This Little Piggy Went to Transplant…
Xenotransplantation's Big, Uncertain Future
Transplant organs are a rare resource. Using animal donors could go a long way toward addressing a public health crisis--and spell big business for the companies that can make the technology work. But the path to success is fraught with scientific challenges, risk, and no small amount of controversy.
By Vicki Brower
April 27, 2000
In 1997, a pig named Sweetie Pie saved 17-year old Robert Pennington's life. More precisely, it bought the Garland, Texas, teenager a few crucial hours until a liver could be located to replace his dying one. Now 20 years old and healthy, two-and-a-half years ago Pennington was brought to Baylor University Medical Center, jaundiced and declining rapidly as his liver failed. With his only option a liver transplant, and none available at the time, transplant physician Dr. Marlon Levy tried a radical experiment. After getting Pennington's family's permission and the green light from the FDA, Levy sacrificed Sweetie Pie, a 15-week-old, 118-pound transgenic pig raised at Nextran (Princeton, NJ), and removed her liver. Marlon then perfused Pennington's blood through Sweetie Pie's liver and back into the teenager's body. The experiment worked. The pig's liver did what Pennington's own organ could no longer do: purify the blood for six-and-a-half hours until a liver donor was found.
The experiment demonstrated that the human immune system could safely be exposed to animal organs modified with human proteins, giving researchers a certain degree of confidence that using animals' organs as a bridge to solid organ transplantation could provide a solution to the dearth of human organs available for transplant.
"The excellent results of human-to-human transplantation over the past 40 years have also pointed to the possibility that animal-to-human transplantation might be doable," says Dr. Robert Michler, chairman and chief cardiothoracic and transplant surgeon at Ohio State University in Columbus. Nowadays, most transplanted human hearts last for about 10 years before they are rejected, and a recent report showed that the median time of survival of transplanted kidneys has nearly doubled from 11 to 19.5 years because of the development of better, more specific immunosuppressant drugs. The first successful kidney transplant took place only 46 years ago.
However, success with transplantation is counterbalanced by what Michler calls "its Achilles' heel--the epidemiology of transplantation." Each year, he explains, "there are 60,000 new patients in the US who need heart transplants, but only 2,000 are available." And organ availability is actually declining. In 1999, 42,415 Americans were waiting for a kidney transplant, whereas only 12,166 transplants were performed, and 2,295 died while waiting, according to UNOS (United Network for Organ Sharing, the non-profit group contracted by the federal government to manage organ donation and allocation). Supply and demand ratios are similar for other organs--as are the death rates those unbalanced ratios make inevitable.
Xenotransplantation--transplanting organs, cells, and tissues from animals to replace diseased or damaged human ones--is considered to be a potential, if imperfect, solution to the chronic shortage of human donations. For almost a century, doctors and scientists have viewed xenotransplantation as a challenge and a hope, but only in the past decade have strides been made in the science of fighting rejection to begin the make it a real possibility.
Big Need, Big Rewards
If successful, xenotransplantation could be very big business: according to a recent study by Frost & Sullivan, it could bring in $10 billion annually by the end of 2005. High demand for new organs and replacement tissues drew two pharmaceutical giants--Nextran, (owned by Baxter Healthcare of Deerfield, IL) and Imutran, (owned by Novartis of Basel, Switzerland)--into the race in the early 1990s. The companies are competing to develop the first transgenic pig organs that can be implanted within the human body without being rejected.
Numerous biotech companies have also jumped into the race to develop ex vivo "bridge" organs like Sweetie Pie's liver, as well as transgenic animals to serve as permanent solid organ donors and a source for implantable tissues, and drugs to prevent rejection. These companies include Advanced Cell Therapies (Worcester, MA), PPL Therapeutics, (Roslin, Scotland), Diacrin (Charlestown, MA), Genzyme (Cambridge, MA), Biotransplant (Charlestown, MA), and Alexion Pharmaceuticals (New Haven, CT), each with a web of alliances and variety of approaches that aim to deal with the rejection of donor tissue by the recipient's immune system. There are currently about a dozen xenotransplantation clinical trials ongoing, all involving either cell or tissue transplants or ex vivo perfusion experiments--no solid, implanted organs are being tested yet.
