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Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles: an alternative to direct complement regulation

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Abstract

The future of solid organ transplantation is challenged by an increasing shortage of available allografts. Xenotransplantation of genetically modified porcine organs offers an answer to this problem. Strategies of genetic modification have ‘humanized’ the porcine model towards clinical relevance. Most notably, these approaches have aimed at either antigen reduction or human transgene expression. The object of this study was to evaluate the relative effects of both antigen reduction and direct complement regulation on the human-anti-porcine complement dependent cytotoxicity response. Genetically modified animals were created through CRISPR/Cas9-directed mutation and human transgene delivery. Pigs doubly deficient in GGTA1 and CMAH genes were compared to pigs of the same background that expressed a human complement regulatory protein (hCRP). A third animal was made deficient in GGTA1, CMAH and B4GalNT2 gene expression. Cells from these animals were subjected to measures of human antibody binding and antibody-mediated complement-dependent cytotoxicity by flow cytometry. Human IgG and IgM antibody binding was unchanged between the double knockout and the transgenic hCRP double knockout pig. IgG and IgM binding was reduced by 49.1 and 43.2 % respectively by silencing the B4GalNT2 gene. Compared to the double knockout, human anti-porcine cytotoxicity was reduced by 8 % with the addition of a hCRP (p = .032); It was reduced by 21 % with silencing the B4GalNT2 gene (p = .012). Conclusions: Silencing the GGTA1, CMAH and B4GalNT2 genes in pigs achieved a significant antigen reduction. Changing the porcine carbohydrate profile effectively mediates human antibody-mediated complement dependent cytoxicity.

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References

  • Azimzadeh AM, Kelishadi SS, Ezzelarab MB, Singh AK, Stoddard T, Iwase H, Zhang T, Burdorf L, Sievert E, Avon C, Cheng X, Ayares D, Horvath KA, Corcoran PC, Mohiuddin MM, Barth RN, Cooper DK, Pierson RN 3rd (2015) Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein. Xenotransplantation 22:310–316. doi:10.1111/xen.12176

    Article  PubMed  Google Scholar 

  • Brunetti D, Perota A, Lagutina I, Colleoni S, Duchi R, Calabrese F, Seveso M, Cozzi E, Lazzari G, Lucchini F, Galli C (2008) Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts cloning and stem cells 10:409–419. doi:10.1089/clo.2008.0036

    CAS  PubMed  Google Scholar 

  • Burlak C, Paris LL, Lutz AJ, Sidner RA, Estrada J, Li P, Tector M, Tector AJ (2014) Reduced binding of human antibodies to cells from GGTA1/CMAH KO pigs. Am J Transpl Off J Am Soc Transpl Am Soc Transpl Surg 14:1895–1900. doi:10.1111/ajt.12744

    Article  CAS  Google Scholar 

  • Butler JR, Ladowski JM, Martens GR, Tector M, Tector AJ (2015) Recent advances in genome editing and creation of genetically modified pigs. Int J Surg. doi:10.1016/j.ijsu.2015.07.684

    PubMed  Google Scholar 

  • Byrne GW, McCurry KR, Martin MJ, McClellan SM, Platt JL, Logan JS (1997) Transgenic pigs expressing human CD59 and decay-accelerating factor produce an intrinsic barrier to complement-mediated damage. Transplantation 63:149–155

    Article  CAS  PubMed  Google Scholar 

  • Byrne GW, Stalboerger PG, Du Z, Davis TR, McGregor CG (2011) Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation. Transplantation 91:287–292. doi:10.1097/TP.0b013e318203c27d

    Article  CAS  PubMed  Google Scholar 

  • Byrne GW, Du Z, Stalboerger P, Kogelberg H, McGregor CG (2014) Cloning and expression of porcine beta1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen. Xenotransplantation 21:543–554. doi:10.1111/xen.12124

    Article  PubMed  PubMed Central  Google Scholar 

  • Cooper DK, Ekser B, Tector AJ (2015) Immunobiological barriers to xenotransplantation. Int J Surg. doi:10.1016/j.ijsu.2015.06.068

