Can Miniature Pigs Take Flight on the Biotech Surge? Advancing Xenotransplantation Through Gene-Edited Porcine Models

In 2024, xenotransplantation entered the inaugural year of an explosion in clinical research.

In January 2024, the world’s first ex vivo perfusion procedure using a miniature pig liver on a brain-dead patient was successfully completed. The animal liver used in the surgery was derived from gene-edited miniature pigs developed by eGenesis. This pig liver maintained the patient’s hepatic function for up to 72 hours ex vivo, with no signs of rejection observed throughout the process. The patient’s liver exhibited stable blood flow, pressure, and pH levels.

In March of the same year, after obtaining FDA approval for “compassionate use,” Massachusetts General Hospital performed a xenotransplantation of a kidney into a patient. The kidney used was sourced from a miniature pig that had undergone 69 genetic edits. Although the patient unfortunately passed away in May, they experienced a period of good recovery following the xenotransplantation, which undoubtedly brought hope to the field of xenotransplantation.

In China, in March 2024, a team of experts from Xijing Hospital performed the first transplantation of a gene-edited pig liver into a 50-year-old brain-dead male. Twenty-four hours postoperatively, the recipient’s hemodynamics remained stable, the transplanted liver exhibited good bile secretion, ultrasound imaging confirmed adequate blood supply to the graft, and pathological examination revealed no signs of rejection, marking the surgical procedure itself as successful.

As xenotransplanted organs are able to sustain patients’ lives for increasingly longer periods, related research is intensively advancing into clinical trials, and miniature pigs, as key donors for xenogeneic organs, are also emerging as a focal point of attention.

For many years, selectively bred miniature pigs have served as important laboratory animals in life sciences research.

The organ size and physiological structure of experimental miniature pigs are similar to those of humans, making them highly suitable models for studying complex and refractory human diseases. In life science research, miniature pig models are primarily used in studies of human cardiovascular disease models, pharmacodynamics, the immune system, drug tolerance, allergic reactions, drug metabolism, and wound healing.For instance, the anatomical and physiological characteristics of miniature pigs are similar to those of humans, making them widely used as disease models for basic research and drug screening. This application is primarily seen in the development of new drugs for diabetes, cardiovascular diseases, cancer, and other fields. Furthermore, due to the high similarity between miniature pigs and humans in physiological functions and enzyme activity, they are also frequently employed in pharmacokinetic studies of transdermal drug delivery formulations.

However, compared with more established experimental animals such as non-human primates and laboratory mice and rats, these advantages of miniature pigs are insufficient to drive their widespread adoption in life science laboratories. The true surge in the use of laboratory miniature pigs occurred after the introduction of gene-editing technologies. By leveraging gene editing to make targeted modifications in miniature pigs, researchers can enhance their suitability and precision as model organisms.

For instance, in the establishment of a diabetic minipig model, a high-fat diet alone typically induces phenotypes such as obesity and impaired glucose tolerance but generally does not lead to diabetes. Therefore, it is necessary to combine this approach with other methods to disrupt pancreatic β-cells, such as the administration of streptozotocin or alloxan, or the use of gene-editing techniques.

More importantly, because the size, physiological structure, and function of pig organs are similar to those of their human counterparts, pig organs are considered the closest alternative when human donor organs are unavailable.In xenotransplantation, immune rejection remains the most significant challenge. Due to substantial genetic disparities between donor and recipient, the recipient’s immune system recognizes the xenogeneic organ as “non-self” and mounts an attack, thereby triggering a robust rejection response.

Through gene editing, researchers can modify donor organs to reduce or eliminate immune rejection in the human body. Among these, CRISPR-Cas9 is a relatively mainstream gene-editing technology. Specifically, researchers working with miniature pigs used in experiments can utilize CRISPR-Cas9 technology to knock out genes that cause rejection, introduce human genes to enhance compatibility, eliminate endogenous viruses, regulate platelet coagulation, and suppress immune responses.

For example, in the aforementioned xenogeneic kidney transplantation performed at Massachusetts General Hospital, eGenesis conducted 69 genetic edits on Yucatan miniature pigs, knocking out genes associated with hyperacute rejection and porcine endogenous retroviruses (PERVs), while inserting human genes involved in regulating immune rejection. In the xenotransplantation surgery at Xijing Hospital, the partner company Zhongke Aoge used gene-editing technology to knock out three major pig-to-human xenoantigens and inserted two complement regulatory proteins and one anticoagulant protein, thereby reducing immune rejection and making pig organs more suitable as donors for human transplantation.

In addition, through gene editing technologies, scientists are able to modulate the coagulation function of porcine platelets and introduce new genes to suppress immune responses, thereby reducing the risk of post-transplant rejection. Beyond CRISPR/Cas9 technology, researchers have successfully generated miniature pig models with specific gene deletions, such as ApoE and LDLR double-knockout pigs, using their independently developed multi-gene precise editing technology, which can be utilized for further research and applications.

