At a secret location in Cambridgeshire, researchers inject human DNA into a pig embryo. Six months later Astrid, the world’s first transgenic pig, is born – of a virgin, in a sterile stable, on Christmas eve. The hope is that the implanted gene will make pig organs compatible with the human immune system, thus helping to solve one of medicine’s fastest growing problems: the shortage of organs for transplant surgery. Astrid produces offspring, the research gathers pace. But there are problems, too: antivivi-sectionists launch firebomb attacks and medical ethicists get jumpy.
It could be the plot of a TV drama about the future of genetic engineering, but it isn’t. Astrid, the ‘pig with a human heart’ as she is dubbed in headlines, is as real as the surgical aspirations of the British scientists who created her two years ago. Now the transgenic clan has grown to some 200 pigs. And Imutran, the company behind the project, is taking the next step – testing what happens when human blood is pumped through hearts taken from some of Astrid’s descendants.
This week, Imutran’s research director, David White reports the first findings to delegates at an international conference on ‘xenotransplantation’ in Washington DC. ‘It’s absolutely clear that hearts from transgenic pigs work better than those from normal pigs and show fewer signs of immune rejection,’ he told New Scientist beforehand. ‘But what isn’t clear is whether that result will correspond to better survival rates of xenografts in primates or humans.’
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If it does, the rewards – financial as well as medical – could be considerable. Last year in the US alone some 2800 people died waiting for human organs to become available, and in Britain at present about 25 per cent of heart patients die waiting. Such figures are set to climb next century as conventional transplantation techniques improve and patients who are now considered too old or sick to benefit from new organs are put on waiting lists.
All being well, Imutran expects trials in humans to begin in 1996. But hard on its heels are two American biotechnology companies replete with glossy brochures featuring transgenic pigs, some designed to function as organ donors. Moreover, the American companies have already started transplanting genetically-altered pig tissue into primates – experiments whose outcome will be vital to persuading ethics committees that there is a case for proceeding to trials in humans.
SURGEON’S DREAM
A bountiful supply of designer organs sounds like a transplant surgeon’s dream. But before it can be turned into reality, Imutran and its rivals must clear some towering obstacles. First, nobody has yet begun to draw up guidelines on the ethics of using genetically-engineered animal organs in surgery, and especially how to preempt any needless experimentation on humans. Falter here, and the pig engineers could find the public set against them. Secondly, the law on patenting transgenic animals is still desperately unclear, raising the prospect of a legal dispute over commerical rights. But the biggest question of all rests with biology: can genetic engineering really deliver animal organs that look like ‘friend’ rather than ‘foe’ to the human immune system?
At the moment, everyone is focusing on making pigs with genes designed to disarm a powerful immune response known as hyperacute rejection. After conventional transplants, organ rejection can, in most cases, be prevented with drugs – such as cyclosporin – that act to ‘handcuff’ aggressive white blood cells known as T cells. But when organs are transplanted from species to species, rejection is too fast and violent to be pacified with drugs alone. The immune system treats the graft much as it would a solid clump of bacteria, unleashing agents that destroy the epithelial cells at the surface of the grafted organ while triggering a massive clogging of the arteries supplying it with blood. Within hours, the graft is reduced to a blackened mess.
Much of the damage is caused by a team of hostile blood proteins called the complement cascade. If antibodies and white blood cells are the ground troops of the immune system, the complement cascade is its air force. Normally reserved for attacking microorganisms, its proteins can punch lethal holes in cell membranes, producing an effect which, in White’s words, is ‘like a bomb going off’.
It was a desire to defuse this bomb that led the Cambridge researchers to inject human DNA into pig embryos in August 1992. Human cells are spared from attack because they carry markers – molecular ‘white flags’ – that can pacify complement proteins. The researchers reasoned that if they could transfer genes for these white flags into pigs, the complement cascade might be fooled into holding its fire. One candidate for the job was a gene encoding a protein called Decay Accelerating Factor. In human tissue, it was clear that DAF molecules stuck out of the surfaces of cells, warding off complement proteins. Could they do the same in pig tissue?
The latest results on perfusing trans-genic hearts with human blood, reported this week in Washington DC, suggest the answer is a qualified ‘yes’. ‘We see little or no signs of any hyperacute rejection of pig hearts expressing DAF,’ says White, who nonetheless stresses the limitations of perfusion tests as a measure of immune compatibility.
