A turning point in xenotransplantation research

The donation shortage that affects many countries, where Switzerland has long nurtured hopes of finding alternative solutions to cure patients with heart, liver or kidney problems, dooming patients to heavy treatments or even certain death.

In the last ten years, research on xenotransplantation – the transplantation of an organ of animal origin into a human – has experienced very rapid progress thanks to the advent of new genetic technologies that allow modifying the genome of pigs to make them more compatible. advances who, last December, persuaded the US Medicines Agency (FDA) to urgently authorize a group of scientists in Baltimore to embark on an unprecedented and risky venture: transplanting a genetically modified pig heart into David Bennett, a man of 57 years old seriously ill, ineligible for human donation and convicted.

With his agreement, the man was transplanted on January 7, under the spotlight of the entire world. Two months later, he dies; his surgeons have been working ever since 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 ongoing investigation, published Wednesday in the journal New England Journal of Medicine. For the scientists, the transplant was a success, but several ‘hiccups’ contributed to the heart failure.

The organ implanted in David Bennett 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 human transplant purposes. With the 2012 discovery of CRISPR molecular scissors – winning the Nobel Prize in Chemistry for co-discoverers Emmanuelle Charpentier and Jennifer Doudna in 2020 – targeted DNA modification has taken a huge leap forward. It is now possible to efficiently and cheaply remove or add genetic sequences to the genome of cells.

This innovation allowed to relaunch research on xenotransplantation, hitherto delayed by rejection problems, despite the advent of immunosuppressants. Scientists in the field manipulated the genome of pigs – animals favored 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 Freiburg Cantonal Hospital and of 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 clotting processes on the surface of blood vessels, otherwise there is a risk during xenotransplantation.” The animal ends up becoming a little less pig and a little more human.

The Revivicor company’s specialist farm has provided surgeons in Baltimore with the heart of a pig carrying 10 genetic manipulations. Cloned, selected, separated from its mother and congeners over two generations, this animal should be clean, ie virus free, 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 known virus called porcine cytomegalovirus (CMV) in blood samples taken from David Bennett, who was doing very well. On the 50th day after the transplant, his condition deteriorated rapidly despite the battery of treatments he received, including antivirals. Your heart is failing, blood circulation and oxygenation 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 doubled in size. But no typical signs of rejection were observed, according to the American team. The presence of the swine virus in the patient’s heart and blood may have played a role in heart failure. “The detection of swine CMV was unexpected given the rearing conditions, the negative pre-transplant PCR test, and the use of antivirals for prophylaxis, the report authors write. 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 xenotransplantation of organs from pigs into primates,” recalls Nicolas Müller, a specialist in immunology in human transplants at the University of Zurich. Immunosuppression in primates reactivated swine viruses that were dormant and the infection led to transplant failure due to a clotting 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 enough because it can be negative and still carry the virus. It would be necessary to carry out a serology to look for antibodies against CMV in the blood, but these tests are not developed for the pig. Everyone underestimated the difficulty of having a ‘clean’ pig”.

In addition to the swine virus, another element may have compromised the experiment. American doctors reacted quickly, perhaps too quickly, after the virus was detected in the blood. To fight 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 carried out by the University of Maryland. “This is precisely what we try to avoid in the case of a xenograft, mainly 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 to heart maintenance.

a mixed result

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

A rather mixed success and a decisive step forward for the xenotransplantation scientific community, which is also on an unprecedented path. “The heart was the first to arrive at the clinic in the United States and it was a good idea because the pre-clinical results in baboons were good,” confirms Eckhard Wolf, a specialist in xenotransplantation at Ludwig University. the first in December 2018 to keep monkeys transplanted with genetically modified pig hearts alive for nearly six months. These German scientists had already warned in 2020 about the deleterious effects of swine CMV that caused grafts to fail in two of their primates who died before the others. “Among the members of our association, we believe that the case of David Bennett could put the FDA in tension in the future”, comments Léo Bühler. In 2018, the agency expressed reluctance to test the heart and favored the kidney. This organ is not vital and if there is rejection, the patient can return to dialysis. The next order will certainly have a better chance of success if it is a kidney xenograft.”

Kidney transplant trials in people with brain death have multiplied in recent months with very promising results, obtained mainly by three teams in the United States. New York University surgeons announced in September 2021 that they had successfully operated on a Revicor pig kidney on two brain-dead patients. The kidney that produced the urine was ‘connected’ to the human body, that is, connected to its general circulation, for 54 days. A similar experiment was performed in January 2022 by surgeons at the University of Birmingham, Alabama, with a kidney held for 74 hours in a femoral connection and with no signs of rejection. The Baltimore team is also working on pork kidney xenotransplantation.

“The enthusiasm of the scientific community has continued to grow in recent years,” notes Raphaël Meier, a transplant surgeon and researcher at the University of Maryland in Baltimore. I remember a conference in Japan, 10 years ago, on xenotransplantation, the room was almost empty. At the last convention in Boston, between 300 and 400 people packed the room. Public perception is also positive. One of my patients, who cannot receive a human kidney transplant and is sentenced to multiple dialysis every week, told me, ‘whenever you want, I can apply for an animal kidney!’ There are few options for these patients, and few survive. Xenotransplantation always brings hope.” A hope tempered by obstacles that still need to be overcome.

Xenotransplantation timeline 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.
  • 1960: 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 rapidly depleting.
  • 1984: Baboon heart transplant by surgeon Leonard Bailey on 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 pig pancreas islets into humans.
  • 2012: Discovery of CRISPR-Cas9 molecular scissors by researchers Emmanuelle Charpentier and Jennifer Doudna. This revolutionary tool makes DNA modification easy.
  • 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: Genetically modified pig heart transplant from David Bennett in the United States. The patient dies two months after the operation.

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