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.
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.”
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.