MOUSE HEARTS
A similar picture is emerging from Imutran’s rivals in the US. About 18 months ago, Boston-based DNX Corporation began producing mice and pigs carrying not only DAF but human genes encoding two other ‘white flag’ proteins, CD46 and CD59. Experiments involving perfusion with human blood show that transgenic mouse hearts are not attacked by the complement cascade, says John Logan, DNX’s vice-president of research.
The latest company to join the race, a Yale firm called Alexion Pharmaceuticals, is hoping to gain ground with a genetically-engineered protein designed to combine the talents of both DAF and CD59. Having just filed for a patent on the protein, Alexion is cagey about details but decidedly upbeat about clinical potential. ‘We’ve taken engineered pig epithelial cells that produce high levels of protein and transplanted them into primates,’ says Stephen Squinto, the company’s programme director. ‘Normal pig cells are destroyed by hyperacute rejection within minutes. The engineered cells survived for hours.’
Alexion expects to be transplanting designer pig organs into primates this autumn. ‘If we can get decent survival, we’ll move on to humans,’ says Squinto. ‘We’ll probably start with high risk patients and will most likely transplant hearts. The heart is a priority because there’s little you can do with dying patients. You can’t put them on dialysis.’
If the Cambridge researchers are anxious about this competition, they are certainly not showing it. ‘Everything is beginning to hot up,’ says White, ‘but we like to think we’re still in the lead.’
Even those who normally preach caution on xenotransplants seem to be caught up in the excitement. Mindful of past failures to transplant baboon organs into humans, Roy Calne, a pioneer of kidney transplantion and a surgeon at Addenbrooke’s Hospital in Cambridge, warned researchers at a conference in Cambridge last autumn against rushing into more xenotransplants on humans without getting a firmer grip on the biology that causes such transplants to be violently rejected. However, in the next breath, and only half in jest, Calne conjured up a new era in transplant surgery based on the ‘self-pig’.
One day, the vision goes, transgenic technology may be so cheap and easy that we may all take the precaution of paying for the creation and upkeep of a custom-made transgenic pig, an immunological twin in porcine clothing that would come to the rescue in the event of an accident or disease. Contract hepatitis, and self-pig would provide a new liver; develop Alzheimer’s disease, and a supply of personalised pig neurons would be at hand. Heart failure? No problem.
It sounds far fetched, and for the time being it is. Yet a decade from now, self-pigs may fall within the reach of genetic engineers. But whether such animals will ever see the light of a sanitised sty is another matter.
The genes that enable our immune systems to distinguish ‘self’ tissue from foreign tissue, and which make each of us immunologically unique, are encoded by a vast tract of DNA known as the ‘major histocompatibility complex’. To make self-pigs, you would have to disable each animal’s MHC genes and replace them with copies of those belonging to each human ‘twin’. But until recently that would have been unthinkable on technical grounds. Gene ‘knock out’ techniques were too laborious and imprecise, and it was possible to transfer only relatively short pieces of DNA from animal to animal.
Now things are quietly changing. Researchers bent on reprogramming animal genomes are discovering the benefits of ‘yeast artificial chromosomes’, which can be used to transfer stretches of DNA as long as 500 000 base pairs or more. And that could revolutionise the whole business of making transgenic pigs.
Take the case of Astrid and her siblings. White and his colleagues created them by injecting an embryo with an artificially ‘edited down’ version of the natural DAF gene. Partly because of its size the gene is expressed somewhat erratically: not all organs in all pigs bear the DAF white flag. If nothing else, say the researchers, YACs could help to solve that problem by allowing the insertion of a much fuller version of the gene.
But scientific feasibility is only part of the equation. Just as important is commercial viability. And this is where the idea of self-pig could fall into the trough. To prospective backers, donor animals that must be genetically tailored for each and every patient years in advance of any medical problem would surely seem more like a legal and financial nightmare than a life-saving innovation. Or as Squinto puts it: ‘I don’t see how you could market such animals.’
Nor, sadly for fans of self-pig, does there seem to be any middle ground between creating ‘generic’ organ donors – animals that could be used by everyone – and creating animals with genes specific to individual patients.
