title: Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.
abstract: Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - 'gene stacking', and cointegration of multiple engineered large vectors - 'combineering', to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Konrad
authors.initials: K
authors.lastname: Fischer
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Kraner-Scheiber
authors.affiliation: Institute of Farm Animal Genetics, FLI, Mariensee, Neustadt. Rbge, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Petersen
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Rieblinger
authors.affiliation: Medizinische Hochschule Hannover, Transplantations labor, Hannover, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Buermann
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Flisikowska
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Flisikowski
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Christan
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Marlene
authors.initials: M
authors.lastname: Edlinger
authors.affiliation: Medizinische Hochschule Hannover, Transplantations labor, Hannover, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Baars
authors.affiliation: Chair of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-Universität München, Oberschleissheim, Germany.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Kurome
authors.affiliation: Chair of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-Universität München, Oberschleissheim, Germany.
authors.forename: Valeri
authors.initials: V
authors.lastname: Zakhartchenko
authors.affiliation: Chair of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-Universität München, Oberschleissheim, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kessler
authors.affiliation: Robert Koch Institute, Berlin, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Plotzki
authors.affiliation: Department of Genetics and Animal breeding, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: Izabela
authors.initials: I
authors.lastname: Szczerbal
authors.affiliation: Department of Genetics and Animal breeding, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Switonski
authors.affiliation: Robert Koch Institute, Berlin, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Denner
authors.affiliation: Chair of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-Universität München, Oberschleissheim, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
authors.affiliation: Medizinische Hochschule Hannover, Transplantations labor, Hannover, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schwinzer
authors.affiliation: Institute of Farm Animal Genetics, FLI, Mariensee, Neustadt. Rbge, Germany.
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kind
authors.affiliation: Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Schnieke
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000276:immunology; D064113:CRISPR-Cas Systems / Q000235:genetics; D003165:Complement System Proteins / Q000235:genetics* / Q000276:immunology; D000072669:Gene Editing; D006084:Graft Rejection / Q000235:genetics / Q000276:immunology; D064593:Heterografts; D006801:Humans; D013552:Swine / Q000235:genetics / Q000276:immunology; D014183:Transplantation, Heterologous / Q000379:methods*
chemical_list: D003165:Complement System Proteins