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Transplant Immunology

Special Issue 3 - December 2023 - Transplant Immunology

Animal engineering for xenotransplantation

Authors

Key words: pig, genetic engineering, CRISPR/Cas9, somatic cell nuclear transfer, xenotransplantation
Publication Date: 2023-10-04

Abstract

Xenotransplantation involves the transplantation of organs, tissues, or cells from animals to humans. The increasing need for organ transplantation due to longer life spans and improved medical care cannot be met by human donors. Pigs are considered suitable donors due to their physiological similarities to humans and ease of genetic modification. However, there are scientific, immunological, and ethical challenges associated with xenotransplantation. The severe immune rejection response from human recipients poses a major hurdle, which researchers are trying to or have partially overcome through genetic engineering and immunosuppression therapy. To generate the animals from genetically engineered cells somatic cell nuclear transfer is of paramount importance. Safety concerns include the transmission of infectious diseases from animals to humans, primarily porcine endogenous retroviruses (PERVs), although there have been no reports of transmission to date. Ethical considerations revolve around the welfare and rights of animals involved in research. Genome editing techniques based on programmable nucleases, such as Zinc- Fingers, TALENS and CRISPR-Cas9, have facilitated the modification of pig genomes to address immunological and physiological barriers. However, further research is needed to ensure safety, efficacy, and to develop novel immunosuppression therapies that is second pillar of xenotransplantation. Scientists are also exploring other avenues of research looking at pig-human chimeric organs, where pig embryos with human cells are generated to create organs more tolerable to the human immune system. Challenges include making cells of different species talking to each other and preventing human cells from contributing to other organs in the pig. To ensure the success and ethical implementation of xenotransplantation there is the need for rigorous scientific investigation, regulatory oversight, and public engagement.

INTRODUCTION

Improved medical care and better living standards are increasing the life span of people around the world. Living longer however has an increased incidence of cell, tissue or organ loss of function or failure. This has opened the way to new medical disciplines, such as organ transplantation and more recently regenerative medicine. Xenotransplantation, a pioneering field at the intersection of biology, medicine, and ethics, holds the promise of revolutionizing organ transplantation and addressing the critical shortage of organs for transplantation. Xenotransplantation refers specifically to the transplantation of organs, tissues, or cells between different species, most commonly from animals to humans. For decades, organ transplantation has been a life-saving procedure for patients suffering from end-stage organ failure. However, the demand for organs significantly exceeds the available supply, resulting in extensive waiting lists, prolonged suffering, and unfortunate deaths. Xenotransplantation presents a potential solution to this problem by utilizing organs from genetically engineered animals, primarily pigs, for transplantation into humans.

Pigs have been chosen as the most suitable donors due to their physiological similarities to humans, organ size compatibility, the ease of breeding and genetic modification 1,2, and the availability of a high resolution map of the genome 3. Pigs are also very efficient at reproduction and have a relatively short generation interval. Other livestock species are also being used as a source of biological materials for xenotransplantation or as bioreactors for products to be used in therapeutic or biomedical applications 4-6. Furthermore, other valid alternative options, such as cattle to produce bioprosthetic heart valves can also be envisaged 7. Nevertheless, xenotransplantation faces numerous scientific, immunological, and ethical challenges that must be overcome to ensure its success and widespread acceptance 8,9. One of the primary hurdles in xenotransplantation is the severe immune rejection response triggered by the human recipient’s immune system against the foreign pig organs 10. Researchers have been diligently working to develop innovative strategies such as genetic engineering techniques to produce pigs with organs that are less likely to be rejected by the human immune system and on the other side to develop more effective immunosuppression therapy 11. These genome modifications involve the removal or alteration of specific genes responsible for the synthesis of molecules eliciting an immune response or the addition of specific human genes to make the organs more compatible with the human immune system. An example of xenotransplantation already in clinical use are bioprosthetic heart valves of animal origin, manufactured with wild type tissue 12 but they undergo calcification as a consequence of immune response 13. Pig islet xenotransplantation from wild type animals has entered clinical trials 14. It is likely that the clinical outcome could be significantly improved with the use of appropriately engineered animals as source materials. Life supporting solid organs transplanted into nonhuman primates, however, still do not survive long enough to warrant implementation of clinical trials 15 although heterotopic heart transplantation in a primate model has now resulted in the remarkable survival of almost 3 years 16 and in orthotopic transplantation for 195 days 17. Several immunological hurdles have been identified (Tab. I) and these are currently being addressed at multiple levels through genetic engineering. Moreover, ensuring the safety of xenotransplantation procedures is of paramount importance. Concerns about the transmission of infectious diseases, particularly viruses, from animals to humans, such as porcine endogenous retroviruses (PERVs) 18 or porcine cytomegalovirus (PCMV) 19 have been a subject of intense scrutiny. Extensive research is being conducted to address these concerns and develop comprehensive screening and monitoring protocols to minimize the risk of cross-species infections 20. However to date there has never been a report of PERV transmission to humans patients following tissue xenotransplantation 21-23. Despite this context pig solid organs with several genome edits are recently being transplanted to human brain-dead decedent patients 24,25 and for the first time to a patient on a compassionate use 26. Despite the extensive screening of the donor pig possible failure after 60 days was likely attributable to the presence of PCMV (porcine cytomegalovirus) in the heart of the donor pig 27. Ethical considerations also surround xenotransplantation. It raises questions about the welfare and rights of animals involved in the research and potential long-term consequences for animal populations. Striking a balance between the potential benefits to human health and the ethical treatment of animals remains a critical challenge that must be addressed through robust regulations and ethical frameworks. While xenotransplantation holds tremendous promise, it is essential to approach this field with cautious optimism, ensuring rigorous scientific investigation, regulatory oversight, and public engagement. If successful, xenotransplantation has the potential to revolutionize organ transplantation, significantly alleviate human suffering, and enhance the quality of life for countless individuals in need of life-saving interventions. With the advent of somatic cell nuclear transfer 28 and now the spectacular development of highly specific synthetic programmable endonucleases 29 the generation of genetically engineered pig lines has grown exponentially in the last 5 to 10 years. Genome editing of pigs for xenotransplantation, specifically using the CRISPR-Cas9 system in various declinations 30, has been a topic of significant scientific research and discussion. The primary objective of genome editing in this context is to modify specific genes in the pig’s genome to address the immunological and physiological barriers that exist between humans and pigs. By making targeted genetic modifications, scientists hope to create pigs that are more compatible with human recipients and reduce the risk of rejection or transmission of diseases. The genetic engineering of animals’ genome to be realized requires the use of advanced assisted reproduction techniques to generate the animals starting from engineered cells. The enabling technique for this purpose has been SCNT (Somatic Cell Nuclear Transfer) better known as cloning 31. Several edits are required at the same time or are added by re-cloning of already generated animals with a multi-stacking approach 32,33. However for single or simple edits microinjection into zygotes is considered a viable option in some circumstances other than xenotransplantation 34. It’s important to note that genome editing for xenotransplantation is still an active area of research, and there are significant challenges and ethical considerations that need to be addressed before xenotransplantation becomes a viable clinical option. While progress has been made in modifying pig genomes, further research is required to ensure the safety and efficacy of xenotransplantation procedures and develop novel immunosuppression therapies. Further down the road another route, that is being actively explored is aimed at generating pig-human chimeric organs with greater scientific and ethical challenges 35. If a pig organ is made up of human cells, the human immune system should better tolerate it. In this scenario, defective pig embryos for one target organ can be generated by genetic engineering and SCNT technology and then aggregated with pluripotent stem cells (PSCs) of human origin (blastocyst complementation), and thus during the development of the resulting animal the defective organ will be generated by the PSCs 36,37. The question still to be addressed however is how to prevent that human PSC will contribute to other organs of the pig like the brain for example. This review will address the different steps and challenges that need to be addressed to generate a viable animal starting from the selection of the target gene, introduction into the genome of a somatic cell to the cloning and birth of the animals.

