Gene Editing’s Extra DNA Problem: Déjà Vu All Over Again

UC Davis researcher Alison Van Eenennaam described the experience of learning that the “poster animals for the gene-editing revolution” do not, after all, comprise the “same outcome [that] could be achieved by breeding in the farmyard,” like she and her collaborators at Recombinetics had been claiming for years, to Antonio Regalado of the MIT Technology Review by saying simply that “We were surprised, but when you get new information, you proceed ahead, that is what science does.”

My reaction back in the early 1990’s to hearing similar news about extra, unintended DNA being inserted into Flavr Savr™ tomatoes, the poster GMOs for the first generation of genetic engineering technology, was somewhat more intense. My boss at Calgene, Inc. at the time, Bill Hiatt, gave me the news as we were traveling on the subterranean moving walkway system that connects the United Airlines terminals at Chicago’s O’Hare airport. I stopped in my tracks (although I kept moving). It just couldn’t be true, I said to myself; the flashing lights and recurrent United theme song intensifying the surrealism I felt at that moment. (Thinking about it now still gives me a queasy stomach.) I remember asking myself whether genetically engineered (GE; my definition covers gene-editing as well) products were really ready to bring to market when we, evidently, still had a lot to learn about how the technology worked and what unintended changes it could produce.

Like Van Eenennaam’s Recombinetics collaborators, we at Calgene had also initially informed US regulators at FDA and USDA that the changes we had engineered into our tomatoes were very precise, and that we knew exactly what DNA sequences we had inserted into them. And like Van Eenennaam and her animal scientist collaborators, we plant scientists only learned the truth about plasmid vector “backbone” DNA sequences getting unintentionally inserted into our GE products–in addition to the genes we intended to insert–through our interactions with scientists at FDA.

In our case, FDA had asked Calgene to provide experimental evidence to prove our claim that only our genes of interest would be inserted into our GE products, a claim which had been based on 10 years of published reports. It was the results of those experiments that Hiatt had conveyed to me at O’Hare airport. I, with a couple of Calgene colleagues, then published our findings in the Plant Cell in 1994. (Our paper might have been published in a more widely read, general science journal had it not been for the fact that one of its anonymous peer-reviewers seemed not to believe our results.)

In the case of Recombinetics’ GE cows, which had been engineered using one of the new gene-editing methods (TALENs) to prevent them from growing horns, FDA scientists were the ones who conducted the experiments that provided the incriminating evidence. FDA then informed Van Eenennaam and Recombinetics, as well as the Comissão Técnica Nacional de Biossegurança (CTNBio) in Brazil. (CTNBio had been planning–in conjunction with Recombinetics–to create a herd of gene-edited, hornless dairy cows; FDA’s results put an end to those plans.) FDA scientists are now in the process of publishing their results; a preprint describing them was posted online on July 28, 2019.

Will Publishing “Contradictory Results” Help?

It’s important that scientists publish findings like these so that developers of other GE products are aware of them and can, as mentioned by FDA scientists Norris et al. in their paper describing this latest GE “surprise,” employ “screening methods suited to reliably detect the unintended integration of plasmids and multiple template copies.” Because only then, after developers identify engineered/edited organisms that contain DNA they did not intend to insert, can informed decisions be made about whether or how to commercialize those products; for example, in cases like Recombinetics’ cows, in which the unintended DNA contains genes that convey resistance to antibiotics like ampicillin and neomycin/kanamycin, I believe the decision should be to not commercialize.

But publishing is apparently not enough. Some 20 years after we at Calgene published our paper about the “extra DNA problem” associated with the Agrobacterium method of inserting genes into plants, a former Calgene colleague of mine informed me that the scientists at the ag biotech company she was then working for had not been checking their potential products for vector “backbone” DNA that could have been unintentionally inserted during the genetic engineering process…until she brought them up to speed on the issue.

Similarly, papers documenting “unwanted” insertion of plasmid/vector DNA sequences when using zinc-finger nucleases to modify genomic DNA targets in recipient cells had been in the scientific literature for half a decade by the time scientists at Recombinetics published their results on gene-edited cows (see references cited in Norris et al.). Yet those published papers describing unintended insertion of DNA, as well as our paper describing the phenomenon in plants, were apparently not read or otherwise taken into account by the authors of the Recombinetics paper.

The preprint posted by FDA scientists Norris et al. cites additional, more recently published papers that also document unintended integrations of foreign DNA, as well as scrambled DNA at target sites and off-target effects, in various GE organisms that have occurred via use of various gene-editing/engineering techniques, including the CRISPR/Cas9 system.

