A turning point in xenotransplantation research

The shortage of donations which affects many countries, where Switzerland has long nurtured the hope of finding alternative solutions to cure patients whose heart, liver or kidneys malfunction, condemning the sick to heavy treatment or even certain death.

Over the past ten years, research on xenotransplantation – the transplant of an organ of animal origin into a human – has experienced very rapid progress thanks to the advent of new genetic technologies making it possible to modify the genome of pigs to make them more compatible. Advances who, last December, convinced the United States Medicines Agency (FDA) to urgently authorize a group of scientists in Baltimore to embark on a risky and unprecedented enterprise: to transplant a genetically modified pig’s heart into David Bennet, a 57-year-old seriously ill patient, ineligible for human donation and sentenced.

With his agreement, the man was transplanted on January 7, in the spotlight of the whole world. Two months later, he dies; his surgeons have since worked to understand the causes of his death. Is it the graft that didn’t take? Did other factors come into play? The answer is complex, according to the first results of this still ongoing investigation, published on Wednesday in the journal New England Journal of Medicine. For scientists, the transplant was a success but several ‘hiccups’ contributed to heart failure.

The organ implanted in David Bennet did not come from any pig, but from an animal “manufactured” by the American company Revivicor, which specializes in the production of genetically modified pigs for the purpose of transplants into humans. With the discovery in 2012 of the CRISPR molecular scissors – earning the Nobel Prize in Chemistry for co-discoverers Emmanuelle Charpentier and Jennifer Doudna in 2020 – targeted DNA modification took a huge leap forward. It is now possible to efficiently and inexpensively remove or add genetic sequences in the genome of cells.

This innovation has made it possible to relaunch research on xenotransplantation, hitherto slowed down by problems of rejection despite the advent of immunosuppressants. Scientists in the field have manipulated the genome of pigs – favorite animals for their physiological similarity to us – to remove porcine genes recognized by the immune system while adding other genes that promote graft acceptance.

“We have to turn off the genes of different sugars present in pigs against which there are natural antibodies in humans,” explains Léo Bühler, professor of surgery at the Cantonal Hospital of Friborg and the Hirslanden group, member and former president of the International Xenotransplantation Association, which did not participate in the American experiment. We must also introduce human genes, transgenes, to modulate, among other things, the processes of coagulation on the surface of blood vessels, otherwise there is a risk during xenotransplantation.” The animal is ultimately made a little less pig and a little more human.

The specialized farm of the Revivicor company provided surgeons in Baltimore with the heart of a pig carrying 10 genetic manipulations. Cloned, selected, separated from its mother and its congeners over two generations, this animal was supposed to be clean, that is to say without virus, nasal PCR test in support. But the precautions were insufficient. On the 20th day after the transplant, analyzes revealed the presence of DNA from a well-known virus called porcine cytomegalovirus (CMV), in blood samples taken from David Bennett, who was doing quite well. On the 50th day after the transplant, his condition deteriorated rapidly despite the battery of treatments he received, including antivirals. His heart is failing, circulation and oxygenation of the blood must be supplemented by an extracorporeal machine. He died on March 8.

A heart twice as big and a virus

Cardiac ultrasound on days 19 and 49 revealed thickening of the lining of the ventricles of the heart, and analysis of cardiac tissue after biopsy showed deterioration after day 50 with approximately 40% necrosis of cardiac muscle cells. When David Bennett died, the heart had doubled in size. But no typical signs of a rejection were observed, according to the American team. The presence of the porcine virus in the patient’s heart and blood may have played a role in the heart failure. “The detection of porcine CMV was unexpected given the rearing conditions, the negative PCR test before transplantation and the use of antivirals for prophylaxis, write the authors of the report. Further testing is ongoing as human herpes virus 6 was found in the patient’s lungs and may have reacted with CMV.”

This virus problem should have been avoided. “This case reminds me of what we demonstrated in the 2000s, when we studied the xenotransplantation of pig organs in primates, recalls Nicolas Müller, specialist in immunology in human transplantation at the University of Zürich. Immunosuppression in primates had reactivated porcine viruses that were latent and the infection had led to the failure of the transplant due to a coagulation problem causing bleeding. According to the specialist, the preventive tests carried out on the pig were not complete. “The search for viral DNA in a nasal sample by PCR, in a healthy animal is not sufficient because it can be negative and still carry the virus. It would be necessary to do a serology for the search for antibodies against CMV in the blood, but these tests are not developed for the pig. Everyone has underestimated the difficulty of having a ‘clean’ pig”.