Admittedly, no gene-editing technology alone is sufficient to resolve all the complex challenges associated with xenotransplantation. However, as gene-editing techniques become increasingly mature in the precise modification of laboratory miniature pigs, they have undoubtedly propelled this bold medical innovation of xenotransplantation a significant step closer to clinical implementation.

In recent years, overseas, the genetic editing of animals as a source of food and drugs, or directly as food and drugs, has become an undeniable trend in health innovation. Some genetically edited animals have obtained relevant qualifications and officially entered the regular market. According to statistics, the FDA has approved four products related to genetically edited animals for marketing so far.

First, gene-edited animals as bioreactors.2In 2009, the FDA approved Atryn, a medication for hereditary antithrombin deficiency, marking the global debut of an approved recombinant antithrombin product. Developed by GTC Biotherapeutics, Atryn utilizes gene-edited goats as bioreactors. By employing gene-editing techniques, GTC Biotherapeutics researchers introduced the human antithrombin gene into goat mammary cells, enabling these goats to produce human antithrombin protein. This protein acts as a natural blood thinner in the human body, preventing and treating acute and chronic thrombosis in patients with antithrombin deficiency.

Atryn is also the first drug approved for production by genetically edited animals, opening up the possibility of using genetically edited animals as future drug factories.

Subsequently, in 2015, the FDA approved a genetically modified chicken for the production of Kanuma, a drug for rare diseases. Kanuma, developed by Alexion Pharmaceuticals, is the first therapeutic agent for patients with lysosomal acid lipase deficiency (LAL-D). It is extracted from the egg whites of genetically engineered chickens. Its function is to replace the defective enzyme in the human body, thereby restoring the ability to break down lipid molecules within cells. Alexion is highly optimistic about the commercial prospects of Kanuma, projecting that it could eventually generate annual sales exceeding $1 billion, which would help sustain the company’s growth in the rare disease sector.

Secondly, gene-edited animals themselves can also be directly approved for market as food (and potentially as pharmaceuticals in the future).In 2015, the FDA approved AquAdvantage Salmon for market release, making it the first genetically engineered food animal in history to receive such approval. In the United States, genetically modified foods must be approved by the FDA before they can be marketed. Developed by AquaBounty Technologies, AquAdvantage Salmon has a growth hormone gene from Chinook Salmon and an antifreeze protein gene from Ocean pout inserted into its genome. Following genetic modification, AquAdvantage Salmon can reach market weight in a shorter period, requires no additional feed, and has a reduced environmental impact. Data show that AquAdvantage Salmon reaches market size in 18 months, whereas conventional non-GMO salmon require three years.

Despite the U.S. Food and Drug Administration (FDA) confirming, after a rigorous 20-year review, that AquAdvantage salmon is as safe and nutritionally equivalent to conventional Atlantic salmon, and despite Health Canada approving its sale in Canada in 2016, the commercialization of AquAdvantage salmon has faced significant challenges. Due to public opposition, the promotion of AquAdvantage salmon in the U.S. market has been restricted. In 2018, AquaBounty Technologies incurred losses exceeding $100 million and repeatedly faced the brink of bankruptcy.

By December 2023, the FDA had increased its support for gene-edited animals by approving the market release of a gene-edited pig suitable for both human consumption and medical applications. This gene-edited pig, named GalSafe Pig, was developed by Revivicor. Revivicor previously gained widespread recognition for providing the gene-edited pig heart used in the world’s first pig-to-human heart transplant. In January 2022, a team from the University of Maryland School of Medicine transplanted a pig heart into a patient. Although the patient survived only 60 days, the transplanted heart functioned well during the postoperative period.

Following gene editing, GalSafe pigs lack the alpha-galactose (Alpha-Gal) molecule that triggers allergic reactions. On one hand, GalSafe pigs can be consumed directly as food by patients with alpha-gal syndrome, thereby avoiding severe allergic reactions. On the other hand, GalSafe pigs serve as bioreactors to produce heparin that is safer for individuals with alpha-gal syndrome. Notably, since alpha-galactose may cause rejection in transplanted organs, GalSafe pigs, with this gene knocked out, hold significant promise for organ transplantation.

In xenotransplantation, gene-edited miniature pigs essentially serve as bioreactors for target organs. However, the process of bringing gene-edited porcine organs into clinical application is more complex, involving not only fundamental breakthroughs in biotechnology but also multifaceted issues related to animal welfare, medical ethics, and clinical techniques. Nevertheless, the successive market approval of products derived from gene-edited animals may offer a regulatory framework that can serve as a reference for the development of xenotransplant organs.