And so I ask again the question I asked myself in the tunnel between United terminals at O’Hare airport back in 1993: are we really ready to bring gene-edited/engineered products to market when, as described by Norris et al., “our understanding of the unintended alterations” produced via these technologies is still evolving?

Regulation of all GE products–including those that have been gene-edited–should be based on the fact that they were produced using lab-based techniques that we still don’t understand very well

Norris et al. make recommendations for technical ways genetic engineers might rectify what these authors refer to as a “blind spot” in GE product development that was highlighted by their discovery of unintended insertion of vector/plasmid DNA into Recombinetics’ cows.

But FDA should go further. I hope that Recombinetics’ blunder will also convince US regulators that the agricultural biotechnology industry–including developers using the new gene-editing techniques–requires more regulating, not less (as opposed to claims made previously by Recombinetics). Developers of GE products should be required to adequately screen their potential products for these newly discovered failures of precision in the processes of genetic engineering/gene-editing.

For one thing, requiring that all GE products be regulated in this way will ensure that all developers of GE products become aware of this lack of precision in their methods.

For another, regulatory requirements for all GE products could save the ag biotech industry from itself since these kinds of mistakes reflect badly on the entire sector as noted in this August 26th Tweet by Chris Thompson @centrekicker82: “Between Theranos, and now this mess, the impending biotech revolution sure seems a bit… incompetent?”

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U.S. Grain Industry Believes USDA APHIS’s “New Proposed Rule [for regulating GMOs] Is Fundamentally Flawed”

I’ve got to give credit to the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) for persistence. USDA APHIS has proposed new rules for regulating genetically engineered (GE) crop plants (AKA genetically modified organisms: GMOs) on three different occasions over the last decade: in October of 2008, January of 2017 and again a couple of months ago.

There is no doubt that USDA APHIS needs new regulations for GMOs. Rather than based on science or risk assessment, the current rules are based on whether a GMO was engineered using a vector, vector agent, or DNA from an organism on USDA’s plant pest list. Consequently, the rules don’t apply to all GMOs and GMOs that likely don’t pose a “plant pest” or “noxious weed” risk are being regulated while others that could pose such risks are not being regulated at all.

But despite the agency’s efforts to update its regulations, the 2008 and 2017 proposals were withdrawn and–based on public comments it received on its most recent proposal–I expect (or at least hope) that the newly proposed regulations will be withdrawn as well.

Most of the comments on the new regulations proposed in the June 6, 2019 Federal Register (I estimate greater than 90% of them) were from individual citizens who were understandably unhappy that APHIS was planning to let developers of new GMOs determine for themselves whether their products required regulatory review or not. But citizen consumers were not the only ones worried about foxes guarding the henhouse…a group of U.S. grain and oilseed organizations submitted a pretty harsh critique of the new proposed rules as well.

Leaders of the National Grain and Feed, Corn Refiners, National Oilseed Processors, North American Export Grain, and North American Millers’ associations all believe that “APHIS’s largely deregulatory approach” would not only “undermine consumer confidence in the U.S. regulatory system” but also “create further impediments” to their industry’s ability to market grains and oilseeds in global markets because it “directly contradicts the well-established international norms of case-by-case risk-assessment” and is thus “largely out of step with key international markets and governments.” They also noted (as an aside) the aviation industry’s current “significant turmoil and scrutiny over the safety certification process where the federal government leaned heavily on self-determination by an aircraft manufacturer.”

They additionally called out APHIS for exempting some engineered crops from the new regulations “because they could be produced through traditional plant breeding techniques and thus are unlikely to pose a greater plant pest risk than traditionally bred crops, which APHIS has historically not regulated.” [Emphasis added by the grain industry leaders.]  This statement by APHIS, these industry leaders believe, provides “insufficient scientific justification for granting broad exemptions.” (As an aside, I note that assumptions about pilots being able to handle any failures or misfires associated with the new technology on board the Boeing 737 MAX, and about the technology’s failure being “unlikely to result in death or the loss of the plane” [my emphasis], contributed to the aviation industry’s current turmoil referred to above.)

The grain industry leaders also noted that “APHIS would be ill-advised to issue a final rule” until USDA had coordinated its efforts on “rules and guidance on genome editing and other forms of plant breeding innovation” with FDA and EPA. After all, the U.S. approach to regulating GMOs is supposed to comprise a “Coordinated Framework for Regulation of Biotechnology;” therefore, these grain industry leaders recommend that the efforts of USDA APHIS, FDA and EPA “should all be compatible with each other and be coordinated through the White House Office of Science and Technology Policy.”

