RNAs in corn, which is a culturally significant staple to my people and a (co-mingling) threat to species grown by Indigenous peoples for millennia. Your comment "That is because these plants are making small RNAs that do not exist in nature, so we have not co-evolved with these RNAs" is profoundly meaningful. Dr. Folta's strawberry work is a threat as well. Why does Dr. Folta need to genetically improve strawberry sweetness? Why does he have to publicly berate our colleagues, like Dr. Huber? Why does he participate in online trainings to teach trolling pointers and bullet points on how to disrupt social media dialog about GMO labeling?

 

Voracious Worm Evolves to Eat Biotech Corn Engineered to Kill It By Brandon Keim http://www.wired.com/2014/03/rootworm-resistance-bt-corn/

 

Thanks Dr. Vance for your complete answer. It sounds intriguing and I'm looking forward to additional follow up. Just to clear the misinformation, NativeGrl59, the always off-the-mark Valerie Goodness, once again chimes in on something she knows nothing about. My "strawberry work is a threat"? Of course, there are no GM strawberries and all of our improvements are made through marker-assisted breeding. Oops. I'm really interested because these small RNAs could be a huge threat through traditional breeding as well. There are many plants that have never been used for human consumption that are being introgressed into elite varieties by breeding. Mutation breeding also has the potential to produce new miRNAs. We really need some definitive research in this area. RNAi in GM plants is not a very common technology at this point, outside of viral sequences that are there in the first place. It will be important for us to understand these issues in all cases of plant genetic improvement, transgenic, traditional breeding or other methods.

 

REPLY TO THIS COMMENT

Small addition: If anyone wants to have a paper I wrote/distributed on RNAi for the ISBGMO meeting, I can email it to you. PLease dont ask unless you think you will understand the technical language but it covers off-target effects of small RNAs and the issue of dsRNAs. dsRNAs, as expressed in (future) transgenic RNAi plants, are elicitors of vertebrate antiviral defences and there is a substantive and informative but semi-forgotten literature on their hazards, that we recovered.

I can be reached from the website:

www.bioscienceresource.org

best wishes

 

 

Since sequencing is a problem because of the small size of RNAi molecules why has nobody thought of proving the knockdown of the target proteins by either western blots? It could also be done by knocking down a reporter protein in mice (eGFP?). That would be the best indicator of if miRNA or siRNA reaches the cells.

 

@Jens Hjrleifsson An extensive suite of experiments on the effects of the LDLRAP1 gene were done in the Zhang et al paper mentioned in the article. He monitored expression changes in human tissue culture cells and in mice. If you have access to it, well worth a read. If you don't have a subscription, we review his findings in depth in our open access paper here: Environment International, Volume 55, May 2013, Pages 43-55

 

Thanks Dr. Vance for your complete answer. It sounds intriguing and I'm looking forward to additional follow up. Just to clear the misinformation, NativeGrl59, the always off-the-mark Valerie Goodness, once again chimes in on something she knows nothing about. My "strawberry work is a threat"? Of course, there are no GM strawberries and all of our improvements are made through marker-assisted breeding. Oops. I'm really interested because these small RNAs could be a huge threat through traditional breeding as well. There are many plants that have never been used for human consumption that are being introgressed into elite varieties by breeding. Mutation breeding also has the potential to produce new miRNAs. We really need some definitive research in this area. RNAi in GM plants is not a very common technology at this point, outside of viral sequences that are there in the first place. It will be important for us to understand these issues in all cases of plant genetic improvement, transgenic, traditional breeding or other methods.

 

"There are many plants that have never been used for human consumption that are being introgressed into elite varieties by breeding." Absolutely. But the answer to this is to advocate for risk assessment of novel dsRNAs rather than advocate against risk assessment of novel dsRNAs that might be in GMOs just because there might be novel dsRNAs in both.

 

 
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