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Codon optimized membrane cofactor protein expression in α 1, 3 galactosyltransferase knockout pig cells improve protection against cytotoxicity of monkey serum

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Abstract

In this study, we attempted to upgrade GT−MCP/−MCP pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT−MCP/−MCP pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT−MCP/−MCP/mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival.

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Abbreviations

2A:

Porcine teschovirus-1 2A

bp:

Base pair

GTKO:

Alpha-1,3-galactosyltransferase homozygous knockout

Icam2:

Porcine intercellular adhesion molecule 2 (Icam2)

MCP:

Membrane cofactor protein

pEF:

Porcine ear skin fibroblasts

pAEC:

Porcine aortic endothelial cells

SCNT:

Somatic cell nuclear transfer

TBM:

Thrombomodulin

References

  • Adams DH, Kadner A, Chen RH, Farivar RS, Logan JS, Diamond LE (2001) Human membrane cofactor protein (MCP, CD 46) protects transgenic pig hearts from hyperacute rejection in primates. Xenotransplantation 8(1):36–40

    Article  CAS  PubMed  Google Scholar 

  • Agashe D, Martinez-Gomez NC, Drummond DA, Marx CJ (2012) Good codons, bad transcript: large reductions in gene expression and fitness arising from synonymous mutations in a key enzyme. Mol Biol Evol 30(3):549–560

    Article  PubMed  PubMed Central  Google Scholar 

  • Anastasiou G, Gialeraki A, Merkouri E, Politou M, Travlou A (2012) Thrombomodulin as a regulator of the anticoagulant pathway: implication in the development of thrombosis. Blood Coag Fibrinol 23(1):1–10

    Article  CAS  Google Scholar 

  • Conway EM (2012) Thrombomodulin and its role in inflammation. In: Seminars in immunopathology, 2012. vol 1. Springer, pp 107–125

  • Crikis S, Zhang X, Dezfouli S, Dwyer KM, Murray-Segal L, Salvaris E, Selan C, Robson S, Nandurkar H, Cowan P (2010) Antiinflammatory and anticoagulant effects of transgenic expression of human thrombomodulin in mice. Am J Transplant 10(2):242–250

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Frelin L, Ahlen G, Alheim M, Weiland O, Barnfield C, Liljeström P, Sällberg M (2004) Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene. Gene Ther 11(6):522

    Article  CAS  PubMed  Google Scholar 

  • Gao H, Chen P, Wei L, Xu J, Liu L, Zhao Y, Hara H, Pan D, Li Z, Cooper DK (2017) Angiopoietin-1 and angiopoietin-2 protect porcine iliac endothelial cells from human antibody-mediated complement-dependent cytotoxicity through phosphatidylinositide 3-kinase/AKT pathway activation. Xenotransplantation 24(4):e12309

    Article  Google Scholar 

  • Hurh S, Cho B, You D-J, Kim H, Lee EM, Lee SH, Park SJ, Park HC, Koo OJ, Yang J (2013) Expression analysis of combinatorial genes using a bi-cistronic T2A expression system in porcine fibroblasts. PLoS ONE 8(7):e70486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hwang S, Oh KB, Kwon D-J, Ock S-A, Lee J-W, Im G-S, Lee S-S, Lee K, Park J-K (2013) Improvement of cloning efficiency in minipigs using post-thawed donor cells treated with roscovitine. Mol Biotechnol 55(3):212–216

    Article  CAS  PubMed  Google Scholar 

  • Iwase H, Ekser B, Satyananda V, Bhama J, Hara H, Ezzelarab M, Klein E, Wagner R, Long C, Thacker J (2015) Pig-to-baboon heterotopic heart transplantation–exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens. Xenotransplantation 22(3):211–220

    Article  PubMed  PubMed Central  Google Scholar 

  • Iwata K, Seya T, Yanagi Y, Pesando JM, Johnson PM, Okabe M, Ueda S, Ariga H, Nagasawa S (1995) Diversity of sites for measles virus binding and for inactivation of complement C3b and C4b on membrane cofactor protein CD46. J Biol Chem 270(25):15148–15152

    Article  CAS  PubMed  Google Scholar 

  • Ko N, Lee J-W, Hwang SS, Kim B, Ock SA, Lee S-S, Im G-S, Kang M-J, Park J-K, Jong OhS (2013) Nucleofection-Mediated α1, 3-galactosyltransferase Gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation. Animal biotechnology 24(4):253–267

    Article  CAS  PubMed  Google Scholar 

  • Kuwaki K, Tseng Y-L, Dor FJ, Shimizu A, Houser SL, Sanderson TM, Lancos CJ, Prabharasuth DD, Cheng J, Moran K (2005) Heart transplantation in baboons using α1, 3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med 11(1):29

    Article  CAS  PubMed  Google Scholar 

  • Kwon D-J, Kim D-H, Hwang I-S, Kim D-E, Kim H-J, Kim J-S, Lee K, Im G-S, Lee J-W, Hwang S (2017) Generation of α-1, 3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes. Transgenic Res 26(1):153–163

    Article  CAS  PubMed  Google Scholar 

  • Lee G, Park S, Lee H, Ji S, Lee J, Byun S, Hwang S, Kim K, Ock S, Oh K (2017) Development of α 1, 3-galactosyltransferase inactivated and human membrane cofactor protein expressing homozygous transgenic pigs for Xenotransplantation. J Embryo Transf

  • Lee S, Kim J, Chee H, Yun I, Park K, Yang H, Park J (2018) Seven years of experiences of preclinical experiments of xeno-heart transplantation of pig to non-human primate (cynomolgus monkey). In: Transplantation proceedings, vol 4. Elsevier, pp 1167–1171