In summary, the leaders of these U.S. grain associations “believe that APHIS’s new proposed rule is fundamentally flawed.” Two years ago they “jointly urged APHIS to go back to the drawing board in response to its 2017 proposed rule to modernize its biotechnology regulations….” Now they “strongly urge APHIS to amend” its newly proposed rule to “require all technology providers to notify the agency in advance before introducing gene-edited or other plant breeding innovation traits for commercialization–even those within APHIS’s expressly exempted categories–so that the agency can issue an official letter for all traits attesting that they do not present a plant pest risk.” [Emphases added by the grain industry leaders.]

I agree whole-heartedly with these agricultural industry leaders. It’s time for USDA APHIS (together with FDA and EPA) to go back to the drawing board and try again to craft effective regulations for GMOs.

Let’s hope APHIS has the persistence to get these regs right on the fourth try.

 

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Disappointment in the First 25 Years of GMO Foods

The world’s first, commercially available GMO (genetically modified organism) created for human or animal food was first sold in U.S. grocery stores twenty-five years ago today.

I can still remember the excitement my colleagues and I at Calgene, Inc. felt as we ushered into the marketplace that first genetically engineered (GE) whole food (AKA GMO) available to consumers. We told each other it was something we’d tell our grandchildren about.

Granted, I’d gone into the agbiotech industry with hopes of helping solve the world’s problems by contributing to projects like engineering maize (corn) to fix its own nitrogen, and the Flavr SavrTM tomato had not been created with that lofty ideal in mind. Instead, these tomatoes had been genetically engineered so that they softened more slowly, an attempt to allow them to remain on the vine longer, to let them develop their true tomato flavor as they would in one’s own garden, yet remain firm enough to truck them, en masse, to market.

Still, I rationalized back then, this tomato would open the door for the technology that produced it, allowing others to solve more important agricultural problems. And so at Calgene, in May of 1994, we celebrated what we considered to be a huge, important milestone in agricultural history.

Now, 25 years later, I certainly don’t feel the same excitement or satisfaction about that achievement.

In fact, my principal emotion now is one of disappointment. Profound disappointment.

Not only are we still not able to engineer corn or any other non-leguminous crop plant to fix its own nitrogen (which could reduce use of nitrogen fertilizers which would, in turn, benefit smallholder farmers and reduce the fertilizer run-off into rivers that creates “dead zones” in the world’s oceans), but the agbiotech industry hasn’t produced much of anything else that might help solve significant agricultural problems either.

GE Golden Rice still isn’t available, for example, due to both project-specific technical difficulties and agbiotechnology-induced mutations in potential products. And even when it finally becomes available to those it was designed to help, it will remain to be determined how effective it may be at reducing vitamin A deficiency. (An initial study with children in China was retracted due to inappropriate collection of participant consent information, among other issues.)

The introduction into Burkina Faso of cotton varieties genetically engineered to produce their own insecticide turned into another disappointment recently. Instead of overall farmer savings associated with reducing the number of pesticide applications yet protecting the crop and thereby increasing yields, the poor quality of the cotton fiber from those GE varieties has contributed to economic problems for the smallholder farmers there. (A Google search today indicates that perhaps the GE cotton situation in Burkina Faso may be improving; I hope so.)

And the Republic of South Africa apparently refused to approve a GE “drought-tolerant” (DT) maize/corn for production and sales in that country recently. The government’s regulatory authorities indicated that the available data didn’t demonstrate that DT GE corn was significantly different from conventional, non-DT varieties in terms of drought-tolerance…and they weren’t pleased with the DT GE crop’s grain yield either.

Overall, most of the GE crops grown around the world today have been engineered to produce their own pesticide (usually in every part of the plant—including the edible parts), or to be tolerant of herbicides (predominantly glyphosate, which was classified an animal carcinogen and a probable human carcinogen in March 2015), or both. Perhaps as a consequence, most of the GE crops grown anywhere on our planet today are fed to animals.

Anywhere, that is, except in the United States. Here it has been estimated that 70 to 80% of processed foods in grocery stores contain GE ingredients, and some varieties of sweet corn and crookneck squash are GE as well.

Why this difference between the U.S. and other countries? As opposed to more than 60 other countries throughout the world, the U.S. has not—for the past 25 years since GE food products first entered the marketplace—required GE foods or ingredients to be labelled as such (although producers will have to comply with a recently enacted federal labelling statute in the near future). The likely difference, therefore, is that Americans simply haven’t been given a choice in the matter.

