Recent progress in xenotransplantation – a new way to fight donor organ shortage

by Masin Abo-Rady and Jörg Schneider

The biggest problem of patients suffering from end-stage organ failure is the increasing shortage of organ transplants from deceased donors. The demand of viable donor organs exceeds the supply by far. In 2020 150,000 patients were registered on organ waiting lists in the 47 member states of the Council of Europe. 41,000 patients were transplanted and 48,000 newly registered on waiting lists (see here). With an ageing society, this trend is set to continue and alternatives for human organ transplants are urgently required. Xenotransplantation of genetically modified pig organs offers a promising potential solution to overcome the shortage of donor organs (see Figure 1).

Transplantation of wild-type pig organs to non-human primates (NHP) or humans resulted in rapid and violent hyperacute rejection due to molecular differences between donor and recipient species. It became clear that in order to succeed, the donor organs needed to be humanized and the recipient´s immune and coagulation system reactions needed to be suppressed. The past 35 years of xenotransplantation research defined the pathobiological barriers, and to date, 50 different beneficial modifications have been identified, and the list is growing (1, 2). Pathways requiring modification are complement regulation, differences in glycosylation, targets of cellular immune responses, expression or deletion of genes involved in coagulation, inflammation and apoptosis.

A crucial step to prevent hyperacute transplant rejection is the deletion of α1,3-galactosyltransferase (GGTA1) to eliminate galactose-alpha-1,3-galactose on the surface of the pig cells, against which humans have an antibody-mediated immune response. A combination of CRISPR/Cas9 technology and transposon constructs promises to accelerate the process of pig xenotransplant humanization at an unprecedented speed. In a recent publication by Yue et al (3) nine human xenoprotective transgenes designed to inhibit complement activation (hCD46, hCD55, hCD59), blood coagulation and platelet aggregation (hTHBD, hTFPI, hCD39), natural-killer-cell activation (hB2M, HLA-E) and macrophage activation (hCD47) were combined in three multi-cistronic expression cassettes all contained within one transposon vector to direct integration at a single genomic locus. The vector was transfected into wild-type porcine fibroblasts together with the transposase and expression vectors for Cas9 and guide RNAs targeted to GGTA1 and also CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) and B4GALNT2 (beta-1,4-N-acetyl-galactosaminyltransferase 2), which are responsible for the two major non-Gal glycan antigens (the sialic acid Neu5Gc and the blood-group antigen SDa). The researchers used cell clones with the desired genotype for somatic-cell nuclear transfer to create, in a single step, triple-knockout pigs with nine human transgenes. All transgenes were functional in vitro in a pig cell line in which all 25 PERV copies were inactivated via genome editing.

In addition to the modification of functional pathways, there are still further safety aspects to address when considering to use pigs as donor organisms for xenotransplants. The genomes of jawed vertebrates contain endogenous retroviruses and the evolutionary divergence of pigs and humans resulted in distinct species-specific viruses. Transmission microorganisms and of porcine viruses such as cytomegalovirus, hepatitis E virus and single-stranded DNA viruses must be prevented to avoid potential induction of diseases (zoonoses) in the recipients. While the role of human (HERV) and porcine (PERV) endogenous retroviruses is being characterized, the impact of cross-species infection remains to be elucidated and therefore represents a potential safety risk. Concern has been raised that the transfer of PERVs to humans might be pathogenic, or that recombination between PERVs and HERVs may give rise to a new virus that may be pathogenic. Current opinion is that PERVs are not likely to be pathogenic to humans, but it would be possible to prevent activation of these viruses by genetic engineering of pigs (1). For example, PERV activation can be suppressed by small interfering RNA technology (4) or could be eliminated from the genome. Recently, all PERVs in a porcine primary cell line were inactivated (5).

Finally, besides microbiological safety and genetic modification, further general challenges need to be addressed. Physiological function and size have to match with donor requirements, which is addressed by selection of appropriate donor animals such as Auckland Island pigs or miniaturized German Landrace pigs. Additionally, a supportive medication regimen is needed to provide sufficient immunosuppression and overgrowth of specific organs.

As of today, pig-to-NHP xenotransplantation has already been successful (6), while crucial progress has been made to enable safe use of pig organs in human patients. It is likely that this acceleration of genetic engineering combined with the continued high demand in donor organs will move us closer to viable xenotransplants in the near future. Recently, the FDA approved an intentional genomic alteration (IGA) in a line of domestic pigs, referred to as GalSafe pigs (for details, see here). These pigs lack GGTA1 and may be used for food or human medicinal products which is perhaps more relevant for preventing the alpha-gal syndrome, an allergy to red meat from food producing mammals and other products containing mammalian-based materials. Although organs from GalSafe pigs cannot be used for xenotransplantation without additional approval yet, the IGA approval by the FDA may pave the way for future approval of cells and organs generated in extensively modified pigs.

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References

1. Cooper DK, Ekser B, Ramsoondar J, Phelps C, Ayares D. The role of genetically engineered pigs in xenotransplantation research. The Journal of pathology. 2016;238(2):288-99.

2. Fischer K, Schnieke A. Extensively edited pigs. Nature Biomedical Engineering. 2021;5(2):128-9.

3. Yue Y, Xu W, Kan Y, Zhao H-Y, Zhou Y, Song X, et al. Extensive germline genome engineering in pigs. Nature Biomedical Engineering. 2020:1-10.

4. Dieckhoff B, Karlas A, Hofmann A, Kues WA, Petersen B, Pfeifer A, et al. Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors. Arch Virol. 2007;152(3):629-34.

5. Niu D, Wei H-J, Lin L, George H, Wang T, Lee I-H, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 2017;357(6357):1303-7.

6. Längin M, Mayr T, Reichart B, Michel S, Buchholz S, Guethoff S, et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature. 2018;564(7736):430-3.