| Agricultural biotechnology is in retreat. To win the perception wars, the industry must have products that consumers want. It also needs better technology for creating these products. Both are at hand, but will companies take advantage? |
Agbiotech is under siege. No new genetically modified (GM) crops have been approved by the European Union since April 1998, and a de facto moratorium on new GM products is in place. Brazil remains off limits, and Japan will require labeling of GM foods beginning next year. Baby food makers H.J. Heinz Co. and Gerber have rejected GM ingredients, and agribusiness giant Novartis AG is phasing out GM ingredients from all its food products due to consumer pressure. This year, for the first time, GM corn and cotton acreage in this country declined, as farmers realized that the markets for these crops were disappearing fast.
Stunned by these setbacks, life sciences companies are jettisoning their agricultural assets. Novartis and AstraZeneca plc are on the verge of spinning off their combined agribusiness divisions as Syngenta AG. Pharmacia Corp., which just merged with Monsanto Co. at the end of March this year, is now about to sell 13.7 percent of Monsanto in a public offering. And rumors persist that Aventis SA will spin off its crop protection business. “It wouldn’t surprise me if they went ahead and did it,” says A.G. Edwards & Sons Inc. analyst Alex Hittle. “Pharmaceutical businesses get much higher valuations, so by spinning off these [agricultural] businesses, you leave behind the pharmaceuticals.” The model, pioneered by Monsanto, of the integrated life sciences company, using biotechnology to help create both new drugs and GM foods, appears dead. “That vision, in Wall Street’s eyes, failed,” says Hittle.
The stock market’s 1997-98 infatuation with agbiotech now seems a distant memory. “We’ve gone from euphoria to dysfunction,” says Sano Shimoda, president of Bioscience Securities Inc. in Orinda, CA. “The industry thinks the worst is over. I think there’s going to be a lot more downside here.”
Life sciences companies, convinced that GM crops were safe, failed to anticipate the European backlash. “It was a question of mindset,” says Shimoda. “The companies were coming from a science mindset. They failed to put enough emphasis on the consumer acceptance issue.” In retrospect, commercializing herbicide-resistant and pesticide-engineered crops first was a mistake. “The first GM crops produced were all ones where the benefit accrued to farmers,” says Hittle. Even if industry thinks there's no real risk, he adds, "the consumer takes the risk and all the benefits go to the farmer."
The industry, in at least one important instance, also rejected technology that could have blunted a major objection to GM crops. Although USDA researchers demonstrated a method to remove antibiotic-resistance genes from transgenic plants in 1991, only recently -- prompted by new rules from the European Union -- have companies acted to avoid the theoretical danger of causing antibiotic resistance in microorganisms. Although there is no evidence to date that such “horizontal” gene transfer from plants to bacteria takes place outside the laboratory, companies had the opportunity to head off the issue, and chose not to.
The good news for the agbiotech sector is that it now can profit from these lessons. Stunning advances in our understanding of micronutrient pathways in plants have set the stage for new products -- Golden Rice is only one -- to attack nutritional problems in both the industrial and developing worlds. Consumers, in theory, would have a reason to buy these products. And advances in plant transformation technologies, especially targeted gene transfer, should address concerns about multiple, random gene integration, as well as make new product development more efficient. Still, returns wouldn’t come for years, so it remains to be seen if an industry in crisis will aggressively pursue nutritional enhancement and targeted gene transfer. But the science has arrived.
In the nine months since Ingo Potrykus’ paper on beta-carotene rice appeared in Science (X. Ye et al. Science 287, 303 [2000]), the Swiss molecular biologist has been on the cover of Time Magazine, and his Golden Rice has become famous, even though it’s years away from market. In agbiotech circles, the story of how Potrykus’ team at the Swiss Federal Institute of Technology in Zurich engineered genes for three enzymes into rice endosperm, in order to synthesize the beta-carotene that’s normally confined to the bran (typically discarded in developing countries), is now well-known. Potrykus must still demonstrate the ability to cross-breed his strain of japonica rice with indica rice -- the dominant strain in the developing world -- but that work should soon begin. Although progress halted due to what Potrykus calls a "frightening" patchwork of patents on the genes and methods he employed, those issues, he says, have now been resolved. “As we have achieved ‘freedom to operate,’ the rice breeders will soon be able to begin with their work,” Potrykus told Signals.
