Published On January 18, 2022
On January 10, a team of surgeons at the University of Maryland School of Medicine announced that, three days earlier, a pig heart had been successfully transplanted into a human. The patient was a 57-year-old man with heart failure who did not qualify for a human heart transplant. The surgery was hailed as a milestone, and some marked it as a first step toward the end of a chronic shortage in donor organs.
That event happened at the end of a very long scientific relay race. “There is this sort of aha moment right now, but people have been working on related studies for 20 to 30 years,” says Jay Fishman, the director of the Transplant Infectious Diseases and Compromised Host Program at Massachusetts General Hospital.
Making a pig organ that was safe for the human body and protected from the human immune system required a cascading series of advances in genetics and immunology, many of which unveiled new, knotty problems along the way. “The research efforts to unravel these have gradually brought our knowledge about the whole field of xenotransplantation”—the field of transplanting organs across species—”to the point where this kind of feat is possible,” says Fishman.
Here are four areas of discovery that led to the historic event:
Tweaking the Genes of Donor Animals
The biggest puzzle was also the oldest—why exactly human bodies might reject a heart or kidney from another species. Modern efforts to transfer animal organs into humans date from the 1960s, and the first heart transplant was performed in 1964 using a chimpanzee as the donor. The field saw some limited success using organs from baboons and chimpanzees before the Food and Drug Administration imposed a moratorium—in part because of the possibility of infectious diseases and in part because of ethical considerations.
In the 1990s, the field decided to use organs from pigs instead. One of the earliest and most formidable barriers was hyperacute rejection. When a pig organ is transplanted into a person or a baboon, it turns black and fills with blood clots within minutes or hours—“quite a discouraging and dramatic thing to see up close,” says Richard Pierson, cardiac surgeon and transplant immunologist at MGH. The same happens when pig organs are perfused outside the body with human blood.
Clues into the biology of hyperacute rejection soon led to important breakthroughs. A research team in Oklahoma led by David Cooper, now at MGH, identified the Gal alpha-1,3-galactose sugar as the main target of the anti-pig antibodies in human subjects. The sugar, commonly called Gal, is expressed on the surface of cells and blood vessels in pigs and many other animals. Humans lack this sugar. So after a transplant, the human immune system identifies the tissue as foreign and rallies defenses against it.
To counter this “preformed antibody” response, xenotransplant scientists used the early tools of genetic engineering. In 2002, one team at the University of Missouri, in partnership with scientists from MGH, and another team at PPL, the company that created Dolly the cloned sheep, announced nearly simultaneously that they had created pigs in which the enzyme encoding the Gal gene had been deleted, or “knocked out.” The Missouri team had also cloned four piglets with this altered genome. The tissues of these pigs, without the Gal sugar, reduced up to 85% of the antibody response and that proved, in various test systems, to significantly protect these organs from an immune response from the recipient.
In parallel, in the mid-1990s a team of scientists in Cambridge, England, used genetic engineering to add another gene—an anti-inflammatory “complement regulatory” gene—to otherwise normal pigs. Organs from these transgenic pigs survived for weeks or even months in monkey models. Later, that modification was incorporated with the Gal knockout gene alteration to create a line of “MGH miniswine”—a line of pigs, distinct from the Revivicor “10-gene” pig used by the Maryland team, that is currently being developed by a team led by MGH’s David Sachs, now based primarily at Columbia University.
The process of cloning to get pigs that express the same gene edits is still widely used. “Once you have a live pig that contains the desired genes, cloning can make many copies of that pig. You can also start the process with just living pig cells. Wait three months, three weeks, and three days”—the gestation period of a sow—”and, hopefully, you get a litter of identical pigs expressing the genes you want,” says Pierson.
Making Pig Organs More Human
But work with both the Gal knockout pigs and single-gene transgenic’ pigs created new obstacles. Monkeys and baboons were used as stand-ins for humans during the research process, and in them, the new pig organs that lacked the Gal gene weren’t usually viable for more than a few weeks.
The next major step was identifying that two additional sugars—Neu5Gc and beta4Gal—were also important targets of preformed anti-pig antibody. Fortunately, accurate and versatile CRISPR-Cas9 gene editing technology was becoming available in the mid-2010s. CRISPR-Cas9 enabled both creation of a “triple knockout” pig—one that essentially neutralized the preformed human anti-pig antibody response— and also allowed introduction of additional human “transgenes,” modifications that addressed other biological incompatibilities between humans and pigs. Some of these genes were related to blood clotting and inflammation.
In the pig-to-human transplant in Maryland, 10 genes in all were altered in the pig donor. The edits knocked out the three key sugars, added two human “transgenes” to inhibit blood clotting and four others to reduce inflammation. Each was intended to reduce a specific mechanism known to injure pig cells and organs. A final nudge reduces expression of a growth hormone receptor, with a goal of preventing the pig heart from growing too large for its human host. That same edit would be used for other pig donor organs, such as a kidney.
