# News this Week

Science  13 Dec 2002:
Vol. 298, Issue 5601, pp. 1894
1. BIOTECHNOLOGY

# Retreat From Torrey Mesa: A Chill Wind in Ag Research

1. Gretchen Vogel*
1. With reporting by Andrew Lawler and Eliot Marshall.

A pacesetter in plant genomics, the Torrey Mesa Research Institute (TMRI), will close its doors by the end of January, an apparent victim of tightening research budgets. TMRI's owner, the Swiss-based agribusiness giant Syngenta, announced on 4 December that it intends to shutter the 4-year-old San Diego, California, institute, which led the company's efforts to sequence the rice genome and developed the first gene chips for Arabidopsis and rice.

The closure is part of a restructuring that will bring together Syngenta and Diversa, a San Diego-based biotech company that focuses on isolating genes from microbes in extreme environments. As many as 76 of TMRI's 180 employees will move to Diversa; 30 more will relocate to Syngenta Biotechnology Inc. (SBI) in Research Triangle Park, North Carolina. Steven Briggs, TMRI's chief executive, will go to Diversa as senior vice president of research and development platforms. He sees the new partnership as “an extremely exciting opportunity,” given Diversa's experience in prokaryotes and Syngenta's work in eukaryotes and genomics.

The move reflects the “maturity” of TMRI's research, says David Jones, Syngenta's head of plant science in Basel, Switzerland. “The work that has been achieved at TMRI has been quite outstanding. We are very pleased with our investment,” he adds. But others see the closure as part of an industry-wide scale back on research funding. “It is a big retraction in Syngenta's apparent willingness to invest in basic research,” says Chris Sommerville of the Carnegie Institute of Washington's department of plant biology in Stanford, California. Biologist Alan Jones of the University of North Carolina, Chapel Hill, says he sees similar belt tightening throughout the agbiotech industry: “They are hunkering down.”

Plant scientists had long speculated about a reorganization, but many were surprised by the TMRI decision. “Every kind of rumor has been flying around for the past few weeks, and the one that seemed to be completely paranoid was that they would shut down TMRI—and it turned out to be true,” says Jeff Harper of the Scripps Research Institute in La Jolla, California. One senior university scientist said he was “flabbergasted” because “Briggs had an academic style of research and was doing powerful work.”

TMRI took shape during California's technology boom of the late 1990s. The Novartis Research Foundation recruited plant geneticist Briggs in 1998 to direct its newly formed Novartis Agricultural Discovery Institute. Then in 2000, Novartis spun off its agricultural arm to form Syngenta, which acquired the San Diego institute and renamed it TMRI.

Besides coordinating the sequencing of the rice genome (Science, 5 April, p. 32), TMRI developed a collection of 100,000 Arabidopsis mutants and made them available—with certain commercial restrictions—to outside researchers. The institute also collaborated with Affymetrix to develop a gene chip containing about half the rice genome—still the only gene chip available for a cereal plant, notes plant physiologist Russell Jones of the University of California (UC), Berkeley. The closure will likely delay the availability of the full-genome chip, which scientists had expected early next year.

Syngenta's Jones says the company will continue its collaborations with academic researchers through SBI in North Carolina, where the Arabidopsis mutant collection, the rice genome databases, and the gene chip platforms will move. “There will be no change except geography,” he predicts.

Briggs blames the “very somber experience” of TMRI's closing on the agricultural economy. “We've been in a state of contraction for the past 5 years,” he says. “There's no indication of a turnaround … and so there's a danger of significant reductions in research budgets—the focus has to be on generating revenues. You sacrifice your research.”

