Extensive germline genome engineering in pigs

Abstract

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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Fig. 1: The engineering of PERVKO·3KO·9TG pigs.
Fig. 2: PERVKO·3KO·9TG pig engineering and validation of the 3KO and 9TG edits at the genomic level.
Fig. 3: Validation of 3KO and 9TG in 3KO·9TG pigs and PERVKO·3KO·9TG pigs at the mRNA and protein levels.
Fig. 4: Functional validation of PERVKO·3KO·9TG pigs in mitigating human antibody binding, complement toxicity and NK cell toxicity, and modulating coagulation function.

Data availability

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).

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Acknowledgements

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).

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Contributions

L.Y., G.M.C. and Y.G. envisioned and supervised the whole project; H.-J.W. and H.-Y.Z. supervised pig cloning and production. Y.Y., W.X. and Y.K. designed the experiments and wrote the manuscript. Y.Y., Y.K., Y.Z., X.S., L.Lamriben, J.W., J.X., M.X., Q.Z., Y.L., J.V.L., M.L., V.P., M.E.Y., Z.S., Y.D., W.W., H.D., L.S., X.W., L.Le, X.F, H.G., R.A. and S.Y.W. performed experiments. W.X., D.G., M.Y. and M.G. analysed the data. J.G., S.M., D.J., T.D.N. and Z.L. performed pig cloning and generated pigs. J.M., W.Q. and W.F.W. revised the manuscript.

Corresponding author

Correspondence to Luhan Yang.

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Competing interests

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

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