Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.
Animals
Antibodies, Heterophile
/ metabolism
CRISPR-Cas Systems
Cells, Cultured
Complement System Proteins
/ metabolism
Galactosyltransferases
/ genetics
Graft Rejection
/ immunology
HLA Antigens
/ immunology
Heterografts
/ immunology
Histocompatibility Antigens Class I
Humans
Kidney Transplantation
Mixed Function Oxygenases
/ genetics
N-Acetylgalactosaminyltransferases
/ genetics
Swine
Transplantation, Heterologous
B2M
B4GALNT2
CMAH
GGTA1
MHC-I
SLA-I
complement regulators
multiplex CRISPR/Cas9 gene editing
xenotransplantation
Journal
Xenotransplantation
ISSN: 1399-3089
Titre abrégé: Xenotransplantation
Pays: Denmark
ID NLM: 9438793
Informations de publication
Date de publication:
01 2020
01 2020
Historique:
received:
16
04
2019
revised:
16
09
2019
accepted:
17
09
2019
pubmed:
9
10
2019
medline:
21
5
2021
entrez:
9
10
2019
Statut:
ppublish
Résumé
Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.
Sections du résumé
BACKGROUND
Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation.
METHODS
Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro.
RESULTS
Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect.
CONCLUSION
We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.
Substances chimiques
Antibodies, Heterophile
0
HLA Antigens
0
Histocompatibility Antigens Class I
0
Complement System Proteins
9007-36-7
Mixed Function Oxygenases
EC 1.-
CMPacetylneuraminate monooxygenase
EC 1.14.18.2
Galactosyltransferases
EC 2.4.1.-
N-Acetylgalactosaminyltransferases
EC 2.4.1.-
alpha-1,3-galactosyltransferase 1, porcine
EC 2.4.1.-
beta-1,4-N-acetyl-galactosaminyl transferase 2
EC 2.4.1.165
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e12560Informations de copyright
© 2019 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.
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