Syngenta's troubles could affect other centers. Sources close to the controversial partnership between Syngenta and UC Berkeley say that their 5-year agreement likely will not be renewed in 2003, when the $25 million deal expires. Briggs says no decision has been made, but he adds that if “we had to renew now, we wouldn't.” 2. AIR POLLUTION # Counting the Cost of London's Killer Smog 1. Richard Stone LONDON—In December 1952, an acrid yellow smog settled on this city and killed thousands of people. The catastrophe, known as the “Big Smoke,” was a turning point in efforts to clean up polluted air in cities across the Western world. It has taken half a century, though, for some of the fog to clear around the death toll from the roiling sulfurous clouds. New research suggests that the U.K. government might have underestimated the number of smog-related deaths by a factor of 3. Experts agree that the foul fog, which descended on London for a weekend in December 1952, killed roughly 4000 people that month alone. But researchers are now sparring over the cause of death of another 8000 Londoners in January and February 1953. Fresh analyses, debated at a conference here earlier this week to mark the 50th anniversary of the Big Smoke, suggest that these people succumbed to delayed effects of the smog or to lingering pollution. Other analyses insist that many of the “excess” deaths in early 1953 were caused by influenza, a view that the government has always supported. The debate reveals how much is unknown even today about the effects of smog, which continues to menace big cities, particularly in developing countries with weak air-pollution laws. Even in a city legendary for its “pea-soup” fogs, the Big Smoke is the stuff of legend. In early December 1952, an area of high pressure settled over London. Residents kept piling sulfur-rich coal into their stoves to keep warm in the near-freezing temperatures. In the still air, the smoke from these stoves and from coal-fired power plants in the city formed a smog laden with sulfur dioxide and soot. On Friday, 5 December, schools closed and transportation was disrupted. On Saturday night, a performance of the opera La Traviata had to be abandoned after smog obscured the stage. It wasn't until Tuesday, 9 December, that winds finally swept away the fouled air. By then, it was clear that a disaster was unfolding, as scores upon scores of people succumbed to respiratory or heart ailments. In 1953, the Ministry of Health concluded that the deaths of 3500 to 4000 people—nearly three times the normal toll during such a period—could be attributed to the smog. But officials decreed that any deaths after 20 December had to be from other causes. During the first 3 months of 1953, there were 8625 more deaths than expected. Officials put 5655 down to flu and listed 2970 as unexplained. A few years ago, epidemiologist Devra Davis, a visiting professor at the London School of Hygiene and Tropical Medicine, which sponsored the conference, and Michelle Bell, then a graduate student at Johns Hopkins University in Baltimore, decided to test the idea that flu caused all the deaths. In 1953, influenza was not a disease that doctors were obliged to report to health authorities. But examining public health insurance claims, hospital admissions, and news accounts of the flu outbreak, Davis and Bell concluded that most of the excess deaths in early 1953 could not have been from flu. “Nothing we found said the flu outbreak was huge,” says Bell. Often-listed causes of death such as pneumonia and bronchitis, they claimed, had to be from the Big Smoke or from persisting pollutants. “It's a very interesting study to disentangle these deaths,” says Ross Anderson of St. George's Hospital Medical School in London. Nevertheless, he suspects that influenza deaths were two to 10 times greater than reported, and that there might have been “the possibility of interaction” between pollution and flu. Frederick Lipfert, an environmental consultant in Northport, New York, presented his own analysis suggesting that flu was a bigger killer than Davis and Bell acknowledge. Another study put an upper limit on flu deaths. Epidemiologist Klea Katsouyanni of the University of Athens Medical School reported that data from recent flu outbreaks, analyzed by a pan- European pollution project, suggested at most 2650 flu victims in early 1953—although the real, unknowable tally, she says, was probably far lower. The debate is more than academic. Although London smogs are now more legend than reality, air pollution continues to smother big cities. In a new analysis presented at the meeting, the World Health Organization estimates that bad air kills about 600,000 people worldwide each year. “Some lessons of the Big Smoke still haven't been learned,” Davis says. 3. HIGH-ENERGY PHYSICS # Particle Trap Confirms Antimatter Shuffle 1. Charles Seife The neutrino has just become a little less mysterious. The first results from a Japanese experiment that measures antineutrinos streaming away from nuclear reactors show that antineutrinos behave just like their counterparts, neutrinos. The study, announced last week, also dispels uncertainties about earlier experiments that used neutrinos from the sun. “It's a profound result,” says John Bahcall, a physicist at the Institute for Advanced Study in Princeton, New Jersey. “It dots the i's and crosses the t's for the interpretation of what happens with solar neutrinos. It's an incredible achievement.” The experiment, based at a zinc mine in Kamioka, Japan, and dubbed KamLAND, is one of several underground experiments studying some of nature's hardest-to-capture particles. But whereas most of the other efforts detect neutrinos coming from the sun and from the atmosphere, KamLAND looks for antineutrinos created by 17 nuclear reactors that dot the Japanese landscape. Like neutrinos, antineutrinos come in three varieties—electron, muon, and tau—named after other particles with which they are associated. The antineutrinos are generated by the decay of radioactive elements within the reactor. The detector itself is a 1000-ton sphere full of mineral oil and an organic solvent known as pseudocumene. When an electron antineutrino strikes a hydrogen nucleus—a proton—in the liquid, both particles change identities. The proton becomes a neutron, and the antineutrino becomes an antielectron in a process known as inverse beta decay. The scientists detect the flashes caused by the newborn antielectron and neutron, which signals that an antineutrino has met its demise. In 6 months of observations, a team led by Atsuto Suzuki of Tohoku University in Sendai, Japan, detected 54 electron antineutrinos—significantly fewer than the 87 or so that the team should have seen, given the output of the reactors and the sensitivity of the detector. The deficit implies that electron antineutrinos change into muon or tau antineutrinos after they leave the reactor, just as electron neutrinos from the sun change into muon or tau neutrinos before reaching Earth (Science, 26 April, p. 632). “When you do it with the antiparticle instead of the particle, you get a consistent result,” says John Learned, a KamLAND collaborator at the University of Hawaii, Manoa. “This is what everyone expected, but nature could have fooled us,” says Bahcall. If neutrinos and antineutrinos behaved differently, they would violate an important principle in particle physics known as CPT symmetry. Physicists have tested the principle in the sector of physics that has to do with the strong force (and quarks and gluons), but KamLAND's result is the first to verify it with any degree of accuracy in the realm of the weak force (and neutrinos). Furthermore, because the antineutrinos are created in nuclear reactors rather than in the core of the sun, physicists needn't worry that incorrect assumptions about the sun's inner workings might mess up their calculations. “There was a possibility that [the sun's] magnetic fields were flipping the spins of the neutrinos,” says Learned. “The caveats about solar neutrino measurements are all eliminated in one grand stroke.” With more data and a refined understanding of the detector's properties, KamLAND scientists should be able to pin down the difference in mass between two species of neutrinos, says Giorgio Gratta, a KamLAND team member at Stanford University. That's one of the key parameters that dictate their properties. But even the first results are narrowing the possible range of the mass difference, Gratta says: “I'm very happy.” The KamLAND team hopes eventually to spot neutrinos coming from deep inside Earth. They are the product of the decay of radioactive elements that keep the planet's center so hot. The number of neutrinos from these nuclear reactions will give geologists a direct measure of the amount of radioactive material buried in the heart of the planet. 4. GENETIC MODIFICATION # Europe Prepares for Arrival of GM Foods 1. Philipp Weis BRUSSELS—The European Union (E.U.) appears set to lift its 4-year ban on foods made from genetically modified organisms (GMOs), following the drafting of a new directive on food labeling late last month. The European Parliament is expected to give its final approval of the new rules next March, and the European Commission last week put in place the mechanism to make the new system work: the European Network of GMO Laboratories (ENGL). The new rules will require a GMO label on any food containing more than 0.9% GMO material, a threshold designed to allow for some accidental contamination. “These are among the tightest regulations [on GMOs] in the world,” says Barry Mc Sweeney, director-general of the commission's Joint Research Centre (JRC). JRC's main laboratory in Ispra, Italy, will coordinate the ENGL network of more than 45 institutes in the 15 E.U. member states and 10 countries that are expected to join in 2004. These labs will randomly test foodstuffs to ensure that they are GMO-free if they claim to be, or that they contain only approved GMO materials. “We need harmonized procedures and methods to ensure that we get the same results” all over Europe, says Guy van den Eede, coordinator of ENGL. In the future, any food or feed company that wishes to market a new GMO will have to submit reference material and a specific testing method to the Ispra ENGL lab. ENGL will validate the test and, if approved, it will be registered as an international standard. All the ENGL labs will then use the test in their countries. The idea behind the new legislation is to allow consumers to choose GMO-free food if they wish, while allowing biotech companies to market their wares. Although most environmental organizations welcome the strict regulations, one proposal drew fierce opposition. For a 3-year period, the commission wants to allow foods containing 0.5% of “GMO material unauthorized in the E.U., but which has undergone a favorable risk assessment.” Peter Riley of Friends of the Earth in the U.K. says unlicensed GMOs should be completely banned: “If the U.S. is growing crops that are not accepted worldwide, it is their problem.” But E.U. Research Commissioner Philippe Busquin says the network “provides us with an important tool to ensure that we harvest the potential that biotechnology holds for consumers in a responsible way.” 5. CLIMATE # River Flow Could Derail Crucial Ocean Current 1. Erik Stokstad Some of the biggest rivers in the world are dumping 7% more water into the Arctic Ocean than they were in the 1930s—an increase of 128 cubic kilometers per year. The finding, reported on page 2171, fits well with climate-model predictions that precipitation at high latitudes will increase as global temperature climbs. If the warming trend continues, the influx of fresh water could have a major impact on ocean circulation and northern climate. But many experts caution that too little is known to make any solid predictions about such effects. “I would call this intriguingly important,” says Bert Semtner, an oceanographer at the Naval Postgraduate School in Monterey, California. To get the results, ecosystem scientist Bruce Peterson and colleagues at the Marine Biological Laboratory in Woods Hole, Massachusetts, teamed with hydrologists from the University of New Hampshire, Durham, and the State Hydrological Institute in St. Petersburg, Russia, to analyze discharge records for six major Eurasian rivers. The records spanned 64 years, about twice as long as comparable records for major Arctic rivers in North America. Figures for each river varied widely from year to year but, on average, the total annual runoff increased by 2 cubic kilometers each year. Global warming is likely to be causing the increase, climatologists say. Higher temperatures mean more evaporation, especially in the subtropics. Warmer air can hold more moisture, which then precipitates as air masses move to high latitudes, leading to an increase in river discharge. “There is no other measure of change in [the] Arctic freshwater budget that's as accurate and comprehensive,” Peterson says. The researchers estimate that for each degree of global warming, these six Eurasian rivers would pour an extra 212 km3 per year into the Arctic Ocean. If global temperature rises by 5.8 degrees Celsius by 2100—the upper end of estimates from the Intergovernmental Panel on Climate Change's (IPCC's) 2001 report*—the rivers might increase freshwater flow to the Arctic Ocean by 1260 km3 per year. “It's a worrying number,” says co-author Stefan Rahmstorf, a climatologist at the Potsdam Institute for Climate Impact Research in Germany. Increasing river runoff, he explains, might put the brakes on an important current in the North Atlantic called the thermohaline circulation (THC). Under present conditions, cold, salty surface waters sink to great depths and then move south, while warmer water on the surface moves northward. Any freshening of the surface waters in the North Atlantic would reduce the seawater density and slow the THC. Climate models by Rahmstorf and colleagues at Princeton University suggest that the IPCC's worst-case warming scenario would put discharge in the ballpark of the amount needed to bring the THC to a halt. Contributions from other Arctic rivers, precipitation onto the Arctic Ocean, and melting ice (such as that on the Greenland ice cap) could push the THC across the threshold. That would put a damper on warming near the North Atlantic, Rahmstorf says. THC shutdowns have had severe consequences in the past, he points out: 11,000 years ago, a sudden, massive pulse of freshwater into the North Atlantic chilled Europe. “It's not just an odd thing that happens in models,” Rahmstorf says. Much remains to be learned. “I would be very careful about anything more than very loose speculation on the influence of the runoff and changes in the overturning of the North Atlantic,” says Knut Aagaard, an oceanographer at the University of Washington, Seattle. Some researchers, for example, question how much influence additional discharge could have on the THC. Semtner says that factors such as direct warming of the ocean surface might have more sway in weakening the THC. That's one question that may be clarified by researchers participating in the Coupled Model Intercomparison Project. They are now running six major climate models, all including a major pulse of freshwater from the Arctic Ocean. Results are expected to be released next spring. • *IPCC, Third Assessment Report—Climate Change 2001. 6. # Report Seeks Answers to Marine Mystery 1. David Malakoff A sleek sea lion with a hefty appetite for fish could become the centerpiece of a massive ecological experiment. A panel of the National Academies* last week recommended that the U.S. government run a decade-long test off Alaska to determine whether commercial fishing is a threat to Alaska's dwindling Steller sea lion population. The advice, requested by Congress, could help settle a high-stakes dispute over catch restrictions in one of the world's most valuable fisheries. “You need to do something at this [large] scale if you want to understand what's driving the [population] decline,” says panel member Larry Crowder, a fisheries biologist at Duke University in Durham, North Carolina. “But it's not an easy thing to pull off.” Steller sea lions once dotted North Pacific shores from California to Japan, with an estimated 70% living in Alaskan waters. Over the last 30 years, however, Alaskan populations have plummeted by 80%, to fewer than 70,000 animals. Scientists have long debated the cause. Whereas some blame fishing boats for taking too much of the mammals' prey, others finger climate change, predators, disease, or poaching. Two years ago, federal biologists concluded that commercial fishers catching pollock and other groundfish posed a serious threat to the sea lion's recovery, and they imposed major restrictions on Alaska's$1 billion fishery. That alarmed Senator Ted Stevens (R-AK) and other members of Congress, who ordered the academies' study.

In a preliminary report issued 4 December, a panel led by zoologist Robert Paine of the University of Washington, Seattle, concludes that fishing probably isn't the major cause of recent population losses. But it says that commercial catches can't be ruled out as a significant problem. In hopes of resolving the issue, the committee recommends setting up four experimental zones in Alaskan waters. Fishing would be banned for up to 50 nautical miles around two sea lion breeding colonies and permitted near two others. Biologists would then compare sea lion population trends. The experiment “is the only approach that directly tests the role of fishing in the decline” while controlling for other factors, such as climate change, the panel says.