As a scientist, I’m sorely disappointed that this powerful technology hasn’t been used more carefully and judiciously and to better the human condition, instead of for purposes like selling herbicides/poisons and supporting unsustainable industrial agricultural systems.

As a U.S. citizen, I’m sorely disappointed that—after the initial introduction of Calgene’s GE tomato—the biotech industry chose to deny the American people their right to know what is in their food.

And as a citizen of Planet Earth, I am appalled at the tactics, both alleged and exposed (see here and here), that have been used by Monsanto, until recently the largest producer of GE crop products in the world, to promote (what I consider) disappointing products.

Nowadays, I wonder what I’d tell my grandchildren about bringing the first GE whole food to market. I guess I’d tell them that Calgene was commendably transparent during the process, that we told the FDA and USDA about the technology’s positives as well as its negatives. And that we labelled our GE tomatoes—at least initially.

But then I’d have to admit to them that Calgene failed as a company. Monsanto purchased it and, soon thereafter, the world’s first GE whole food was eliminated from Monsanto’s Roundup Ready-prevalent product line.

Perhaps I’ll go on to tell my (currently hypothetical) grandkids that I made an effort, at least virtually with this blog, to fill in some of the gaps in transparency about agricultural genetic engineering and its products —the things not expressed by the agbiotech industry and academic scientists who support it—for consumers shopping for food for themselves and their families.

In any case, whatever I tell them, the story won’t have the worldwide, problem-solving ending I was hoping for 25 years ago—unless things drastically change for the better in the near future.

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UC Professors Weigh in on Glyphosate (AKA Roundup) and Carcinogenicity

Glyphosate, the “most widely used herbicide in the world,” is in the news a lot lately, largely because the first of thousands of lawsuits, filed by folks who used Monsanto’s Roundup (or other glyphosate-based herbicide products) for decades–who assumed it was as safe as the company claimed it was–and then developed cancer, are now coming to trial.

To those of you who might be interested in learning the lengths to which Monsanto apparently went in defending its glyphosate-based product(s), I highly recommend that you follow the Courthouse News Service articles on these trials.

The opening statement in the third case to go to trial, for example, by the attorney for a California couple who were both diagnosed with non-Hodgkin lymphoma within four years of one another after having sprayed Roundup on their residential properties for 35 years, could serve as the basis for a great movie plot (IMHO).

As described in Roundup Trial: Monsanto Used Fake Data to Win Over Regulators, Monsanto “seemingly planted one of its employees at a contract lab…in the 1970s to fake negative mouse carcinogenicity data for…glyphosate that were to be used to win regulatory approval for the weed killer in 1975; planned an attack to discredit the World Health Organization’s (WHO) cancer research agency, anticipating the agency would classify glyphosate as a probable human carcinogen in 2015 [which, nevertheless, the agency did]; and exploited ‘deep connections’ within the U.S. Environmental Protection Agency to classify glyphosate as non-carcinogenic.”

You may have heard some of these allegations before. Bloomberg reported on the Monsanto-EPA connection in “EPA Official Accused of Helping Monsanto ‘Kill’ Cancer Study” (published in March of 2017), for example.

But maybe not others, like the much older scandal at the now defunct contract lab. (This 1983 article in the Washington Post provides some general information regarding inadequate or falsified data on over 200 pesticides–including Roundup–tested by the specific contract lab mentioned in the current trial.)

Jurors serving on the current trial are also learning about a new study, published in Mutation Research in February 2019, that found “a compelling link between exposure to glyphosate-based herbicides and an increased risk of non-Hodgkin lymphoma.” The first author of this new scientific paper, which comprises a meta-analysis of multiple previously published studies that were designed to determine whether glyphosate exposure increases cancer risk, is an adjunct professor of toxicology with the School of Public Health at the University of California (UC) in Berkeley named Luoping Zhang.

Dr. Zhang, along with two of her co-authors on the Mutation Research paper, had been members of EPA’s 2016 scientific advisory panel on glyphosate. But “Zhang was ‘so outraged’ by the EPA’s failure to follow its own herbicide-assessment guidelines” that she resigned from the advisory panel to carry out the meta-analysis herself. The resulting study is focused on the evidence pertaining to groups of people most highly exposed to glyphosate, and also takes into account the most recent publication on the Agricultural Health Study, a large and long-term study of the effects of pesticides on U.S. farmers that was initiated in 1993. One of Monsanto’s complaints about the WHO’s 2015 determination that glyphosate is a probable human carcinogen had been that the agency hadn’t taken the most recent results from the Agricultural Health Study (AHS) into consideration. (WHO hadn’t taken them into consideration because those results hadn’t been published yet.) But after considering all the available published data–including the latest data from the AHS–Zhang et al.‘s results still indicated that high exposure to glyphosate increased a person’s risk of developing non-Hodgkin lymphoma.