GENETIC ENGINEERING (GE) OF THE CELL LINE FOR SOMATIC CELL NUCLEAR TRANSFER (SCNT)

Primary cell lines have a finite lifespan in vitro, undergoing senescence after a certain number of population doublings. This window of time is however sufficient to introduce one round of genetic modifications.

The last twenty years have seen the advent and development of programmable nucleases for precise editing of the genome 29,38-40. What was used in the past to achieve genetic modification essentially exploited the cell DNA repair mechanisms (NHEJ, non-homologous end-joining or HDR, homology directed repair), taking advantage of the double strand DNA breaks occurring spontaneously at a very low pace throughout the genome. With the use of current technology the frequency of DNA breaks is enhanced by a few logs times by the precise cutting ability of the programmable nucleases at selected target sequences. Amongst the programmable nucleases used today for genome editing (Tab. II), CRISPR/Cas9 is the most widely used because of its ease to use, flexibility 41 and low cost and more variants will become available in the future 30. The full exploitation of the technologies requires accurate DNA sequencing data as well as the software tools necessary for nuclease design, target site selection and experimental validation 42-44 to ensure efficiency and avoid undesired side effects in other genomic loci.

These types of nucleases have all been used to successfully edit the genome in a variety of organisms including livestock species both for agricultural 45,46 and biomedical applications 47,48 to mention only a few. In the biomedical arena one species of long standing interest for genetic modification has been the pig for xenotransplantation research, usually targeting one specific locus 49 or two 50. When inactivation of a specific endogenous gene(s) is needed then the knockout (KO) approach is required. Firstly this has been the case for the galactose 1-3 galactose epitope by genetic inactivation of the enzyme responsible for its expression (GGTA1, Alpha-1,3-galactosyltransferase) 51, followed by the KO of the enzyme CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) responsible for the synthesis of Neu5Gc antigen 50,52-54 and more recently by the KO of B4GalNT2 (1,4-N-acetylgalactosaminyltransferase) gene, done simultaneously with the KO of GGTA1 and CMAH 55. More recently CRISPR/Cas9 has been implemented to a degree of efficiency to obtain the multiple simultaneous mutation of 3 xenoantigens in the pig 54,55. In additional developments of the CRISPR system the cytosine base editor (CBE) has been used to convert C to T with high efficiency without causing DSBs. This technique has been developed to silence endogenous genes through directly induced nonsense mutations, which is much safer than ZFN or Cas9 56,57. Because this process is carried out on cells cultured in vitro, there are large margins at very limited cost to select amongst many the cell clones carrying the exact desired mutation. Then, by using SCNT, an animal originating from that genotype can be obtained in a relatively short time. Although the direct injection of CRISPR/Cas9 into zygotes can also work in creating GE animals 58,59, when a multiplexed GE is required, the efficiency of HDR mediated KI (Knock In) is low. In addition there is the risk of having a mosaic animal, due to editing taking place at later cleavage stages of the embryo, not in all blastomeres and as a consequence may not be transmitted to the offspring 60. This possibility represents a too high risk in livestock species that have long generation intervals compared with the direct derivation of an animal by SCNT from the selected and validated cell clone carrying the desired modification.