Yet commercial promise alone will not be enough to speed generic pig donors from the laboratory to the clinic. For a start, militant antivivisectionists in Britain are unlikely to call off their campaign of threats. Even moderate animal rights groups will continue to lobby for a European moratorium on the genetic manipulation of animals, or at least for restrictions on the patenting of such animals. In the case of transgenic pigs, they fear that tampering with immune genes could harm the animal, perhaps by causing immune disorders or a loss of resistance to infections that could be inherited.
Others also see this kind of genetic engineering as something of a slippery slope. ‘You’re not treating the animal as an end in itself but as a means to an end,’ says Richard Nicholson, editor of the Bulletin of Medical Ethics. And has anyone bothered to consider the psychological impact on patients of using animal organs in transplant surgery?
Xenograft researchers react to such concerns with the air of an elephant staring down the barrel of a peashooter. There is no evidence of ill-effects in any of the transgenic pigs and mice produced so far, insist all three companies. And, as if to outface the worriers, conferences on xenografting seldom run seminars on ethics. Instead, one view on animal rights is invariably chanted like a mantra from the podium: the idea of using pigs as organ donors is on a moral par with eating bacon. Speakers concede that primate donors, with their social hierarchies and seemingly richer emotional lives, might never be acceptable to the public. But who could question using an animal that is bred by the million for food and whose heart valves are already being inserted into humans?
PLAYING GOD
This line on ethics (by strange coincidence) harmonises perfectly with the practicalities. Baboons are slow breeders and are difficult to keep free from viral infections, some of them potentially lethal to humans. Pigs, by contrast, are about the same size as humans and can more easily be bred in sterile conditions. And surely the prospect of saving thousands of human lives justifies slotting the odd human gene into a porcine chromosome?
Perhaps. Yet even if proponents of xenografting triumph over animal rights (as seems likely), that won’t be the last of the social obstacles. Just as worrying for the public is the surgeon-playing-God sce-nario, the fear that transplant teams armed with genetically-engineered animal organs will indulge in reckless experiments on human patients.
In the past decade in the US, there have been three attempts to transplant baboon organs into human patients, all failing, and all generating storms of controversy. Despite that, international guidelines on xenotransplants are still nowhere in sight, and decisions about operations remain in the hands of individual hospital or regional ethics committees. National medical guidelines, it is true, stress the need for ‘reasonably informed consent’ in all medical experiments. But what this would amount to in the case of a xenograft experiment is far from clear.
At last year’s conference in Cambridge, delegates were shocked to hear that surgeons at Cedar-Sinai Hospital in Los Angeles had already attempted to transplant a pig’s liver into a 26-year-old woman. Within a day the organ had been rejected. ‘What you saw in this experiment was what you would predict from 30 years of literature – blockage of arteries and a mass of hyperacute rejection,’ says Squinto. ‘Without agents to block the hyperacute response, the experiment was premature.’ The condemnation seems unanimous. ‘Before doing this kind of trial on humans you have to show that the grafts can survive in animals,’ says Logan. ‘But that wasn’t the case.’
Will it be the case when surgeons want to experiment with transgenic pig organs? Might not the intense commercial competition encourage recklessness? Imutran and its rivals insist that a premature transplant with a negative outcome is the last thing they want. ‘We wouldn’t want to go into clinical trials until we can be sure of getting survival rates similar to those for human-to-human transplants – a 70 to 75 per cent chance of surviving for more than a year,’ says White. Any trade in, and clinical use of, transgenic organs could be adequately policed by government watchdogs such as the Food and Drug Administration in the US, argues Logan
A plentiful supply of transgenic animal organs might even help to reduce some long-running ethical concerns about transplantation surgery, say some researchers. The current shortage of human organs requires surgeons to make tough decisions about who should be put on waiting lists. Should organs go to the sickest patients, or those who can most benefit from them? And what about age? In Britain, there are no official age limits, but the vast majority of heart recipients are under 60.
Animal organs could change that. And in the long run, say their proponents, they could also lead to patients being spared some of the unpleasant and debilitating side-effects of immune suppression. ‘Xenotransplantation swings the focus away from interfering with the host immune system to interfering with the organ,’ says Squinto. ‘Why use drugs to produce broad suppression of the host’s immune system when you can modify the graft?’