Another UC Berkeley professor, Elena Conis of the Graduate School of Journalism and Center for Science, Technology, Medicine and Society, recently wrote an overview of the history of how pesticides have been regulated in the U.S. that sheds some light on how we got to this point where pesticides “are innocent and on the market until…enough independent science has produced evidence of harm….” In her Perspective piece in the Washington Post, Dr. Conis mentions that the EPA classified glyphosate as a carcinogen in 1985 but “reversed course after six years of correspondence with Monsanto executives;” that since then Monsanto has asked EPA to base its decisions on Monsanto-commissioned science; and that “[i]n one instance, the EPA ceded to industry requests to remove a certain scientist from a glyphosate safety review panel.”

On the other hand, I, for one, am grateful to Dr. Zhang for removing herself from the 2016 glyphosate review panel so that she could conduct the kind of independent science that is so desperately needed to protect humans from capitalists who put their bottom lines ahead of our collective health. U.S. regulatory agencies need to correspond more with scientists who are not working for, or otherwise aligned with, the companies which make money by developing poisons (which is what pesticides were called until after WWII) to spray on our food crops.

In the meantime, while the safety of glyphosate is being debated in the courts, there are steps you can take to limit your exposure to it from the food you eat.

Traditional crops die if glyphosate is applied to them, but crops genetically engineered (GE) to tolerate glyphosate do not. So avoiding GE fruits, vegetables and grains (AKA GMOs) should reduce your intake of glyphosate.

However, some non-GMO crops, like wheat and oats, are now sprayed with glyphosate just prior to harvest to speed up their desiccation–and the regulatory agencies have responded to this new farming practice by increasing the legally permissible levels of glyphosate residues on those crops (see Zhang et al. and references therein for more information on exposure to glyphosate). Consequently, the best way to avoid glyphosate in these crops is to buy products from crops that have been organically grown; glyphosate use is not allowed in organic farming per the current USDA organic standards.

 

 

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“Modified” the Film: Upcoming Screenings

A new film dubbed “A Food Lover’s Journey Into GMOs” was released recently and I highly recommend it…both as an interesting, beautiful and poignant story of its own and also as a means toward filling in gaps in the GMO story as told, for example, in the film Food Evolution (which has been called propaganda by many).

It’s called Modified. Thus far, it’s received 13 film festival awards (including four Audience Choice awards) and well known chef Jacques Pépin calls it “a very personal, tender, touching tribute and a well-researched, enlightening and powerful documentary.”

Canadian Aube Giroux, who wrote, directed, edited and produced Modified, tells me that “audiences respond very strongly and favourably to the film.” I can see why. As described on its website, Modified intimately portrays a “mother-daughter quest for answers” about GMOs, interweaving a “visual celebration of food”, cooking, gardening and family legacy with an investigation into the “extent to which the agribusiness industry controls our food policies.” Modified makes a “strong case for a more transparent and sustainable food system.”

Check out the 2019 schedule for screenings of Modified. It’s being shown in Nevada City on Feb. 24 at Nevada Theatre, for example. And it will be screened during the San Luis Obispo International Film Festival March 12-17 and during the Phoenix Film Festival April 4-14.

IMHO, Modified does a much better job of laying the groundwork for a more civil and productive societal debate about GMOs than efforts like Food Evolution. But view it for yourself and see if you agree.

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New Salk Institute Study Reveals More Detailed Picture of “Scrambled Nature” of Gene Insertions in GMOs

Plant molecular biologists have known for decades that the techniques they use to genetically engineer plants are error prone and can result in GMOs with unintended:

  • mutations in host plant genes,
  • insertion of vector backbone DNA sequences,
  • small duplications or deletions of inserted or host DNA, and/or
  • large rearrangements of host plant chromosomes.

Other unintended changes are also possible; for example, Monsanto’s commercially available NK603 corn unintentionally produces a hybrid RNA molecule derived from sequences of the foreign DNA inserted into that product and sequences from the recipient corn variety’s DNA adjacent to the insertion site.

