The door to xenotransplantation has been further opened, and key technical issues such as xenotransplantation safety, functional consistency, and immune rejection have been solved.
Written by | Yan Xiaoliu
Source | "Medical Community" public account
Recently, the world's first pig heart transplant was successfully carried out, which caused a sensation in the medical community.
On January 12, local time, the patient, 57-year-old David Bennett, successfully removed the extracorporeal membrane oxygenation device (ECMO).
Due to terminal heart failure, he was treated at the University of Maryland Medical Center (UMMC) and had previously survived for 45 days. (For more information, please click "World First Case!) Transplanting pig hearts to patients, it was finally done")
According to reports, since the transplant surgery was completed on January 7, Bennett is in stable condition, has no ultra-acute immune rejection, and can now breathe and speak quietly on his own.
"There is a lot of discussion about this in China, but there are not many doctors familiar with xenotransplantation, and they think it is exciting and far from the clinic." Wang Wei, chief expert of the Third Xiangya Hospital of Central South University (hereinafter referred to as the Third Xiangya Hospital) and director of the Hunan Xenotransplantation Engineering Technology Research Center, told the "medical community" that the UMMC incident represents that the door of xenotransplantation has been further opened, and key technical issues such as xenotransplantation safety, functional consistency and immune rejection have been solved. All that remains is to test the medium- and long-term effects.
Photo caption: David Bennett's son (center), left, visits his father at the hospital on January 12, local time. /University of Maryland Medical Center
xenotransplantation,
When can I say "success"?
Over the past few decades, the global medical community has been exploring the possibility of xenotransplantation, including the transplantation of animal organs, tissues, cells, etc. into the human body.
Islets are the first successful xenografts. Wang Wei told the "medical community" that unlike the heart, kidneys and other substantial organs, there is no ultra-acute rejection after xenopanic islet transplantation.
For most organs, ultraacute rejection is a natural defense mechanism for species to maintain germline continuation.
For example, the vascular endothelial cells of pigs have a glycoprotein molecule called alpha-gal. Primate organisms, including humans, have an antibody that attacks the glycoprotein molecule, forming blood clots widely, and transplanted organs quickly lose function and die.
Studies have shown that hyperacute rejection generally occurs within 48 hours of surgery. In the past, the academic community has studied some drugs and tried to control and deal with this situation without success.
In this pig heart transplant, no ultra-acute immune response occurred nearly 1 week after the operation. Wang Wei believes that it shows that we may have effectively swept away the huge barrier of xenotransplantation.
Proven gene-editing techniques are key. "We can knock out specific genes so that the lining of the pigs' blood vessels does not produce alpha-gal glycoprotein molecules."
At the end of the last century, gene editing theory took shape. Since then, the scientific community has done a lot of animal experiments, including the National Institutes of Health researchers who anastomated the heart of a genetically modified pig with an aortic branch of the experimental baboon. This "partially connected" heart survives for more than 900 days.
In animal experiments with in situ heart transplantation, the hearts of transgenic pigs can completely replace the baboon hearts and survive for nearly 1 year.
Kidney transplantation in genetically modified pigs also achieved good results. In October 2021, the Transplant Institute of New York University Langone Medical Center transplanted a genetically modified pig kidney to a brain-dead patient, and no ultraacute rejection was observed for the next 54 hours, and normal urinary function was demonstrated.
This time the pig heart transplant, standing on the shoulders of the predecessors, took another step forward: the recipient is a conscious person.
Patients know that xenotransplantation is the only hope of survival, and the surgery is also granted an "emergency authorization" from the U.S. Food and Drug Administration (FDA) based on the provisions of expanded access (compassionate use).
Arthur L. Caplan, chair of the department of bioethics at New York University's Langone Medical Center, said in an interview that the results remain uncertain. "In the coming weeks, Bennett is still at risk of developing immune rejection and viral infections."
Wang Wei said that judging whether the operation of xenotransplantation to a human is "successful" depends on two major indicators.
First, on the basis of overcoming ultra-acute rejection, whether it is possible to control the acute rejection of acquired immunity (including T cell-mediated acute rejection) and chronic rejection to achieve long-term survival. This can occur within weeks, months, or years after a clinical transplant, or lead to necrosis of the transplanted organ.
Second, test whether the pig heart can completely replace the human heart in function and withstand the daily activities of the human body.
Observing whether the human nerve-humor regulation system can regain control of the transplanted heart is one of the secondary indicators of evaluation. Under the action of human nerve-humoral regulators, the heart rate increases during exercise and agitation, and the heart rate slows down when sleeping. However, the transplanted heart is controlled by the myocardium autonomously and will deviate from the above regulatory system to some extent.