Ingo Potrykus |
A year before Potrykus' paper, Science published a less dramatic but potentially more influential one. In 1998 Dean DellaPenna, then at the University of Nevada, Reno, demonstrated that over-expression of a single enzyme could elevate Vitamin E content in Arabidopsis seed oil by a factor of nine (D. Shintani and D. DellaPenna Science 282, 2098 [1998]. Arabidopsis is a small plant in the mustard family that's become the model organism for studying the molecular genetics of flowering plants.) Although complete Vitamin E deficiencies are relatively rare, this lipid-soluble antioxidant might have protective effects against heart disease, cancer and degenerative diseases when taken in larger amounts. But DellaPenna’s tour de force was not putting the gene into a plant -- that part was routine -- but finding it in bacteria in the first place.
Genomics made it all possible. DellaPenna's gene-finding journey took him from human to plant to bacteria -- then back to the plant. First, he found one enzyme, the one that initiated the tocopherol (vitamin E) pathway in Arabidopsis (HPPDase), by using the human homologue. “We were able to clone this gene without ever having touched the protein, basically by making hypotheses in the computer and testing them in the wet lab,” says DellaPenna.
Then DellaPenna used a clever genomics approach to find a second enzyme, the key one in the pathway. He guessed that it might be found on the same bacterial operon, or gene cluster, as the gene for his first enzyme. The genome of the photosynthetic bacterium Synechocystis had recently been sequenced. DellaPenna identified a likely candidate in the computer, and knockout and over-expression experiments verified that his gene candidate was gamma-tocopherol methyltransferase (gamma-TMT), which catalyzes the methylation of gamma tocopherol to form alpha-tocopherol, the form of tocopherol with the highest Vitamin E activity. Finally, finding a homologous sequence in Arabidopsis was easy, and over-expressing the gene yielded the spectacular results reported in Science.
Arabidopsis
“The entire project, from thinking about making high vitamin E plants, to actually having high vitamin E Arabidopsis, took about sixteen months,” DellaPenna says. “[That's] an amazingly fast period of time.” Soon after, DellaPenna coined the term “nutritional genomics” to describe his approach, and it’s now catching on among scientists. “It’s basically all the things that we normally do -- biochemistry, physiology, genetics, molecular biology -- but… interweaving them very tightly using genomics as the common thread,” he says. “What genomics has really allowed us to do is to speed up our experiments, both the successes and failures.”
DellaPenna, who moved to Michigan State University this fall, believes that nutritional genomics will quickly help uncover how the other vitamins are made in plants. “I see no reason why, in the next three years or so, all the vitamin pathways in plants won’t be worked out, to a large extent,” he says. About three billion people worldwide, he points out, suffer from chronic micronutrient deficiencies. “We’re in a unique position to actually do something beneficial,” he says. “This won’t cure malnutrition, but it will make a dent in the problem. And that’s all we can hope for.”
Iron is also a major target, since as many as two billion people suffer from iron deficiency anemia, including many in the U.S. Rice, for example, is notoriously low in iron. What’s worse, most iron is stored not in the seed that’s eaten, but in the plant’s shoot. Michael Grusak, a plant physiologist at the USDA’s Children’s Nutrition Research Center in Houston, is working on iron transport from the shoot and leaf to developing seeds. He’s identified a pea plant mutant that puts three- to four-fold higher levels of iron in the seed, and is trying to isolate the gene (or genes) responsible. “Our hope, if we can identify how this [pea] mutant is doing it,” says Grusak, “is to then use that information and move into, for instance, rice.”