In addition to the porcine virus, another element could have compromised the experiment. American doctors reacted quickly, perhaps too quickly, after the virus was detected in the blood. To combat a potential viral infection, they gave David Bennett two doses of broad-spectrum human antibodies, some of which can recognize and neutralize porcine proteins, according to tests performed by the University of Maryland. “This is precisely what we try to prevent in the case of a xenograft, in particular with genetic modifications so that the immune system does not turn against the animal graft, comments Léo Bühler. This could have been problematic and counterproductive for the maintenance of the heart.

A mixed result

In the end, the death of David Bennett would have been caused by diastolic heart failure (inability of the heart to relax after contraction and to fill with blood), the origin of which is still unexplained, according to American scientists. Probably the poor general state of health of the patient at his treatment, the presence of the porcine virus, the administration of very concentrated antibodies, played a role in the final heart failure. “We consider this experiment a success and that we have learned a lot,” concluded Muhammad Mohiuddin, director of the cardiac xenotransplantation program at the University of Maryland and co-author of the report, before the participants at the American Transplant Congress, who is held in early June in Boston.

A rather mixed success and a decisive step for the xenotransplantation scientific community, which is also on an unprecedented path. “The heart was the first to reach the clinic in the United States, and it was a good idea because the preclinical results in baboons were good,” confirms Eckhard Wolf, a xenotransplantation specialist at Ludwig University. Maximilian from Munich, whose team had been the premiere in December 2018 to keep alive monkeys transplanted with genetically modified pig hearts for almost six months. These German scientists had already warned in 2020 the deleterious effects of porcine CMV which had caused the grafts to fail in two of their dead primates before the others. “Among the members of our association, we think that the case of David Bennett could tense the FDA in the future, comments Léo Bühler. In 2018, the agency expressed reluctance for a trial with the heart and favored the kidney. This organ is not vital and if there is rejection, the patient can return to dialysis. The next request will surely have a better chance of succeeding if it is a kidney xenograft.”

Kidney transplant trials in brain dead people have multiplied in recent months with rather promising results, obtained mainly by three teams in the United States. New York University surgeons announced in September 2021 that they had successfully to operate a Revivicor pig kidney in two brain dead patients. The kidney that produced urine had been ‘attached’ to the human body, i.e. connected to its general circulation, for 54 days. A similar experiment was conducted in January 2022 by surgeons at the University of Birmingham in Alabama, with a kidney maintained for 74 hours in femoral connection and without signs of rejection. The Baltimore team is also working on pig kidney xenotransplantation.

“The enthusiasm of the scientific community has continued to grow in recent years, observes Raphaël Meier, transplant surgeon and researcher at the University of Maryland in Baltimore. I remember a congress in Japan, 10 years ago, on xenotransplantation, the room was almost empty. At the last convention in Boston, between 300 and 400 people filled the room. Public perception is also positive. One of my patients, who cannot receive a human kidney transplant and is condemned to several dialyses every week, said to me: ‘when you want, I sign up to receive an animal kidney!’. There are few options for these patients, and few survive. Xenotransplantation always brings hope.” A hope tempered by the obstacles that remain to be overcome.

Timeline of xenotransplantation in the 20th and 21st centuries

  • 1906: Pig kidney transplant in a patient by Lyon surgeons Matthieu Jaboulay and Alexis Carrel. Immediate immune rejection of the graft.
  • 1960s: Attempts at xenotransplantation with chimpanzee kidneys in patients, by surgeon Keith Reemtsma in the United States. Most grafts lasted between 4 and 8 weeks. One patient died nine months after the operation.
  • 1980s: With the discovery of immunosuppressants, human-to-human transplants take off. Organ donations are running out fast.
  • 1984: Baboon heart transplant by surgeon Leonard Bailey in Stephanie Fae Beauclair, a newborn with congenital heart syndrome. She died 21 days after the operation due to rejection, despite immunosuppressants.
  • 1993: First attempt to xenotransplant islets from pig pancreas into humans.
  • 2012: Discovery of CRISPR-Cas9 molecular scissors by researchers Emmanuelle Charpentier and Jennifer Doudna. This revolutionary tool makes it easier to modify DNA.
  • 2018: Transplantation of genetically modified pig hearts into baboons in Germany. Animals survive for up to six months after transplantation.
  • 2021: Genetically modified pig kidney transplants in brain-dead humans in the United States.
  • 2022: Transplant of a genetically modified pig heart at David Bennet in the United States. The patient dies two months after the operation.

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