Abstract
In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.
Highlights
Pigs are similar to humans with respect to their genetics, anatomy, and physiology, making them the best donor of biological materials for xenotransplantation (Butler et al 2015; Cooper et al 2017; Hryhorowicz et al 2017)
These findings suggest that cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene knockout (KO) is necessary to prevent further rejection of xenografts (Park et al 2012; Park et al 2011)
The ear fibroblast cells isolated from GTKO piglets were simultaneously transfected with the CRISPR/Cas9 vectors for CMAH and A3GALT2 KO
Summary
Pigs are similar to humans with respect to their genetics, anatomy, and physiology, making them the best donor of biological materials for xenotransplantation (Butler et al 2015; Cooper et al 2017; Hryhorowicz et al 2017). Despite this similarity, there is a considerable phylogenetic distance between pigs and humans, which can cause problems with the immune system following xenotransplantation (Hryhorowicz et al 2017). Neu5Gc is synthesized from N-acetylneuraminic acid (Neu5Ac) by cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) It is expressed in the tissues of most mammals, including pigs and monkeys, but humans have an inactivated CMAH gene, so only Neu5Ac is present on human cells (Breimer 2011). Butler et al reported that GGTA1/CMAH double KO pig livers
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