But, according to the new study published by Jupe et al., “detailed understanding of structure and epigenomic status of insertion events [had been] limited” by the technologies available to researchers; “plant scientists didn’t have the molecular tools” that could enable them to “see” the multiple effects that inserting pieces of foreign DNA into a host plant’s genome have had on those genetically engineered (GE) genomes in a single “picture” until recently.

For this new study, conducted under the supervision of Joseph R. Ecker, a highly respected academic plant molecular biologist at the Salk Institute for Biological Studies, researchers used “state-of-the-art long-range sequencing technologies…to provide new insights into the structural impact of inserting foreign [DNA] fragments into plant genomes….”

These authors, primarily associated with the Salk Institute but including a scientist from the Donald Danforth Plant Science Center and two others from the J. Craig Venter Institute, utilized the state-of-the-art techniques to elucidate unintended changes in the genomes and epigenomes of Arabidopsis thaliana plants that had been genetically engineered using the Agrobacterium-based method. [Arabidopsis is a model plant organism used by many academic researchers. The Agrobacterium-based method of genetic engineering involves the intended transfer of a specific piece of DNA (the transferred DNA or “T-DNA”); this method is good enough at mutating host plant genes that it was used by plant scientists in an effort to mutate every gene in the Arabidopsis genome. Many commercially available GMOs were created using the Agrobacterium-based method of genetic engineering.]

The new study revealed the “scrambled nature of T-DNA and vector backbone insertions and rearrangements in high detail” and “uncovered various effects of T-DNA insertions on the adjacent chromatin landscape.” Unintended “intra- and interchromosomal rearrangements” were discovered, for example, as well as unintended “alterations of chromatin and thus chromatin structure and functionality.” With “unprecedented detail” this study reveals “novel insights into the impact of these events on plant genome/epigenome integrity.”

The authors point out that this kind of “Knowledge of structure variations induced by transgene insertions…as well as evidence for epigenetic changes to the host genome is crucial from scientific as well as regulatory perspectives.”

I agree. We just don’t know enough about genomes and epigenomes, and how randomly inserting foreign pieces of DNA into them and scrambling them will affect them. Therefore, I think U.S. regulators should take a more precautionary approach to regulating GMOs, and require developers of GMOs to provide information about the integrity of the genomes and epigenomes of their products prior to commercializing them.

But the FDA has never required developers of GMOs to provide the kind of crucial information about structure variations induced in their products by transgene insertions that Jupe et al. addresses…even though older techniques for revealing most of them have been available for decades. And some who have provided information about the “scrambled” genomes in their GMOs to the FDA voluntarily–like Monsanto did for NK603–have gone ahead and commercialized those scrambled products anyway.

Jupe et al. conclude the Discussion of their results by stating that “Our findings pave the way for structural genomic studies of transgenic crop plants….”

Maybe. But will developers of GMO crops follow this newly paved path? Will FDA make them?

Or will GMOs with scrambled genomes and epigenomes continue to be commercialized?

 

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Golden Rice: Of Good Intentions, Insertional Mutants, Human Error, and the Need for Better Regulation of GMOs

After learning that the genetically engineered (GE) Golden Rice “event” being bred into varieties of rice favored by Indian farmers turned out to have a mutation—a mutation created as a result of the genetic engineering process itself (you can check out my first post on this subject here)—I was curious to learn more about the history of this genetic engineering project undertaken for humanitarian purposes.

The Golden Rice project is an attempt to use genetic engineering to alleviate vitamin A deficiency, a major cause of blindness, illness and death—mostly among young children—in developing countries where diets can consist primarily of rice and sometimes little else in the way of vegetables or other foods that contain beta-carotene, a compound that human beings can then convert into vitamin A. The project was initiated in the early 1990s and was widely publicized in 2000 when a photo of Ingo Potrykus, one of its “founding” scientists (along with Peter Beyer), appeared on the cover of Time magazine.

But it’s been 18 years since then and Golden Rice is still not available to the world’s poor who might benefit from it. Why is it taking so long to deliver this humanitarian product?

A very revealing paper by a member of the Golden Rice Humanitarian Board, Adrian Dubock, provides some answers. In it, Dubock asks “What can Golden Rice’s development history and trajectory teach us?”

SPOILER ALERT! My answer to that question—which is essentially diametrically opposed to his—is this: Check early in your GE project to identify any unintended insertional mutations in your prototype crops that were caused by the genetic engineering process, and then throw any GE “events” that have such a mutation out of your product development pipeline! (Dubock’s answer, on the other hand, might be summed up as: international regulation of GE crops, in the form of the Cartagena Protocol, impeded our progress every step of the way; more on whether regulation of GE foods is currently adequate at the end of this post.)