Part of the reason for this is that the scientific obstacles and risks for successful xenotransplantation are more formidable for solid organs and than they are for tissues and cells, according to Michler. There are four known types of rejection that typically occur in solid organ transplantation of any kind. The first is hyperacute rejection (HAR), which involves an immune response cascade that occurs within minutes after transplant. The second, acute vascular rejection, occurs within days to weeks after xenotransplantation, and is caused by antibodies attacking vascular tissue in the transplant. The third type, cell-mediated rejection, may happen from weeks to months after transplant, and involves T-cell activation. It may occur along with the fourth type, chronic rejection, which involves the ongoing attack of the foreign organ by the body's immune cells, the condition for which transplantation patients must take immunosuppressive drugs on a continuing basis. Xenotransplantation technology has not progressed enough to even tangle with these last two complications, says Michler, but it has made some progress against the first two.
"Hyperacute rejection is a major barrier that has largely been overcome by both Imutran's and Nextran's transgenic pigs," says Michler. "They are engineered to express human complement proteins, which are not recognized by the human recipient as 'foreign' and thus do not set off the complement cascade that leads to [immediate] rejection." Other companies, like Alexion, have also dealt with HAR by complement inhibition.
Solid animal organs will be the most difficult to replace because of the complex blood supply needed and ability of antibodies to target and attack blood vessels, according to Michler, referring to the second cause of rejection. There is as yet no clear solution to this problem, he adds. He thinks a greater short-term likelihood of success will be in the area of cell transplants--which include BioHybrid's (Shrewsbury, MA) artificial pancreas that sequesters porcine cells with membrane technology, and Genzyme's NeuroCell PD for Parkinson's disease, a porcine neural cell implant for which patients are given anti-rejection drugs (see sidebar).
Transplanted cells do not always require their own blood supply and therefore are less likely to elicit acute vascular rejection--although this remains a matter of controversy. Some cells may still express an alpha-gal surface sugar and thus cause essentially the same immune response, says Dr. Julia Greenstein, CSO of Biotransplant. PPL's David Ayares, director of R&D at the company's Blacksburg, VA operation, for example, believes that the obstacles for successful tissue and cell xenotransplantation are equally as formidable. Unlike other companies in the field, PPL has resolved not to move into human trials until it has resolved all four types of rejection.
Diversifying the Risk: A Business Strategy
It is telling that few companies are working solely in the area of xenotransplantation. Rather, most have extended their main technology platforms to include xenotransplantation, starting from developing immunosuppressive transplantation drugs, like Novartis, or devices for dialysis, like Baxter. Others, including Alexion and La Jolla Pharmaceuticals (La Jolla, CA), are developing drugs to treat autoimmune diseases or inflammation, which address the complement cascade also implicated in organ rejection. Their move into xenotransplantation is a natural extension of this technology. In other words, in this very experimental field, most companies working toward xenotransplantation are diversifying their risk by using their technology for other therapeutic areas.
BioTransplant is one example of this strategy. "Success in xenotransplantation is not a requirement for our success," says CEO Elliot Leibowitz, whose company is one of the more advanced and committed to the xenotransplantation field. He remarks that the company's AlloMune program, used to foster acceptance of an organ following standard transplant surgery, is an easier sell to investors than XenoMune, the same technology applied to animal organs. He attributes that in part to the perception of xenotransplantation as a high-risk pursuit, which Leibowitz believes will turn out to be illusory. BioTransplant funds its extensive xenotransplantation program with strong partnerships rather than relying on investors, who are less supportive of this area.
Here Come the Clones!
