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New Scientist 18 Oct 97

EFFORTS to breed pigs which are free of viruses that infect people appear doomed to failure. The finding heightens concerns about the safety of using genetically engineered "humanised" pig livers as organs for transplants. And it comes as doctors in the US step up a trial to test these livers as a temporary "bridge" for patients awaiting a human liver. Companies on both sides of the Atlantic, including Imutran in Cambridge and Nextran in Princeton, New Jersey, have engineered pigs to carry human proteins on the surface of their cells. The idea is that their organs will not immediately be rejected by the human immune system, and so can be used to tackle the shortage of human livers for transplant (see Figure). But serious concerns about the safety of pig livers were raised earlier this year, when two research groups showed that pigs can carry at least two retroviruses, one of which clearly had the potential to infect human cells (This Week, 1 March, p 6). These viruses incorporate themselves into pig DNA, and so are difficult to remove. However, those who support transgenic pigs as organ donors have argued that it should be possible to find pigs without the viruses, and then breed a virus-free strain. But in this week's Nature (vol 389, p 681), researchers at the Institute of Cancer Research and the National Institute of Medical Research (NIMR), both in London, suggest that breeding virus-free pigs will be extremely difficult, if not impossible. The researchers used sensitive molecular probes to search for copies of the two viruses in pigs from a range of breeds. They found multiple copies of the viruses in all the pigs tested. And they now have evidence that both viruses are able to reproduce in human cells. "The existence of 20 to 30 copies per cell will make it very much harder to remove the virus from pig cells," says Jonathan Stoye of the NIMR, who led the research. "It may actually be impossible." Although the viruses infect human cells in the lab, nobody knows whether they would cause disease in people. Until this is investigated, Stoye is against pressing ahead with pig-to-human xenotransplants. He says: 'We ought to know more about the pathogenic potential of these viruses." However, a clinical trial of Nextran's humanised pig livers is already under way in the US. The trial, which has the approval of the Food and Drug Administration, does not involve actual transplants. Instead, blood from patients with liver failure is fed through a pig liver kept alive in a sterile container. The technique is akin to kidney dialysis and should keep patients alive until a human donor is found. Last week, Northwestern University Medical School in Chicago became the second centre to join the trial, after Baylor University Medical Center in Dallas. Ten patients will take part in this first phase of the trial. They will be hooked up to a fresh pig liver for 24 hours at a time. However, this may need to be repeated twice a week over many weeks. "If you're going to hook someone up to a pig liver outside their body for a long period, I don't think it's much safer than an actual transplant," says Stoye. John Logan, Nextran's vice-president of research and development, agrees that the pig livers which will be used in the trial are likely to contain the retroviruses. But he believes the trial is justified. "If we stopped and worried about everything that could go wrong in medical experiments we'd never achieve anything," he says. "Xenotransplantation could save tens of thousands of lives each year. That's a tremendous medical opportunity." Michael Day


The engineered salmonella

Tumours Fall to a Trojan Horse New Scientist 18 Oct 1997

TODAY salmonella might make you sick or worse; tomorrow it could cure your cancer. Researchers from Connecticut have found that mice with an aggressive form of skin cancer lived twice as long when they engineered their bacteria to carry cancer-fighting genes and congregate in tumour cells. "We get accumulations of bacteria 1000 to 10 000 times as high in tumour tissue as in normal tissue," says David Bermudes, associate director of biology at Vion. S. typhimurium preferred the company of tumour cells because John Pawelek and Brooks Low at Yale had made it hard for the bacterium to survive in normal cells. It lacked the building blocks to make DNA, RNA and protein because the researchers had deleted three vital genes. The fast-growing cancer tissue, however, is especially rich in these nutrients. The team also equipped the growth bacterium with a gene that makes an enzyme which turns a harmless substance called ganciclovir into a drug lethal to cells. By restricting production of the enzyme to tumour cells where the bacteria collect, ganciclover only kills these tumour cells.

Early experiments reported this week in Cancer Research (vol 57, p 4537) have shown that the high concentrations of bacteria both stunt tumour growth and reach secondary tumours in the body. "The bacteria find multiple tumours without knowing where they are," says Bermudes. The researchers also found that the mice suffered no side effects, despite being injected with a million times as many bacteria as would normally kill them. Bermudes stresses that a cancer treatment is still a long way off. Many more experiments need to be done to refine the technique. But he hopes the approach will work, not least because the bacteria clump in other types of tumour cell, including those from cancers of the lung, colon, breast, liver and kidney. He hopes that human trials might begin in about 15 months' time. The researchers hope the bacteria will prove a more effective cancer therapy than the genetically engineered viruses used in most trials so far. Unlike the bacteria, the viruses must be injected directly into tumours and can't reach secondary growths. Andy Coghlan

