Weighing the Risks and Benefits of Xenotransplantion

By Jason Socrates Bardi

If thou art privy to thy country's fate, Which, happily, foreknowing may avoid, O, speak!

——Shakespeare, Hamlet, Prince of Denmark, 1600

The patient is put under general anesthesia for the duration of this major operation.

When he was a teenager, this patient developed Type 1 ("insulin dependent") diabetes—one of the most prevalent chronic diseases among children in the United States. For reasons that are not completely understood, his immune system began destroying a certain type of cell in his pancreas called "islets." These islets are the body's only source of insulin, a protein responsible for regulating blood glucose levels. Without the islets, the insulin disappears from the bloodstream, and without insulin, the glucose in the bloodstream increases and is maintained at levels much higher than normal, causing damage to the body's organs.

For his entire adult life, he has injected insulin every day. Now he is getting a more advanced treatment—one necessitated by years of damage to his internal organs. He is getting a new, healthy pancreas from a human donor.

A team of surgeons work several hours to place the pancreas in the abdomen, connecting it to the major artery and major vein that bring it blood, and to the intestine.

Some 1,500 to 1,800 pancreatic transplants are performed every year in the United States, and most are successful. Seventy-five percent of the grafts are viable five years after the operation.

The operation is a significant scientific and medical breakthrough, because when it is over, the patient's new pancreas begins producing insulin and monitoring the body's blood sugar automatically. But to fully recover from this major procedure, a patient like the fictional one portrayed here usually must take at least two weeks in the hospital and another three months at home.

The Islets Have It?

A much less invasive and safer alternative involves injecting "islet" cells isolated from the pancreas into the patient.

In this experimental procedure, a team of doctors removes the pancreas from the donor and insufflates it with collagenase, a proteolytic enzyme that breaks down the collagen in the tissue. They then supervise a controlled digestion of the pancreas that releases the islets, after which the islets are purified using a specially designed density gradient and a centrifugal cell separator device originally developed to purify bone marrow stem cells.

Then, says Daniel R. Salomon, associate professor in The Scripps Research Institute (TSRI) Department of Molecular and Experimental Medicine, you deliver them via a single injection into the liver. It is a much faster and less invasive procedure, and the patient's recovery time is quicker than in the whole organ transplant.

However, islet transplantation and whole pancreas transplantation are both limited by the severe shortage of available human donor organs—there are only about 5,000 available per year in the United States. And because of this shortage, the islet operation tends to be done much less than the whole organ tansplant. Islet transplantation has just not been around long enough for there to be sufficient data supporting the long-term survival and normal function of these cell transplants.

"What are you going to do if you have a pancreas available and a patient who has waited months to get it?" asks Salomon. "You are not going to use it on an experimental procedure."

Still, pancreatic islet transplantation has the potential to make a huge difference in the future because there are far more diabetes patients than there are pancreata and if pancreatic islets could be recovered from another source, many more patients could be treated.

Towards this end, some U.S. scientists are trying to find a way to grow and expand pancreatic islets in culture, for instance. Other groups are looking at the potential of using gene therapy to transfect insulin-producing genes into a patient's liver or intestine cells. Some are looking at the possibility of engineering islets from adult or fetal stem cells and then transplanting these into the patient.

These are tantalizing ideas, but they may never pan out, and their application is years away even if they do.

"Therapeutic gene delivery—don't even think about it for 10 years," says Salomon. "Human stem cells—even without the political barriers—don't even think about it for 10 or 15 years."

"The best short term bet for developing a clinically viable therapy appropriate for treating tens of thousands of patients in the next five to ten years," he adds, "is to use animals as the source of the islets. Pig insulin works very well in human patients and has been used for many years."

The Cases For and Against Xenotransplantation

The argument for using animal organs is a simple one—the alleviation of human suffering. Type 1 diabetes afflicts about 1.5 to 1.8 million Americans, and accounts for 30,000 newly diagnosed cases each year. But only about 5,000 whole pancreata are available for transplantation in a given year.

