Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

Fischer, Konrad; Rieblinger, Beate; Hein, Rabea; Sfriso, Riccardo; Zuber, Julia; Fischer, Andrea; Klinger, Bernhard; Liang, Wei; Flisikowski, Krzysztof; Kurome, Mayuko; Zakhartchenko, Valeri; Kessler, Barbara; Wolf, Eckhard; Rieben, Robert; Schwinzer, Reinhard; Kind, Alexander; Schnieke, Angelika (2020). Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2. Xenotransplantation, 27(1), e12560. Wiley 10.1111/xen.12560

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR)
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Herz und Gefässe
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Handchirurgie
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Sfriso, Riccardo and Rieben, Robert

Subjects:

600 Technology > 610 Medicine & health
500 Science > 570 Life sciences; biology

ISSN:

0908-665X

Publisher:

Wiley

Language:

English

Submitter:

Marla Rittiner

Date Deposited:

12 Dec 2019 15:06

Last Modified:

08 Feb 2020 01:32

Publisher DOI:

10.1111/xen.12560

PubMed ID:

31591751

Uncontrolled Keywords:

B2M B4GALNT2 CMAH GGTA1 MHC-I SLA-I complement regulators multiplex CRISPR/Cas9 gene editing xenotransplantation

BORIS DOI:

10.7892/boris.136305

URI:

https://boris.unibe.ch/id/eprint/136305

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