Pierson says understanding the genetic pathways involved in rejection was its own moonshot, a massive undertaking by teams around the world, and all of that work informed the decision to alter each of the genes in the Maryland pig. Pierson’s team, then at the University of Maryland, did many of the “proof of principle” experiments validating these gene choices. Their model used pig lungs perfused with human blood, which gave a picture of how well each gene modification was working.
“Sorting out the pathways has been slow and messy,” says Pierson. “Each pig with gene modifications gave us information not only about whether or not the problems we already thought were important were effectively addressed, but also frequently revealed new problems looming on the horizon.”
Eyes on a Lurking Threat
One potential problem lies in hiding in pigs’ DNA. All animals carry stretches of genetic material called endogenous retroviruses, which are likely left over from ancient encounters with contagious retroviruses. At some point in evolution these retroviruses lost the ability to reproduce and spread within the host species and, instead, got woven into the host’s DNA, where they are copied and passed along to offspring. This largely happens without obvious consequences to the host. Pig endogenous retroviruses (or “PERVs”) don’t appear to harm the pigs and mostly stay hidden in genetic material. In the 1990’s, however, scientists discovered circumstances under which PERV genes could recombine with each other to yield virus particles. Those, in turn, could infect human cells.
Xenotransplantation researchers and regulatory bodies worried that the PERVs might cause infections in humans. An infectious PERV might not only make the recipient sick, but also infect the recipient’s close contacts or even spread to other people, potentially causing a pandemic like HIV or SARS-CoV2.
In 2015, a team led by George Church at Harvard Medical School announced that it had knocked out all 62 known copies of the PERV genome using CRISPR-Cas9 technology. While the pig used in the Maryland case did not include PERV modifications, this development reinforced growing enthusiasm in the xenotransplant community that pig organ transplants could be made safe with respect to the PERV risks if PERV infections do occur. Meanwhile other groups, including an MGH team lead by Fishman, showed that antiretroviral drugs currently being used to treat HIV infections are quite effective against PERV, providing another margin of safety.
A Treatment Regimen for Recipients
Xenotransplant research teams knew that, despite efforts to make a genetically suitable organ, the human immune system would still put up a fight. Even transplant organs from other humans require a faithful regimen of immunosuppressive drugs, and heart transplant recipients still experience rejection rates of 10% to 20%. Improved techniques to treat these complications, however, and steady advances in immunosuppressant drugs have enabled much better long-term survival of both grafts and patients, according to Fishman.
One major advance has been a class of drugs that targets “costimulation”—a family of signaling pathways that immune cells use to communicate with each other. When one or more costimulation pathways are blocked, the cells that cause rejection don’t receive key secondary signals necessary to respond effectively. That mutes the rejection of the “foreign tissue” of the pig organ. These newer drugs target molecules in the costimulation communication pathway, including CD40, CD154 and CD28/B7.
Since the 1990s, transplant teams at MGH have pioneered the use of anti-CD154 and anti-CD40 in models of transplant surgery. A CD28/B7 costimulation pathway-blocking drug, Belatacept, was approved for clinical use in 2011, and it significantly helped people with kidney transplants become less reliant on conventional immunosuppressive drugs.
For pig-to-primate procedures, a blockade of the CD40/CD154 costimulation pathway helped to minimize the recipient’s immune response, according to work from a number of xenotransplant teams. Researchers expect the same to hold true for transplants from pigs to people. “It turns out that the CD40/CD154 pathway is the critical one in xeno,” says Pierson. In Maryland, the team around the human recipient is using a monoclonal antibody against CD40. This approach, according to previous work in primates who have received a pig organ, should be safe and effective.
These four advances positioned xenotransplantation to take the historic first step into a human trial. For their part, though, researchers are cautious not to declare victory yet. Although the recipient is reported to be recovering well, questions remain about the risks he faces. For instance, no one knows whether the heart’s genetic modifications will continue to protect it from biologic incompatibilities with the host. The anti-CD40 treatment regimen has also never before been used for a human transplant recipient, so the drugs’ ability to safely prevent immune injury to the pig heart remains uncertain.
The threat of pig-to-human infections may still pose a risk, for the patient and perhaps to others. “Some infection problem could still occur in the recipient of a pig organ that we simply don’t know about,” says Pierson. “That is one of the big hurdles that the Maryland group had to clear—to persuade the FDA that they had a surveillance mechanism in place to track all the close contacts of the patient as well as the patient himself,” he says. The group is actively banking blood samples from the patient, his family and his caregivers, to allow infectious disease experts like Fishman to track for new viral threats.
Meanwhile, the world is watching. If the procedure leads to an enduring success, it may open a new path forward. People in desperate need of a new heart or kidney may not have to endure the long waits they currently face for donor organs, and decades of patient research will pay dividends in countless saved lives.
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