A pig that can “help”

If your relative or you need an organ transplant, would you mind if the organ comes from a pig?

In December 2020, the U.S. Food and Drug Administration (FDA) approved Revavicor’s gene-edited pig GalSafe to be marketed, which can be used in the food and medical fields. Unlike conventional pigs, the researchers used genetic engineering techniques to knock out the α-galactose molecules on the surface of pig cells.

Alpha-galactose molecules are widely present on the cell surface of “red meat” mammals such as pigs, sheep, and cattle. For some sensitive people, eating red meat containing alpha-galactose can cause allergic reactions, ranging in severity from urticaria to difficulty breathing. After knocking out this molecule, the allergic reaction to food can be resolved.

According to the FDA, GalSafe pigs can not only be used directly as food, but can also be used as a bioreactor to provide a source of pork material for the production of alpha-galactose-free medical products, such as the blood thinning drug heparin. However, what Revavicor most wants to achieve with GalSafe is to produce organs that can be used for xenotransplantation.

According to data from the US Department of Health, in 2019, more than 100,000 people in the US alone are waiting for suitable organ donations. According to a research progress report on organ transplantation, around the world, due to organ shortages, 90% of patients in need of organ transplantation died while waiting. Seeking alternative organs other than the human body has become the research direction to solve the shortage of organs.

Theoretically, choosing primates that are closely related to humans as the source of transplantation is the best option. However, the number of primates is small and the organs are also small. It is precisely because of the closeness that it is easier to cause ethical problems dispute. Pig organs are close to human organs in size and function, and their genes are easy to modify and modify. Pigs that are usually used for food are now a potential source of organ transplantation.

Of course, when transplanting pig organs into humans, immune rejection is still an important obstacle. The alpha-galactose knocked out of GalSafe pigs is not only allergic, but also one of the main reasons for immune rejection. Because after animals evolve into primates, galactose is no longer in their bodies.

In a report released in 2016, researchers from Revavicor and the National Institutes of Health stated that they were able to keep the heart of GalSafe pigs alive in baboons for two and a half years, and their kidneys survived more than 6 in monkeys. month. However, it is worth noting that Revivicor’s xenotransplantation of pig hearts was not orthotopic transplantation. Baboons still retain their own hearts, but pig hearts were transplanted into the baboon’s abdomen. This method is called heterotopic transplantation. The circulatory system of the recipient animal is connected to see if it will be rejected by the recipient animal.

Revivicor is not the only organization that uses genetic pigs for organ transplants. South Korea’s Chungnam University and MGEN have cloned mini pigs with human immune genes. In 2018, the Bruno Reichart team of the Walter Brendel Experimental Medical Center in Munich, Germany, also transplanted gene-edited pig hearts to baboons. Unlike Revavicor, this team did an orthotopic transplantation experiment, that is, a pig heart was completely replaced. Baboon heart. In that year, Bruno Reichart’s team achieved the longest survival record of 195 days, breaking the previous record of 57 days in the scientific community.

This team has made several contributions to the research progress of genetic pig organ transplantation: First, the researchers improved the preservation of pig hearts in vitro to extend the preservation time of organs. At the same time, the researchers also used the drug rapamycin to inhibit the development of pig heart. Rapamycin has immunosuppressive and anti-proliferative effects. It is usually used to prevent rejection after organ transplantation, and as a coating on coronary stents in percutaneous coronary artery treatment (PCI) surgery to prevent Vascular restenosis. In addition, in 2017, the research team headed by eGenesis of the United States stated that they have knocked out PERV (endogenous retrovirus) in the genome of pig populations, and PERV is also regarded as a major obstacle to preventing pigs from providing organs for humans.

The name Revivicor may not be well-known, but its predecessor, PPL Therapeutics, is famous-the creator of the world’s first cloned sheep “Dolly”.