Even so, reassuring the public may require an openness about data that conflicts with commercial ambitions. Eager to protect the interests of their shareholders and backers, all three companies breeding transgenic pigs have filed for patents. As night follows day, an unseemly courtroom battle is now on the cards and a veil of secrecy hangs over many experimental details. For example, DNX declined to explain its gene constructs and experiments on primates two weeks ago citing ‘commercial sensitivity’.
To pursue their commercial rights, the American companies may have to challenge a patent filed by the Cambridge researchers in 1989 which embraces the whole concept of using genes and proteins to protect animal tissue from attack by human complement. There are broader problems looming, too. For lawyers, the awkward thing about pigs is that they procreate. If company A makes a transgenic animal using techniques owned by company B, then it is certainly infringing the patent. But is that still the case if company A breeds offspring from this animal and sells them? And what happens if company A tries to sell a kidney from these offspring? ‘The issue is at best cloudy,’ says White.
Big though these social and commercial obstacles may be, they might ultimately be dwarfed by a more fundamental question: can animal organs ever be refashioned sufficiently to be fully compatible with the human body? Certainly, say die-hard optimists such as Stephen Grundy, a surgeon at the Medical Center of Loma Linda University, where the first baboon-to-human transplant was carried out a decade ago: ‘There are no biological barriers to xenotransplantation, just a series of small steps.’
Others are more measured in their analysis. It is not just a question of immunology, says Calne. In addition to looking like a ‘friend’ to the immune system, a transplanted organ must function properly too. ‘Even proteins produced by close species such as the baboon are different from their human counterparts,’ he notes despondently.
Even when it comes to immunology, the science of xenografting is still desperately young. ‘Five years ago it had the status of alchemy,’ quips one surgeon. It could take researchers years to hit on exactly the right combination of drugs, antibodies and transgenic donors to reach their final goal of producing complete tolerance.
A key worry is whether blocking the complement cascade will be enough to prevent hyperacute rejection of pig organs in humans. The complement cascade is certainly important: animals born with genetic defects in complement genes are unusually tolerant to xenografts. But it might prove to be just the first of many hurdles. There is increasing evidence, for instance, that antibodies also contribute to hyperacute rejection. Everyone seems to carry antibodies in their blood that can attack pig tissue, and over the past two years it has become clear that a main tar-get of these antibodies is a sugar molecule called gal(alpha-1,3)gal which is found on the surfaces of pig epithelial cells.
CELLS OF WRATH
Removing this sugary target could be vital to eliminating the hyperacute response, says Squinto. One approach would be to disable, or ‘knock out’, the pig gene that encodes one of the enzymes needed to tether the sugar to the surfaces of cells. Making trans-genic pigs of this kind is no easy task as it requires special manipulations of embryonic stem cells. But with Alexion and others now poised to try, there seems little doubt that ‘knock-out’ pigs will be among the donor animals of choice next century.
And if they are, genetic engineers won’t necessarily stop at removing the pig’s troublesome sugar molecule. For even if the threat of hyperacute rejection can be completely silenced that way, xenografts will still be subjected to the gentler wrath of T cells – just as human-to-human transplants are. In conventional surgery, hostile responses of T cells are suppressed with drugs. But with pig organs, those responses could turn out to be much stronger. In which case, say researchers, it may be necessary to identify exactly which pig molecules trigger responses in human T cells, and eliminate them with genetic engineering. 91É«ÇéƬing on the work still to be done, White says: ‘At the moment we’re making a Model T Ford. But everyone would like to make a Ferrari.’
But in the end, even a Ferrari-style donor may not be quite enough. To produce tolerance to animal organs, it may still be necessary to manipulate the patient’s immune system; to administer ‘friendly’ monoclonal antibodies that bind to antibodies that would otherwise attack pig tissue; even to transplant bone marrow tissue from a pig to the patient.
Bone marrow cells being the progenitors of all the body’s immune cells, a pig-to-human transplant of this kind could – in theory – produce a host immune system with a conveniently split personality: enough donor immune cells to induce specific tolerance to donor tissue; and enough host immune cells to sustain normal human immunity. Some see this as a recipe for chaos in the immune system. But this week in Washington DC Elliot Lebowitz and his colleagues at BioTransplant in Charles-town, Massachusetts, report what they claim are promising results on rodents and primates.
