As we start a new calendar year, it is interesting to look back on the strides that have been made in the field of xenotransplantation since the introduction of the earliest genetically engineered pig to be used as donors. Progress at times seem slow but 20 years ago the transplantation of a pig organ to a primate resulted in graft loss within a few minutes or hours due to hyper acute rejection (HAR). The earliest genetically engineered (GE) pigs added human complement regulatory proteins to overcome HAR. This led to survival of days to weeks until delayed xenograft rejection, which was found to be antibody mediated.

The big breakthrough in GE pigs was the development of alpha 1,3-Galactosyltransferase gene knock-out (GTKO) pigs, which eliminated the predominant pig carbohydrate to which humans had preformed antibodies against. This has led to the identification of several correspondingly lesser but still potentially important carbohydrates that contribute to graft rejection. Initially, methods of cloning and gene editing were slow and cumbersome, but ever more sophisticated methods such as CRISPR-Cas9 have allowed for more and quicker modifications. McGregor et al reported pig-to-baboon heterotopic cardiac graft survival of up to 113 days in 2004 using pigs transgenic for hCD46. The following year Yau-Lin et al reported 179-day graft function using GTKO pigs. A decade later the group of Mohiuddin et al has the place of honor, reporting graft survival of 945 days using GTKO/hCD46/hTBM pigs in an heterotopic (non-life-supporting) pig heart grafts in baboons.

In 2018, a German group described the first instance of 75% survival for up to 195 days of a life-supporting orthotopic pig heart in baboons using triple genetically-modified porcine organs, well exceeding the key requirement for clinical application of cardiac xenotransplantation as defined in 2002 by the ISHLT International Advisory Board. In the kidney, graft survival has reached almost 500 days using GTKO/CD55 pigs. Other organs have shown impressive results in nonhuman primates using multi-GE pigs as well. The synergy between the scientists generating genetically engineered pigs and those studying graft survival in xenogeneic animal models brings us to the present, where we now have the ability to utilize pigs transgenic for many more modifications, opening new realms of possibilities that did not exist just a few decades ago. Slow but steadily advancing successes lead us to anticipate clinical trials to begin perhaps as soon as the coming year. In 20 years, scientists may look back on our work today as the pioneering a new age of clinical xenotransplantation.

On December 14, the US Food and Drug Administration (FDA) approved the use of GalSafeTM pigs, for both human consumption and as a source for potential therapeutic uses. The first dual use approval of its kind ever and a new landmark in developing genetically engineered pigs.

The FDA granted approval of the GalSafeTM pigs to Revivicor, a company based in Blacksburg, Virginia, USA that first generated (1,3) galactosyltransferase Knock-Out (GTKO) cloned pigs via nuclear transfer using adult cells to provide tissues and cells for xenotransplantation.

David Ayares, PhD, Executive Vice President and CSO of Revivicor, shares his enthusiasm for the approval of the Revivicor GalSafeTM pigs, a process that required not only advanced technology in genetic engineering but also a rigorous approach to ensure all regulatory aspects of the production complied with FDA mandates. The whole process has taken several years and significant efforts.

What is the need for such pigs?

GalSafeTM pigs lack the enzyme galactosyltransferase, which is necessary to express galactose-α-1,3-galactose (alpha-Gal) a sugar that is found on cells of all mammals with the exception of Old World monkeys and humans. In Old World monkeys and human’s exposure to alpha-Gal elicits natural anti-alpha-Gal antibodies (IgM and IgG), which are responsible for hyperacute rejection of xenografts.

The extent and consequences of the evolutional differences in alpha-Gal expression, however, turned out to be more relevant to human health than initially comprehended. In the last 5 years a body of evidence has emerged showing that alpha-Gal exposure can also elicit IgE production, thus predisposing the immune system to develop harmful allergic reactions to even minute doses of alpha-Gal, in this case a powerful allergen.

Allergies to alpha-Gal can occur via consumption of red meat, in combination with a tick bite, which then sensitizes the patient to the allergen. Known by various names such as “red meat allergy”, “tick bite allergy” or “alpha-Gal syndrome”, the consequences of this allergic response can be serious, and often life threatening. Recent studies estimated that millions of individuals in the US, EU, and Asia suffer from such conditions, and that there are areas in the world where as much as 20% of the population develop allergies to alpha-Gal, especially among individuals that spend long hours outdoors in non-urban areas. Alpha-Gal syndrome can also cause serious allergic reactions, such as anaphylaxis, to mammalian-derived medicines like heparin, or medical devices including heart valves. Consumption of GalSafeTM meat, now FDA approved, provides an alternative to traditional un-manipulated alpha-Gal expressing products, while avoiding allergic reactions to alpha-Gal.

What is the relevance of GalSafeTM approval for the xenotransplantation scientific community?

The use of genetically engineered pig donors proved to be a crucial factor in improving compatibility between donor and recipient thus enhancing graft survival in pre-clinical studies, with better results achieved using pigs with multiple genetically manipulations (currently up to 10 gene edits are under consideration for whole organ transplants). Dr. Ayares thinks that, even though GalSafeTM pigs carry only one genetic modification, the steps required for FDA approval of GalSafeTM should help set the stage to facilitate approval of multiple genetically engineered pigs for future medical endeavors. Regardless, GalSafeTM pigs may have immediate utility for xenotransplantation of heart valves, skin, neurons and pancreatic islets, which would require separate approvals as human therapeutics.

We may expect that in the near future GalSafeTM meat will be available on the market and the first question will likely be “how does it taste?” Compositional analysis (ie. protein, lipid, nutritional contents), as well as sensory studies (taste, texture), performed under GLP protocols, showed equivalence of GalSafeTM to unmodified pork, thus answering that question.