The FDA greenlights α-Gal allergy-safe meat, but its makers have organs for transplants in their sights.
The first genetically engineered pig products could soon be coming to a dinner plate—or pharmacy—near you. Late last year, the US Food and Drug Administration (FDA) authorized a facility in northern Iowa to raise hogs that lack the gene needed to produce galactose-α-1,3-galactose (α-Gal), a sugar molecule found naturally on the surface of porcine cells. Trademarked under the name ‘GalSafe’, the pigs could now provide a source of meat for people who develop tick bite–induced allergic reactions to the sugar, a condition known as α-Gal syndrome. Byproducts of pork production could also be harvested to make allergy-free pharmaceuticals and medical implants. The porcine tissue could help overcome deficiencies in the donor supply of skin and nerve grafts.
The pigs were never made with any of those applications in mind, though. In the early 2000s, following failed attempts to use unmodified porcine tissue for skin grafts, pancreatic islet cell transplants and outside-the-body blood perfusions, David Ayares and his colleagues at Scotland’s PPL Therapeutics used a combination of recombinant DNA and cloning technologies—first described in Nature Biotechnology—to create the pigs as a source of solid organs for pig-to-human xenotransplantation. Soon thereafter, PPL spun out Revivicor as a standalone company.
Revivicor, now a subsidiary of United Therapeutics, is still angling to transplant pig kidneys and hearts into human patients—just not with GalSafe-derived organs. According to Ayares, who serves of CSO of Revivicor, the company is hoping to launch clinical trials with organs from more elaborately engineered pigs within the next year or two. And he views the landmark approval of first genetically modified pigs as “an important stepping-stone” toward that goal.
“All the pieces that went into the FDA approval of the GalSafe pigs”—safety evaluations, environmental impact assessments, molecular characterizations of the genetic alteration—“those same pieces are going to be required” for approval of other engineered pigs intended for xenotransplantation purposes, he says.
The regulatory path is thus better defined now—and not just for Revivicor. Other xenotransplantation companies and academic groups hope to build on Revivicor’s success with the FDA to advance their own modified pigs and cross-species transplant products. Some companies are also partnering with Revivicor to develop GalSafe-based products for burns and peripheral nerve injury. Axonova Medical, for example, uses neurons from GalSafe embryos as the starting material for the company’s tissue-engineered nerve grafts. Earlier this year, research director Kritika Katiyar and her colleagues reported that the grafts elicited axon regeneration in rats. Pig-to-pig studies are planned next.
Meanwhile, XenoTherapeutics, the company behind the first human trial of tissue from a genetically engineered porcine donor, had previously used skin from a different α-Gal-deficient pig—miniature swine bred by Immerge BioTherapeutics, a rival to PPL, in the early 2000s—to provide temporary wound closure for patients with severe burns. According to XenoTherapeutics cofounder and CEO Paul Holzer, that six-person, phase 1 trial “truly went better than I could have hoped for,” and the company plans to apply for accelerated approval on the basis of the small study’s unpublished results later this year. However, it may seek approval with GalSafe skin, rather than the miniature swine hide, now that the Revivicor animals have passed regulatory muster. (The GalSafe pigs are also larger and more fecund than the miniature swine, thus offering more tissue for transplantation.)
Although Revivicor has no plans to commercialize the GalSafe pigs themselves, Ayares says his team is looking for more licensing opportunities with partners interested in selling allergy-free pork or pig derivatives. For people living with α-Gal syndrome, a group that counts at least 34,000 individuals in the United States alone, such food products, if made available for purchase, would be a “game-changer,” says Scott Commins, an allergist at the University of North Carolina-Chapel Hill who specializes in treating the condition. “The food use is part of the story,” he says, “but in my mind it’s arguably not as important as the other medical and implantable uses of the GalSafe pig.”
“The food use is part of the story, but in my mind it’s arguably not as important as the other medical and implantable uses of the GalSafe pig.”