Ingo Potrykus and Paola Lucca have also been working on iron. Their approach, complementary to Grusak’s, is three-fold. First, over-express the iron-storage protein ferritin; second, add a gene that codes for a protein rich in cysteine, an amino acid that helps the human digestive tract absorb iron; and third, inactivate phytate (a.k.a., inositol hexaphosphate), a molecule that ties up dietary iron and keeps the human body from absorbing it. Lucca has now succeeded, says Potrykus, and their paper will soon be published in Theoretical and Applied Genetics. Potrykus hopes to cross the iron strain with his beta-carotene rice to combine both improvements in a single plant. But, he admits, “increasing iron alone may not be effective.” Since plants can’t make iron -- it must all be absorbed from the soil through the roots -- transport of iron to the seed is critical. Hence the importance of Grusak’s work.
None of this is a sure thing. For example, reducing phytates in plants helps humans absorb the nutrients, but it could hurt the plant. “The developing seedling needs that phosphorus to get started,” Grusak notes. “What are the tradeoffs there?” Altering any metabolic pathway involves costs, often unknown. “You make a transgenic plant, you try to make more of compound X. What does that do to the broader biochemistry of the plant?… The plant only has so much raw material to work with.”
But, overall, Grusak is hopeful. “Ten years ago, people weren’t considering this,” he says. “Most people weren’t focusing on nutritional quality of foods, and plant foods, and what we could do to manipulate that.” Besides vitamins and minerals, there’s a third major target for nutritional enhancement: products of plant “secondary metabolism,” the 80,000-plus known compounds that don’t seem to play a direct role in plant growth or development, but could be of great nutritional value in humans. One example is isoflavones, the weakly estrogenic soy compounds that seem to protect against heart disease and certain cancers. Earlier this year, a group at DuPont cloned the gene for isoflavone synthase, a key enzyme in isoflavone biosynthesis. “That really opens up that whole pathway for study,” says DellaPenna.
DuPont is also making nutritionally enhanced “designer oils,” which are much further along. Unlike the convoluted biosynthetic pathway for vitamins, the oil pathway is straightforward and easy to manipulate. And changes in oil content tend not to affect seed metabolism or affect the viability of the plant -- major hazards in GM crop development.
DuPont has had a high oleic acid soybean ready in the pipeline for years. (Elevation of oleic acid stabilizes cooking oil, eliminating the need for hydrogenation and the generation of unhealthy “trans” fatty acids.) DuPont scientist Tony Kinney, by adding an extra copy of the gene that converts oleic acid to linoleic acid, silenced its expression, causing oleic acid to accumulate in the seed. (Such “gene silencing” is thought to be a natural plant defense mechanism.) DuPont soybean products on the way include trans-free cooking oil, soy protein with improved taste, and industrial applications like improved lubricants. (Novartis is also working on a high oleic acid soybean.)
But DuPont ran into a roadblock. “The earlier lines contained antibiotic marker genes, which are objectionable to some non-U.S. regulatory agencies,” Kinney explained. “Thus commercialization was delayed somewhat until marker gene-free lines were created.” Introduction of transgenic high-oleic acid soybeans, at least outside the U.S., must wait until the markers are gone.
DuPont’s problem is not unique. Last July, the European Union proposed new rules banning antibiotic resistance genes in GM crops. This will be a major headache for all agbiotech companies, which routinely incorporate such markers in the gene cassette to select for transformed plants at the very first stage of development. Afterwards the markers are no longer needed, but they remain in the genome as useless relics. The European Union’s concern is that these genes will move from food to bacteria in the human gut and confer antibiotic resistance to disease-causing pathogens.
That concern is strictly theoretical. Although lab experiments have demonstrated horizontal gene transfer, no one has shown that native bacteria will take up antibiotic resistance genes under natural conditions. The United Nation’s Food and Agriculture Organization and the World Health Organization, in a recent report (http://www.who.int/fsf/GMfood/FAO-WHO_Consultation_report_2000.pdf), concluded that “there is no evidence that the markers currently in use pose a health risk to humans or domestic animals.” But the FAO/WHO report added that “the possibility of transfer and expression of these genes is a risk that warrants their avoidance in the genome of widely disseminated genetically modified plants.”