According to the International Steering Committee on Xenotransplantation, the goal of a xenotransplantation clinical trial is to "permanently replace the original organ, and more than 50% of the recipients must survive longer than 6 months"
In this regard, the heart xenotransplantation technology has made a revolutionary breakthrough, but it is still on the road to maturity, and whether it can further benefit all sentient beings still needs to be observed and studied.
Photo caption: Bennett (3rd from left) with his family, infographic. /AP
The cross-species infection barrier has been resolved
In China, on the road of xenotransplantation research, Wang Wei has been moving forward for more than 20 years.
China is one of the first countries in the world to successfully carry out human transplantation of pig islet cells for the treatment of diabetes. As early as 1999, Wang Wei and the team of Xiangya Third Hospital were approved by the former Ministry of Health to start the Phase I clinical study of human transplantation of pig islets. They used 6 years to do 22 cases of type 1 diabetes patients, confirmed the safety of pig islet transplantation into the human body, and also saw a certain effect.
In 2008, the "First Global Symposium on Clinical Research Specifications for Xenotransplantation" led by the World Health Organization (WHO) was held in Changsha, China. Taking the achievements of Wang Wei's team as a reference, the meeting formulated and released the changsha declaration, the world's first programmatic document for xenotransplantation.
Four years later, in accordance with the "Changsha Declaration" and the relevant regulations of the former Ministry of Health of China, Wang Wei's team established a special "pig without designated pathogen (DPF pig)" for xenotransplantation, and launched phase II clinical trials, involving 10 cases of type 1 diabetes patients. The results showed that compared with the transplanted pig islets, the overall insulin use of the patients was reduced by more than 50% on average, and the best effect was reduced by 88%.
Xenotransplantation uses pigs as donors, stemming from the WHO norm, "Pigs are currently the only xenotransplanted donor animals that allow clinical research... Clinical studies must use DPF pigs as donors."
On the one hand, this is because the physiological tissues of organs such as pig hearts, kidneys, lungs, islets, and corneas are similar to those of humans.
Zhou Mi, deputy chief physician of the Department of Cardiac Surgery at Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, told the "medical community" that his team had done a lot of surgery with pigs as experimental objects.
"The anatomy of the pig heart and the human heart is basically the same, only the angle is different, the human heart is on the left side, the tip of the heart is facing the lower left, and the pig heart is directly behind the sternum and the tip of the heart is down. Under the same weight conditions, the size of the pig heart and the human heart are also similar, and the kinetics of the two are similar. If the pig heart matches the human body, there is no special technical obstacle. Even if the size of the anastomosis port is slightly mismatched, this is common in allogeneic heart transplants (that is, heart transplants in the usual sense), so surgery has some suturing techniques that are enough to trim the donor heart to a 'match'. ”
Zhou Mi pointed out that from the existing allogeneic heart transplantation, the one-year survival rate of transplant patients is about 88%-90%, and the 10-year survival rate is about 70%. In the past, the life expectancy of pigs was artificially interfered with, but the "strong pigs" in Sichuan lived to be teenagers. Therefore, the age of use of transplanted pig hearts may be similar to that of human-derived organs.
On the other hand, a large number of previous studies have proved that there are relatively few cross-transmitted diseases between pigs and humans. For example, pigs are not infected with human hepatitis virus, avian influenza virus, HIV virus (AIDS) and other diseases, nor will they have high-risk infectious diseases such as Ebola virus infection. This improves the biosecurity of xenotransplantation.
Less does not mean nothing. There are still a variety of potential risks of exogenous and endogenous infections.
In 2005, for biosecurity reasons, the WHO said it would stop all clinical trials of xenotransplantation until a solution was found.
Breeding DPF pigs through a high barrier environment has become an effective means to exclude exogenous infectious factors. China offers successful technologies and examples in this area. At the end of 2012, Changsha, China, built the first medical grade (DPF) donor pig breeding center in Asia and the second in the world.
China's relevant efforts have also been endorsed by the WHO and the International Association for Xenotransplantation (IXA) and have been included in the international Xenotransplantation Clinical Research Specification "The 2018 Changsha Communique".
The risk of endogenous infection lies in the fact that there are some normally present porcine endogenous retroviral (PERV-C) sequences in the pig genome. This does not express toxicity in pigs. But when pig cells come into contact with human cells, the PERV-C sequence runs the risk of being transferred or exchanged into the human genome.
The solution that Wang Wei thought of was the "screening method". He firmly believes that there must be pig breeds without PERV-C in the body, but they need to be deliberately searched for.
The team has visited more than a dozen provinces and remote villages to screen more than 10,000 samples of dozens of types of pigs. Eventually, the team anchored a pig breed and bred it in inbreed to produce a pig breed with a stable genetic phenotype and complete perv-C deletion. It was named Xeno-1.