"It doesn't require a degree in rocket science to be able to do the math," Salomon says. "If you want something that's over the horizon to deal with the millions of diabetes victims, xenotransplantation is a good direction to go in."

Despite acknowledging the potential advantages of xenotransplantation, Salomon is also one of the leading voices in acknowledging its potential dangers. And his opinions are often sought. He is chair of the National Institutes of Health's Center for Research Resources Consortium for Clinical Islet Transplantation, chair of the Food and Drug Administration's Biological Response Advisory Committee, and member of the Secretary of Health's Advisory Committee for Xenotransplantation.

Basically, the dangers come down to risk of infection and risk of rejection.

As the recent case of three patients contracting West Nile virus from transplanted organs from a single donor dramatically demonstrates, the possibility of acquiring emerging infections via transplantation is very real. However, in xenotransplantation, this could be addressed with special precautions. Pigs could be raised away from pathogens, in sterile conditions, the way that smallpox vaccine used to be grown on the legs of cows in sterile settings.

The risk of rejection is also a very real danger, since immune systems have to be suppressed even when a patient receives an "allo" transplant (from another human). Tissues used for xenotransplants, like pig pancreata or islets, are even more likely to be rejected.

But germlines could be transgenically altered to remove genes that cause the tissue to be rejected. In fact, the primary molecule responsible for eliciting a human immune response against porcine tissue has been identified (a galactose sugar, which pig cells carry on their surface) and can be removed by knocking out a metabolic gene (the a-1,3-galactosyl transferase).

And one could clone this new breed of donor animal to ensure each organ was nearly identical.

And this has already been accomplished by the same Scottish company that cloned the sheep Dolly. A few years after Dolly, they announced their success in cloning five piglets from an adult sow.

Because of these advances, Salomon says, there was a lot of excitement about xenotransplantation about eight years ago, accompanied by a large infusion of dollars into research in the field.

"Many felt that [these advances] would change a xenotransplant into an allotransplant [a transplant from one person to another]," says Salomon. "I think that with continued engineering of the pigs, that this is quite possible. However, like many first approaches to major challenges in medicine, the hype was far ahead of the reality and there has been a very appropriate cooling of enthusiasm in the last three years."

However, even with super clean, cloned, and sugar-free cells, xenotransplantation is still a potential risk because of the danger of infection with what is known as porcine endogenous retroviruses (PERV).

Endogenous retroviruses, are unlike viruses like West Nile in that they do not cause disease in their animal hosts. They are the remnants of viral infections that long ago infected the species and long ago retired into dormancy. The hypothesis is that they are remnants of past epidemics where the viruses moved into the entire species, actually becoming part of the germ cell line. For instance, gibbon ape leukemia virus, a retrovirus endogenous to that species, was most likely introduced into the gibbon ape from Asian mouse populations. All mammalian species have these endogenous retroviruses.

"It is estimated that from three to eight percent of our genome is endogenous retroviral sequence," says Salomon. "It is not functional, but our genome is filled with these sequences."

Many of these are defective, but some of them can code for a complete and infectious viral particle. The fear with xenotransplantation is that the viral genes may come out of retirement.

"A group of us said, 'Wait a minute, what about the potential of [PERV] infections being introduced into humans by xenotransplantation and then spreading to the public at large?'" says Salomon. "We were not very popular at the time."

Around the same time, in 1997, Robin Weiss and his colleagues at Oxford reported to Nature that porcine endogenous retroviruses could infect human cells in vitro. That result sparked a great deal of debate and led some to call for a moratorium on xenotransplantation.

Salomon rejected those calls and instead advocated for proceeding with clinical trials, but cautiously and in carefully defined, highly controlled, and well monitored trials. And if there is any evidence of real and productive infection, he adds, then everything should be closed down. Xenotransplantation is a new field and with any new technology there are potential risks. If we close down new directions in medicine every time there is a theoretical risk identified than there will be little progress made.