The European Commission rules, in any case, make the issue moot. Industry must comply, and that means long delays. New lines of marker-free transgenic crops must be created from scratch, moved into breeding programs, and field-tested -- an expensive process that takes years.
The problem, however, was completely avoidable. In 1991, David Ow’s group at the USDA reported a marker removal system using Cre-lox technology. (A natural gene-splicing tool that allows researchers to "edit" DNA. The marker is placed between two lox sites and snipped out by expressing Cre recombinase.) Ironically, DuPont, which holds the patent, now faces product launch delays from failure to utilize it.
Compared to other obstacles facing agbiotech, “I don’t think it’s very important,” says Alex Hittle. “[But] I do think it’s indicative of the industry. The evidence that there’s been any migration, or effect of the antibiotic resistance gene being consumed is very weak. They’re scientists, and they have that view of the world.” Industry, for years, scoffed at the objections. “[They said] ’Huh? Wait a minute -- animals are fed antibiotics all the time. If you’re worried about antibiotic resistance, there are bigger fish to fry here.’ Industry was unwilling to bend, [and] was not very smart in terms of PR.”
The Consumer Policy Insitute’s Michael Hansen, who asked the FDA back in 1992 to mandate antibiotic-resistance gene removal (the FDA refused), puts it more strongly. “The problem is one of hubris,” Hansen says. “It’s scientific arrogance.”
Now industry has rediscovered marker gene removal. For instance, DuPont is finally developing versions of Cre-lox for this purpose. Monsanto, citing its pending IPO, won’t say what it’s doing, but it probably has a Cre-lox cross-licensing deal with DuPont. And in March, Novartis unveiled a new system, called Positech, that enables transgenic plant selection without the use of antibiotic resistance marker genes. (Inserting the phosphomannose isomerase gene into plants permits them to grow on mannose, a carbon source, while non-transformed plants die.) But nobody is admitting there’s a problem beyond one of public perception. “While Novartis stands by the safety of other marker genes, Positech provides an alternative,” explains the company’s website.
Novartis and the others now realize that perception is everything when it comes to consumer products. That realization took years. “Unfortunately, outside of the laboratory, public perception is reality,” says the USDA's Ow, who agrees there’s minimal risk. “GMO plants are going to be treated like any other product in the marketplace. They will have to cater to consumer preferences.”
Industry has an even bigger PR problem: the specter of “unexpected effects.” The argument is that genes randomly inserted into plants might alter expression of the plant’s other genes, slashing nutrient production or pumping up toxin levels. “We just don’t know enough,” says the Consumer Policy Institute’s Hansen.
This position may be self-serving, but it’s not entirely fanciful. All three plant transformation methods are undeniably crude. Particle bombardment, or “biolistics,” involves shooting microscopic particles (such as gold) covered with DNA into plant tissues with a gene gun. Electroporation exposes plants to an electric field, enabling them to take up DNA from the surrounding medium. The preferred method uses Agrobacterium (the original bacterial strain for genetic engineering in plants) as a vector to infect and transform the target plant cutting. In all cases, the new gene is randomly integrated into the plant’s genome, often in multiple copies in multiple locations. This can lead to “gene silencing” -- the inactivation of genes, probably as a defense mechanism against foreign DNA -- and other unpredictable metabolic effects.
All three methods can be so disruptive that the vast majority of transformed plants must be discarded because of stunted growth or other developmental problems. Once that selection takes place, the transformed laboratory variety must be back-crossed repeatedly with an “elite” field variety, incorporating as much of the field variety’s genome as possible. Then a battery of biochemical tests is performed, checking for toxins and monitoring nutrient levels. But these can’t check for everything.
“It’s nice not to splatter your genome up with bits and pieces of DNA all over the place; the regulators don’t like it,” says Purdue University plant biologist Stanton Gelvin, who works on Agrobacterium-mediated transformation. “Agro… tends to do that much less than these other methods do.”
But Agrobacterium doesn’t work well, so far, for crops like wheat, corn, barley and oats. And it’s still random integration. Unfortunately, the Holy Grail -- targeted gene replacement -- remains elusive. Homologous recombination, the spontaneous alignment and replacement of identical sequences, works often enough in mammals to make knockout mice possible. But it’s about two orders of magnitude rarer in plants, so isn’t yet practical. “Plants just seem to prefer a random integration, for whatever reason,” says Gelvin.
