Driving Pig Organ Xenotransplantation Towards Clinical Trials

Use of pig organs, or xenografts, is viewed as a promising near-term solution to the acute shortage of human organs. The current supply of human cells and organs is insufficient to meet the needs of an ever-growing list of individuals who could benefit from lifesaving transplants.

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Cutting-edge research at Massachusetts General Hospital is revolutionizing the field of xenotransplantation.

Addressing the Shortfall in Available Donor Organs

Gradual increases in life expectancy, driven by steady improvements in disease diagnosis and treatment, have translated into growing numbers of patients whose length and quality of life are limited by end-stage organ failure.

Organ transplantation has emerged over the past 80 years as the paradigm of effective treatment for acute or chronic kidney, heart, lung, and liver failure. However, for each of these organs, the imbalance between organ supply and demand significantly constrains the number of patients who can benefit.

As of December 2022, there were 105,235 individuals in the United States on the waiting list for an organ transplant, a number that has increased steadily since the 1970's. Only ~40,000 transplants were performed in 2022, and because of the organ donor shortage more than 6,000 patients on transplant waiting lists die annually before receiving a transplant.

'Xeno' is the Greek word for 'foreign.' 'Xenotransplantation' describes the transplantation of cells, tissues, or organs from a 'foreign,' non-human animal source to a human recipient.

Mass General investigators feel that the use of pig cells and organs represents the most promising near-term solution to the current shortfall in available human organs.

A recipient's response to a cell, organ, or tissue xenograft depends largely on the degree of phylogenetic dissimilarity between the donor and recipient species. For example, transplants between closely related species, such as from non-human primates (NHPs) to humans or foxes to dogs, are less likely to elicit a rapid and destructive immune response compared to those between more distantly related species. Moreover, in the case of transplants from pigs into humans, the presence of preexisting 'natural' antipig antibody in the blood of humans, coupled with other biologic incompatibilities, presents an especially formidable barrier to successful organ xenotransplantation.

The types of host rejection of transplanted tissue can be broadly represented in three categories:

• Hyperacute rejection (HAR) occurs within minutes to hours of transplantation and involves the rapid destruction of grafted tissue by the host immune system. This was seen historically in transplant recipients who had preformed antibodies in their blood that attacked and destroyed a transplant from another person. HAR almost always occurs when 'normal' unmodified pig organs are transplanted into humans
• Delayed xenograft rejection (DXR) describes a phenomenon that occurs days to weeks after xenotransplantation despite the use of conventional or experimental immunosuppression. While in some cases, DXR is attributable to antibody-mediated disruption of the function of transplanted cells, in other cases the cause is less clear. DXR has been linked to innate immune activation as well as physiologic incompatibilities between the coagulation pathways of pigs and humans
• Acute cellular rejection (ACR) and antibody-mediated rejection (AMR) are mediated by cells associated with the adaptive immune system, specifically T and B lymphocytes. ACR and AMR, alone or in combination, was typically observed days to weeks after human-to-human organ transplantation before effective immunosuppressives were found. However, they are usually well controlled (prevented or reversed) by current 'conventional' immunosuppressive drug treatments. ACR is unusual in xenotransplant preclinical models, while AMR is often implicated in late failure of cell and organ xenografts

While similar responses can occur with human-to-human allotransplants, overcoming an immune response to tissue transplanted from a different species represents a particularly significant hurdle.

"Although broadly complex, the challenges associated with xenotransplantation tend to be organ-specific," explains Richard N. Pierson III, MD, a cardiac surgeon at Mass General and scientific director for the Center for Transplantation Sciences (CTS). "Treatments and gene modifications that can consistently protect a heart or kidney xenograft appear to be insufficient to translate into similar results for lung or liver xenografts."

Engineering an Optimal Xenotransplantation Model

Although their phylogenetic similarity to humans would predict that their transplanted cells and organs could function normally and be protected by 'conventional' immunosuppression, NHPs are unsuitable as cell or organ donors for xenotransplantation. NHPs such as chimpanzees, whose organs at adult size are similar to humans, are relatively scarce or endangered, and their use for biomedical research has been halted owing to ethical considerations.

Consideration of other NHPs, such as baboons, has been halted due to the potential for cross-species transmission of endogenous retroviruses between closely related species, unfavorable breeding and husbandry characteristics, and disparities in organ size. The pig was selected as the most appropriate xenograft organ donor species based on relatively large litter size, short maturation period, and amenability to modifications by genetic engineering that are needed to develop organs resistant to rejection.

"We remain focused on two major research areas," says Dr. Pierson. "The first is the continued development of genetically modified pigs as preclinical models to improve the compatibility of their organs with human physiology. The second is optimizing drug-treatment regimens for post-transplant immunomodulation to maximize xenotransplant success."