    Google Scholar 

  • Diaz TM, Moscoso I, Centeno A, Lopez-Pelaez E, Ortega D, Domenech N (2004) Flow cytometry complement-mediated cytotoxicity assay detects baboon xenoantibodies directed to porcine epitopes undetected by hemolytic assay. Transpl Immunol 13:313–317. doi:10.1016/j.trim.2004.09.001

    Article  CAS  PubMed  Google Scholar 

  • Estrada JL, Martens G, Li P, Adams A, Newell KA, Ford ML, Butler JR, Sidner R, Tector M, Tector J (2015) Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes. Xenotransplantation. doi:10.1111/xen.12161

    PubMed Central  Google Scholar 

  • Higginbotham L, Mathews D, Breeden CA, Song M, Farris AB 3rd, Larsen CP, Ford ML, Lutz AJ, Tector M, Newell KA, Tector AJ, Adams AB (2015) Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model. Xenotransplantation 22:221–230. doi:10.1111/xen.12166

    Article  PubMed  PubMed Central  Google Scholar 

  • Iwase H, Liu H, Wijkstrom M, Zhou H, Singh J, Hara H, Ezzelarab M, Long C, Klein E, Wagner R, Phelps C, Ayares D, Shapiro R, Humar A, Cooper DK (2015) Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date. Xenotransplantation 22:302–309. doi:10.1111/xen.12174

    Article  PubMed  PubMed Central  Google Scholar 

  • Kuwaki K, Tseng YL, Dor FJ, Shimizu A, Houser SL, Sanderson TM, Lancos CJ, Prabharasuth DD, Cheng J, Moran K, Hisashi Y, Mueller N, Yamada K, Greenstein JL, Hawley RJ, Patience C, Awwad M, Fishman JA, Robson SC, Schuurman HJ, Sachs DH, Cooper DK (2005) Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med 11:29–31. doi:10.1038/nm1171

    Article  CAS  PubMed  Google Scholar 

  • Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002) Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089–1092. doi:10.1126/science.1068228

    Article  CAS  PubMed  Google Scholar 

  • Li P, Estrada JL, Burlak C, Montgomery J, Butler JR, Santos RM, Wang ZY, Paris LL, Blankenship RL, Downey SM, Tector M, Tector AJ (2015) Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection. Xenotransplantation 22:20–31. doi:10.1111/xen.12131

    Article  CAS  PubMed  Google Scholar 

  • Lublin DM, Coyne KE (1991) Phospholipid-anchored and transmembrane versions of either decay-accelerating factor or membrane cofactor protein show equal efficiency in protection from complement-mediated cell damage. J Exp Med 174:35–44

    Article  CAS  PubMed  Google Scholar 

  • Lutz AJ, Li P, Estrada JL, Sidner RA, Chihara RK, Downey SM, Burlak C, Wang ZY, Reyes LM, Ivary B, Yin F, Blankenship RL, Paris LL, Tector AJ (2013) Double knockout pigs deficient in N-glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation 20:27–35. doi:10.1111/xen.12019

    Article  PubMed  Google Scholar 

  • Martin MJ, Rayner JC, Gagneux P, Barnwell JW, Varki A (2005) Evolution of human-chimpanzee differences in malaria susceptibility: relationship to human genetic loss of N-glycolylneuraminic acid. Proc Natl Acad Sci USA 102:12819–12824. doi:10.1073/pnas.0503819102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McGregor CG, Ricci D, Miyagi N, Stalboerger PG, Du Z, Oehler EA, Tazelaar HD, Byrne GW (2012) Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts. Transplantation 93:686–692. doi:10.1097/TP.0b013e3182472850

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Menoret S, Plat M, Blancho G, Martinat-Botte F, Bernard P, Karam G, Tesson L, Renaudin K, Guillouet P, Weill B, Chereau C, Houdebine LM, Soulillou JP, Terqui M, Anegon I (2004) Characterization of human CD55 and CD59 transgenic pigs and kidney xenotransplantation in the pig-to-baboon combination. Transplantation 77:1468–1471