Moreover the possibility to target the transgenes of human origin, controlling complement activation, coagulation or inflammation, at a specific location in the pig genome, in so called “safe harbor” loci, such as ROSA-26, 61-63, or in new characterized loci 64, AAVS1 or CCR5 identified in the mouse and human genomes 65,66, can control undesirable side-effects, ensure single integration and sustain expression through successive generations. Indeed for xenotransplantation work the “safe harbor” can be the same target for knock out (KO) of xenoantigens like the GGTA1 67 or the CMAH genes. The targeting at a specific permissive locus can facilitate single copy integration, allow transcription without disruption of endogenous genes. Furthermore, under circumstances of possible lethal effect on embryo development, inducible systems 68 could be used and in general more sophisticated technologies could be implemented to control gene functionality.

If the transgene, because of its biological activity, might require tissue specific expression in endothelial cells for example 69 or in insulin producing cells 70, side-effects of the transgene are reduced and GE is compatible with the life of the animal. Another approach to the control of transgene expression is the use of inducible promoters that can be activated by the administration to the animal of the required activator like tetracycline 71 or doxycycline 68. Under these circumstances, transgene expression can be triggered when required during the lifespan of the animal or after organ harvesting and transplantation to the recipient. A third approach involves RNA interference technology. This has been used to reduce expression of porcine endogenous retroviruses (PERVs) 72,73, because they are present in multiple copies in the porcine genome, or to reduce expression of the pig Tissue Factor 74 because the KO is not compatible with the survival of animals. In these contexts, siRNA is the best option available as it can reduce the expression of a single gene by up to 95% or more but does not eliminate it completely. When using commercial line of pigs one of the problems in the long term is the size of the organ that continue to grow after xenotransplantation creating obvious consequences to the recipient. Solution to this problem can include the selection of breeds of minipigs or to edit the genome of the farmed pig to knock out the receptor for the GH (growth hormone) that together with the size reduction might unfortunately result in a diseased phenotype 75,76.

SCNT AND BIRTH OF ANIMALS

Cell line preparation and selection

Cell line source is the first key variable in the process of SCNT embryo production and still one of the black boxes responsible for success or failure. Culture conditions, doubling numbers, oxygen tension 77 etc. can all contribute to the selection during in vitro culture of a particular cell population or a sub-population that influence the status of the chromatin and most importantly its susceptibility to be reprogrammed after nuclear transfer. Therefore, from a practical point of view, the identification of cell lines with a high SCNT efficiency can lead to astonishing results as opposed to cell lines that deliver huge failures. The most used cell types are fibroblasts from skin biopsies if the animal to be cloned should be of known genotype/phenotype. If this is not the case, then fetal fibroblasts are the most used cell type especially for GE. There are many reports with claims on the most efficient cell type to be used for cloning pigs 78,79 but this choice might be conflicting with the need to use a particular cell line required for a specific project. GE of the cell line to be used for SCNT generally does not change its ability to be used successfully for generating offspring and a slight reduction was observed in the case of gene KO (knock-out) experiments 79,78. All cell lines can easily be cryopreserved at early passages before GE, ensuring that the same cells can be used repeatedly in nuclear transfer rounds while controlling for a key variable in the procedure. In the case of GE because of the clonal selection required for screening for the correct mutation, the cells undergo to a high number of population doublings bringing the cells to the limit of senescence that might reduce SCNT efficiency.

Embryo production

Over the years the technique of cloning by nuclear transfer in livestock has not changed in the basic principles pioneered by Willadsen 80 and further developed with somatic cells 28. The first step is the preparation of a matured enucleated oocyte whereby the metaphase plate is removed from matured oocyte by micromanipulation. In a second step a nucleus coming from a somatic cell that carries the desired mutations is transferred to the enucleated oocyte. Finally in the third step, electrical or chemical activation is induced to resume the cell cycle in the oocyte. The reconstructed embryos are either transferred at one cell stage to the oviducts of synchronized recipients or cultured to the blastocyst stage that can then be transferred to the uterus. The large number of metaphase II oocytes required for embryo production in these species can easily be sourced from slaughterhouses at very low cost and in respect of the 3R principle. The procedures to mature oocytes and culture embryos are well established in the pig 81 and the same are used for SCNT as well. The micromanipulation work is still a bottleneck of the technology as it is labor intensive, it requires specialized equipment and above all experienced embryologists. The metaphase plate can effectively be visualized with Hoechst staining and UV light exposure because the cytoplasm of livestock species is rich in lipids, making them dark compared to the mouse. A scaling up of the nuclear transfer procedure can be to some extend implemented with what is known as Handmade Cloning 82 or in its various declinations 83 by removing the zona pellucida to facilitate enucleation. However, from a practical point of view, being without zona pellucida it requires that the embryos are cultured in vitro, in special dish to avoid sticking them together prior to transfer until the blastocyst stage. Pre-implantation SCNT embryos do have a reduced potential to develop to term, despite having normal morphology. Developmental competence is depending on another “black box” that is cellular reprogramming, i.e how the nucleus of the donor cell is reset to direct normal embryo development. At present it is a very inefficient process that has slowly been re-winded essentially in the mouse 84 and it is only at the beginning for livestock species to be fully understood 85,86. In mouse, significant improvements in livebirth rates have been obtained with the use of Trichostatin A (TSA), a histone deacetylase inhibitor, during the first few hours of culture of the reconstructed embryo after nuclear transfer to help demethylation of the chromatin to favor reprogramming 87. Similar approaches with a variety of demethylating agents have been explored successfully in some laboratories in the pig 88.