"The bottom line is this: we need to advance our understanding of PERV to make an informed prediction of risk to the human patient and human population," he says. "[And] we need to assess the risk based on good science and not on dramatics."

A Hierarchy of Questions

Weiss's 1997 study demonstrated that a risk exists, but did not determine how much of a risk it is.

Would a pig cell transplanted into a human make a virus? If it made a virus, would the virus be transmitted to the human cells? If the transmission occurred, would the virus become productive or would it stay dormant? If the virus were productive, would it spread to other cells and organs? If the virus spread, would it cause disease in the person? If it caused disease, would it also be transmissible? If so, how? What would be the consequences to public health?

And even if one were able to answer all these questions, determining how to shape policy based on them may not be so straightforward. If, for instance, a person's xenotransplant will cause disease, he/she can still make an informed consent to receive it. After all, having the transplant and living with the risk of disease is better than not having it and dying with no disease. However, if there is also a risk that the transplant recipient could infect others, then the solution is not so straightforward. One cannot consent to placing someone else at risk.

"It is the transition from individual risk to public health risk where the real controversy comes," says Salomon.

Salomon has spent the last several years trying to work out some of the science-based answers to this hierarchy of risk questions.

Productive Infection

Two years ago Salomon and his colleagues demonstrated that pig islets make infectious virus and infect mouse and human cells that have been implanted into in vivo models. In other words, when scientists transplanted pig islets under conditions similar to what might be seen in the clinic, they saw PERV replicate and infect other cells.

"You have to face the fact that there is virus coming out of that transplanted pig tissue and that [the virus] is replication competent and infectious," he says.

He recently cloned, with investigator Clive Patience at Immerge Biotherapeutics and investigator Robin Weiss and his colleagues at Oxford, two previously unknown genes that encode human receptors of PERV. They are in the process of transfecting the receptors into the germline of mice and demonstrating whether they are able to get productive infection.

"If it works, we will have a new model," he says. This will allow them to study the pathology of PERV in living tissue—since in pigs, the viruses cause no disease.

Even before they finish these models they have already demonstrated that the human receptor works well when transfected into mouse cells. They are currently defining the functionality of the receptors, expression in different human tissues and studying their binding mechanisms in order to ascertain what is happening.

This is all simply establishing whether the possibility exists for productive infection, which is an important issue because the family of retroviruses to which PERV belongs are all leukemia viruses—diseases that cause hematological malignancies.

"We're still working our way through the hierarchy," cautions Salomon. "We haven't talked about disease yet and we haven't talked about potential public health risks. But our understanding of how PERV enters cells and produces productive infection has increased significantly."


But patients are not waiting for these answers. Shortly before agreeing to an interview, Salomon had established contact with a doctor in Mexico who has already transplanted pig islets obtained from a company in New Zealand into 12 adolescents and is planning to expand his trials to include 24 more.

Would people go to Mexico or some other country for the transplant and then return to the United States or elsewhere the procedure is not yet approved?

There is already a booming black market in organ transplants, which Salomon has witnessed first-hand in the clinic. Unlike transplant tourism, though, what Salomon has called "xenotourism" is a far greater concern because of the unanswered questions about possible dangers of disease and infection caused by PERV and other animal pathogens that might be introduced into humans especially in uncontrolled trials."

"It is in no way exaggerated to raise the concern," Salomon says. In fact, he is now working with the staff of the U.S. Secretary of Health to formulate a plan to disseminate basic information on the potential danger of xenotourism and, hopefully, discourage U.S. citizens from participating in such foreign adventures.



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Investigator Daniel Salomon acknowledges both the potential advantages and potential dangers of using pig pancreata to treat human diabetes patients. Photo by Mark Dastrup.






















A confocal micrograph (630X) of SIRC cells expressing a C-terminal EGFP-tagged PERV receptor. Expression of this engineered fluorescent receptor is one tool being used in experiments to study the cellular distribution of receptor before and after exposure to infectious virus.