Fishing industry representatives appear relieved that the academies didn't put the blame squarely on them. And ecologists are pleased by a renewed call for what they say would be one of the biggest real-world ecological experiments ever, albeit a tardy one. “It should have been done 10 years ago; it's a shame we've waited this long,” says Andrew Trites, a marine mammal specialist at the University of British Columbia, Vancouver.

The National Marine Fisheries Service must approve any test, and no decision is expected until sometime next year. The panel will shortly release a final version of its report.

7. COMPARATIVE GENOMICS

# Tunicate Genome Shows a Little Backbone

1. Elizabeth Pennisi

This week, researchers are unveiling the DNA code of one of the most unusual creatures sequenced to date: the sea squirt. These tiny marine animals have long captivated biologists because even though the adults are typical, squishy invertebrates, their larvae might be modern doppelgängers of the ancestor to the vertebrates.

The genome sequence of the sea squirt Ciona intestinalis “will help us unzip several evolutionary changes that occurred at the transition between invertebrates and vertebrates,” says Paolo Sordino of the Zoological Station in Naples, Italy. Ciona's branch on the evolutionary tree puts it closer to humans than are other invertebrates whose genomes have been sequenced, such as nematodes and fruit flies, but farther away than mice are. As such, it offers a different view of the history of human DNA.

Sea squirts have puzzled biologists for more than a century. The adults live attached to pilings, rocks, and boat bottoms. Their bodies are cloaked in a leathery sheath, or tunic—hence their scientific name, tunicates. Charles Darwin thought they were relatives of mollusks. In the mid-1800s, Russian biologist Alexander Kowalevsky countered that the mobile tunicate tadpole, with its dorsal cartilaginous column resembling a spine, should be grouped with vertebrates and not clams and snails—even though tunicates never develop a backbone. His view stuck: Now any species that even temporarily possesses a dorsal nerve cord, notochord, primitive brain, and a few other traits is considered a chordate, a member of the phylum that includes vertebrates.

Some evolutionary biologists once argued that tunicates gave rise to backboned critters. That view has been abandoned in favor of a history in which the two simply share a common ancestor. Nonetheless, this sequence “is an opportunity to peek into the [early] chordate condition from a genomic point of view,” says Sean Carroll, an evolution and development (evo-devo) researcher at the University of Wisconsin, Madison.

In early 2001, sea squirt researchers Nori Satoh of Kyoto University in Japan and Michael Levine and Daniel Rokhsar of the University of California, Berkeley, convinced the sequencers at the Department of Energy Joint Genome Institute in Walnut Creek, California, to take on the creature. Eighteen months later, about 50 biologists and bioinformaticists spent a week poring over the newly assembled draft genome. They identified as many genes as they could and compared the sea squirt sequence to existing genome information, focusing on how various types of genes had changed through time. The results of this effort are reported on page 2157.

The sea squirt's 117 million bases sequenced include 16,000 genes. Among the genes are single and double copies of genes that have multiplied several times in the mouse and human genomes. The sea squirt shares about 60% of its genes with the nematode and fruit fly, whereas about 5% have matches only in the human, mouse, and puffer fish genomes. And about 20% seem unique to Ciona, including several involved in the production of a stiff starch called cellulose, the main ingredient in its tunic.

“Every genome is like a history book,” explains Peter Holland, an evo-devo biologist at the University of Oxford, U.K. “But the problem is [that] there don't seem to be any dates” for when different genes appeared over the course of evolution. The new sequence, however, should enable biologists to distinguish which genes arose in vertebrates and which predate the split between vertebrates and sea squirts. For example, Ciona lacks many vertebrate neural genes and certain immune system genes, suggesting that these came after the split between tunicates and early vertebrates. These additions, says Levine, made possible “probably the most spectacular [vertebrate] innovations”: the complex nervous and immune systems.

To Holland, the fact that many genes have multiplied in vertebrates but not in Ciona “suggests that something dramatic happened to vertebrates” that caused this great expansion. Take Smad genes, which typically help regulate bone development. Ciona has five; mice have eight. This change in gene number is “a recurring theme in the analysis of the Ciona genome,” Rokhsar points out.

Some genes' origins can be pinpointed to a time when the common ancestor to vertebrates and sea squirts thrived. For example, thyroid hormones and receptors are not found in other invertebrates but are present in Ciona. “We don't know what they are doing,” says Rokhsar, but he suspects that they are involved in the transition from tunicate tadpole to sedentary adult, as they are in frogs' metamorphosis.

Meanwhile, Ciona's unique genes for making cellulose “come out of nowhere,” Carroll points out; they have not been found in other animals. Levine sees the genes as “very compelling evidence for remarkable horizontal gene transfer between bacteria and Ciona,” as the sea squirt seems to have adopted bacterial enzymes needed to use cellulose.

Even more genomes must be sequenced before researchers can say whether Levine is right about the horizontal transfer. And the more genomes the better, evolutionary biologists argue. “As each complete genome unfolds,” says Carroll, “we are getting a bigger and better picture of patterns of gene evolution and of gene families.”

8. RESEARCH INFRASTRUCTURE

# NSF Urged to Boost Spending on Facilities

1. Jeffrey Mervis

Nobody's talking about changing the National Science Foundation's (NSF's) name to Need to Support Facilities. But the foundation must spend a larger share of its $5 billion budget on research infrastructure to maintain U.S. leadership in science, declares a new report from its oversight body. An internal survey of NSF's disciplinary offices yielded a wish list of almost$2 billion a year through 2012 for scientific tools ranging from computing networks and research vessels to telescopes and synchrotrons (see table). That's double NSF's current spending level. “The need is greater than we can address with our normal budget mechanisms, and it won't go away,” says John White, chancellor of the University of Arkansas, Fayetteville, and chair of the National Science Board task force that produced the 41-page draft report posted this week (www.nsf.gov/nsb; 02–190).