The main product of PPL Therapeutics was the genetically modified mammary gland bioreactor, which allowed some biologically active substances to be expressed using goat milk or milk as a carrier. These substances can be used to treat diseases. But in the end, its products were not approved for clinical use. In 2002, PPL Therapeutics went bankrupt. The following year, the American team of PPLTherapeutics established Revavicor, a regenerative medicine company that produces alternative tissue sources for the treatment of degenerative diseases. The product line includes insulin production for diabetes, and alternative pig kidneys and pig hearts for organ transplantation. , Pig liver organs.

The introduction of Revivicor’s official website deliberately wrote: “The next generation of genetic pigs that regulate T cell gene expression is currently being produced and tested.” Allograft rejection is caused by T cell attack, which is part of the human immune defense system. Identify special external signals. In allogeneic transplantation, immune rejection is mainly controlled by continuous drug therapy. Revivicor’s method is to add or reduce genes that affect T cell responses in pigs.

Lai Liangxue, a researcher at the Guangzhou Institute of Biomedicine and Health of the Chinese Academy of Sciences, has also been doing research on xenotransplantation of genetic pigs very early. In 1998, he went to the University of Missouri to overcome gene-editing pig technology. Like what Revivicor did, Lai Liangxue’s team is also working on galactose knockout pig products. In January 2002, Lai Liangxue published an article in the authoritative magazine “Science” as the first author, announcing the successful breeding of the world’s first gene knockout pig. Lai Liangxue told China Business News that the University of Missouri team used small pigs, and Revavicor used large pigs. But the purpose of the two teams is the same, both are to solve the problem of immune rejection after transplanting pig organs into humans.

At that time, gene editing had not been developed as fast as it is now, and Lai Liangxue’s team used relatively traditional homologous recombination technology. This technology was developed based on the principle of DNA homologous recombination in the mid to late 1980s, using embryonic stem cell microinjection to realize the recombination between or within DNA molecules of homologous sequence. “At that time, there was no efficient gene editing technology, and the success rate of gene editing was very low.” Lai Liang said.

According to Lai Liangxue’s introduction, genetic pigs mainly include ordinary transgenic and genetic targeting. Ordinary transgenic pigs usually place foreign genes into pig cells, allowing the pig to express genes that it does not have, or genes that it has but weakly expressed; gene targeting refers to “cutting” specific locations of pig genes. Gene targeting has two main functions: one is gene knockout, that is, to remove a certain gene; the other is gene knock-in, that is, to place a certain gene in a certain place.

South Korea has bred genetic pigs with human immune genes for organ transplantation.

The classic gene targeting technology is inefficient and difficult. Until around 2009, new gene editing tools based on nucleases came out one after another, such as ZFN, TALEN, and CRISPR/Cas9, which greatly improved the efficiency and throughput of gene editing operations. These three technologies were called first-generation and second-generation technologies, respectively. Generation and third generation gene editing technology. With the change of technology, they are increasingly used to find therapies for a variety of genetic diseases, to make performance modifications such as disease resistance, and to improve new species of animals and plants to increase their production efficiency.

Although advances in gene editing tools have greatly improved the accuracy and efficiency of genetic modification, the application of xenotransplanted organs still faces great challenges. Relatively simple organs such as cartilage and cornea may be applied relatively quickly, while advanced organs such as the kidney and heart with complex functions will be slower.

The prerequisite of the International Xenotransplantation Steering Committee for the introduction of xenotransplantation research into the clinic is that “60% of the animal experiment recipients survive for more than 3 months, and there are at least 10 consecutive transplant experiments.” The goal of clinical trials is: to permanently replace the original organs, more than 50% of the recipients must survive longer than 6 months.

“There are many end-of-organ failure patients who may die soon if they do not receive organ transplants in time. One of the ideas is that if allogeneic organ transplantation is done first, even if it can only help extend the life of the patient by half a year, the patient may be able to wait until the end. Suitable human organs, and then transplantation, this may be the market for pig organ transplantation.” Zhao Jianguo, a researcher at the State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, told China Business News.

However, to realize the efficient transformation of pig genome, the more important basic work is to strengthen the understanding of pig genome information and to dig out more genetic information that affects traits. “Our understanding of the pig genome is far from enough.” Zhao Jianguo said.