The application of CRISPR technology greatly accelerated making candidate alterations to the pig genome intended to overcome the known barriers to pig-to-human xenotransplantation. Gene edits have both knocked in human genes capable of modulating immune activation, and knocked out pig genes encoding carbohydrate enzymes unique to pigs but absent from the human genome. Removal of the latter was particularly important, given that human preformed antibodies against just three carbohydrates accounts for almost all of the immediate and devastating immune response leading to xenograft HAR.

Evaluation of the organs and immunosuppressive regimens has proceeded in NHP as pig organ xenograft recipients owing to NHP's close physiological and phylogenetic relationships to humans. Recent results demonstrate that transplantation of hearts and kidneys from pigs bred with specific genetic backgrounds virtually eliminated HAR events. DXR was delayed or prevented when pigs with advanced genetics were combined with various novel immunosuppressive drug treatment regimens, particularly blockade of the CD154 costimulation pathway.

"Our findings specifically with hearts and kidneys show great promise," explains Dr. Pierson. "We have achieved sustained post-transplant survival in NHPs ranging from six months to two years following transplantation of kidneys from pigs with specific genetic backgrounds combined with a tailored, clinically applicable immunosuppressive regimen. This work has benefited from close collaboration within our Mass General research teams, and with the companies that make genetically modified pigs and key immunomodulatory molecules."

A Groundbreaking Achievement in the Field

In January 2022, a 57-year-old patient with terminal heart disease underwent the first successful xenotransplant of a genetically modified pig heart at the University of Maryland Medical Center. Although ultimately succumbing to heart failure associated with multiple factors, the patient survived for two months.

Importantly, autopsy results highlighted the transmission of porcine cytomegalovirus (pCMV) as a primary factor associated with the outcome. In addition, increasing amounts of antibody against the donor pig was detected in the patient's blood over the last month of his life, suggesting that the immunosuppressive treatment regimen used, which was minimized because of recurrent infectious complications, may not have been sufficient to prevent immune injury to the xenograft. Dr. Pierson emphasizes the importance of acknowledging and learning from both the positive and negative findings.

"On the positive side, they clearly demonstrated that a pig heart can sustain human life for at least one month and in a patient who was already in a physiologically depleted state. On the negative side, the findings revealed the harm of conveying pCMV in the grafted organ. This case underscores the attention to detail necessary in the care and screening of donor animals, and the importance of patient selection, so as to allow fair evaluation of this new technology."

Joren C. Madsen, MD, DPhil, director of the Mass General Transplant Center, acknowledges this achievement as a game-changing advance in the field. "Overall, this represents a resounding success, in that it answered longstanding questions concerning the procedure, not least of which is whether a pig heart could actually support the life of a human being."

Driving Innovation to Save Lives

The FDA has suggested to sponsors that they expect demonstration of consistent one-year survival in a preclinical pig-to-human model as the basis for approving an Investigational New Drug or Device (IND) for human clinical trials of xenotransplantation. This bar is extremely high since, until recently, one-year survival has only occasionally been accomplished in preclinical xeno models.

Acknowledging that overcoming difficulties in preclinical pig-to-NHP transplant models will serve to increase the degree of confidence before translation to human recipients, it is likely that success may be easier to achieve in pilot clinical experiments than in preclinical models.

For example, pigs designed for use in humans with 'triple carbohydrate gene knockouts' (TKO) unveil a new carbohydrate target recognized by monkeys and baboons (but not humans), making TKO organs difficult to test in NHPs.

Patients consenting to participate in xenotransplantation trials will be motivated to comply with recommended treatments and can take advantage of sophisticated diagnostic and therapeutic options that are unavailable in the preclinical research environment. As such, preclinical testing is ongoing in parallel with trial design of clinical 'pilot' studies.

"Our goal is to demonstrate sufficient improvements in the preclinical models to ultimately justify a transition to formal IND-supported clinical trials in humans," explains Dr. Pierson, who acknowledges Mass General as an incredibly supportive environment and ideal setting for the cutting-edge research necessary to make xenotransplantation a reality.

"We are fortunate to have some of the top experts in the field here at Mass General engaged in these efforts," Dr. Madsen adds. "This mission is fully supported by constant efforts by our leadership to recruit and keep the best people and provide the best facilities possible."

"We represent one of the only, if not the only, institution in the world actively engaged in evaluating the xenotransplantation of pig heart, lung, liver, and kidney organs and pancreatic islets," says Dr. Madsen. "The extraordinary people engaged in this work are what make this a truly premier hospital in terms of both its clinical and scientific contributions."

Learn more about the Heart Failure and Cardiac Transplantation Program at Mass General

Refer a patient to the Corrigan Minehan Heart Center