Also significant when considering these companies' future prospects and competitors is the overlap of their goals and technologies with companies developing stem cell therapies and nuclear transfer technology. For example, Geron BioMed, formed a year ago from the merger of Geron (Menlo Park, CA) and Roslin BioMed (Roslin, Scotland), is working with nuclear transfer, telomerase, and human embryonic stem cells with the goal of producing autologous replacement tissues and, eventually, solid organs that could obviate the need for xenotransplantation. That possibility is not on the foreseeable horizon, but mid-term, Geron BioMed plans use these same technologies for xenotransplantation, presumably by creating identical, engineered animals for organ harvest.
PPL, Genzyme and Infigen (DeForest, WI) are using transgenic technology and nuclear transfer to breed animals to produce human proteins in their milk for use as therapeutics, in addition to creating transgenic animals for xenotransplants. Other companies focusing solely on stem cells, such as Osiris (Baltimore, MD), Layton Biosciences (Sunnyvale, CA) and StemCell (Sunnyvale, CA) hope to develop tissues and organs from human cells to entirely circumvent the need to use animals as donors.
Another interesting characteristic of the field is the "cross-fertilization" of technologies--meaning that many alliances and collaborations exist between competing companies, such as between BioTransplant and Novartis, and Infigen and Imutran, which could mean that as the technology matures, companies will consolidate.
Five Cloned Piglets Move Xenotransplantation Forward
In March, PPL made a long stride toward commercializing xenotransplant technology when it announced that it had successfully cloned the first pigs ever, from an adult cell, using a method different from the one employed to clone Dolly the sheep. Now able to produce identical transgenic pigs, PPL said it could reach the clinic with transplantable organs within four years. Pigs have been notoriously resistant to nuclear transfer due to a number of technical challenges: porcine ova are very fragile, and pregnancies must support numerous fetuses to result in any live births, says PPL's David Ayares. But pigs are very attractive as potential organ donors because many of their organs are similar in size, shape, and function to human organs.
PPL has also succeeded in producing porcine "knock-out" cells in which the alpha 1-3 gal transferase gene, which is responsible for producing a sugar that triggers hyperacute rejection in humans, is deactivated. The next step is to repeat the pig cloning experiment to produce knock-out cloned pigs.
The company also says that it has made progress in dealing with the three other known causes of transplant rejection, and that it will introduce three human genes into pigs to control more downstream causes of delayed xenograft rejection (a single-chain antibody gene against the porcine VCAM adhesion molecule, and two novel anticoagulant genes--TFPI and huridin). PPL plans to give potential xenotransplant patients a transfusion containing modified cells, taken from pigs that will supply the organs, which will "tolerize" the patient and thereby induce immune recognition without rejection. It is now combining its various strategies into one male and one female pig, and breeding from those. Advanced Cell Therapies (ACT) is also working toward cloning pigs, said vice president Robert Lanza, knocking out the alpha gal sugar molecule, and replacing it with human type O blood protein.
"We don't expect to be first in the clinic but we believe, because we are trying to address all forms of rejection, that our organs will be superior to others," says Ayares. PPL's goal is to greatly reduce the need for post-transplant immunosuppressive drugs to the same level as needed in human allografts.
Nextran is currently testing its ex vivo liver perfusion device in humans, and recently completed a Phase I trial. It hopes to file for an in vivo solid liver xenotransplant trial within the next 18 months, said Baxter spokesperson Deborah Spack. The company is also testing pig kidneys and hearts in baboons, and has reported that it has been able to keep pig hearts alive in baboons for 39 days. Imutran is knocking out the gene for decay accelerating factor (DAF) on human tissue to prevent complement activation.
Imutran says it has surpassed Nextran's organ survival time in non-human primates by an additional 60 days. BioTransplant has reported transplant survival time as long as one month in primates. "We are shooting for three to six months as an indicator that we could get long-term xenograft survival in man," says Biotransplant's Greenstein. The three- to six-month timeframe marks an important threshold, Greenstein explains, because it mirrors the low-end of the period that standard organ transplants are supported in small primate models. As such, transplant survival beyond this period indicates that long-term acceptance is possible.