Pregnancy

Upon transfer, the ability of SCNT embryos to establish pregnancies is by and large lower than that of embryos obtained by fertilization and this has an economic impact because of the cost of carrying recipients not being pregnant or loosing the pregnancy. This can be partially compensated especially in the pig by the transfer of an excess of embryos as SCNT embryo production usually is not a limiting factor. In the pig this is well tolerated, even the transfer of over 100 embryos, since this species can adjust for the number of fetuses developing by physiological reabsorption of the excessive number of embryos developing. Another issue with SCNT pregnancies is the prolonged gestation period usually requiring, induction of parturition and or caesarian C section.

Offspring

Depending on how the success rate is calculated, on the reconstructed embryos or on the transferred blastocyst, development to term can be up to 16% 89, although many variables are responsible of this rate including the pig line, the type of genetic modification introduced into the cells, etc. making comparisons impossible 79,90. In general, SCNT offspring at birth are more fragile animals and have a higher perinatal mortality. To optimize the survival of SCNT derived offspring, special attention should be given to farrowing and neonatal care. Once the first few days or weeks are over, then the cloned animals have a normal life. They are also fertile and, most importantly, possible phenotypical deviation observed are not observed in their offspring 91-94. This is an important aspect to be taken into consideration for the commercial application of this technology. A contributing factor to the successful generation of viable animals that can then be successfully bred by natural means is how many genome edits can be tolerated/necessary 32, if those edits are compatible with the homeostasis of a normal animal 95 and that the expression of transgenes inserted are maintained at the desired level. This requires a confirmation by a genotyping and phenotyping of the newborn animals. What exact genetic modifications do we need in the organ-source pig should be fully considered in the future and might require a more systematic approach to assess one edit at a time before going further. An example of excessive, maybe not necessary genome engineering, was the knock-out of all 64 copies of the PERVs present in the genome of one pig line 18 since there is no evidence that there has been in past xenotransplantation experiments transmission to humans 21-23, taking also into consideration that PERV might have physiological role in the genome 96,97 that has yet to be unraveled.

CONCLUSIONS

Significant progress has been made in pig genetic engineering to create pigs with organs suitable for transplantation into humans. Researchers have used gene-editing techniques such as CRISPR/Cas9 to modify pig genes associated with organ rejection, viral transmission, and immunological compatibility. This has led to the generation of many genetically modified pigs with reduced immunological barriers and increased compatibility with human recipients. One of the main challenges in xenotransplantation is the immunological response triggered by pig organs in human recipients. Pigs possess certain genetic elements that can trigger a severe immune response in humans, leading to organ rejection. Genetic engineering aims to address this barrier by modifying or eliminating these elements. While progress has been made, further research is needed to ensure long-term graft survival and prevent immune-mediated rejection. Another critical concern in xenotransplantation is the risk of transmitting porcine viruses or other pathogens to humans. However, comprehensive testing and monitoring protocols must be established to ensure the safety of xenotransplantation in terms of viral transmission. The genetic engineering of pigs for xenotransplantation raises ethical considerations regarding animal welfare and the potential consequences of modifying animal genomes. It is essential to ensure that the welfare of genetically modified pigs is safeguarded, and rigorous ethical frameworks are in place to guide their creation and use. Xenotransplantation involving genetically modified pigs is a complex and highly regulated field. Before xenotransplantation can become a routine clinical practice, extensive preclinical studies, safety assessments, and regulatory approvals are necessary. Clinical trials are required to evaluate the safety and effectiveness of pig organ transplantation in humans. In conclusion, pig genetic engineering for xenotransplantation shows promising potential to address the organ shortage crisis. While significant progress has been made, further research is needed to overcome immunological barriers, minimize the risk of pathogen transmission, address ethical considerations, and navigate regulatory and clinical challenges. Continued scientific advancements, robust safety measures, and thoughtful ethical considerations will be crucial for the successful translation of pig genetic engineering into safe and effective xenotransplantation therapies.

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This review did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethical consideration

Not applicable.