Are Other Developers of GE Crops Neglecting to Discard the Unintentional Mutants Created by the Genetic Engineering Process?

Golden Rice’s history also begs the questions: how many other developers of GE crops have neglected to screen their GE prototypes for insertional mutants? Are there insertional mutants among the GE crops currently on the market? (I’ve tried, unsuccessfully, to determine whether Monsanto’s NK603 GE corn was screened for this potential problem, for example.)

But I’m getting ahead of myself….

Timeline of the Development of Golden Rice

What follows is a timeline of the development of Golden Rice focused on events relevant to the problem of the potential for unintentionally creating GE mutants, a problem still faced by all developers of GE crops using the Agrobacterium-based and biolistics methods. This timeline is based on the information contained in “The present status of Golden Rice” by Adrian Dubock. (The information I mention from Dubock’s article can be found on the pages of it indicated in parentheses below; emphases are mine.)

2000: Ye et al. publish a paper describing the first “prototype” Golden Rice; the inventors of Golden Rice trade “commercial rights in the technology to Zeneca [soon to be Syngenta], in return for the companies [sic] support for the inventor’s humanitarian vision;” Zeneca is granted exclusive rights to Golden Rice technology “free of charge for humanitarian applications, but royalty bearing for commercial applications” (p.71).

The “Golden Rice Humanitarian Board is created “to advise the inventors” during its development and one of its “first agenda items was to hear the advice of a Zeneca biotechnology regulatory specialist about the molecular characteristics required of a genetically transformed GMO-crop to ensure that it would be able to be registered for use under the regulations to be derived from the very recently (2000) published, but not yet in force (2003) Cartagena Protocol on Biosafety” (p.73); the US biotech industry pays for television advertising “implying fields of golden rice were [already] growing in US fields…” and Gordon Conway, President of the Rockefeller Foundation, joins “anti-GMO activist Vandana Shiva in agreeing that ‘the public relations uses of golden rice have gone too far’” (pp.72-3).

2001: A license agreement is signed by inventor Potrykus and Dr. Ron Cantrell, director of the International Rice Research Institute (IRRI) in the Philippines, and samples of the Golden Rice prototype are hand-delivered to IRRI two days later (p.73).

2002: The Humanitarian Board asks the “network of public sector rice research institutions” to create more than 1000 additional transformation events “from which it was hoped an improved transformation event could be selected collaboratively, to be taken forward as the one lead event by all Golden Rice licensees” (p.73).

2003: New GE “events” of Golden Rice [GR1], the best of which accumulates 13 micrograms (μg) total carotenoids per gram (g) of rice, as opposed to the 1.6 µg/g in the prototype, were created and plans to conduct field and human feeding trials with some of them are made; an additional version of Golden Rice [GR2], with even higher levels of beta-carotene, looks promising in Syngenta’s labs (pp.73-4).

2004: Field trials of GR1 Golden Rice are conducted in the US; Syngenta donates GR1 and 13 GR2 “events,” at least the latter “of which were considered by [Syngenta Biotechnology, Inc.] scientists and regulatory specialists to be ready for and capable of complying with regulatory studies and standards” (p.76), to the Golden Rice Humanitarian Board and announces that it has “no commercial interest in the Golden Rice project” (p.75); the Humanitarian Board decides to concentrate efforts on 6 of the 13 GR2 individual transformation “events” donated by Syngenta (p.76).

2005: Paine et al. publish a paper describing the development of the GR2 events.

2006: IRRI requests, and Syngenta provides, molecular data on the 13 GR2 events (p.76).

2008: The field phase of a feeding trial with Chinese children is conducted; IRRI plants the first confined field trial of events of GR1 and GR2 Golden Rice in Los Banos, Philippines (p.77).

2009: A meeting of the Humanitarian Board is held in March to choose the top 2 Golden Rice events for product development; results of the feeding trial with human adults in the US are accepted for publication and presented at the meeting (p.77); agronomic data on 3 GR1 and 6 GR2 events, introgressed into 4 different indica rice varieties, were considered and Dr. S.R. Rao (Dept of Biotechnology, Government of India) asked “if there was any molecular data available to support the decision making. No such data was forthcoming (although IRRI had received it in 2006, it appeared to have been forgotten)” (p.78); nevertheless, based on the data the Board did have at hand, event GR2G was selected as the “Lead Transformation Event, with event GR2R as a back-up event if needed” (p.78); by December 1st scientists at IRRI had examined the molecular data on the GR2 events (provided by Syngenta back in 2006) and realized that the DNA inserted into GR2G, the lead event and the one used in the human bioconversion trials, contained an unexpected, unintended deletion of ~400bp; the newly found/examined molecular data also indicated that in both GR2G and GR2R the genetic engineering process had created mutations at the site in the rice genome where the foreign DNA had been inserted; but, “[d]espite the summary slide provided by IRRI, The Board had not reviewed, nor did most have the training, to ‘review all sequence data’ in any meaningful way, and it is unclear which other individuals had or the level of scrutiny afforded to it,” the decision was nevertheless made to drop the GR2G event and “bring forward” the GR2R event as the lead event (p.79).