The team then bred pigs without designated pathogens (DPF) according to the Changsha Declaration. The whole growth process does not require antibiotics, no vaccinations, and relies on air, water, diet, etc. to form a protective barrier and strictly cut off all sources of infection.
Compared with Wang Wei, the cultivation methods in Europe and the United States are more "fashionable". On September 21, 2020, Nature Biomedical Engineering published an article introducing the use of CRISPR-Cas9 gene editing technology to breed "Pig 3.0".
"Pig 3.0" has 13 independent genes modified, including knocking out genes and solving the problem of immunity and coagulation incompatibilities between pigs and humans. At the same time, the potential risk of virus transmission is eliminated by genome-wide inactivation and removal of PERV in the genome.
The achievements and concerns of gene editing
Wang Wei once admitted that there are too many genetic modification points in "Pig 3.0".
At the 2018 WHO meeting, world experts agreed that if you want to use gene editing technology to modify the source of xenotransplantation, it is safer to control the modification gene points in the range of as few as possible.
"Before the lack of sufficient evidence of biosafety, the upper limit of gene editing should not exceed 15, and safety should be placed before effectiveness." Wang Wei explains.
On Bennett's transplanted pig heart, the researchers completed 10 gene edits. Knock out 3 genes in the pig that cause ultra-acute rejection in the human body, and transfer to 6 genes that help the human immune system accept pig organs. In order to prevent pig heart overgrowth, 1 growth gene is also knocked out.
Wang Wei said that it is not clear that the specific modified gene loci may include genes that promote the secretion of immunomodulatory proteins in pigs' hearts and block the activation of T cells.
After surgery, Bennett still needs to use anti-rejection drugs. "If through gene editing, no anti-rejection drugs are used at all, it is equivalent to having an anti-rejection drug production workshop in the human body." In the event of changes in the body and immunosuppression becoming a health problem, the body's own anti-immune rejection protein secretion function will be harmful. Wang Wei explained.
Too many genes can also introduce new risks. One of them comes from the "off-target effect" of CRISPR-Cas9 technology, which is commonly understood as "cutting in the wrong place". The more edits, or the more errors.
This can disrupt the basic function of the cell, leading to 3 outcomes. The best case scenario is that the cells die and are no longer inherited. The second is to cause severe malformations, or genetic phenotypic differences, so that breeding pigs will be eliminated.
The academic community is most worried about the third condition: inducing malignant tumors. Therefore, gene-edited pigs must undergo multi-generational reproduction to count individual and population genetic phenotype differences and assess whether tumor incidence is higher than that of surrounding populations.
At present, Wang Wei's genetically modified pigs have been bred to the fourth generation, with a total of more than 200 heads. Second-generation sequencing of more than 100 heads, the population genotype is very stable, there is no malignant tumor gene problem. The oldest pigs are 8 years old (equivalent to about 40 years old in humans) and have not shown tumorigenesis.
Photo caption: In 2012, Changsha, China, built the first medical grade (DPF) donor pig breeding center in Asia and the second in the world. /Huasheng Online
Ethical thresholds are not difficult
Currently, global human organ donors can only meet about 10% of transplant demand.
"With such a groundbreaking operation as [Bennett], I can almost imagine the ethical controversy it could cause. But I think that once the technical barriers to xenotransplantation are completely overcome, the ethical resistance will definitely be washed away, and how many people will choose to die in the face of the choice of undoubted death and having a pig heart? Li Qingchen, a surgeon and author of "The Legend outside the Heart", wrote an article.
In Wang Wei's view, the "lowest level" representing the patient and the "top layer" representing the competent department have all received xenotransplantation. "In the 10 years from 2008 to 2018, the WHO held three workshops on clinical research specifications for xenotransplantation, and continuously promulgated and updated international standards. The issues raised in the past have been largely implemented and conclusions have been drawn. ”
The WHO attaches great importance to and promotes xenotransplantation, mainly due to the current shortage of donors worldwide, and xenotransplantation is considered to be a possible solution to donor shortages.
On June 11, 2020, at the fourth "China Organ Donation" theme activity, former Minister of Health Huang Jiefu said that there are about 300,000 patients suffering from organ transplantation in China every year, and the number of organ transplants in the same period is only about 20,000 cases, "the gap is very large".
Another important reason is "quality control". Human donors have shortcomings such as uncontrollable organ quality, and biosecurity is difficult to guarantee. Organ donors vary in age and physical condition, and some may have chronic diseases or have certain latent viral infections. These all pose a greater safety risk to patients undergoing organ transplants.
In contrast, the advantages of xenotransplantation are obvious. Through industrialization, standardized breeding, strict diet control and environmental management, and cutting off the transmission route of various infectious diseases, high-quality and relatively sufficient donor organs can be obtained.