In one experiment, the researchers used the bone marrow approach to transplant kidneys between primates of different blood groups, an operation that can be as risky as xenografting. The animals have survived for over a year, says Lebowitz – and without needing to have their immune system suppressed with drugs. The key to success, he says, is to kill off ‘mature’ T cells in the recipient and remove troublesome antibodies before injecting the bone marrow cells and transplanting the donor tissue. That way, he adds, ‘a new immune system can be generated that accepts both the donor tissue ands self tissue’.
Lebowtitz is convinced the dream of producing tolerance to xenografts can become a reality. He is not alone. ‘It’s time again for xenotransplantation in the clinic,’ said Grundy uncompromisingly in Cambridge last autumn. An evangelist for the cause, Grundy likes to confront sceptics with a slide show of transplant heroes: a group of ‘xeno-goats’, alive and well despite their sheep hearts, and a grinning baboon called Max who survived 502 days with a heart from a rhesus monkey and a little help from immunosuppressive drugs. How long before this gallery includes the face of a human being, alive and well and living with a pig’s heart?
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Transplantation’s Catch 22
Is there a genuine need for animal organs? Couldn’t the organ shortage crisis be cured simply by persuading more people to carry donor cards, or (as is the custom in the US) to state a willingness to donate on driving licences?
Superficially, the statistics suggest it could be. The formal gap between supply and demand today could be closed if the tens of thousands of potential donor organs that go up in smoke each year ended up in organ banks. But that may never happen, say organ banks and transpant services. While more than two in three people in Britain and the US say they would be happy to donate their organs, at least 30 per cent of families deny consent after death and some even override the wishes expressed on donor cards or driving liciences.
But there may be bigger reasons for turning to animal donors. Even if the current shortage could be cured with more human donors, transplant surgery is caught in a Catch 22: the more it advances technically, the greater the number of patients who stand to benefit, and the more acute the organ shortage becomes. Between 1986 and 1993, the number of people awaiting a transplant in the US leapt threefold, while the number of donors inched up woefully from 3990 to 4549, according to figures from the US transplant agency UNOS, in Richmond, Virginia.
Moreover, legislation on motor cycle helmets and seat belts mean that fewer people are dying in road traffic accidents. In Britain, the proportion of donor organs from ‘RTAs’ fell by about 30 per cent between 1989 to 1993. The great paradox of organ transplantation – that it takes a death to save a life – leaves it vulnerable to measures that improve health care and safety in society as a whole.
Some surgeons and patient groups have long campaigned for ‘presumed consent’, wherein people are presumed to have given consent for organ donation unless they expressly say otherwise. Instead of opting in to organ donation with a donor card, you opt out with the equivalent of an anti-donor card. When Belgium began such a scheme in 1986, the number of organ donors increased markedly. But opponents of presumed consent are unimpressed.
‘It’s uncertain whether it was the legislation or the coincidental publicity that produced the increase in Belgium,’ says John Faber, President of the British Transplant Society. ‘Presumed consent might even adversely affect the number of donors. If surgeons ever acted against the wishes of a family, it would generate bad publicity and possibly a backlash. There could be accusations of coercion, of potential organ donors not being given the best possible treatment.’
In the US, meanwhile, some patient groups and hospitals are looking for other ways of encouraging consent. Despairing with the limitations of altruism, they have begun to consider paying ‘death fees’, amounting to two or three thousand dollars, to families of potential donors. That may produce more donors. But it will also add to medical costs that are already spiralling out of control.
The Transplant Center of the University of Pittsburg, world famous for transplanting livers, runs a fleet of aircraft and employs scores of star surgeons – and it all has to be paid for by private or state medical insurance. In 1990, Medicare paid out $10 million for liver transplants. This year it is expected to pay more than $120 million. The growing shortage of donors, say organ procurement agencies, means they have to spend more money chasing each organ just to maintain the supply as it now is.
But there are deeper concerns about death fees. As it now stands, buying human organs is against the law in the US just as it is in most countries. Any cash-for-organs policy is likely to foment intense fears of abuse – of terminally ill patients feeling pressured to sign away organs for the good of their relatives, of the ‘wallet biopsy’ determining who will or won’t get a new heart, of a black market trade blossoming in organs of undisclosed origins (such as the corpses of prisoners executed in distant dictatorships).