Eric Kmiec |
Chimeraplasts -- RNA/DNA chimeric oligonucleotides, pioneered by Eric Kmiec of Thomas Jefferson University -- offer some hope. In theory, mutations can be introduced precisely using these oligos, taking advantage of the cell’s natural DNA repair machinery. But, so far, chimeraplasty in plants has only worked for single nucleotide changes, and it may never work for large chunks of DNA. Some researchers are working on homologous recombination in plants, but have little to show. “I don’t think we will see homologous recombination in plants for a while,” says Ow.
But Ow has a spectacular interim solution. In 1995, he demonstrated precise, single-copy integration of DNA in tobacco using Cre-lox. Ow has been able to create multiple transgenic plants, all with a single copy of the transgene in the identical location. This isn’t perfect targeting, since the original recombination site is randomly created by Agrobacterium-mediated gene transfer. But once the site’s in place, new DNA will go there and integrate.
Ow has not stopped there. He envisions an “operating system” to engineer future generations of GM crops. In the future, “we will probably be targeting DNA into a specific site,” he predicts. “The targeting will also allow precise insertion of a single copy… No longer will the argument be valid that you’re randomly putting things in there, and they’re scattered throughout the genome, and you don’t know what you’re disrupting.” The system can be combined with marker removal, eliminating that problem as well.
Once the best recombination sites are selected and well-characterized (for the optimal expression patterns, and to regulators’ satisfaction), they can be bred out into field varieties using traditional methods. “But you only do that once,” Ow stresses. “Afterwards, all transformation that’s done in the laboratory variety would then move out there within a few generations, not six to ten back-crosses.” That’s because precise, site-specific recombination should take place in the very first few back-crosses, avoiding “linkage drag,” the tendency for undesirable traits to persistently travel with the transgene and remain in the crop, generation after generation. Ow thinks his system should cut breeding time in half or more.
That should be alluring to industry -- especially since it takes care of the “unexpected effects” issue, at least in great part, and the marker issue. “Public perception is one aspect that we should all deal with,” says Ow. “But, most importantly, we’re dealing with efficiency.” Still, Ow wonders if companies will make the long-term investment needed to put the system into place. “This is research that’s probably going to be initiated in the public sector,” he predicts.
Will agbiotech learn from its errors of the past? The industry now knows that it needs to sway public opinion. This summer the Biotechnology Industry Organization (BIO) launched a $50 million-plus PR blitz, including high-profile TV ads. “BIO basically shook down its members and said, ‘We’re going to get creamed here if we don’t do something,” says Alex Hittle. But ads aren’t the answer, says Hittle. “I personally don’t think it’s terribly effective for them to be jumping up and down and saying, ‘It’s safe,’” he says. “Because who’s going to believe it? They’re always going to be at a credibility disadvantage.” Hittle thinks agbiotech needs a broader defense from the independent scientific community.
But it would also help to have consumer-friendly products, made with cleaner methods. “If the industry could snap its fingers, and we could have Golden Rice and other products to try, then this industry would be pulling itself out,” Shimoda says. “Unfortunately we don’t have these products.” But the technology is quickly coming on line, and it’s up to industry to adopt it. So far, the work of Potrykus, DellaPenna and Grusak has been almost entirely supported by foundation funding and government grants.
But even engineering vitamins, minerals and isoflavones into food won’t be enough for industry to win the perception wars. Golden Rice has been a wonderful PR tool for the industry, but it has not caused Europe or Japan to embrace GMOs. Nor will it mollify critics of plant transformation. "I’ve got some basic problems with anything that’s transgenic, because of the transformation technologies, the unexpected effects,” says Hansen. Committed opponents like Hansen are probably unappeasable, but the public is still up for grabs. Maybe the industry leaders should just wait out the storm. But it might be wise, in the meantime, to pursue the best technology -- whether or not they think they need it.
|