Looking for a Little Tolerance
For its research program, BioTransplant is breeding mini-swine, which it believes are better matched in size to humans than regular-sized pigs. The company is in preclinical testing with its ImmunoCognance technology, designed to enable long-term acceptance of cells, tissues and organs by "re-educating" the patient's immune system to recognize donor tissue as "self" instead of "other," explains Greenstein.
This approach to creating tolerance in the recipient was pioneered by David Sachs at Massachusetts General Hospital, and is based on mixing elements of the donor's immune system with that of the recipient, to establish recognition of donor tissue as "self." This creates a "chimeric" or mixed immune system without the use of immunosuppressants. Using a chimeric system both in human non-matched transplants and xenotransplants, BioTransplant believes that this could reduce or even eliminate the need for life-long anti-rejection or immunosuppressive drug therapy. In its xenotransplantation program, BioTransplant is placing porcine bone marrow cells into primate recipients which, after bone marrow engraftment, should be recognized by the thymus as "self."
A second approach to creating tolerance in a recipient pioneered by Sachs in conjunction with BioTransplant involves transplanting an entire thymus onto a transplanted kidney capsule. Recently, his MGH team reported that they induced graft tolerance in thymectomized pigs transplanted with such a "thymokidney"-- a kidney with vascularized autologous thymic tissue under its capsule--despite a significant mismatch between donor and recipient in major histocompatability complex, the family of antigens that help distinguish foreign from autologous tissue.
La Jolla Pharmaceutical's xenotransplantation drug, LJP-920, addresses tolerance by a different means of immune modulation: decreasing the levels of antibodies to alpha gal and antibody-producing B cells. One of a number of "Toleragen" drugs, LJP-920 was recently reported to be effective in inducing B cell tolerance, shutting down the production of antibodies by B cells in mice. Those receiving the drug showed low levels of IgM and IgG alpha gal antibodies--the antibodies associated with HAR organ rejection--and fewer B cells producing antibodies to alpha gal than the control group. The drug also worked the same way in primates, said director of business development Andrew Wiseman.
PERVs and Problems
The issue of endogenous and exogenous pathogens in porcine donor animals, and whether they can cause infection in humans is far from settled, many experts believe. Last August, Novartis' Imutran published a landmark study in Science that concluded there was no evidence of porcine endogenous retrovirus, or PERV, infection in 160 patients previously treated with various porcine tissues, including 36 who had been immunosuppressed. Patients who had received implants up to 12 years earlier were tracked down, who include those receiving skin grafts for severe burns, pancreatic islet cells for diabetes, and patients whose blood had been perfused ex vivo through pig spleens, livers or kidneys. Three types of tests were conducted to determine whether patients had been infected by pig viruses: Western blot, PCR and retrovirus PCR.
Twenty-three patients did show evidence of circulating pig cells, or "microchimerism," but no infection. According to Novartis, "this finding demonstrates that pig tissue can survive in the human body with no ill effects." At the recent BIO International meeting in Boston, MA, Corinne Saville, Novartis COO, indicated that she considered the study to be conclusive. However, according to Jonathan Allen, a virologist at Southwest Foundation for Biomedical Research in San Antonio, TX, and member of the FDA Xenotransplantation subcommittee, the fact that there is no active infection now doesn't mean that there is no risk that the recipient could become actively infectious at some point in the future.
One strain of PERV is able to infect human cells in vitro, but there is no evidence of in vivo infectivity, he says. PPL's David Ayares explains that while all pig cells contain PERVs because they are incorporated into the pig's genome, the viruses have never been expressed or become functional in humans because they cannot reproduce. "An event of recombination would have to occur for infection to take place," Ayares said. Some companies, including PPL will try to delete certain conserved PERV genes from the pig genome to increase safety.