The top spending priority, according to the board, should be advanced cyberinfrastructure —not just more powerful computers but also better storage, analysis, visualization, and distribution tools—to benefit the entire scientific community. This is a broad program, “not just bigger machines at a few places,” says board member Anita Jones of the University of Virginia, Charlottesville. But certain disciplines also have big needs, the board says. NSF would have to triple its annual spending on large research facilities—to $350 million—just to eliminate a backlog of detectors, telescopes, and other projects that the board has approved but Congress has yet to fund (Science, 14 September 2001, p. 1972). There's also a problem with “mid-sized” facilities—those costing tens of millions of dollars—that are too pricey for individual programs yet too small to rank as a major research installation. Midsized crisis. There's a growing need for moderately priced facilities. View this table: NSF now spends 22% of its budget on tools, a fraction that “is too low,” according to the report. The task force would like to see it grow to about 27%, says board member Robert Richardson, vice provost for research at Cornell University. The report, 2 years in the making, expresses the hope that a growing NSF budget will provide “the majority of these additional resources.” That's a reference to a projected doubling of NSF's budget over 5 years, a concept that Congress endorsed last month in passing a bill that reauthorizes NSF's programs. Should the pie not expand rapidly enough, however, NSF officials might have to revisit the thorny issue of striking the right balance between “big” and “little” science. “I think that the PI [principal investigator] community could see it as a threat,” says one science policy analyst who had not yet seen the report. At the same time, a university lobbyist speculates that the needs outlined in the report could be used by some federal legislators to help push for a broader economic stimulus package. The board hopes for feedback from the community before issuing a final version of the report this winter. “The proposed changes are not radical, but they are significant,” Richardson told board members before they signed off on the draft report. “And I think people should pay attention.” 9. PATENTING LIFE # Canadian High Court Rejects OncoMouse 1. Wayne Kondro* 1. Wayne Kondro writes from Ottawa. OTTAWA—Canadian researchers don't have to worry about paying licensing fees for the use of transgenic animals. The nation's top court ruled last week that higher life forms aren't patentable. In a 5-4 decision, the Supreme Court of Canada ended Harvard University's 17-year quest to obtain Canadian patent protection for its OncoMouse, ruling that the cancer-prone rodent can't be owned. The court said that OncoMouse, developed by Philip Leder of Harvard Medical School in Boston, isn't an invention under a 1869 Canadian law that protects “any new and useful art, process, machine, manufacture or composition of matter.” Although the court prohibited the patenting of OncoMouse, it did allow Harvard to proceed with applications to protect the process by which the animal is engineered. “We're going to do our best to squeeze all the protection we can out of this judgment,” says Ottawa lawyer David Morrow, who represents the university. Morrow believes that the decision leaves room for patents on cells, cell cultures, plasmas, and other aspects of OncoMouse, which is licensed to researchers through E. I. du Pont de Nemours and Co. of Wilmington, Delaware. Writing for the narrow majority, Justice Michel Bastarache made a philosophical argument for the court's ruling, which stands in contrast to patents granted by 17 nations, including France, Germany, Japan, and the United Kingdom. “A complex life form such as a mouse or a chimpanzee cannot easily be characterized as ‘something made by the hands of man,’” he wrote. Nor is OncoMouse a “composition of matter,” he added. “Higher life forms are generally regarded as possessing qualities and characteristics that transcend the particular genetic matter of which they are composed,” Bastarache noted. “A person whose genetic makeup is modified by radiation does not cease to be him or herself. Likewise, the same mouse would exist absent the injection of the oncogene into the fertilized egg cell; it simply would not be predisposed to cancer.” Although Canada has granted patent protection to lower life forms such as yeasts, Bastarache wrote, it's up to Parliament to sift through the thorny ethical issues and decide whether higher life forms should be patentable. Should Parliament decide that, it won't be easy to draw the line between what is patentable and what isn't, Bastarache warned: “There is no defensible basis within the definition of invention itself to conclude that a chimpanzee is a ‘composition of matter’ while a human being is not.” BIOTECanada president Janet Lambert says the decision has “sent a pall” through the burgeoning industry and could stifle new investment. But Matthew Spence, president of the Alberta Heritage Foundation for Medical Research in Edmonton, thinks that most Canadian researchers and companies will not be affected by the ruling. Although some small firms might move south out of fear that their intellectual property could be plundered, he says, others might favor the current environment, “because we're not getting delays due to patent considerations.” Some predict that the ruling might even stimulate research. “The patent only gives the patent holder the right to exclude others,” says Arnold Naimark, director of the University of Manitoba's Centre for the Advancement of Medicine in Winnipeg and chair of the federal government's arm's-length Canadian Biotechnology Advisory Committee. “If there is no patent in Canada, there is no restriction on people being able to do research on the Harvard OncoMouse if they get a hold of it.” 10. GENE THERAPY # RAC's Advice: Proceed With Caution 1. Jocelyn Kaiser BETHESDA, MARYLAND—The cancer that appeared earlier this year in a patient who took part in a French gene therapy trial appears to have been caused by a rare combination of factors, a panel of experts concluded at a meeting here last week. The risk of a second occurrence seems sufficiently remote, the panel agreed, that this trial and others like it should go forward. This review by the National Institutes of Health's (NIH's) Recombinant DNA Advisory Committee (RAC) was the second major U.S. inquiry into an adverse event in a trial of therapy for severe combined immunodeficiency (SCID) at the Necker Hospital for Sick Children in Paris. Nine of 11 children in the study have shown remarkable improvement. But after one boy developed a leukemia-like disorder in September, lead investigator Alain Fischer and co-workers halted the study out of concern that the therapy might have triggered cancer. Agencies in several countries, including the U.S. Food and Drug Administration (FDA), put other SCID trials on hold. The French team, working with Christof von Kalle, a molecular biologist at Cincinnati Children's Hospital Medical Center in Ohio and the University of Freiburg, Germany, concluded that the retrovirus they used to shuttle working genes into the patient's cells had inserted into a gene called LMO2 that has been linked to T cell leukemia (see diagram). A single γδT cell with this insertion then began proliferating, and the child's T cell count soared. In mid-October, an FDA advisory committee decided that three pending U.S. SCID trials should go forward, but it asked for changes to monitoring plans and informed-consent documents (Science, 18 October, p. 510). Investigators are still working on the revisions. SCID trials remain on hold in Italy and Japan but were not suspended in the United Kingdom. Germany last month decided to resume some halted retrovirus trials. At last week's meeting, experts noted that they have suspected “for decades” that a retrovirus could insert in the wrong place, but the risks have been theoretical until now. Said Theodore Friedmann of the University of California, San Francisco (UCSF), RAC's chair: “This event represents kind of a watershed event in the field of gene therapy.” The case also might be unique. At the meeting, Alexander Rakowsky of NIH's Office of Biotechnology Activities reported that OBA has now looked for other unexpected cancerlike disorders in 181 trials, registered with the office since 1988, that used retroviruses. NIH found eight suggestive reports but no evidence that any cancers were caused by gene therapy. Von Kalle reported that the leukemic cells from the affected child in the French trial contained another anomaly—a copy of part of chromosome 6 attached to chromosome 13—that does not appear to have been caused by the gene therapy. Fischer and Von Kalle's group postulates that this 6;13 translocation somehow contributed to the cells' cancerous morphology. The child, who is being treated with chemotherapy, no longer has detectable T cells with morphology typical of leukemia, von Kalle said, even though cells carrying the altered LMO2 gene are still present. The child's extended family had an unusual occurrence of two cases of a particular childhood cancer. This led many of the 17 RAC panelists to conclude that this predisposition and other factors, possibly involving the 6;13 translocation, worked in concert with the LMO2 insertion to produce leukemia. “Given this family's history, we may never see another leukemia,” said cancer researcher David Sidransky of Johns Hopkins University in Baltimore, Maryland. Not everyone agreed, however: Maxine Lineal of the Fred Hutchinson Cancer Research Center in Seattle warned, “It wouldn't surprise me if in 20 years, we were seeing tumors in more of these children.” Unlike the FDA panel that met in October, RAC concluded that the gene therapy was “a cause” of rather than “likely caused” the child's leukemia, and that “other predisposing factors may have contributed.” Although other SCID trials should proceed, RAC concluded, federal guidelines should be revised to include a suggestion that gene-therapy researchers do more intensive monitoring for signs of cancer and archive tissue so that molecular events can be traced. “We would know nothing about the French patient if they had not archived samples,” says UCSF's Diane Wara. RAC is scheduled to vote on further recommendations at its next meeting in March, when it will expand the discussion to other gene-therapy studies that use retroviruses. “This is just the beginning,” Friedmann says. 11. PLANETARY SCIENCE # How a Pair Marries for the Eons 1. Richard A. Kerr Planets have satellites. Asteroids have satellites. So astronomers weren't too shocked to discover in 1998 that Kuiper belt objects—icy remnants from the solar system's formation—have them, too. But they were mystified nonetheless: The pairs of Kuiper belt objects (KBOs) spotted out beyond Pluto seemed impossibly large and widely separated; companions are of comparable size and hundreds if not thousands of times their own diameters distant from each other. Collisions between asteroids suffice to loft bits of rock into close orbit, but how could so much mass get so far from its companion? In this week's issue of Nature, three astrophysicists more likely to be working on stars and galaxies than nearby ice balls suggest a way that such binary KBOs might have come into existence in the earliest days of the solar system without the help of collisions. According to the new calculations, the relationships developed slowly. Newborn KBOs would have interacted through their gravitational pulls, first forming loosely bound, unstable pairs during close encounters and then being stabilized by gravitational interactions with other KBOs. The proposed mechanism predicts that, as astronomers sharpen their view of the Kuiper belt, they'll find that most KBOs are tightly bound binaries, or even triplets, and that the standoffish partners seen so far are outliers. “It's an exciting paper,” says planetary dynamicist William Bottke of the Southwest Research Institute (SwRI) in Boulder, Colorado. “It's a really creative mechanism.” But it's not the only proposed solution. A closer look at the Kuiper belt should determine the winner, or winners. In the past, theorists have come up with viable explanations for binaries of all kinds. In the 1980s and early '90s, studies of terrestrial craters began suggesting that about 15% of impactors were actually doubles. So in 1996, Bottke and planetary dynamicist Jay Melosh of the University of Arizona, Tucson, argued that if collisions have battered all but the largest and the very smallest asteroids into flying piles of rubble, Earth's tidal forces could pull apart any rubble-pile asteroid that flew within 10,000 kilometers or so—much as Jupiter ripped apart comet Shoemaker-Levy 9. Sometimes, the ruptured rubble pile would form a binary that would later hit Earth. Discoveries in the last few years confirm that about 15% of near-Earth asteroids are