Still, any implantable product more elaborate than, say, a heart valve or short-term skin graft would need further genetic tweaks to prevent organ rejection. Some say only a couple more knockouts of pig carbohydrate antigens should do the trick. But decades of painfully slow progress in large-animal transplantation studies suggest the elimination of a few pig glycans will be insufficient; more steps, such as introducing human transgenes or removing porcine endogenous retroviruses (PERVs) are likely to be needed to protect porcine kidneys, hearts, livers and lungs from injury in human recipients.
In the 1990s, following reports that PERVs embedded in the pig genome could take up residence in cultured human cells, many researchers and regulatory bodies worried about the potential infectious complications of pig-to-human transplants. This was an important concern—one of many regarding safety— that put the brakes on the field for most of the 2000s. With the advent of CRISPR–Cas9, Harvard Medical School geneticist George Church, his former student Luhan Yang and their colleagues used gene editing to inactivate every single PERV in their first-generation pig. Yang’s logic: “If it’s safer and does not impact the health” of the pig, she says, why not get rid of the PERVs?
But other researchers think that step is unnecessary to progress to clinical xenotransplantation. “We have amassed a plethora of data” over the past 20 years, and “to date, on no occasion has anyone found any evidence that PERVs have caused infection in human recipients who have been exposed to xenotransplantation,” says Linda Scobie, a virologist who studies the safety of xenotransplantation at Glasgow Caledonian University, UK. “PERV is considered to be low risk, and a more pressing concern is regarding unknown or emerging pathogens.”
Opinions differ, however, and the genetic sweet spot for the number of alterations to pig cells sufficient to support safe xenotransplantation remains a matter of intense scientific debate. “We have to find a balance between what’s just right and what’s too much and unnecessary,” Scobie says.
Building on the foundation of an α-Gal-lacking pig, xenotransplantation researchers have eliminated more pig-specific antigens that were contributing to interspecies incompatibilities. Disrupting two genes in particular—cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and β-1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2)—proved critical to that effort. When researchers knocked out CMAH and B4GALNT2 alongside α-1,3-galactosyl transferase (GGTA1), the enzyme responsible for synthesizing the α-Gal epitope, cells from the ‘triple knockout’ (TKO) pigs bound substantially fewer human antibodies.
“This suggests that organs from TKO animals will be far less prone to antibody-dependent mediated rejection when transplanted into humans,” says Agnes Azimzadeh, president of the International Xenotransplantation Association and a researcher at the Massachusetts General Hospital’s Center for Transplantation Sciences in Boston. That TKO construct, she adds, is now the “backbone of any pig” intended for use as an organ donor.
On top of that backbone, some teams have also inserted human genes involved in tempering immune responses to the transplant. These include genes encoding components of the complement activation pathway (CD46, CD55 and CD59), regulators of platelet coagulation (thrombomodulin, tissue factor pathway inhibitor and endothelial protein C receptor) and others with immune-quelling effects such as heme oxygenase-1 and CD47. Last year, for example, scientists at Qihan Biotech and eGenesis—affiliated companies both founded by Church and Yang—described pigs carrying the triple knockout plus nine of these human transgenes.
Revivicor, meanwhile, has engineered pigs with six human transgenes plus a fourth knockout in the porcine growth hormone receptor gene, a change designed to prevent organs slated for transplantation from getting too large in the donor pigs. The resulting ‘ten-gene’ pigs will form the basis of the company’s pending clinical applications, Ayares says.
Some scientists, though, say that the Revivicor and Qihan/eGenesis pigs are overengineered. Christopher Burlak, for one, worries about whether the companies can achieve consistent transgene expression in porcine organs from one transplant to the next. “The more complicated the genetic engineering of these pigs is, the more difficult it is to produce animals reliably,” says Burlak, a xenotransplantation researcher at the University of Minnesota in Minneapolis.
And as Joseph Tector, an abdominal transplant surgeon at the University of Miami Miller School of Medicine, points out, cross-species transplants typically fail because of antibody-mediated rejection. “Other issues like coagulation regulation and complement regulation—those are all downstream effects of the antibodies binding to the cell,” he says. At his company, Makana Therapeutics, a subsidiary of Recombinetics, Tector is thus moving ahead with basic TKO pigs, no transgenes involved. As with human-to-human allotransplants, he thinks that cross-match methods and human leukocyte antigen (HLA) typing should be sufficient to allow successful xenotransplants from TKO pigs.