Figures and tables

Problem Possible cause Possible solution
Hyperacute rejection (HAR) Pre-formed antibodies against Galactose 1-,3-galactose and other non-Gal antigens (Neu5Gc); activation of the complement cascade. KO of 1-3 galctosyltransferase, CMAH, B4GALNT2, iGb3S and other non-Gal antigens Expression of hCRP (CD55, CD46, CD59)
Acute humoral xenograft rejection (AHXR) De novo antibodies against Galactose 1-,3-galactose and other non-Gal antigens (Neu5Gc); activation of the complement cascade. Endothelial cell activation; Thrombotic microangiopathy Consumptive coagulopathy hTBM, hEPCR, hA20, TFPI, CD39,HMOX1
Immune cell-mediated rejection (ICMR) NK and T-cell activation hTRAIL, CTLA4Ig, HLA-E, hu 2m, CD47, SLA class I
Instant Blood-Mediated Inflammatory Reaction (IBMIR) Surface proteins, complement mediated, innate immunity, platelets and leucocytes activation All of the above genetic modifications
KO: Knock Out; Neu5Gc: N-Glycolylneuraminic acid; CMAH: CMP-N-acetylneuraminic acid hydroxylase; B4GALNT2: Beta-1,4-N-Acetyl-Galactosaminyl Transferase 2 ; iGb3S : isogloboside 3; hCRP: human complement regulatory proteins; hEPCR: human endothelial protein C receptor; TFPI: tissue factor pathway inhibitor; TRAIL: human tumor necrosis factor related apoptosis inducing ligand.
Table I. Immunological barriers to xenotransplantation that can be abrogated through genetic engineering (from Perota and Galli, 2016, mod.).
Zinc finger nuclease TALEN CRISPR/Cas9
Recognition site Typically 9–18 bp per ZFN monomer, 18–36 bp per ZFN pair Typically 14–20 bp per TALEN monomer, 28–40 bp per TALEN pair 22 bp (20-bp guide sequence + 2-bp protospacer adjacent motif (PAM) for Streptococcus pyogenes Cas9); up to 44 bp for double nicking
Specificity Small number of positional mismatches tolerated Small number of positional mismatches tolerated Positional and multiple consecutive mismatches tolerated
Targeting constraints Difficult to target non-G-rich sequences 5 targeted base must be a T for each TALEN monomer Targeted sequence must precede a PAM
Ease of engineering Difficult; may require substantial protein engineering Moderate; requires complex molecular cloning methods Easily re-targeted using standard cloning procedures and oligo synthesis
Immunogenicity Likely low, as zinc fingers are based on human protein scaffold; FokI is derived from bacteria and may be immunogenic Unknown; protein derived from Xanthamonas sp. Unknown; protein derived from various bacterial species
Ease of ex-vivo delivery Relatively easy through methods such as electroporation and viral transduction Relatively easy through methods such as electroporation and viral transduction Relatively easy through methods such as electroporation and viral transduction
Ease of in vivo delivery Relatively easy as small size of ZFN expression cassettes allows use in a variety of viral vectors Difficult due to the large size of each TALEN and repetitive nature of DNA encoding TALENs, leading to unwanted recombination events when packaged into lentiviral vectors Moderate: the commonly used Cas9 from S. pyogenes is large and may impose packaging problems for viral vectors such as AAV, but smaller orthologs exist
Ease of multiplexing Low Low High
Table II. Comparison of different programmable nuclease platforms used in livestock genome editing (adapted from Cox et al., 2015, mod. with permission from the Publisher) 38.

