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



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.
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA.
James R
JR
Butler
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA.
Gregory R
GR
Martens
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA.
Jose L
JL
Estrada
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA.
Luz M
LM
Reyes
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA.
Joseph M
JM
Ladowski
Avantea Laboratorio Di Tecnologie Della Riproduzione, Cermona, Italy.|Department of Veterinary Medical Sciences, University of Bologna, Bologna, Ozzano Emilia, Italy.
Cesare
C
Galli
Avantea Laboratorio Di Tecnologie Della Riproduzione, Cermona, Italy.
Andrea
A
Perota
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA.
Conor M
CM
Cunningham
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA.
Matthew
M
Tector
Department of Surgery, Indiana University School of Medicine, UH 4601 550 N. University Blvd, Indianpolis, IN, 46202, USA. jtector@uab.edu.
A
A
Joseph Tector
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000276:immunology; D064113:CRISPR-Cas Systems / Q000235:genetics; D003165:Complement System Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D005700:Galactosyltransferases / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics*; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics*; D016377:Organ Transplantation; D013552:Swine / Q000276:immunology; D014183:Transplantation, Heterologous
D003165:Complement System Proteins; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine; C071413:beta-1,4-N-acetyl-galactosaminyl transferase 2
Antibody binding; Antibody-mediated cytotoxicity; Complement regulation; Xenotransplantation