The clinical applicability of porcine xenotransplantation—a long-investigated alternative to the scarce availability of human organs for patients with organ failure—is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR–Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs’ immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.
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The main data supporting the findings of this study are available within the paper and its Supplementary Information. Data from the RNA-seq analyses are available at figshare (https://doi.org/10.6084/m9.figshare.12841418.v1). The raw data generated during the study are available at the China National GeneBank, with the accession code CNP0001254. The pig reference genome (Sus scrofa 11.1) sequence was obtained from Ensembl (ftp://ftp.ensembl.org/pub/release-91/fasta/sus_scrofa/dna). The pig transcript isoform information was obtained from the APPRIS database (http://appris.bioinfo.cnio.es/#/seeker).
Sykes, M. & Sachs, D. H. Transplanting organs from pigs to humans. Sci. Immunol. 4, eaau6298 (2019).
Cooper, D. K. C., Ekser, B. & Tector, A. J. Immunobiological barriers to xenotransplantation. Int. J. Surg. 23, 211–216 (2015).
Denner, J. & Tonjes, R. R. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clin. Microbiol Rev. 25, 318–343 (2012).
Patience, C., Takeuchi, Y. & Weiss, R. A. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3, 282–286 (1997).
Shin, J. S. et al. Minimizing immunosuppression in islet xenotransplantation. Immunotherapy 6, 419–430 (2014).
Niu, D. et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357, 1303–1307 (2017).
Cooper, D. K. Modifying the sugar icing on the transplantation cake. Glycobiology 26, 571–581 (2016).
Byrne, G., Ahmad-Villiers, S., Du, Z. & McGregor, C. B4GALNT2 and xenotransplantation: a newly appreciated xenogeneic antigen. Xenotransplantation 25, e12394 (2018).
Song, K. H. et al. Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation. Biochem. J. 427, 179–188 (2010).
Estrada, J. L. et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes. Xenotransplantation 22, 194–202 (2015).
Phelps, C. J. et al. Production of α1,3-galactosyltransferase-deficient pigs. Science 299, 411–414 (2003).
Lai, L. et al. Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089–1092 (2002).
Martens, G. R. et al. Humoral reactivity of renal transplant-waitlisted patients to cells from GGTA1/CMAH/B4GalNT2, and SLA class I knockout pigs. Transplantation 101, e86–e92 (2017).
Yamada, K. et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of α1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat. Med. 11, 32–34 (2005).
Kuwaki, K. et al. Heart transplantation in baboons using α1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat. Med. 11, 29–31 (2005).
Cooper, D. K., Ekser, B., Ramsoondar, J., Phelps, C. & Ayares, D. The role of genetically engineered pigs in xenotransplantation research. J. Pathol. 238, 288–299 (2016).
Mohiuddin, M. M. et al. B-cell depletion extends the survival of GTKO.hCD46Tg pig heart xenografts in baboons for up to 8 months. Am. J. Transpl. 12, 763–771 (2012).
Zhou, C. Y. et al. Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor. Xenotransplantation 12, 142–148 (2005).
Griesemer, A., Yamada, K. & Sykes, M. Xenotransplantation: immunological hurdles and progress toward tolerance. Immunol. Rev. 258, 241–258 (2014).
Lilienfeld, B. G., Crew, M. D., Forte, P., Baumann, B. C. & Seebach, J. D. Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity. Xenotransplantation 14, 126–134 (2007).
Ide, K. et al. Role for CD47-SIRPα signaling in xenograft rejection by macrophages. Proc. Natl Acad. Sci. USA 104, 5062–5066 (2007).
Siegel, J. B. et al. Xenogeneic endothelial cells activate human prothrombin. Transplantation 64, 888–896 (1997).
Lee, K. F. et al. Recombinant pig TFPI efficiently regulates human tissue factor pathways. Xenotransplantation 15, 191–197 (2008).
Choi, C. Y. et al. Pig tissue factor pathway inhibitor α fusion immunoglobulin inhibits pig tissue factor activity in human plasma moderately more efficiently than the human counterpart. Biotechnol. Lett. 39, 1631–1638 (2017).
Robson, S. C., Cooper, D. K. & d’Apice, A. J. Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation 7, 166–176 (2000).
Ji, H. et al. Pig BMSCs transfected with human TFPI combat species incompatibility and regulate the human TF pathway in vitro and in a rodent model. Cell. Physiol. Biochem. 36, 233–249 (2015).
Kopp, C. W. et al. Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors. Transplantation 63, 749–758 (1997).
Iwase, H., Ezzelarab, M. B., Ekser, B. & Cooper, D. K. The role of platelets in coagulation dysfunction in xenotransplantation, and therapeutic options. Xenotransplantation 21, 201–220 (2014).
Miwa, Y. et al. Potential value of human thrombomodulin and DAF expression for coagulation control in pig-to-human xenotransplantation. Xenotransplantation 17, 26–37 (2010).
Mohiuddin, M. M. et al. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft. Nat. Commun. 7, 11138 (2016).
