Abstract
Two sgRNAs were designed to target the region of exon 2 of the pULBP1 gene by microinjection. The co-injection of modified Cas9-D10A nickase with a pair of sgRNAs into the zygote’s cytoplasm easily and efficiently generated biallelic modification of the pULBP1 gene in one step. Five out of nine F0 generation piglets showed insertions or deletions in the targeting site of the pULBP1 gene, indicating that pULBP1 mutation efficiency reached about 56% (5/9). Quantitative determination of pULBP1 showed approximately a 1.53-fold reduction in the amount of protein ULBP1 on the cell surface (ELISA). A human NK-cell cytotoxicity test leads to the conclusion that higher cell viability is observed for −/− ULBP1 (survival rate 85.36%) compared to +/+ ULBP1 (69.58%). ULBP1-KO pigs will provide a more progressive xenograft source for further research studies, especially those measuring the effects of abolishing the gene function in terms of the complexity of the immunological interactions.
Highlights
A donor shortage is the main limitation for conducting transplantations to save patients or improve their quality of life by treating severe organ failure
We present a generation of pigs with pULBP1 gene modified by CRISPR-Cas9 and their potential use as donors in xenotransplantation
Seventy-three fertilized egg cells from three donors were subjected to intracytoplasmic microinjection. 100% of the microinjected zygotes were qualified for transfer after a morphological examination, which suggests that sgRNAs/Cas9D10A and the microinjection manipulation had no negative influence on the early embryonic development
Summary
A donor shortage is the main limitation for conducting transplantations to save patients or improve their quality of life by treating severe organ failure. The main cause of hyperacute rejection is an antigen present in the glycolipids and glycoproteins of the endothelial cells surface of pigs (not present in humans nor in NHPs). Carbohydrate residue Galα1,3Galβ-R (αGal) synthesis is catalysed by α1,3-galactosyltransferase (α1,3GT, EC2.4.1.151) and results from adding galactose residue to N-acetyllactosamine Another significant problem is the incompatibility of porcine complement regulatory proteins (CRPs) on donor endothelium with the human complement, which results in uncontrolled complement activation. HAR silencing by immunosuppressive therapies and the organs from genetically modified pigs with α1,3-galactosyltransferase knockout (GTKO) or/and expressing human α1,2-fucosyltransferase (hHT), or/and galactosidase (GLA), or/and expressing human complement regulatory proteins (hCRPs) will reveal cellular and/or humoral epiphenomena causing delayed responses, including xenograft rejection (DXR), called acute humoral xenograft rejection (AHXR) (Cooper 2012, Puga Yung et al 2017). According to Pierson et al, strategies to overcome NK-cell activity include human MHC class I molecules expression on the surface of swine endothelium and blocking of molecular events leading to NK recruitment (Pierson et al 2009)
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