In fact, before GalSa fe pigs, there have been cases of gene-edited animals on the market in the United States. In 2015, F DA approved AquaBounty’s AquAdvantage genetically modified salmon to be marketed for consumption. The growth rate of this fish is about twice that of traditional farmed salmon-it takes at least 3 years for traditional salmon to grow to market size.

AquaBounty’s development of genetically modified salmon is mainly based on economic factors. According to the company’s response to China Business News magazine, AquAdvantage salmon not only grows faster, but also raises more efficiently, and can get a larger output in each breeding cycle. And it can be bred near consumption areas on land, thereby reducing transportation costs.

But faster and cheaper salmon products have not gained market recognition. Although it has been approved by regulatory agencies, the product has not been listed in the United States for a long time because of public opinion’s resistance to genetically modified foods. In 2016, the U.S. Congress issued a directive to the FDA to prohibit any product containing genetically modified ingredients from being sold in the U.S. market unless the product is labeled to inform consumers of genetically modified information. This ban was not lifted until 2019.

GalSafe pigs and AquaBounty’s salmon products both use genetic engineering technology, but the difference is that genetic modification involves inserting foreign genes into the target gene. Products such as GalSafe pigs are genetic modifications to themselves, only through gene knockout or The single-base substitution method “addition and subtraction” to gene fragments does not bring in foreign genes, which is why the researchers believe that the product has no additional risks.

Each country has slightly different policies for different types of gene-edited organisms. Japan, Brazil, Argentina and other countries have relatively looser and more flexible supervision on the listing of gene-edited organisms, and they are more open to the application of the differentiated management of gene-edited organisms and genetically modified organisms that have not been introduced into foreign genes. The EU and New Zealand still regard the former as genetically modified organisms and implement strict supervision.

China has not yet issued a specific management policy for gene-edited organisms. All related products can only be managed in accordance with genetically modified organisms. For example, the gene-edited yellow catfish developed by Nanjing Yaoshunyu Biotechnology Co., Ltd. is applying for “long” safety. Evaluation review. Yaoshunyu Biology uses gene editing technology to knock out the growth-suppressing genes of yellow catfish, so that the fish can grow rapidly. “The current management model of genetically modified organisms is more complicated and strict. It is necessary to evaluate ecological safety and see if there are changes in surrounding species. This will take 4 to 5 years.” Wang Mingkai, the company’s vice president, told China Business News magazine.

Many people in the field of gene editing biology have said that gene editing and genetic modification should be managed separately to give gene editing technology more space. If classified according to the source of gene mutation, gene mutation can be divided into three types: natural mutation, artificial mutation by physical and chemical methods, and biological mutation including gene editing technology. Researchers believe that the new varieties mutated through gene editing technology are essentially similar to those naturally mutated in nature. However, some questioning parties expressed concern about the uncontrollability of gene-edited organisms. For example, after multiple generations of gene-edited animals and plants, they may have irreversible pathogenic traits and other unpredictable consequences.

The FDA’s review document also analyzed the food safety issues of GalSafe pigs: the feeding conditions of GalSafe pigs are much stricter than those of traditionally raised pigs. Galsafe does not have animal safety issues in the well-managed commercial raising conditions. However, the modification of the target gene may also affect the expression of other genes, such as whether a new protein will be expressed. Therefore, “before the product is put on the market, it is still necessary to evaluate the possible risks.”

The approval of Galsafe pigs is of course a positive signal in the field of gene editing, but Revavicor said that pork production is not the company’s main business direction. Its approved products will not be sold in supermarkets, and Revivercor will not directly participate in sales. Consumers who are allergic to pork with α-galactose molecules need to place orders with a third party to buy Revivercor’s pork products. .

Xenotransplantation of pig organs is the focus and future direction of this company.

Before attempting a heart replacement, Revavicor plans to start with a kidney transplant. They are waiting for formal human trials, and if it goes well, clinical trials will begin this year. A company from Boston is already testing GalSafe pig skin transplantation to see if it can be used as a treatment option for burn patients to regenerate their own skin. “Our ultimate goal is to truly realize the’unrestricted supply of organs.” Revivecor CEO David Ayares once said.