References

  1. Yue Y, Xu W, Kan Y. Extensive germline genome engineering in pigs. Nat Biomed Eng. 2021;5:134-143. doi:https://doi.org/10.1038/s41551-020-00613-9
  2. Walters E, Wolf E, Whyte J. Completion of the swine genome will simplify the production of swine as a large animal biomedical model. BMC medical genomics. 2012;5. doi:https://doi.org/10.1186/1755-8794-5-55
  3. Groenen M, Archibald A, Uenishi H. Analyses of pig genomes provide insight into porcine demography and evolution. Nature. 2012;491:393-398. doi:https://doi.org/10.1038/nature11622
  4. Kuroiwa Y, Kasinathan P, Choi Y. Cloned transchromosomic calves producing human immunoglobulin. Nat Biotechnol. 2002;20:889-894. doi:https://doi.org/10.1038/nbt727
  5. Kues W, Niemann H. The contribution of farm animals to human health. Trends Biotechnol. 2004;22:286-294. doi:https://doi.org/10.1016/j.tibtech.2004.04.003
  6. Vanhove B, Duvaux O, Rousse J. High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2. Eur J Immunol. 2021;51:1412-1422. doi:https://doi.org/10.1002/eji.202049072
  7. Perota A, Lagutina I, Duchi R. Generation of cattle knockout for galactose-alpha1,3-galactose and N-glycolylneuraminic acid antigens. Xenotransplantation. 2019;26. doi:https://doi.org/10.1111/xen.12524
  8. Reichart B, Cooper D, Langin M. Cardiac xenotransplantation: from concept to clinic. Cardiovasc Res. 2023;118:3499-3516. doi:https://doi.org/10.1093/cvr/cvac180
  9. Cooper D, Pierson R. Milestones on the path to clinical pig organ xenotransplantation. Am J Transplant. 2023;23:326-335. doi:https://doi.org/10.1016/j.ajt.2022.12.023
  10. Griesemer A, Yamada K, Sykes M. Xenotransplantation: immunological hurdles and progress toward tolerance. Immunol Rew. 2014;258:241-258. doi:https://doi.org/10.1111/imr.12152
  11. Vadori M, Cozzi E. Immunological challenges and therapies in xenotransplantation. Cold Spring Harb Perspect Med. 2014;4. doi:https://doi.org/10.1101/cshperspect.a015578
  12. Naso F, Gandaglia A, Bottio T. First quantification of alpha-Gal epitope in current glutaraldehyde-fixed heart valve bioprostheses. Xenotransplantation. 2013;20:252-261. doi:https://doi.org/10.1111/xen.12044
  13. Senage T, Paul A, Le Tourneau T. The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration. Nature Med. 2022;28:283-294. doi:https://doi.org/10.1038/s41591-022-01682-w
  14. Coe T, Markmann J, Rickert C. Current status of porcine islet xenotransplantation. Curr Opin Organ Transplant. 2020;25:449-456. doi:https://doi.org/10.1097/MOT.0000000000000794
  15. Tector A, Adams A, Tector M. Current status of renal xenotransplantation and next steps. Kidney360. 2023;4:278-284. doi:https://doi.org/10.34067/KID.0007152021
  16. Mohiuddin M, Singh A, Corcoran P. Critical need of continuous co-stimulation blockade with anti CD40 antibody (2C10.R4) for long-term maintenance of GTKO.HCD46.hTBM pig cardiac xenograft survival in baboons. Xenotransplantation (Abstracts of the IPITA-IXA-CTS 2015 Joint Congress November 15-19, 2015, Melbourne, Australia). 2015;22:S121-S184. doi:https://doi.org/10.1111/xen.12206
  17. Langin M, Mayr T, Reichart B. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature. 2018;564:430-433. doi:https://doi.org/10.1038/s41586-018-0765-z
  18. Niu D, Wei H, Lin L. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 2017;357:1303-1307. doi:https://doi.org/10.1126/science.aan4187
  19. Denner J. Porcine endogenous retroviruses and xenotransplantation, 2021. Viruses. Published online 2021. doi:https://doi.org/10.3390/v13112156
  20. Fishman J. Risks of infectious disease in xenotransplantation. N Engl J Med. 2022;387:2258-2267. doi:https://doi.org/10.1056/NEJMra2207462
  21. Scobie L, Padler-Karavani V, Le Bas-Bernardet S. Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts. J Immunol. 2013;191:2907-2915. doi:https://doi.org/10.4049/jimmunol.1301195
  22. Wang W, Mo Z, Ye B. A clinical trial of xenotransplantation of neonatal pig islets for diabetic patients. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2011;36:1134-1140. doi:https://doi.org/10.3969/j.issn.1672-7347.2011.12.002
  23. Morozov V, Wynyard S, Matsumoto S. No PERV transmission during a clinical trial of pig islet cell transplantation. Virus Res. 2017;227:34-40. doi:https://doi.org/10.1016/j.virusres.2016.08.012
  24. Montgomery R, Stern J, Lonze B. Results of two cases of pig-to-human kidney xenotransplantation. N Engl J Med. 2022;386:1889-1898. doi:https://doi.org/10.1056/NEJMoa2120238
  25. Porrett P, Orandi B, Kumar V. First clinical-grade porcine kidney xenotransplant using a human decedent model. Am J Transplant. 2022;22:1037-1053. doi:https://doi.org/10.1111/ajt.16930
  26. Griffith B, Goerlich C, Singh A. Genetically modified porcine-to-human cardiac xenotransplantation. N Engl J Med. 2022;387:35-44. doi:https://doi.org/10.1056/NEJMoa2201422
  27. Denner J. The porcine cytomegalovirus (PCMV) will not stop xenotransplantation. Xenotransplantation. 2022;29. doi:https://doi.org/10.1111/xen.12763
  28. Wilmut I, Schnieke A, McWhir J. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997;385:810-813. doi:https://doi.org/10.1038/385810a0
  29. Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem. 2014;83:409-439. doi:https://doi.org/10.1146/annurev-biochem-060713-035418
  30. Anzalone A, Koblan L, Liu D. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol. 2020;38:824-844. doi:https://doi.org/10.1038/s41587-020-0561-9
  31. Galli C, Lazzari G. 25th Anniversary of cloning by Somatic-cell nuclear transfer: current applications of SCNT in advanced breeding and genome editing in livestock. Reproduction. 2021;162:F23-F32. doi:https://doi.org/10.1530/REP-21-0006
  32. Kemter E, Schnieke A, Fischer K. Xeno-organ donor pigs with multiple genetic modifications – the more the better?. Curr Opin Genet Dev. 2020;64:60-65. doi:https://doi.org/10.1016/j.gde.2020.05.034
