Production and Functional Verification of 8-Gene (GGTA1, CMAH, β4GalNT2, hCD46, hCD55, hCD59, hTBM, hCD39)-Edited Donor Pigs for Xenotransplantation

Abstract

Gene-edited (GE) pig-to-human xenotransplantation continues to make breakthroughs, but which kind of gene combination is suitable for organ-specific transplantation remains unclear. In this study, we utilised CRISPR/Cas9 gene editing technology, PiggyBac transposon system, and serial somatic cell cloning technology to develop GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene-edited cloned (GEC) donor pigs and performed pig-to-non-human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The 8-GEC pigs were obtained by recloning with a 33-day-old 8-GEC fetus with O blood type, which was generated after cell transfection, screening of cell colonies, and somatic cell cloning. Molecular identification at DNA, mRNA, and protein levels confirmed successful 8-gene editing. Three copies of transgenes were identified by droplet digital polymerase chain reaction and whole genome sequencing, which were inserted into the introns of pig RFTN1 and MYO10 genes, as well as the intergenic region between PRLR and LOC110257300 genes of these 8-GEC pigs. The 8-GEC pigs also exhibited the ability of germline transmission when mated with our previously generated 4-GEC male pigs. Moreover, antigen–antibody binding assay and complement-dependent cytotoxicity assay demonstrated that 8-gene editing effectively reduced the immune incompatibility and kidney xenograft from 8-GEC pigs survived for 15 and 17 days in two NHPs, respectively. Postoperatively, the recipient serum antibodies IgA, IgG and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. Serum creatinine was normal within 10 days post operation. However, the kidney xenograft developed active antibody-mediated rejection at necropsy, characterised by the deposition of antibodies IgG and IgM, as well as complements C4d, C3c and C5b-C9, infiltration of CD68⁺ macrophages, and micro-thrombotic embolism of glomerular capillaries, etc. In conclusion, we successfully developed fertile 8-GEC pigs, which effectively alleviated immune rejection and exerted life-supporting kidney function in the recipients.

Keywords

complement regulatory protein / kidney xenotransplantation / pig / thrombomodulin / xenoantigens

References

[1] B. P. Griffith and C. E. Goerlich, “Genetically Modified Porcine-To-Human Cardiac Xenotransplantation,” New England Journal of Medicine 387, no. 1 (2022): 35-44, https://doi.org/10.1056/NEJMoa2201422.

[2] N. Moazami, J. M. Stern, K. Khalil, et al., “Pig-To-Human Heart Xenotransplantation in Two Recently Deceased Human Recipients,” Nature Medicine 29, no. 8 (2023): 1989-1997.

[3] M. Vadori and E. Cozzi, “Current Challenges in Xenotransplantation,” Current Opinion in Organ Transplantation 29, no. 3 (2024): 205-211.

[4] B. M. Kuehn, “Pig-To-Human Xenotransplants Take Another Step Forward,” Kidney News 15, no. 10 (2023): 7-8.

[5] R. A. Montgomery, J. M. Stern, B. E. Lonze, et al., “Results of Two Cases of Pig-To-Human Kidney Xenotransplantation,” New England Journal of Medicine 386, no. 20 (2022): 1889-1898.

[6] P. M. Porrett, B. J. Orandi, V. Kumar, et al., “First Clinical-Grade Porcine Kidney Xenotransplant Using a Human Decedent Model,” American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons 22, no. 4 (2022): 1037-1053, https://doi.org/10.1111/ajt.16930.

[7] J. E. Locke, V. Kumar, D. Anderson, and P. M. Porrett, “Normal Graft Function After Pig-To-Human Kidney Xenotransplant,” JAMA Surgery 158, no. 10 (2023): 1106-1108.

[8] Y. Wang, G. Chen, D. Pan, et al., “Pig-To-Human Kidney Xenotransplants Using Genetically Modified Minipigs,” Cell Reports Medicine 5, no. 10 (2024): 101744.