Wheeler, D. G. et al. Transgenic swine: expression of human CD39 protects against myocardial injury. J. Mol. Cell Cardiol. 52, 958–961 (2012).
Cooper, D. K. C. et al. Justification of specific genetic modifications in pigs for clinical organ xenotransplantation. Xenotransplantation 26, e12516 (2019).
Samy, K. P., Martin, B. M., Turgeon, N. A. & Kirk, A. D. Islet cell xenotransplantation: a serious look toward the clinic. Xenotransplantation 21, 221–229 (2014).
Matsumoto, S., Tomiya, M. & Sawamoto, O. Current status and future of clinical islet xenotransplantation. J. Diabetes 8, 483–493 (2016).
Fischer, K. et al. Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing. Sci. Rep. 6, 29081 (2016).
Yunga, G. L. P., Riebenb, R., Bühlerc, L., Schuurmanc, H. J. & Seebach, J. Xenotransplantation: where do we stand in 2016? Swiss Med. Wkly 147, w14403 (2017).
Langin, M. et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature 564, 430–433 (2018).
Kim, S. C. et al. Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion. Am. J. Transpl. 19, 2174–2185 (2019).
Li, X. et al. PiggyBac transposase tools for genome engineering. Proc. Natl Acad. Sci. USA 110, E2279–E2287 (2013).
Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Laird, C. T. et al. Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury. Xenotransplantation 24, e12294 (2017).
Chen, D. et al. Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins. Transplantation 68, 832–839 (1999).
Fischer, K. et al. Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2. Xenotransplantation 27, e12560 (2020).
Ekser, B., Markmann, J. F. & Tector, A. J. Current status of pig liver xenotransplantation. Int. J. Surg. 23, 240–246 (2015).
Zhao, Y. et al. Skin graft tolerance across a discordant xenogeneic barrier. Nat. Med. 2, 1211–1216 (1996).
Jesus, B. B. D. et al. Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer. EMBO Mol. Med. 4, 691–704 (2012).
Kennedy, E. M. & Cullen, B. R. Gene editing: a new tool for viral disease. Annu Rev. Med 68, 401–411 (2017).
Burkard, C. et al. Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection. J. Virol. 92, e00415-18 (2018).
Yan, Q. et al. Production of transgenic pigs over-expressing the antiviral gene Mx1. Cell Regen. 3, 11 (2014).
Yang, L. et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101–1104 (2015).
Wei, H. et al. Comparison of the efficiency of banna miniature inbred pig somatic cell nuclear transfer among different donor cells. PLoS ONE 8, e57728 (2013).
Tomii, R. et al. Production of cloned pigs by nuclear transfer of preadipocytes following cell cycle synchronization by differentiation induction. J. Reprod. Dev. 55, 121–127 (2009).
Kurome, M. et al. Production efficiency and telomere length of the cloned pigs following serial somatic cell nuclear transfer. J. Reprod. Dev. 54, 254–258 (2008).
Costa, C. & Manez, R. Xenotransplantation: Methods and Protocols 335 (Springer, xiHumana Press, 2012).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
McKenna, A. et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).
Zhu, L. J., Holmes, B. R., Aronin, N. & Brodsky, M. H. CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. PLoS ONE 9, e108424 (2014).
McLaren, W. et al. The ensembl variant effect predictor. Genome Biol. 17, 122 (2016).
Rodriguez, J. M. et al. APPRIS: annotation of principal and alternative splice isoforms. Nucleic Acids Res. 41, D110–D117 (2013).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
We thank G. Yang of Harvard University for reading our manuscript; Q. Tang and P. O’Connell for their advice; and Y. Yang and Q. Yang from Third Affiliated Hospital of Sun Yat-sen University, H. Liu from Henan Chuangyuan Biotechnology, and colleagues at Qihan Bio and eGenesis for their technical assistance and discussions. The pig cloning work was supported by National Key R&D Program of China (grant no. 2019YFA0110700).
Y.Y., W.X., Y.Z., X.S., M.Y., J.W., J.X., M.X., Q.Z., Y.L., H.D., L.S., X.W., L.Le, X.F., Y.G. and L.Y. are employed by Qihan Bio Inc. Y.K., D.G., L.Lamriben, J.V.L., M.L., V.P., M.E.Y., H.G., R.A., S.Y.W., W.F.W. and W.Q. are employed by eGenesis Inc. M.G. is a consultant to Qihan Bio Inc. and eGenesis Inc. J.M. is an advisor on the scientific advisory board of Qihan Bio Inc. and eGenesis Inc. G.M.C. is the cofounder and scientific advisor of Qihan Bio Inc. and eGenesis Inc. Y.K., M.G., W.Q., Y.G. and L.Y. are listed as inventors on a provisional patent application pertaining to the results of the paper.
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Yue, Y., Xu, W., Kan, Y. et al. Extensive germline genome engineering in pigs. Nat Biomed Eng (2020). https://doi.org/10.1038/s41551-020-00613-9
The CRISPR Journal (2020)