  33. Fischer K, Kraner-Scheiber S, Petersen B. Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing. Scientific Rep. 2016;6. doi:https://doi.org/10.1038/srep29081
  34. Lee K, Uh K, Farrell K. Current progress of genome editing in livestock. Theriogenology. 2020;150:229-235. doi:https://doi.org/10.1016/j.theriogenology.2020.01.036
  35. Wolf E, Reichart B, Moretti A. Designer pigs for xenogeneic heart transplantation and beyond. Dis Model Mech. Published online 2023. doi:https://doi.org/10.1242/dmm.050177
  36. Matsunari H, Watanabe M, Hasegawa K. Compensation of disabled organogeneses in genetically modified pig fetuses by blastocyst complementation. Stem Cell Rep. 2020;14:21-33. doi:https://doi.org/10.1016/j.stemcr.2019.11.008
  37. Das S, Koyano-Nakagawa N, Gafni O. Generation of human endothelium in pig embryos deficient in ETV2. Nat Biotechnol. 2020;38:297-302. doi:https://doi.org/10.1038/s41587-019-0373-y
  38. Cox D, Platt R, Zhang F. Therapeutic genome editing: prospects and challenges. Nature med. 2015;21:121-131. doi:https://doi.org/10.1038/nm.3793
  39. Doudna J, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346. doi:https://doi.org/10.1126/science.1258096
  40. Urnov F, Rebar E, Holmes M. Genome editing with engineered zinc finger nucleases. Nature Rev. 2010;11:636-646. doi:https://doi.org/10.1038/nrg2842
  41. Zhang X, Tee L, Wang X. Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy Nucleic Acids. 2015;4. doi:https://doi.org/10.1038/mtna.2015.37
  42. Lee C, Cradick T, Fine E. Nuclease target site selection for maximizing on-target activity and minimizing off-target effects in genome editing. Mol Ther. 2016;24:475-487. doi:https://doi.org/10.1038/mt.2016.1
  43. Graham D, Root D. Resources for the design of CRISPR gene editing experiments. Genome Biol. 2015;16. doi:https://doi.org/10.1186/s13059-015-0823-x
  44. Naito Y, Hino K, Bono H. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. 2015;31:1120-1123. doi:https://doi.org/10.1093/bioinformatics/btu743
  45. Tan W, Proudfoot C, Lillico S. Gene targeting, genome editing: from Dolly to editors. Transgenic Res. 2016;25:273-287. doi:https://doi.org/10.1007/s11248-016-9932-x
  46. Lillico S, Proudfoot C, King T. Mammalian interspecies substitution of immune modulatory alleles by genome editing. Scientific Rep. 2016;6. doi:https://doi.org/10.1038/srep21645
  47. Wang X, Zhou J, Cao C. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Scientific Rep. 2015;5. doi:https://doi.org/10.1038/srep13348
  48. Wang Y, Du Y, Shen B. Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA. Scientific Rep. 2015;5. doi:https://doi.org/10.1038/srep08256
  49. Hauschild J, Petersen B, Santiago Y. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A. 2011;108:12013-12017. doi:https://doi.org/10.1073/pnas.1106422108
  50. Lutz A, Li P, Estrada J. Double knockout pigs deficient in N-glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation. 2013;20:27-35. doi:https://doi.org/10.1111/xen.12019
  51. Phelps C, Koike C, Vaught T. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003;299:411-414.
  52. Concordet J, Judor J. Generation of CMAH-/- piglets on GAL-/- genetic background. Xenotransplantation. 2013;20:370-371.
  53. Kwon D, Lee K, Kang M. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs. Scientific Rep. 2013;3. doi:https://doi.org/10.1038/srep01981
  54. Li P, Estrada J, Burlak C. Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection. Xenotransplantation. 2015;22:20-31. doi:https://doi.org/10.1111/xen.12131
  55. Estrada J, Martens G, Li P. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes. Xenotransplantation. 2015;22:194-202. doi:https://doi.org/10.1111/xen.12161
  56. Komor A, Kim Y, Packer M. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533:420-424. doi:https://doi.org/10.1038/nature17946
  57. Xie J, Ge W, Li N. Efficient base editing for multiple genes and loci in pigs using base editors. Nature Comm. 2019;10. doi:https://doi.org/10.1038/s41467-019-10421-8
  58. Menchaca A, Dos Santos-Neto P, Mulet A. CRISPR in livestock: from editing to printing. Theriogenology. 2020;150:247-254. doi:https://doi.org/10.1016/j.theriogenology.2020.01.063
  59. Lee K, Farrell K, Uh K. Application of genome-editing systems to enhance available pig resources for agriculture and biomedicine. Reproduct Fertil Develop. 2019;32:40-49. doi:https://doi.org/10.1071/rd19273
  60. Yen S, Zhang M, Deng J. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Develop Biol. 2014;393:3-9. doi:https://doi.org/10.1016/j.ydbio.2014.06.017
  61. Kong Q, Hai T, Ma J. Rosa26 locus supports tissue-specific promoter driving transgene expression specifically in pig. PloS One. 2014;9. doi:https://doi.org/10.1371/journal.pone.0107945
  62. Li S, Flisikowska T, Kurome M. Dual fluorescent reporter pig for Cre recombination: transgene placement at the ROSA26 locus. PloS One. 2014;9. doi:https://doi.org/10.1371/journal.pone.0102455
  63. Li X, Yang Y, Bu L. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Res. 2014;24:501-504. doi:https://doi.org/10.1038/cr.2014.15
  64. Garrels W, Mukherjee A, Holler S. Identification and re-addressing of a transcriptionally permissive locus in the porcine genome. Transgenic Res. 2016;25:63-70. doi:https://doi.org/10.1007/s11248-015-9914-4
  65. Sadelain M, Papapetrou E, Bushman F. Safe harbours for the integration of new DNA in the human genome. Nature Rev Cancer. 2012;12:51-58. doi:https://doi.org/10.1038/nrc3179
  66. Ruan J, Li H, Xu K. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Scientific Rep. 2015;5. doi:https://doi.org/10.1038/srep14253
  67. Ko N, Shim J, Kim H. A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model. Scientific Rep. 2022;12. doi:https://doi.org/10.1038/s41598-022-13536-z