    Article  CAS  PubMed  Google Scholar 

  • Mujtaba MA, Goggins W, Lobashevsky A, Sharfuddin AA, Yaqub MS, Mishler DP, Brahmi Z, Higgins N, Milgrom MM, Diez A, Taber T (2011) The strength of donor-specific antibody is a more reliable predictor of antibody-mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients. Clin Transplant 25:E96–E102. doi:10.1111/j.1399-0012.2010.01341.x

    Article  PubMed  Google Scholar 

  • Mulley WR, Kanellis J (2011) Understanding crossmatch testing in organ transplantation: a case-based guide for the general nephrologist. Nephrology 16:125–133. doi:10.1111/j.1440-1797.2010.01414.x

    Article  PubMed  Google Scholar 

  • Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199

    Article  CAS  PubMed  Google Scholar 

  • Perez de la Lastra JM, Harris CL, Hinchliffe SJ, Holt DS, Rushmere NK, Morgan BP (2000) Pigs express multiple forms of decay-accelerating factor (CD55), all of which contain only three short consensus repeats. J Immunol 165:2563–2573

    Article  CAS  PubMed  Google Scholar 

  • Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris AJ, Rudert WA, Bottino R, Bertera S, Trucco M, Starzl TE, Dai Y, Ayares DL (2003) Production of alpha 1,3-galactosyltransferase-deficient pigs. Science 299:411–414. doi:10.1126/science.1078942

    Article  CAS  PubMed  Google Scholar 

  • Stief A, Winter DM, Stratling WH, Sippel AE (1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341:343–345. doi:10.1038/341343a0

    Article  CAS  PubMed  Google Scholar 

  • Tseng YL, Kuwaki K, Dor FJ, Shimizu A, Houser S, Hisashi Y, Yamada K, Robson SC, Awwad M, Schuurman HJ, Sachs DH, Cooper DK (2005) Alpha1,3-Galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching 6 months. Transplantation 80:1493–1500

    Article  CAS  PubMed  Google Scholar 

  • van den Berg CW, Rix C, Hanna SM, Perez de la Lastra JM, Morgan BP (2000) Role and regulation of pig CD59 and membrane cofactor protein/CD46 expressed on pig aortic endothelial cells. Transplantation 70:667–673

    Article  PubMed  Google Scholar 

  • White DJ, Yannoutsos N (1996) Production of pigs transgenic for human DAF to overcome complement-mediated hyperacute xenograft rejection in man. Res Immunol 147:88–94

    Article  CAS  PubMed  Google Scholar 

  • Xenotransplantation (2000) Nat Biotechnol 18(Suppl):IT53–IT55

Download references

Acknowledgments

The first author is supported by a Research Fellowship Award from the Association of Academic Surgery Foundation And a Scientist Scholarship from the American Society of Transplant Surgeons. This study was supported by IU Health Transplant Institute. The authors thank LARC (Laboratory animal research center) staff for the care of the animals. This investigation utilized a facility constructed with support from Research Facilities Improvement Program Grant Number C06RR10601-01 from the National Center for Research Resources, National Institute of Health. The Institutional Animal Care and Use Committee (IACUC) approved this study. Work in CG laboratory was supported by EU Grant Xenome (EU FP6-LSHB-CT-2006-No. 037377).

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Authors and Affiliations

  1. Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA

    James R. Butler, Gregory R. Martens, Jose L. Estrada, Luz M. Reyes, Joseph M. Ladowski, Conor M. Cunningham, Matthew Tector & A. Joseph Tector

  2. Avantea Laboratorio Di Tecnologie Della Riproduzione, Cermona, Italy

    Cesare Galli & Andrea Perota

  3. Department of Veterinary Medical Sciences, University of Bologna, Bologna, Ozzano Emilia, Italy

    Cesare Galli

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  1. James R. Butler
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  2. Gregory R. Martens
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  9. Matthew Tector
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  10. A. Joseph Tector
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Correspondence to A. Joseph Tector.

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Butler, J.R., Martens, G.R., Estrada, J.L. et al. Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles: an alternative to direct complement regulation. Transgenic Res 25, 751–759 (2016). https://doi.org/10.1007/s11248-016-9958-0

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  • DOI: https://doi.org/10.1007/s11248-016-9958-0

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