Prior to their discovery and breeding as laboratory animals, miniature pig breeds were extremely scarce in number and had long inhabited isolated natural environments with limited accessibility.Compared with ordinary domestic pigs, miniature pigs have a more consistent genetic background, uniform genetic traits among individuals, and stronger stress resistance. Previously, miniature pigs exhibited favorable reproductive performance and relatively low rearing costs.

Wild miniature pigs must undergo artificial rearing and breeding to be utilized as experimental miniature pigs, which are widely applied in life science fields such as medicine, clinical practice, and pharmaceuticals. The purpose of artificial rearing is to ensure that captive herds of miniature pigs have clear origins and genetic information, and to develop experimental populations—including inbred strains, closed colonies, mutant strains, germ-free colonies, gnotobiotic colonies with defined flora, and specific pathogen-free (SPF) colonies—according to the requirements of life science experiments.

In the late 1940s, American researchers began to explore the breeding of miniature pigs.The Minnesota Miniature Pig was the first artificially bred miniature pig strain developed for use in life science experiments. Since then, new miniature pig breeds have continually been added to the list of laboratory animals, including the Yucatan miniature pig, which is smaller in size and has seen broader application in later years.

In China, germplasm resources of miniature pigs are abundant.China is a major producer of domestic pigs, boasting unique advantages in breed diversity.In the vast mountainous and grassland regions of Tibet, Guangxi, Yunnan, and other areas, there live numerous small pig breeds with distinct characteristics. Specifically, at the current stage, the relatively mature domestically bred and utilized miniature pig breeds include the Tibetan miniature pig, Wuzhishan miniature pig, Bama miniature pig, Guizhou Xiang pig, and Banna miniature pig.

Among these, Bama miniature pigs are widely used in xenotransplantation. In the pig liver transplantation project conducted through the collaboration between Zhongke Aoge and Xijing Hospital, Bama miniature pigs were employed. This is a unique pig breed native to areas such as Bama County and Dongtian County in the Guangxi Zhuang Autonomous Region of China. Research indicates that the breeding base for Guangxi Bama miniature pigs is located at Guangxi University. In 1987, Professor Wang Aide and Professor Guo Yafen from the Animal Genetics and Breeding Laboratory at Guangxi University introduced Bama fragrant pigs from their place of origin in the Bama Yao Autonomous County of Guangxi and carried out closed-colony breeding at the Animal Husbandry Experimental Station of Guangxi University.

At present, whether in China or overseas, the exploration of xenotransplantation using organs from gene-edited miniature pigs remains in its very early stages, still constrained by numerous technical and societal challenges. However, it has been largely confirmed that gene editing can mitigate rejection reactions associated with xenogeneic organs, indicating that this technology has overcome the most significant hurdle to clinical application. As organ transplantation represents the last resort for many patients with critical illnesses, there is a substantial gap between supply and demand. Once the feasibility of xenotransplantation is achieved in more routine medical settings, the value of filling this clinical void will be self-evident.

Currently, gene-edited miniature pigs are becoming hot investment targets in the primary market. As a leading company in overseas xenotransplantation, eGenesis, founded in 2015, has completed four rounds of financing, raising nearly $400 million. Its most recent round was closed in September, securing $191 million in Series D funding led by Lux Capital. The funds will be used to advance its core investigational product, EGEN-2784, into the first-in-human clinical trial for kidney transplantation and to expand production capacity. In China, Zhongke Aoge, established in 2018, has also raised nearly RMB 100 million. Its latest financing round, completed earlier this year, secured nearly RMB 10 million in Series A+ funding.

In China, multiple innovative institutions have established layouts in the field of gene-edited miniature pigs. In addition to Zhongke Aoge mentioned earlier, Gelander Bio, based in Beijing, has developed various xenotransplantation materials—including corneas, skin, livers, kidneys, hearts, islets, dopamine cells, and large blood vessels—based on the Wuzhishan miniature pig inbred line from the Institute of Animal Sciences and Veterinary Medicine of the Chinese Academy of Agricultural Sciences (CAAS). Meanwhile, the Shenzhen Institute of Agricultural Genomics at CAAS successfully generated six pigs with dual knockouts of the ApoE and LDLR genes using its self-developed multi-gene precise editing technology. Over a period of more than six years, it also bred three new strains of miniature pig disease models. These new strains exhibit distinct pathological features, with each strain’s population exceeding 60 animals.

In a sense, the industrialization of gene-edited miniature pig organ transplantation technology is starting from nearly the same baseline both domestically and internationally. Meanwhile, China’s abundant germplasm resources have laid a more solid foundation for the rapidly advancing field of gene-edited miniature pigs. We also look forward to the development of an increasing number of xenotransplantable organs, bringing greater hope for survival to patients with otherwise untreatable conditions.

Writing Reference:

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