indeed closely bound binaries. Out in the main asteroid belt, there's no planet handy, so after the 1994 discovery of tiny Dactyl orbiting Ida, the first asteroid seen to have a satellite, theoreticians quickly came to rely on collisions to explain binaries there. Two chunks of debris from the catastrophic disruption of an asteroid could go into orbit around each other, a glancing blow might set an asteroid spinning so fast that it split into a binary, or ejecta from an impact might go into orbit and agglomerate to form a satellite. But “in the case of KBOs, both tidal fragmentation and collisional formation seem very unlikely,” says Bottke. “We probably need a completely different formation scenario.” That's where astrophysicists Peter Goldreich, Yoram Lithwick, and Re'em Sari of the California Institute of Technology (Caltech) in Pasadena came in. Lacking a convenient planet or the necessary huge collisions, they looked at close, purely gravitational KBO encounters, which would have been far more frequent than collisions were. The Caltech group first calculated how often two large KBOs passing near each other would temporarily fall into orbit around each other before again going their separate ways. They then calculated how often such a transient binary could lose enough energy to become a stable binary, the way retrorockets rob energy from a spacecraft to bring it into orbit. A third large KBO passing nearby in the century or two before breakup could do the trick. More subtly, the cumulative gravitational influence of the sea of small KBOs that the pair is passing through could gently rob energy from it. Together, the two mechanisms would have produced the small percentage of widely separated binaries observable today, according to the Caltech calculations. Collisionless formation of KBO binaries “is definitely promising,” says planetary dynamicist Daniel Durda of SwRI, Boulder, because it overcomes the “need for incredibly huge projectiles” in collisional mechanisms. But there might be another way too, Durda notes. Planetary dynamicist Stuart Weidenschilling of the Planetary Science Institute in Tucson has just published a hybrid mechanism in the November issue of Icarus. He uses a collision, but the two colliding KBOs merge. When their merging takes place within the gravitational influence of a third KBO, the combined body can form a binary pair with that third body. Both the Caltech group and he “did primitive, back-of-the-envelope calculations,” says Weidenschilling. “There's a lot more work needed to compare calculations against observations. It may be both mechanisms are viable.” Goldreich and colleagues disagree. Whatever conditions are assumed for the nascent solar system, they write, the close encounters their mechanism requires will always be far more abundant than are the collisions that Weidenschilling needs. “You never need these collisions,” says Sari. “You just need the third body to be there.” Resolution could come with more detailed observations. The collisionless mechanism predicts that, like most stars, most KBOs are binaries (although today's instruments can't yet distinguish small, closely spaced pairs). KBO pairs would have formed with wider separations but spiraled inward until they were too close for current telescopic observations to separate them. The hybrid mechanism, on the other hand, predicts a decreasing abundance of binaries at smaller separations. At stake are insights into what the early, still-forming solar system was really like, including how Pluto and its huge moon Charon came to be paired. “It's going to be very interesting,” says Durda. 12. GENE THERAPY # The Strange Case of Chimeraplasty 1. Gary Taubes A gene-therapy technique that burst on the scene with enormous promise 6 years ago has turned out to be inconsistent or impossible to replicate in most labs that have tried it The history of gene therapy is filled with promise, hype, and disappointment. Among the more profound failures is that only a tiny fraction of the genes injected into animals or humans reach their cellular targets. And only a tiny fraction of those that do so actually works. On 6 September 1996, however, Science published an article about a technology that promised to change all that (p. 1386). The article described a radical new technology for correcting genetic defects, one that appeared to be a million-fold more potent than previous approaches were. The implications did not go unnoticed: The publication caught the media's attention, launched research projects around the world, and spawned a gold rush as researchers and entrepreneurs moved to stake their claim to the extraordinary promise of the technology. The result has been a 6-year roller-coaster ride of science at its cutting edge and most controversial. In traditional gene therapy, researchers stitch a gene into a virus that then shuttles it into target cells. Once inside, if all goes well, the gene integrates into the cellular DNA and begins churning out proteins to replace those missing or defective. In their Science paper, researchers at Thomas Jefferson University (TJU) in Philadelphia, led by Eric Kmiec, reported that they had corrected genetic defects without using a virus to do so. Instead, they used a synthetic molecule of RNA and DNA, a chimera that could slip into cells, at least in the test tube, and correct the mutation responsible for sickle cell anemia. The technology, which Kmiec called chimeraplasty, appeared to be astoundingly efficient. If it performed as the data implied, correcting the sickle cell mutation in 50% of cells, it could revolutionize gene therapy. It would also have a dramatic impact on genomics, where it could be used as a powerful tool to elucidate the function of genes. The promise was such that gene-therapy pioneer Michael Blaese quit his position running the Clinical Gene Therapy Branch of the U.S. National Institutes of Health (NIH) to become chief scientific officer of Kimeragen, the company founded by Kmiec to commercialize his discovery. By 1999, Kimeragen was talking with the U.S. Food and Drug Administration about using chimeraplasty to treat Crigler-Najjar disease, a rare genetic disorder, and Science itself reported that the technology had passed the all-important hurdle of scientific acceptance (16 July 1999, p. 316). “The beauty of chimeraplasty is that it appears to be a universal process,” Blaese told Science. In February 2000, Kimeragen merged with Valigene, a French biotech firm, to form ValiGen, which had as CEO Douglas Watson, the former head of Novartis, and a scientific advisory board that included J. Craig Venter and Nobel laureate Hamilton Smith of Celera Genomics in Rockville, Maryland. But those heady days are over. In October 2001, Watson resigned; ValiGen closed its Princeton, New Jersey, laboratory, the site of nearly all its chimeraplasty research; and Blaese and his researchers were laid off. According to its representatives, the company was undergoing bankruptcy reorganization in France this summer and has sold the license for chimeraplasty to a small company in San Diego, California, to pursue the technology in plants. “I am still a believer in gene-repair technology,” says Blaese, “but the efficiency that was widely touted has been very difficult to reproduce.” Chimeraplasty has always been considerably less promising and more controversial than media accounts have suggested. Many gene-therapy researchers expressed initial skepticism simply because the results were remarkable and the data less than iron-clad. For some, this skepticism deepened as critics uncovered what they considered to be serious flaws in both the Science paper and another key paper Kmiec published in the Proceedings of the National Academy of Sciences (PNAS). The procedure itself has turned out to be fickle at best. Although at least nine laboratories scattered around the world have published reports confirming some ability of chimeraplasty to effect gene conversion, two of those have since moved on to other research projects, and dozens of others—including some of the most experienced in the world in gene repair—have tried to replicate the experiments and failed. Only three of these negative results have been published, but word of their existence spread through the community. “We live and die on reproducibility,” says Harry Orr, director of the Institute of Human Genetics at the University of Minnesota, Twin Cities. “And the scientist in me says to be very dubious of something that cannot be uniformly reproduced.” The ongoing controversy illustrates how publication in a prominent journal, followed by a few confirmations against a much larger but unpublished background of failures, can give life to a remarkable claim. When both journals and journalists attend to the positive signal and ignore the negative background, the result can be a distorted view of reality that can take years to clarify. ## Correcting mistakes For Eric Kmiec, chimeraplasty represented his reemergence after a decade of struggle to rebuild a career that was launched with enormous promise and then descended into controversy. In the 1980s, Kmiec accomplished the noteworthy feat of publishing eight articles in Cell based on his graduate and postdoctoral research. The central findings of the first four, however, published with his doctoral adviser William Holloman, now at Weill Medical College of Cornell University, have never been independently replicated; those of the fifth and sixth, also published with Holloman, were publicly refuted. The remaining two Cell papers, published as a postdoc with biologist Abraham Worcel of the University of Rochester in New York, were retracted in 1988 by Worcel, whose own lab failed to replicate the results after they were challenged by outside researchers. Kmiec, who has continued to stand by his early papers, struggled for the better part of a decade to build his career before reemerging in 1996 with chimeraplasty. Kmiec says the idea for chimeraplasty grew out of his graduate studies on homologous recombination, the process in which chromosomes exchange or “recombine” DNA. Over the years, researchers had tried to enlist homologous recombination for gene therapy or genomics. Although they've had some success, the “conversion efficiency” has remained so low—converting genes in perhaps one in every 1000 or every 10,000 targeted cells—that the techniques have seen limited use. In the late 1980s, for instance, University of Rochester biologist Fred Sherman demonstrated that small, single strands of DNA could induce specific changes in the genomic DNA of yeast via homologous recombination. But the efficiency rate was excruciatingly low—“10 to a minus big number,” says Sherman. In 1993, Kmiec explained in a 1999 issue of American Scientist, his research convinced him that RNA could facilitate recombination reactions. Instead of relying on a single strand of DNA, Kmiec decided to add a second strand consisting of five nucleotides of DNA sandwiched between two longer stretches of RNA that were intended to provide stability to the molecule. The two strands would then be joined together at the ends into a racetrack shape, to avoid dangling nucleotides that might be attacked and degraded by cellular enzymes (see diagram, p. 2116). Kmiec theorized that chimeraplasty might correct genetic defects by artificially inserting an error that homologous recombination would naturally correct. The first step required the synthesis of a short, artificial string of nucleotides—made from the building blocks of DNA, adenine (A), thymine (T), guanine (G), and cytosine (C)—that would be flanked by the RNA. This RNA-DNA “oligonucleotide,” or RDO, would be designed to seek out the genetic region of interest. Specifically, nucleotides bind to their complement (A with T, G with C), so that an RDO that has, say, a string of A's will seek out and bind to a complementary string of T's. As Kmiec conceived it, chimeraplasty would repair a genetic defect by tricking the error-correcting mechanisms of homologous recombination into fixing the error introduced by his RDO. Imagine a stretch of gene that should read AAAAA, but instead reads AATAA. Kmiec reasoned that a complementary RDO of TTTTT would bind to the target sequence, bulging out at the site of the mismatch—where there were T's in each strand—and thus alerting the cell's suite of DNA repair enzymes. These would then remove the “bad” nucleotide from the defective gene and replace it with the correct complement to the one on the RDO. After some encouraging initial results, says Kmiec, he founded Kimeragen in 1994 to pursue the technology, although “with essentially no money.” Kimeragen borrowed research money from TJU, with the expectation that the company would pay back one-quarter of the total ($400,000, according to a TJU press release) every 3 months from investor financing. But that financing was slow to come. Kmiec says he “was constantly at the dean's office or in the tech-transfer office” trying to convince the TJU administrators that Kimeragen would meet its payments.