[9] L. Bernstein, “Pig Kidney Transplant in Brain-Dead Man Marks Advance, NYU Surgeons Say,” Washington Post (2023), https://www.washingtonpost.com/health/2023/08/16/pig-kidney-transplant-nyu.

[10] S. Mallapaty and M. Kozlov, “First Pig Kidney Transplant in a Person: What It Means for the Future,” Nature 628, no. 8006 (2024): 13-14.

[11] M. Langin, T. Mayr, B. Reichart, et al., “Consistent Success in Life-Supporting Porcine Cardiac Xenotransplantation,” Nature 564, no. 7736 (2018): 430-433.

[12] A. K. Singh, C. E. Goerlich, T. Zhang, et al., “Genetically Engineered Pig Heart Transplantation in Non-Human Primates,” Communications Medicine 5, no. 1 (2025): 6.

[13] S. C. Kim, D. V. Mathews, C. P. Breeden, et al., “Long-Term Survival of Pig-To-Rhesus Macaque Renal Xenografts Is Dependent on CD4 T Cell Depletion,” American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons 19, no. 8 (2019): 2174-2185, https://doi.org/10.1111/ajt.15329.

[14] R. P. Anand, J. V. Layer, D. Heja, et al., “Design and Testing of a Humanized Porcine Donor for Xenotransplantation,” Nature 622, no. 7982 (2023): 393-401.

[15] Q. Li and P. Lan, “Activation of Immune Signals During Organ Transplantation,” Signal Transduction and Targeted Therapy 8, no. 1 (2023): 110.

[16] H. Hara, T. Yamamoto, H.-J. Wei, and D. K. Cooper, “What Have We Learned From In Vitro Studies About Pig-To-Primate Organ Transplantation?,” Transplantation 107, no. 6 (2023): 1265-1277.

[17] P. J. Cowan and S. C. Robson, “Progress Towards Overcoming Coagulopathy and Hemostatic Dysfunction Associated With Xenotransplantation,” International Journal of Surgery 23 (2015): 296-300.

[18] K. R. McCurry, D. L. Kooyman, C. G. Alvarado, et al., “Human Complement Regulatory Proteins Protect Swine-To-Primate Cardiac Xenografts From Humoral Injury,” Nature Medicine 1, no. 5 (1995): 423-427, https://doi.org/10.1038/nm0595-423.

[19] R. N. Pierson, A. Dorling, D. Ayares, et al., “Current Status of Xenotransplantation and Prospects for Clinical Application,” Xenotransplantation 16, no. 5 (2009): 263-280, https://doi.org/10.1111/j.1399-3089.2009.00534.x.

[20] M. J. Whitley, J. Suwanpradid, C. Lai, et al., “ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair Through Purinergic Signaling and Is Associated With Metastasis in Human Cutaneous Squamous Cell Carcinoma,” Journal of Investigative Dermatology 141, no. 10 (2021): 2509-2520.

[21] C. Yang, Y. Wei, X. Li, et al., “Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation,” Xenotransplantation 31, no. 4 (2024): e12881.

[22] H. Wei, Y. Qing, W. Pan, et al., “Comparison of the Efficiency of Banna Miniature Inbred Pig Somatic Cell Nuclear Transfer Among Different Donor Cells,” PLoS One 8, no. 2 (2013): e57728.

[23] S. Fisher, A. Barry, J. Abreu, et al., “A Scalable, Fully Automated Process for Construction of Sequence-Ready Human Exome Targeted Capture Libraries,” Genome Biology 12, no. 1 (2011): R1.

[24] D. A. Wheeler, M. Srinivasan, M. Egholm, et al., “The Complete Genome of an Individual by Massively Parallel DNA Sequencing,” Nature 452, no. 7189 (2008): 872-876.

[25] S. Chen, Y. Zhou, Y. Chen, and J. Gu, “Fastp: An Ultra-Fast All-In-One FASTQ Preprocessor,” Bioinformatics (Oxford, England) 34, no. 17 (2018): i884-i890.

