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

Abstract

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.

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

“…However, other xenogeneic antigens, including beta-1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT) [47] and N-glycolylneuraminic acid synthesized by cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) [48,49], also need to be eliminated to prolong organ survival. The triple knockout of porcine GGTA1, CMAH, and B4GALNT2 has been achieved by SCNT and significantly reduces human IgG and IgM antibody binding to porcine peripheral blood monocytes, red blood cells [50][51][52], and the pericardium [53]. In addition, to prevent hyperacute rejection by controlling Fig.…”

Application of Genetically Engineered Pigs in Biomedical Research

“…Recently, pigs carrying functional knock-outs of GGTA1, CMAH, B4GALNT2, and SLA class I with multi-transgenic background (hCD46, hCD55, hCD59, hHO1, hA20) were produced. In vitro study presented that the four-fold knock-out reduced the binding of human IgG and IgM to porcine kidney cells [95].…”

Toward acellular xenogeneic heart valve prostheses: Histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices

“…17,18 The lack of such molecules in kidney cells isolated from four gene knockouts pigs (GGTA1, CMAH, B4GALNT2, and SLA class I knockout) reduced human IgG and IgM binding in vitro, in a more effective way than single or double knockouts. 18 Furthermore, non-self minor histocompatibility antigens, associated with MHC, have been reported to be recognized by T cells, initiating immune rejection. 17,19,20 Gates et al 21 In addition to the above-mentioned protein xenoantigens, carbohydrate antigens seem to play a major role in tissue immunogenicity, due to their large variety within and between species, much greater than that of proteins.…”