This left the research to Kyonggeun Yoon, a chemist whom Kmiec hired from industry. As Yoon recalls, Kmiec told her they would give his idea 3 to 6 months and “then make a decision to kill it or go on.”

Yoon tried Kmiec's RDOs on a variety of cell lines with no success. She then tried an assay that relied on the properties of an enzyme called alkaline phosphatase. If a cell contains “active” alkaline phosphatase proteins, it will turn red when the proper stain is applied. Yoon's idea was to alter a single nucleotide in the alkaline phosphatase gene, leading to an inactive enzyme. Using a plasmid (a circle of bacterial DNA) to carry this defective gene, she would “transfect” it into mammalian cells that otherwise lacked alkaline phosphatase entirely. She would then transfect the cells with RDOs designed to correct the defect. The next day, she would apply the stain and look for the red color that meant the RDOs had corrected at least one of the defective alkaline phosphatase genes and that the genes were producing active enzyme.

For 3 months, Yoon recalls, the RDOs resolutely failed. Then one morning, she arrived at the lab to find that a third of the cells in her latest experiment had turned red. “I couldn't believe my eyes,” she says. “I told my husband, ‘Either something happened, or I'm hallucinating.’”

In the summer of 1995, Yoon and Kmiec wrote a paper and submitted it to Science, which rejected it, Yoon says. She and Kmiec then submitted an article to PNAS, where it was published in March 1996. It claimed that their RDOs had corrected single-point genetic defects “with a frequency approaching 30%.”

Six months later, Kmiec published even more dramatic evidence in Science. In November 1995, Yoon had left Kmiec's lab to take a faculty position at TJU. This left Allyson Cole-Strauss, Kmiec's technician and a co-author on the PNAS paper, to carry out the research. (Cole-Strauss did not return numerous phone calls from Science.) Cole-Strauss, Kmiec, and their co-authors reported that RDOs designed to correct the β-globin mutation responsible for sickle cell anemia appeared to work successfully in 50% of the targeted cells in test tube experiments.

The article was cautiously written, but at those efficiencies or anything close, it was “the answer to everybody's prayers,” says John Wilson, a gene-therapy researcher at Baylor University in Houston, Texas, who later became a member of Kimeragen's scientific advisory board. Despite Science's subsequent publication of two letters strongly critical of Kmiec's sickle cell article (see sidebar above), researchers worldwide considered the efficiencies reported by Kmiec to be reason enough to pursue the technology.

## Chain reaction

The publication of Kmiec's PNAS and Science articles had a dramatic effect on his career. Since leaving Rochester a decade earlier, Kmiec's research had subsisted on grants from the American Cancer Society and the Council for Tobacco Research, a funding organization financed by the cigarette industry. In April 1996, based on the results published in PNAS, Kmiec received his first NIH support since his postdoc years—a 3-year grant for $432,000 to pursue “New Gene Therapy for Connective Tissue Diseases.” In September 1998, he received$850,000 from NIH for 3 years to study “Genetic Repair of the Sickle Cell Anemia Mutation.” And since June 2000, he has received almost $1 million more in NIH funding to study the mechanisms of his gene-correction technology. Kimeragen also benefited, luring Blaese from NIH to be chief scientific officer of the company and raising between$10 million and \$20 million in venture capital. In 1998, Kmiec and Kimeragen parted ways, after Kmiec and the company management, by all accounts, clashed over numerous issues.

The two papers also prompted researchers around the world to try chimeraplasty, given what Blaese called “the enormous promise” if it worked. Last October, at the annual meeting of the American Society of Human Genetics held in Baltimore, Kmiec reported that in the 6 years since his Science publication, more than 30 published papers have reported some success with chimeraplasty. These include work done in bacteria, plants, mice, rats, and a single dog. They reported that the RDOs could trigger gene repair in these systems with efficiencies ranging from 0.0002% to near 50%. By this autumn, nine laboratories had reported some positive result, including Kmiec's at the University of Delaware, Newark, where he moved in 1999, and Yoon's at TJU.

The strongest corroborative evidence has come from Clifford Steer, a University of Minnesota, Twin Cities, medical doctor and liver disease specialist. Before chimeraplasty, Steer told Science, he had never worked in either gene repair or gene therapy. He says he was collaborating with Kmiec on other research when he saw the “scathing” letters to Science on the sickle cell experiments and recalls saying to Kmiec, “if you have any of those chimeric [RDOs] around, why don't you send me one and we'll try testing it in our lab. If we're successful, at least you can tell the general public or the scientific community that another lab independent of yours was able to reproduce the work.” Kmiec sent him the RDOs and within 3 weeks, Steer told Science, he demonstrated that chimeraplasty worked. The subsequent paper was published in Hepatology, co-authored by Kmiec. (Steer's brother was an original investor in Kimeragen and on Kimeragen's board of directors, but Steer says that had no influence on his decision to work with Kmiec or pursue the research.)

Since his first paper with Kmiec, Steer has reported that his RDOs work with astonishing efficiency. In 1999, for instance, Steer reported in The Journal of Biological Chemistry that his RDOs could induce with 48% efficiency a specific mutation in the factor IX gene responsible for hemophilia in the liver of live rats.

Steer says the key to his success is a modified version of a gene-delivery technology that uses polymers known as polyethyleneimines (PEIs) to help plasmids slip into cells. He has reported that his modified PEIs can deliver RDOs and reporter genes to 100% of liver cells in live animals. Researchers such as George Wu of the University of Connecticut at Storrs, Jean-Paul Behr of the University of Strasbourg, France, and Ernst Wagner of the University of Munich, Germany, who work with PEI and similar gene-delivery formulations and who pioneered the technology, told Science that the best they've ever achieved with similar systems is below 1%. Steer's results “boggle the mind,” says one gene-repair expert.

Steer says he welcomes researchers to visit his lab and learn his modified PEI techniques. But he told Science that he knew of no independent researchers who had reproduced his experiments. At least three other labs took up his offer and saw convincing demonstrations that the technology worked in Steer's lab. However, they still failed to reproduce his findings at their own laboratories. Geneticist Thomas Jensen of Denmark's University of Aarhus says his student spent over a month with Steer in the summer of 2000 and then tried for over a year to replicate the experiments. Although this lab seemed to get some positive results, Jensen told Science, the conversion efficiency was so low that “it is difficult to measure.” Wu says his lab “spent a fair amount of time and money” in this pursuit but failed.

Even Kmiec's researchers have been unable to achieve similar results. “We're concerned that Cliff stands out there by himself,” says Howard Gamper, who works with Kmiec at Delaware. “No one has reproduced his work at the efficiencies he reports. This lab has not, and we're not aware of anyone else [who] has had success at that level.”

The difficulty in reproducing chimeraplasty techniques has not been limited to Steer's liver-cell system. In a letter published in the June 2001 issue of Nature Biotechnology, Jim Owens and colleagues at the University of London and the Royal Free and University College Medical School in London reported that they had managed to correct defective apolipoprotein E genes in four different cell types with an efficiency above 25%. Since then, however, as Owens told Science last week, their chimeraplasty experiments have failed persistently. Owens referred to these relentless negative results as the “somewhat sorry situation in our laboratory.” He suggested that the problem might lie with poor-quality RDOs and reagents; his lab is now trying to check that possibility.

Most researchers who tried chimeraplasty failed from the beginning. Science spoke to researchers from over 30 laboratories that had tried the RDOs and failed to produce evidence that they could target and correct dysfunctional genes, either in vitro or in vivo. Researchers at biotech companies such as Epoch Biosciences, Isis Pharmaceuticals, Millennium Pharmaceuticals, and Lexicon Genetics all failed to get chimeraplasty to work in their labs. Experienced gene-targeting researchers at MIT's Whitehead Institute, NIH (including in Blaese's own laboratory), Maine's Jackson Laboratory, and Sweden's Karolinska Institute also saw no effects. Even members of Kimeragen's own scientific advisory panel, such as Baylor's John Wilson, tried it and failed. “Under our conditions,” Wilson says, “we found no correction above background.”

As of last winter, three laboratories had published their negative results, including one from the University of Groningen, the Netherlands, led by Gerrit van der Steege, who saw the technique work in Yoon's TJU laboratory but was unable to replicate it in his Groningen lab. Writing in Nature Biotechnology in April 2001, van der Steege and his colleagues described their “persistent failure” and “complete lack of success” with the RDOs.

The great majority of researchers interviewed by Science say they find the negative results, even though unpublished, more persuasive than the positive ones because they come from independent labs with considerably more experience in gene repair and gene therapy than those that succeeded have. “The people I trusted, the ones I polled who are really good,” says Neal Copeland, for instance, director of the Mammalian Genetics Laboratory at the National Cancer Institute, “invested a lot of time, and none of them got it to work.”

Kmiec and other proponents of chimeraplasty disagree. “The ‘lab-to-lab irreproducibility,’” Kmiec explained in an e-mail to Science, is “overemphasized, and appears to be the consequence of different factors, including incomplete synthesis of the RDO, or a lower frequency of nuclear delivery, or the metabolic state of the cell.” He says the failure of groups such as van der Steege's to get the technique to work in their labs “means only that the same cells can respond differently during each attempt or that differences in equipment, supplies, and even the water can influence the results that are observed.”

## An orphaned technology?

In the past 2 years, the story has taken a peculiar twist. Although Kmiec says, “I believe the chimeraplasty technique is growing in robustness and has never had more potential,” he is now focusing on alternatives. Kmiec says he has turned to single DNA strands because he couldn't afford to buy double-stranded RDOs. Even Steer told Science that he has switched to single-stranded DNA because it is “easier to make” and “a lot less expensive.”