In contrast to the Novartis study, in 1998, Swedish researchers tracking 10 patients who had received fetal pig islets in the early 1990s reported antibodies reactive to swine influenza in all 10 patients, porcine parvovirus in five, and five other pig viruses. Only one was ill with parvovirus.
Another virologist and member of the xenotransplantation subcommittee, Dr. Prem Paul, associate vice provost of Iowa State University in Ames comments that while other infectious agents have been discovered more recently, including the Nipah virus found in Malaysian pigs, circoviruses, and hepatitis E, PERVs remain the main health concern and should stay at the top of the safety agenda--meaning that companies must continue research while testing its recipients and xenograft tissues. "The concern is that like with AIDS, a virus such as PERV could adapt to living in humans," says Dr. Paul.
Both Drs. Paul and Allen, who serve as consultants to companies as well as sit on the FDA subcommittee, believe that trials should continue, with caution. "Trials in xenotransplantation are not a 'yes-no' issue," says Allen. "They are a matter of risk-benefit ratio. I wouldn't want to be the one to deny a patient a last chance to live if his life depended on a xenotransplant." For patients with less life-death urgency, however, more caution and restraint may be in order. Allen agrees with Paul that "companies should be vigorously looking for infectious disease" in xenotransplantation experiments, and recommends more stringent testing.
Allen notes that FDA is responsive to public pressure and the political climate when it comes to xenotransplantation and other matters of an experimental nature. At a January subcommittee meeting, FDA focused on regulations concerning blood donation among xenotransplantation recipients, and there was a good deal of uncertainty and concern about how much risk may be involved for the general population, and for the recipients themselves. There was general agreement that xenotransplantation recipients should not donate blood, but not about how that information was to be disseminated and monitored.
Philip Noguchi, MD, director of the FDA's Center for Biologics Evaluation and Research (CBER), stated at BIO 2000 that the agency plans to intensify xenotransplantation trials' monitoring and oversight in light of the recent tragic death in September's gene therapy trial. Risks for patients in xenotransplantation trials are not unlike those for patients in gene therapy trials, and his agency's goal is to minimize those risks. Back in 1997, as evidence of the existence of PERVs emerged, the FDA placed all clinical trials on hold until it was satisfied with evidence that patients were not being infected. In January 1999, European parliamentarians called for a moratorium on xenotransplantation until matters of disease transfer were clarified.
Since 1997, the year after it issued its guidelines on xenotransplantation, the FDA has been working with other federal agencies--the Centers for Disease Control (CDC) and the National Institutes of Health (NIH)--grappling with issues of safety and oversight and revising those guidelines into a unified body of recommendations, says Dr. Louisa Chapman of the CDC and a member of the US Dept. of Health and Human Services (HHS) Working Group on xenotransplantation. In addition, in October 1999, HHS proposed setting up an advisory committtee, to be known as the Secretary's Advisory Committee on Xenotransplantation (SACX), which would oversee xenotransplantation activities the way the Recombinant DNA Advisory Committee (RAC) does.
Nominations for the committee have been made, and appointments for 15 group members are expected soon, says Chapman. Their mandate will be to review new and extant xenotransplantation protocols, address gaps in scientific knowledge, and provide a sounding board for the public's concerns about the ethical issues around xenotransplantation. The three agencies are also currently developing national patient and donor registry for monitoring and tissue archiving.
These efforts do not satisfy some, however. Alix Fano, head of the Campaign for Responsible Transplantation in New York City, an international coalition representing 86 public interests groups, believes the public health risks to society far outweigh any potential benefits. High on the list of objections is the unanswered questions about risks of infectious disease. "Patients who are asymptomatic today with pig cells circulating in their bodies may become infected in the future," she says. Over 25 diseases in pigs are known to infect humans, including the influenza virus of 1918, which resembled swine flu. And mutations of this are now being seen around the globe. "In 1997, Australian paramyxovirus infected piggery workers with flu-like symptoms," she adds. And, "there is no way to screen for viruses that are not yet known."