2010: The grant to Peter Beyer from the Bill and Melinda Gates Foundation terminates; “Instead the Foundation intended to award a grant for development of Golden Rice itself to IRRI, for management of Golden Rice out of IRRI” (p.80).

2011: By October 3, GR2R seed had only been supplied to research institutes in two countries, India and the Philippines; the “inventors and the public sector Golden Rice licensees in other countries are very frustrated by this slow progress” (p.80).

2012: “Dr. Tang’s research with Chinese children, initially spoken of in 2003/2004 showed that” a bowl of cooked Golden Rice [GR2G?] “can provide ~60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-y-old children;” “Greenpeace issued a press release condemning use of a GMO-crop, Golden Rice, with Chinese children as ‘guinea pigs of American researchers;’” [Tang et al. (2012) was later retracted (and China sacked officials in that country who were associated with the experiment) due to failure of the researchers to follow Tufts University rules regarding securing consent from research participants]; 5 multi-location field trials with GR2R are planted in the Philippines (p.80).

2013: In August one of the field trials was destroyed by anti-GMO demonstrators; data from the other 4 field trials indicated a “yield-drag” in Golden Rice GR2R compared to wild-type rice; in December, the “IRRI Network Coordinator appeared to recall the issue summarized in his December 2009 slide” indicating that the new lead event, GR2R, was an insertional mutant (pp. 80-1).

2014: IRRI posts information on its website about the poor yields with GR2R [but fails to mention the fact that it contains a mutation caused by the genetic engineering process] and that “a decision has been reached to move forward from work solely focused on GR2-R to also include other versions of Golden Rice, such as GR2-E and others” (p.81); Adrian Dubock publishes “The Present status of Golden Rice” in the Journal of Huazhong Agricultural University.

What can this history of Golden Rice teach us?

This is an astounding story. A group of humans with all the best of intentions, who apparently tried to take into account–from the very start of their work together–the regulatory requirements in the various countries for creating, transporting, and marketing a GMO food, and yet years of time and loads of effort and who knows how much money (the Gates Foundation?) were wasted on Golden Rice events that should have been culled from the “production pipeline” by 2005 (or 2006 at the latest). Sad!

Errors were made by multiple humans involved in this story. Why did Syngenta hand over mutant Golden Rice events to the Humanitarian Board in the first place? Why didn’t scientists at IRRI thoroughly examine the molecular data on those events right after they had specifically requested the information from Syngenta? Why show molecular data indicating that 2 of the 3 top Golden Rice events have their foreign DNA inserted into rice genes to a decision-making body incapable of understanding the ramifications of those mutations (that were caused by the genetic engineering process)?

We don’t know whether the insertional mutations in Golden Rice events GR2G and GR2R made the rice grains harvested from those plants unsafe for human consumption. But most mutations are deleterious, and determining whether a particular mutation would cause a problem(s) in rice that would make it unsafe for consumption would require further investigation (of each GE mutant food variety). The most expedient way to deal with insertional mutants caused by the genetic engineering process is to identify them early and eliminate them from commercial development.

Glenn et al., mostly employees of Monsanto, indicate that they now do cull GE insertional mutants in this way. But since they also indicate that they now eliminate GE events with other imprecise manifestations of the genetic engineering process from their commercial pipeline, when Monsanto certainly has not always done that, could there be any GE mutants already in our food supply? And do other developers of GE food crops follow the procedures now used by Glenn et al.?

My take on the Golden Rice story is that we need better, truly science-based regulation of GE foods, regulation that recognizes the imperfections in the technology and regulates its putative products accordingly. Regulators in every country around the world should deny “approval” of any GE food crop that contains a mutation caused by the genetic engineering process. They should do so because genetic engineering is imperfect, and humans–even those with the best of intentions–will continue to make errors, and combining this imperfect technology with error-prone humans could have negative effects on our food supply.

 

 

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