Indeed, both Kmiec and Yoon have reported that DNA single strands, of the kind Rochester's Sherman used in yeast, work better than the double-stranded RNA-DNA chimeras do in some experiments. In November 2000, Kmiec reported in Nucleic Acids Research (NAR) that single DNA strands repair genes with less than 0.02% efficiency in vitro in a cell-free extract, and that this efficiency was three to four times higher than the RDOs. “In that paper,” says first author Gamper, “we're saying that maybe these chimeric RDOs are not so magical.” Kmiec said in an interview in November 2000 that this work implies that RDOs are not necessary to achieve gene repair and that they are difficult to work with, in any event. In October 2001, he reported in NAR that single DNA strands effected gene conversion in yeast with an efficiency of 0.016%, whereas the RDOs achieved 0.0002% efficiency.

To date, the bulk of the research suggests that when either RDOs or single-stranded DNA work at all, they do so at an efficiency rate 1/100 to 1/100,000 of that originally reported and compatible with that of other gene-targeting techniques that rely on homologous recombination. This is also the efficiency reported by researchers who worked with RDOs in plants. “It has now taken 5 years to go from 50% gene correction in human cells to 0.0002% correction in yeast,” says Andrzej Stasiak, a genetic-recombination expert at the University of Lausanne, Switzerland, “and gene targeting in yeast is really easy, so their current, improved method is unlikely to attract a lot of attention.”

The concept still has proponents at half a dozen laboratories, from which positive results occasionally emerge. Kmiec and other chimeraplasty proponents consider these results compelling evidence of what Kmiec calls “the successful application of chimera-based gene repair” and “the normal evolution of scientific knowledge.”

But this argument is not winning many converts. “Once there is some lack of credibility, one has to present a better case,” says Steve Kowalczykowski, a genetic-recombination expert at the University of California, Davis, who was a member of Kimeragen's scientific advisory board. “One more ordinary paper is not convincing. The burden of proof becomes greater.”

After 6 years of research, chimeraplasty still lacks unambiguous data and universal reproducibility. Barring a dramatic turn of events, it seems likely that the technology will pass the way of other potential breakthroughs that garnered their 15 minutes of fame and then vanished slowly into the literature.

13. GENE THERAPY

# Pioneering Papers Under the Microscope

1. Gary Taubes

The two papers that launched chimeraplasty in 1996, published in the Proceedings of the National Academy of Sciences (PNAS) and Science, have attracted some withering scrutiny.

In their PNAS paper, Eric Kmiec and Kyonggeun Yoon, both then at Thomas Jefferson University in Philadelphia, had stitched an alkaline phosphatase gene containing a single-point mutation into a plasmid and “transfected” it into mammalian cells that otherwise lacked alkaline phosphatase genes. They then transfected the cells with chimeric molecules called RDOs designed to correct the point mutation (see main text). If the correction occurred and the cells started producing active alkaline phosphatase, the enzyme should cause a special dye to turn red.

Yoon and Kmiec reported that “approximately one in three” cells had turned red. To corroborate this result, Yoon extracted the plasmid DNA from the cells and put it into bacteria, which grew into colonies. Then she screened more than 400 of these colonies to assess how many included the corrected alkaline phosphatase gene. Kmiec and Yoon reported that the answer was again roughly one in three. This gave them the confidence to state that their experiments “established clearly that sequence correction by the chimeric oligonucleotide occurred in mammalian cells.”

Critics noted, however, that the two tests measured entirely different parameters: The first counted the fraction of cells that contained at least a single healthy gene, whereas the second counted the fraction of corrected plasmid genes themselves in the cells. In interviews with Science, researchers who work with similar technologies said they could think of no scientific reason why those numbers should be identical; indeed, they said, if the RDOs had accomplished what Kmiec and Yoon had claimed, then those numbers should have differed by several orders of magnitude. For example, Phil Felgner, who invented the reagent used by Kmiec and Yoon and is now the chief scientific officer of Gene Therapy Systems in San Diego, California, notes that hundreds of thousands of plasmids carrying the defective gene would enter into each cell under the conditions reported in the PNAS paper, and only a tiny percentage of those—less than 1%—would make it into the cell nucleus, where gene correction by the RDOs could have occurred. So for each cell that contained at least one corrected gene—the minimum necessary to turn a cell red—there could have been several hundred thousand uncorrected genes from plasmids that either never made it to the nucleus or weren't corrected when they did get there.

Experts on the technology, consulted by Science, suggested two possible explanations for how both tests could result in identical numbers. The first—which Yoon also ventured as a possibility when Science asked her about the criticisms—is that cellular enzymes might have degraded most of the plasmids that didn't make it into the nucleus. That would greatly reduce the number of uncorrected copies of alkaline phosphatase genes that the bacteria would take up in Yoon's confirmatory test. But the experts consulted by Science say degradation of the plasmids would be unlikely to happen in the 30 hours reported by Yoon and Kmiec in their paper. And even if such a mass degradation did occur quickly, it would still be a considerable coincidence that the proportion of cells that turned red equaled the proportion of corrected alkaline phosphatase genes that Yoon found in the bacterial colonies.

The other possible explanation was contamination: 30% of the cells may have been contaminated with plasmids that already contained the correct form of DNA. If so, however, the control experiments, which showed no correction of the defective gene, were not contaminated. Yoon told Science that contamination was possible. “It would not be hard [to do],” she said, adding that her colleagues had worried about this possibility. She did not believe this had happened.

When Science asked Kmiec about these criticisms, he responded in an e-mail that he does not accept that a large number of plasmids would necessarily enter each cell. He added that the technology was then in its earliest stage of development, and so “one cannot expect that the data would all be explainable by simple answers.”

The Science paper—in which Kmiec and his colleagues reported correction in 50% of target cells of the defect in β-globin genes that causes sickle cell anemia—came in for equally intense scrutiny. Critics challenged the results in two letters to Science. The first, published on 7 March 1997 (p. 1404), was from gene-therapy pioneer Mario Capecchi and his colleague Kirk Thomas of the University of Utah, Salt Lake City. They suggested that Kmiec and his co-author, Allyson Cole-Strauss, might have inadvertently picked up correct DNA sequences from their RDOs rather than the experimental cells in their tests to determine what fraction had been corrected.

The second letter was co-authored by three veteran genetic-recombination researchers—Andrzej Stasiak of the University of Lausanne, Switzerland, Stephen West of the Imperial Cancer Research Fund in the United Kingdom, and Edward Egelman, then of the University of Minnesota Medical School. They suggested that data in the Science article pointed toward contamination.

Kmiec and Cole-Strauss had sequenced the β-globin genes around the original target mutation and published these sequences in the article. In two of their experiments, these sequences included a second transformation—a dozen nucleotides away from the target—where cytosines had apparently changed to thymines. Stasiak, West, and Egelman pointed out that the RDOs could not have caused this transformation because they only had cytosine at this position. Rather, they suggested, the probable explanation was that the sequencing had been done on a mixture of cells that included wild-type, healthy β-globin genes that happened to be “polymorphic” at this second position; some had cytosine and some thymine. Kmiec and Cole-Strauss's data indicated that their wild-type cells had just that polymorphic mixture. The sequencing data from the control experiments, however, showed no signs of this second transformation.

Science published their letter on 25 July 1997 (p. 460), along with a response from Kmiec stating that contamination of the sort described would not generate the pattern of data he had published. In July 2001, however, Kmiec conceded to Science that such contamination was a likely explanation for the published results. “Of course Stasiak was right,” he said in a telephone interview, suggesting that the sickle cells used in those experiments “probably had various types of other cell types in them or were contaminated by us inadvertently.”

Still, Kmiec insisted that his conclusions were valid because he had other data from similar experiments that could not be explained by contamination. And in an e-mail to Science this fall, Kmiec said that tests on the cells had found no evidence of contamination.

Whatever the correct explanation, says West, the chimeraplasty experiments reported in Science were “very bad science.” After writing the letter and reading Kmiec's response, he says, “we decided that was enough of it. We have better things to do than wave our hands, and we got on with our own work.”

14. FRONTIERS OF SCIENCE

# A Bridge Across the Mideast Divide

1. Jon Cohen

With help from the U.S. National Academy of Sciences, Israeli, Palestinian, and Jordanian scientists are planning to bring together young Middle Eastern scientists in a meeting like NAS's Frontiers in Science symposium

IRVINE, CALIFORNIA—Within 2 hours of meeting at the 14th Annual Frontiers of Science Symposium here last month, a small group of Israeli, Palestinian, and Jordanian scientists hashed out a solution to the Middle East conflict. “Why can we do it in 2 hours, and our people are fighting on this for decades?” asked Israeli Ran Nathan, an environmental ecologist at Ben Gurion University of the Negev in Be'er Sheva.