  68. Klymiuk N, Bocker W, Schonitzer V. First inducible transgene expression in porcine large animal models. FASEB J. 2012;26:1086-1099. doi:https://doi.org/10.1096/fj.11-185041
  69. Cowan P, Shinkel T, Fisicaro N. Targeting gene expression to endothelium in transgenic animals: a comparison of the human ICAM-2, PECAM-1 and endoglin promoters. Xenotransplantation. 2003;10:223-231.
  70. Aigner B, Klymiuk N, Wolf E. Transgenic pigs for xenotransplantation: selection of promoter sequences for reliable transgene expression. Curr Opin Organ Transplant. 2010;15:201-206. doi:https://doi.org/10.1097/MOT.0b013e328336ba4a
  71. Kues W, Schwinzer R, Wirth D. Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs. FASEB J. 2006;20:1200-1202. doi:https://doi.org/10.1096/fj.05-5415fje
  72. Ramsoondar J, Vaught T, Ball S. Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs. Xenotransplantation. 2009;16:164-180. doi:https://doi.org/10.1111/j.1399-3089.2009.00525.x
  73. Dieckhoff B, Petersen B, Kues W. Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs. Xenotransplantation. 2008;15:36-45. doi:https://doi.org/10.1111/j.1399-3089.2008.00442.x
  74. Ahrens H, Petersen B, Herrmann D. siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation. Am J Transplantation. 2015;15:1407-1414. doi:https://doi.org/10.1111/ajt.13120
  75. Hinrichs A, Riedel E, Klymiuk N. Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies. Xenotransplantation. 2021;28. doi:https://doi.org/10.1111/xen.12664
  76. Hinrichs A, Kessler B, Kurome M. Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver. Mol Metab. 2018;11:113-128. doi:https://doi.org/10.1016/j.molmet.2018.03.006
  77. Mordhorst B, Benne J, Cecil R. Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia. Molecul Reproduct Develop. 2019;86:558-565. doi:https://doi.org/10.1002/mrd.23132
  78. Liu T, Dou H, Xiang X. Factors determining the efficiency of porcine somatic cell nuclear transfer: data analysis with over 200,000 reconstructed embryos. Cellular Reprogr. 2015;17:463-471. doi:https://doi.org/10.1089/cell.2015.0037
  79. Kurome M, Geistlinger L, Kessler B. Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set. BMC Biotechnol. 2013;13. doi:https://doi.org/10.1186/1472-6750-13-43
  80. Willadsen S. Nuclear transplantation in sheep embryos. Nature. 1986;320:63-65. doi:https://doi.org/10.1038/320063a0
  81. Redel B, Spate L, Prather R. In vitro maturation, fertilization, and culture of pig oocytes and embryos. Methods Mol Biol. 2019;2006:93-103. doi:https://doi.org/10.1007/978-1-4939-9566-0_6
  82. Vajta G. Handmade cloning: the future way of nuclear transfer?. Trends Biotechnol. 2007;25:250-253. doi:https://doi.org/10.1016/j.tibtech.2007.04.004
  83. Oback B, Wiersema A, Gaynor P. Cloned cattle derived from a novel zona-free embryo reconstruction system. Cloning Stem Cells. 2003;5:3-12.
  84. Matoba S, Zhang Y. Somatic cell nuclear transfer reprogramming: mechanisms and applications. Cell stem cell. 2018;23:471-485. doi:https://doi.org/10.1016/j.stem.2018.06.018
  85. Liu X, Wang Y, Gao Y. H3K9 demethylase KDM4E is an epigenetic regulator for bovine embryonic development and a defective factor for nuclear reprogramming. Development. Published online 2018. doi:https://doi.org/10.1242/dev.158261
  86. Ruan D, Peng J, Wang X. XIST derepression in active X chromosome hinders pig somatic cell nuclear transfer. Stem Cell Rep. 2018;10:494-508. doi:https://doi.org/10.1016/j.stemcr.2017.12.015
  87. Kishigami S, Mizutani E, Ohta H. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem Biophys Res Commun. 2006;340:183-189. doi:https://doi.org/10.1016/j.bbrc.2005.11.164
  88. Zhao J, Ross J, Hao Y. Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod. 2009;81:525-530. doi:https://doi.org/10.1095/biolreprod.109.077016
  89. Lagutina I, Lazzari G, Galli C. Birth of cloned pigs from zona-free nuclear transfer blastocysts developed in vitro before transfer. Cloning Stem Cells. 2006;8:283-93. doi:https://doi.org/10.1089/clo.2006.8.283
  90. Huang Y, Ouyang H, Yu H. Efficiency of porcine somatic cell nuclear transfer – a retrospective study of factors related to embryo recipient and embryos transferred. Biol Open. 2013;2:1223-1228. doi:https://doi.org/10.1242/bio.20135983
  91. Fulka J, Miyashita N, Nagai T. Do cloned mammals skip a reprogramming step?. Nature Biotechnol. 2004;22:25-26. doi:https://doi.org/10.1038/nbt0104-25
  92. Tamashiro K, Wakayama T, Akutsu H. Cloned mice have an obese phenotype not transmitted to their offspring. Nature Med. 2002;8:262-267. doi:https://doi.org/10.1038/nm0302-262
  93. Heyman Y, Richard C, Rodriguez-Martinez H. Zootechnical performance of cloned cattle and offspring: preliminary results. Cloning Stem Cells. 2004;6:111-120.
  94. Cibelli J, Campbell K, Seidel G. The health profile of cloned animals. Nature Biotechnol. 2002;20:13-14. doi:https://doi.org/10.1038/nbt0102-13
  95. Sake H, Frenzel A, Lucas-Hahn A. Possible detrimental effects of beta-2-microglobulin knockout in pigs. Xenotransplantation. 2019;26. doi:https://doi.org/10.1111/xen.12525
  96. Mager D, Stoye J. Mammalian endogenous retroviruses. Microbiol Spectr. 2015;3. doi:https://doi.org/10.1128/microbiolspec.MDNA3-0009-2014
  97. Fu B, Ma H, Liu D. Endogenous retroviruses function as gene expression regulatory elements during mammalian pre-implantation embryo development. Int J Molecul Sci. Published online 2019. doi:https://doi.org/10.3390/ijms20030790

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Cesare Galli - Avantea and Fondazione Avantea Onlus, Cremona, Italy

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[1]
Galli, C. 2023. Animal engineering for xenotransplantation. European Journal of Transplantation. 1, 3 (Oct. 2023), 182–191. DOI:https://doi.org/10.57603/EJT-307.
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