[26] V. Md, S. Misra, H. Li, and S. Aluru, “Efficient Architecture-Aware Acceleration of BWA-MEM for Multicore Systems,” 2019, In 2019 IEEE International Parallel and Distributed Processing Symposium (IPDPS).

[27] A. McKenna, M. Hanna, E. Banks, et al., “The Genome Analysis Toolkit: A MapReduce Framework for Analyzing Next-Generation DNA Sequencing Data,” Genome Research 20, no. 9 (2010): 1297-1303.

[28] S. Bae, J. Park, and J. S. Kim, “Cas-OFFinder: A Fast and Versatile Algorithm That Searches for Potential Off-Target Sites of Cas9 RNA-Guided Endonucleases,” Bioinformatics (Oxford, England) 30, no. 10 (2014): 1473-1475.

[29] Z. Ivics and Z. Izsvák, “Transposons for Gene Therapy!,” Current Gene Therapy 6, no. 5 (2006): 593-607.

[30] J. Q. Zhang, J. X. Guo, X. J. Wu, et al., “Optimization of sgRNA Expression Strategy to Generate Multiplex Gene-Edited Pigs,” Zoological Research 43, no. 6 (2022): 1005-1008.

[31] Z. Li, J. Lan, X. Shi, et al., “Whole-Genome Sequencing Reveals Rare off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer,” CRISPR Journal 7, no. 1 (2024): 29-40.

[32] H. Zhao, Y. Li, T. Wiriyahdamrong, et al., “Improved Production of GTKO/hCD55/hCD59 Triple-Gene-Modified Diannan Miniature Pigs for Xenotransplantation by Recloning,” Transgenic Research 29, no. 3 (2020): 369-379.

[33] Y. Yue, W. Xu, Y. Kan, et al., “Extensive Germline Genome Engineering in Pigs,” Nature Biomedical Engineering 5, no. 2 (2021): 134-143.

[34] S. Y. Alhaji, S. C. Ngai, and S. Abdullah, “Silencing of Transgene Expression in Mammalian Cells by DNA Methylation and Histone Modifications in Gene Therapy Perspective,” Biotechnology & Genetic Engineering Reviews 35, no. 1 (2019): 1-25.

[35] J. E. Cooper, “Evaluation and Treatment of Acute Rejection in Kidney Allografts,” Clinical Journal of the American Society of Nephrology 15, no. 3 (2020): 430-438.

[36] S. Nomura, Y. Ariyoshi, H. Watanabe, et al., “Transgenic Expression of Human CD47 Reduces Phagocytosis of Porcine Endothelial Cells and Podocytes by Baboon and Human Macrophages,” Xenotransplantation 27, no. 1 (2020): e12549.

[37] M. H. Bikhet, C. Hansen-Estruch, M. Javed, et al., “Profound Thrombocytopenia Associated With Administration of Multiple Anti-Inflammatory Agents in Baboons,” Immunity, Inflammation and Disease 10, no. 3 (2022): e588.

[38] U. Martin, V. Kiessig, J. H. Blusch, et al., “Expression of Pig Endogenous Retrovirus by Primary Porcine Endothelial Cells and Infection of Human Cells,” Lancet (London, England) 352, no. 9129 (1998): 692-694, https://doi.org/10.1016/S0140-6736(98)07144-X.

[39] J. Denner, “Porcine Endogenous Retroviruses in Xenotransplantation,” Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association—European Renal Association 39, no. 8 (2024): 1221-1227.

[40] J. A. Fishman, L. Scobie, and Y. Takeuchi, “Xenotransplantation-Associated Infectious Risk: A WHO Consultation,” Xenotransplantation 19, no. 2 (2012): 72-81.

[41] D. Niu, H. J. Wei, L. Lin, et al., “Inactivation of Porcine Endogenous Retrovirus in Pigs Using CRISPR-Cas9,” Science 357, no. 6357 (2017): 1303-1307.

RIGHTS & PERMISSIONS

2025 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.