Fios Therapeutics is similarly working with TKO pigs, but with one key difference: in addition to the triple knockout, the company has incorporated a transgene encoding human CD46, a critical regulator of the complement system that contributes to both innate and adaptive immune responses after organ transplantation. “You have to control complement early on to avoid hyperacute rejection,” says company founder and CEO Christopher McGregor, a cardiac surgeon at the University of Minnesota and University College London.
Others hope to achieve the same immune-dampening effects pharmacologically—although, as surgeon–scientist David Cooper, who co-directs the University of Alabama at Birmingham’s xenotransplantation program, points out: “The standard immunosuppressive drugs are just not up to the task.” Cooper anticipates that newer, experimental therapies directed at the costimulatory protein CD40 or its ligand, CD154, will allow longer survival of xenografts. However, clotting complications have plagued the drug class for decades, and as yet only one experimental agent directed at the CD40 axis has ever progressed past phase 2 development (dapirolizumab pegol, a humanized anti-CD154 IgG1 Fab′ fragment from Brussels, Belgium–based UCB Biopharma in phase 3 testing for treating lupus).
Despite the many outstanding scientific uncertainties, Ricardo Carvajal, an expert in food law and biotechnology at Hyman, Phelps & McNamara in Washington, DC, sees a willing partner in the FDA’s Center for Veterinary Medicine to move the field forward. Consider the center’s Plant and Animal Biotechnology Innovation Action Plan, unveiled in 2018. It includes a pilot project similar to the breakthrough designation for drugs—the Veterinary Innovation Program—that offers extensive hand-holding by FDA experts for sponsors seeking approval of animal cell-based or biotech products. (Revivicor was one of the program’s first participants.) In Carvajal’s opinion, “That’s one way of saying to industry, ‘We hear you. We think this technology has a lot of value to it. We want to try to help you navigate this review and approval process.’”
That’s an important message for xenotransplantation veterans like Cooper, who first identified α-Gal as the major target of the human immune system’s response against pig tissue in the early 1990s and has collaborated with the Revivicor team on pig-to-primate studies for more than 15 years. “We shouldn’t wait for the perfect pig before we start clinical trials,” he says. Once researchers identify the right immunosuppressive drug regimens, build the appropriate pathogen-free facilities and design suitable genetically engineered pigs, human testing should follow. “We’re getting close to all of those,” Cooper says, and the GalSafe approval provides a blueprint to navigate the regulatory thicket.
“PERV is considered to be low risk, and a more pressing concern is regarding unknown or emerging pathogens.”
Outside of the xenotransplantation community, however, the approval is likely to have far less of an impact on the animal biotechnology sector writ large. Under the terms of the FDA’s go-ahead, the Revivicor pigs are limited to one enclosed facility, one herd of no more than 1,000 animals and one abattoir for meat processing—a drop in the industry’s bucket considering there are an estimated 66,000 US swine producers churning out more than 72 million pigs every year.
Other companies hoping to bring genetically engineered pigs to the mass market may have to demonstrate similar levels of molecular characterization to Revivicor’s, but the environmental impact assessments will undoubtedly be more onerous—and those stewardship requirements are “still being designed as we’re going,” says Elena Rice, CSO of Genus, which has used CRISPR gene-editing to make pigs resistant to infection with the virus responsible for porcine reproductive and respiratory syndrome, a costly scourge of the swine industry.
That regulatory ambiguity is a problem for the entire livestock biotechnology sector, says Alison Van Eenennaam, an animal geneticist at the University of California, Davis. “The lack of regulatory certainty and the cost [of R&D] has basically dried up all investment in this field,” she says. Unlike with CRISPR-edited crops, which typically face little to no scrutiny from federal agencies in the United States, with gene-edited animals “the system is set up as if you’re dealing with kryptonite,” Van Eenennaam says.
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Dolgin, E. First GM pigs for allergies. Could xenotransplants be next?. Nat Biotechnol 39, 397–400 (2021). https://doi.org/10.1038/s41587-021-00885-9