GUIDED BY the maxim that where there's muck there's money, a Canadian biotechnology company says it has found a way to raid the treasure trove of potentially useful compounds made by soil-dwelling microorganisms. It also plans to extract drugs from lichens. Every handful of soil contains up to 5000 species of bacteria, but research into their potential to produce antibiotics and industrial enzymes has been hampered by their stubbom refusal to grow in the laboratory. Only I per cent of soil microorganisms can be cultivated artificially. Now, TerraGen Diversity of Vancouver has sidestepped this problem by chopping the DNA of the microorganisms into larger chunks than has previously been possible-and then transplanting them into hosts which are easy to cultivate, such as the bacterium Escherichia coli. The company is also using the method to extract novel compounds from 15 000 lichen species-combinations of algae, bacteria and fungi-which have also defied attempts at laboratory cultivation. At present, TerraGen will only reveal the bare outlines of its technique. First, soil or lichen samples are put through a series of centrifuges to separate the microorganisms from inanimate debris. Samples are treated chemically to destroy soilborne substances, such as humic acid, that would hamper analysis. Once the live organisms have been isolated, TerraGen treats them with restriction enzymes to break the microbial DNA into manageable chunks. After these chunks have been spliced into hoops of DNA called plasmids-which are the way organisms exchange genes naturally-the microbial genes can be transplanted into their hosts. The hosts might be E. coli, fungi or strains of bacteria that make antibiotics. Similar techniques, of expressing alien genes in host bacteria or fungi, are nothing new to the biotechnology industry. Typically, microbiologists transplant chunks containing around 40,000 of the nucleotide bases that form the DNA alphabet. TerraGen claims to have broken new ground by devising a way to transplant especially large strands of bacterial DNA that may contain dozens of genes. The company routinely transplants chunks containing 100,000 nucleotides, and has even inserted chunks of up to 300,000 bases, which account for as much as 5 per cent of a bacterium's genome. The large size of the chunks is important, says Joe McDermott, research director at TerraGen, because it means that several new proteins are likely to be produced in the host. The chunk could even be an entire biochemical production line for a substance that could not be produced if only a couple of genes were transplanted. Searching for the new compounds and proteins is also made easier by TerraGen's transplant method. Because the company is already familiar with the chemicals normally produced by the host, it only has to look for a handful of novel chemicals in each search. "Because we're dealing with the same genetic background time after time, it's easy for us to tell what's native and what's novel," says McDermott. The company has already produced new antibiotics, created by stitching alien genes into the Streptomyces bacteria already used to make antibiotics. It has also isolated chemicals that can influence the way cells respond to messages from their environment. Inadequate or exaggerated responses by cells are thought to be partly responsible for diseases such as cancer and diabetes. TerraGen has also extracted 16 new xylanase enzymes which could find a role in the pulp, paper and textile industries for breaking down and bleaching natural fibres. Andy Coghlan

So far, so good ... New Scientist 8 Aug 98 4

FEARS that novel viruses might wreak havoc in transplant patients who receive pig organs may be groundless, according to findings due to be presented this week in London. The findings, based on screening samples from patients exposed to pig tissue, provide the first compelling evidence that dormant pig viruses do not spread to humans, causing new and incurable infectious diseases. This doomsday scenario has been the main obstacle holding up clinical trials to test "xenotransplants" of pig organs. Robin Weiss of the Institute of Cancer Research in London raised the fears last year when he showed that two pig viruses could spread from pig to human cells in the lab. The viruses, called porcine endogenous retroviruses, have become part of the pigs' own DNA over millennia. Though dormant in the pigs, Weiss's experiments highlighted the possibility that the viruses might reawaken in humans. In the light of Weiss's findings, the US Food and Drug Administration (FDA) temporarily withdrew approval for clinical trials related to xenotransplantation. But since analysing the outcomes of earlier trials, several groups of researchers in the US are starting to dispel the fears. Some of their results were due to be announced this week in London at a closed workshop on pig viruses organised by the UK Xenotransplantation Interim Regulatory Authority, which oversees xenotransplantation proposals in Britain. Last week, it launched guidelines for xenotransplant experiments (see Table). At the meeting, Walid Heinene and Louisa Chapman of the US Centers for Disease Control in Atlanta, Georgia, were due to describe their study of 10 Swedish diabetic patients who received transplants of pig pancreatic islet cells, which manufacture insulin. Even in patients where the cells survived for more than a year, the researchers found no pig virus DNA. Nor could they find any antibodies against pig viruses in their blood, suggesting the blood was free of infection. And there were no signs of viral reverse transcriptase enzymes, a telltale sign of any unidentified viruses. "We looked in blood, lymphocytes and serum, and found nothing," says Heinene. "These are reassuring data." But he cautions against misplaced optimism: "How much we can extrapolate to other types of transplant is not known." Diacrin, a company in Charlestown, Massachusetts, was also due to present encouraging results this week. It has developed treatments for Parkinson's disease and Huntington's disease using brain cells from pig fetuses. The company did not find pig viruses in any of its 24 patients, including one treated three years ago. Other companies reported similar results last month in Montreal at the World Congress of the Transplantation Society, an international organisation. They included Circe Biomedical in Lexington, Massachusetts, a company that has used pig liver cells in a device that extracts toxins from the blood of patients with liver failure. Of the 25 traceable patients receiving the treatment, none has acquired pig viruses. Last week, following approval from the FDA, Circe resumed trials in the US and Europe. Although the negative results for transmission of the viruses are encouraging, caution remains. "Any data that are negative are great, and they mean we can move to the next level of evaluation," says Jon Allen of the Southwest Foundation for Biomedical Research in San Antonio, Texas. But what happens in people who receive an entire organ might be very different, he says. Weiss agrees. "If there are negative results does it mean no one's infected, or that you can't detect it?" he asks. "It's fraught with difficulties." Andy Coghlan