The six Middle Eastern scientists—who do not speak for their respective countries in any official capacity—attended this unusual meeting as observers. They came to explore the possibility of staging a Mideast version of this symposium, in which the U.S. National Academy of Sciences (NAS) gathers together bright North American scientists under age 45 for 3 days to educate one another about their diverse specialties. “Science is a common language, and there are no borders,” said Palestinian Khuloud Jamal Khayyat-Dajani, a public health specialist at Al-Quds University in Jerusalem. “We need to find the common language that can be understood during the hardest of times.” Nathan put it more bluntly: “If we can bring together people who strongly hate each other, we think they'll see that they're human beings despite their terrible stories.”

Physicist Michael Greene of the Policy and Global Affairs division of NAS's National Research Council dubbed their mission “Seeds of Peace for scientists,” a reference to a program that unites Arab and Israeli teenagers. “It's not easy to sell the idea [of such a meeting] in the regions these scientists come from because people don't understand what it's about,” explained Greene, who helped arrange their participation here. “What it's about is bringing together young scientists who will be leaders in the next generation so they'll know one another better.”

Since 1993, NAS has helped sponsor meetings between scientific academies from Israel and its Arab neighbors to help fuel the peace process. The program—known as Science Academies and Councils of the Middle East—has faced some setbacks over the years. Some Arab countries, such as Saudi Arabia and Kuwait, declined from the start to send their scientists, and Egypt played an active role before suddenly canceling a 1995 meeting in Cairo. “It's hard to find out why,” says Greene. “The Egyptians haven't participated, despite a lot of effort to include them.” The Palestinian uprisings, or intifadas, also have brought the program to a grinding halt at various points.

But the societies that stuck with the program have had some tangible successes. A 4-year collaboration between Jordanian, Palestinian, Israeli, and American water scientists led to a well-regarded report, “Water for the Future: The West Bank and Gaza Strip, Israel, and Jordan.” In addition to continuing work on water resources, three committees that sprang from this success have addressed micronutrient deficiencies, biodiversity, and telemedicine. (The micronutrient committee completed a report, but the others shut down because of the second intifada in 2000.)

Following the 11 September attacks last year, NAS invited members of the project to Washington, D.C. At that meeting, NAS president Bruce Alberts and William Wulf, president of the National Academy of Engineering, proposed the idea of holding a Frontiers of Science and Engineering meeting for Middle Eastern scientists, partially funded by NAS.

By the second day of last month's Frontiers of Science meeting, the Middle Eastern observers had optimistic, even dreamy, ideas about what they might do, mixed with starkly realistic and depressing assessments of what they could not accomplish. Holding a similar meeting, said Eitan Reuveny, a biochemist at the Weizmann Institute of Science in Rehovot, Israel, “is probably the first bridge we can rebuild to acquire confidence on both sides, not on a level of scientific cooperation but of scientific exchange.” Similarly, Sami Husein Mahmood, a physicist at Yarmouk University in Irbid, Jordan, said that such a gathering would provide “a great opportunity” to young Middle Eastern scientists, allowing them to meet their neighbors, win recognition for their work, and gain confidence. “I'm hoping this will help us jump over the psychological barriers,” said Mahmood.

But how to organize and stage such a meeting presented some sticky challenges. Foremost among them: where to hold the gathering. Reuveny thought that it might work best in a remote spot, possibly in Israel, that offered a retreat atmosphere and security. But all of the Arab scientists insisted that the gathering could not take place in the Middle East. “In the beginning, it should be done in a neutral country,” said Jihad Al-Sawair, an environmental scientist at the Royal Scientific Society in Amman, Jordan. “But in time, when things become clear and hopefully become better, maybe it will be done in the region.”

Obvious disparities also exist between Israeli scientists, who have a well-established and well-funded scientific culture, and Palestinian scientists, who have few institutions and little support. This makes it difficult to mix the most accomplished young scientists from each country and have them interact as they do in North America. “You can't compare the level of science,” says Reuveny. “They just got started. But maybe they can learn from us: how we get our funding, and how we distribute it.”

To Reuveny, though, these differences are overshadowed by the larger vision. “People who do science on both sides are the most moderate for peace,” he says. “They have been outside. They know peace is the only solution. How do you spread this message?” Gathering them together might not be the ultimate answer, but this modest attempt by Middle Eastern scientists to help resolve their region's intractable conflict is, in its own right, a frontier of science.

15. SEA-FLOOR ECOSYSTEMS

# Trawling's a Drag for Marine Life, Say Studies

1. David Malakoff

Scientists at a recent Florida meeting debated how best to manage fishing practices that affect life on the ocean bottom

TAMPA, FLORIDA—Trawlers are catching more than fish these days. The boats are also capturing the attention of marine scientists, conservationists, and fisheries managers, more than 300 of whom gathered here last month* to discuss, among other topics, new studies on trawling's impact on sea-floor ecosystems and ways to limit the damage. A decade ago, “you couldn't have found enough people interested in trawling impacts to fill a phone booth,” jokes Les Watling, a marine biologist at the University of Maine's Darling Marine Center in Walpole.

The growing interest stems in part from new laws that require governments to protect vulnerable marine habitats. Critics say boats dragging heavy nets across the sea floor kill nontarget species such as corals and harm even commercially valuable populations by flattening habitat (Science, 18 December 1998, p. 2168). But many fishers have argued that the impacts are short lived and that “tilling” the sea floor can augment food supplies for certain prized table fish. That idea took a battering here.

Three years ago, marine ecologist Simon Jennings, biostatistician Mike Nicholson, and their colleagues at the Centre for Environment, Fisheries, and Aquaculture Science in Suffolk, U.K., decided to test claims that trawling helps spur the growth of sole and plaice, two popular flatfish that live in Europe's North Sea. The theory was that frequent trawling had removed larger, slower-reproducing organisms from the sea floor and made room for marine worms and other smaller, faster-breeding competitors favored by flatfish. “Everyone agrees that trawling decimates big animals; the question is what happens further down the food chain,” says Jennings.

The team members analyzed data from ship-mounted monitoring tags to identify 27 sites in the Silver Pit, a popular North Sea fishing ground, that had been trawled heavily, moderately, and lightly between 1994 and 2000. They then counted and weighed every organism in 10 sediment samples from each site. Their conclusion, published in the 13 November Marine Ecology Progress Series: Water depth and sediment type are the major influences on polychaete worm populations. The presence of trawlers had little effect. The study appears to “lay the farming analogy to rest,” says marine ecologist John Steele of the Woods Hole Oceanographic Institution in Massachusetts, who chaired a National Academy of Sciences panel that issued a report last January on trawling impacts.

Jennings and others also raised questions about another popular but largely untested idea: periodically closing certain areas to trawling to allow sea-floor communities to recover. Drawing on several European examples, the researchers showed that current trawling patterns are generally uneven and patchy, allowing organisms from untrawled areas to recolonize nearby trawled zones. But temporary closures can concentrate fleets in smaller areas, increasing pressure on undisturbed sea floors. Over time, a system of rotated closures could also prompt trawlers to venture into previously unfished areas, increasing the total area disturbed unless the number of boats were reduced. “The first impacts of fishing are always more profound than subsequent impacts,” Jennings notes, so “you have to think carefully before you start moving fleets.” That message might complicate pending policy decisions in several regions of the world, including the fish-rich waters off Alaska, where the U.S. government is considering rotating closures to protect potentially sensitive areas.

Conservationists, meanwhile, are pushing for permanent trawling bans in what they say are especially sensitive habitats, such as coral reefs and sponge gardens in deep and shallow waters. Studies presented at the meeting confirmed earlier views that these craggy, high-relief habitats are typically more vulnerable to trawling damage—and slower to bounce back—than are flatter, softer bottoms sifted by strong currents or other natural disturbances. With corals, for instance, the nets can break and crush organisms that are hundreds of years old. “It's become clear that we should be talking about a global ban on trawling in corals,” argued Mike Hirshfield, the science chief of Oceana, a marine conservation group based in Washington, D.C.

Fishing industry representatives didn't disagree, but they expressed frustration at the dearth of information about where such habitats are located. “There is a lot of mapping” and research to be done before large areas are fenced off, said John Gauvin, head of the Groundfish Forum, an industry-backed group in Anchorage, Alaska. The existing trawling literature is “unbalanced,” echoed Jeremy Collie of the University of Rhode Island, Kingston, noting that most studies have focused on the least vulnerable sea-floor types.

The time for information gathering is growing short, however, at least in the United States. Next month, under a 1999 federal court ruling won by Oceana, fisheries managers in Alaska and elsewhere must start releasing draft plans for protecting “essential fish habitat”—including sensitive sea-floor areas—from trawling nets and other fishing gear. Other countries, including Canada and Norway, are also moving to identify and protect sensitive zones.

Well-designed conservation efforts, however, will require more-sophisticated studies, argued several researchers. Scientists have so far focused on changes in the populations of large, relatively easy-to-study organisms, noted Watling. Now, they need to tackle the harder questions of how trawling might change microorganism communities and fundamental sediment and nutrient cycles. As Collie puts it, “we need to go beyond the kill-'em-and-count-'em studies.”

Those finer-scale studies will require additional funding, however, for which the prospects are uncertain. But even making such a request is a sea change in trawling science. “The debate has shifted,” says Hirshfield, “from whether trawling has an impact to how big it is.”

• *“Effects of Fishing Activities on Benthic Habitats,” 12 to 14 November, Tampa, Florida.