  • Loveland BE, Milland J, Kyriakou P, Thorley BR, Christiansen D, Lanteri MB, van Regensburg M, Duffield M, French AJ, Williams L (2004) Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons. Xenotransplantation 11(2):171–183

    Article  PubMed  Google Scholar 

  • Mauro VP, Chappell SA (2014) A critical analysis of codon optimization in human therapeutics. Trends in molecular medicine 20(11):604–613

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McGregor CG, Davies WR, Oi K, Teotia SS, Schirmer JM, Risdahl JM, Tazelaar HD, Kremers WK, Walker RC, Byrne GW (2005) Cardiac xenotransplantation: recent preclinical progress with 3-month median survival. J Thoracic Cardiovasc Surg 130(3):844. e841–844. e849

    Article  Google Scholar 

  • Milland J, Christiansen D, Thorley BR, Mckenzie IF, Loveland BE (1996) Translation is enhanced after silent nucleotide substitutions in A+ T-rich sequences of the coding region of CD46 cDNA. Eur J Biochem 238(1):221–230

    Article  CAS  PubMed  Google Scholar 

  • Miltenyi S, Müller W, Weichel W, Radbruch A (1990) High gradient magnetic cell separation with MACS. Cytometry J Int Soc Anal Cytol 11(2):231–238

    Article  CAS  PubMed  Google Scholar 

  • Miyagawa S, Yamada M, Matsunami K, Koresawa Y, Ikawa M, Okabe M, Shirakura R (2001) A synthetic DAF (CD55) gene based on optimal codon usage for transgenic animals. The Journal of Biochemistry 129(5):795–801

    Article  CAS  PubMed  Google Scholar 

  • Mohiuddin M, Corcoran P, Singh A, Azimzadeh A, Hoyt Jr R, Thomas M, Eckhaus M, Seavey C, Ayares D, Pierson III R (2012) B‐cell depletion extends the survival of GTKO. hCD46Tg pig heart xenografts in baboons for up to 8 months. Am J Transplant 12(3):763–771

    Article  PubMed  PubMed Central  Google Scholar 

  • Mohiuddin MM, Singh AK, Corcoran PC, Thomas III ML, Clark T, Lewis BG, Hoyt RF, Eckhaus M, Pierson III RN, Belli AJ (2016) 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

  • Raab D, Graf M, Notka F, Schödl T, Wagner R (2010) The GeneOptimizer algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst Synth Biol 4(3):215–225

    Article  PubMed  PubMed Central  Google Scholar 

  • Schneider-Schaulies J, Martin MJ, Logan JS, Firsching R, ter Meulen V, Diamond LE (2000) CD46 transgene expression in pig peripheral blood mononuclear cells does not alter their susceptibility to measles virus or their capacity to downregulate endogenous and transgenic CD46. J Gen Virol 81(6):1431–1438

    Article  CAS  PubMed  Google Scholar 

  • Tsai C-J, Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM, Nussinov R (2008) Synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima. J Mol Biol 383(2):281–291

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ward NJ, Buckley SM, Waddington SN, VandenDriessche T, Chuah MK, Nathwani AC, McIntosh J, Tuddenham EG, Kinnon C, Thrasher AJ (2011) Codon optimization of human factor VIII cDNAs leads to high-level expression. Blood 117(3):798–807

    Article  CAS  PubMed  Google Scholar 

  • Yang P, Wei X, Zhang J, Yi B, Zhang G-X, Yin L, Yang X-F, Sun J (2016) Antithrombotic effects of Nur77 and Nor1 are mediated through upregulating thrombomodulin expression in endothelial cells. Arterioscler Thromb Vasc Biol 36(2):361–369

    Article  CAS  PubMed  Google Scholar 

  • Zucchelli E, Pema M, Stornaiuolo A, Piovan C, Scavullo C, Giuliani E, Bossi S, Corna S, Asperti C, Bordignon C (2017) Codon optimization leads to functional impairment of RD114-TR envelope glycoprotein. Mol Therap Methods Clin Dev 4:102–114

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development [Rural Development Administration, Republic of Korea] under Grant [Project Number PJ012022] and (2017) by Academy = Research = Industry Support Program of Rural Development Administration, Republic of Korea.

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Author notes

  1. Heasun Lee and In-sul Hwang have contributed equally.

Authors and Affiliations

  1. Animal Biotechnology Division, National Institute of Animal Science, RDA, 1500, Kongwipatjwi-ro, Wanju-gun, Jeollabuk-do, 55365, Korea

    Heasun Lee, In-sul Hwang, Bala Murali Krishna Vasamsetti, Harikrishna Reddy Rallabandi, Mi-Ryung Park, Sung-June Byun, Hyeon Yang, Sun A. Ock, Hwi-Cheul Lee, Jae-Seok Woo, Seongsoo Hwang & Keon Bong Oh

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  1. Heasun Lee
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  2. In-sul Hwang
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  12. Keon Bong Oh
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Contributions

KBO designed the experiments. HL, BMKV, HY, and SJB performed experiments. KBO, HL, SAO, and HCL analyzed the data. IH, JSW, SH, and MIP performed in vitro culture of oocytes, nuclear transfer, and embryo transfer. HL, IH, and BMKV wrote a draft. BMKV, HRR and KBO edited the draft. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Keon Bong Oh.

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The authors declare that they have no competing interests.

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Lee, H., Hwang, Is., Vasamsetti, B.M.K. et al. Codon optimized membrane cofactor protein expression in α 1, 3 galactosyltransferase knockout pig cells improve protection against cytotoxicity of monkey serum. 3 Biotech 10, 108 (2020). https://doi.org/10.1007/s13205-020-2091-z

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