Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2
Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2
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DOI：
10.1111/xen.12560
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发表时间：
2020-01-01
影响因子：
3.9
通讯作者：
Schnieke, Angelika
中科院分区：
文献类型：
--
作者：
Fischer, Konrad;Rieblinger, Beate;Schnieke, Angelika
背景细胞表面糖类抗原在猪异种移植排斥反应中起重要作用。对于人类受体来说，最重要的是引起超急性排斥的α-1，3-半乳糖(Alpha-1，3-Gal)，以及N-羟基神经氨酸(Neu5Gc)和SD(A)血型抗原，鉴于人类的免疫状况，这两种抗原都可能引发急性血管排斥反应。三个相关基因GGTA1、CMAH和B4GALNT2被敲除的猪细胞，在与人血清孵育后，显示出最小的异种反应抗体结合。然而，人类白细胞抗原(HLA)抗体与猪白细胞抗原I类(SLA-I)发生交叉反应。我们以前证明了将多个异源转基因放置在单个基因组座位上的猪的有效世代。方法采用多重CRISPR/Cas9基因编辑和体细胞核移植技术，获得了携带GGTA1、CMAH、B4GALNT2和SLA I类基因功能敲除的猪，以及来自1~4次敲除动物和具有4重敲除的多个转基因细胞(人CD46、CD55、CD59、HO1和A20)的成纤维细胞，在体外检测了对人免疫球蛋白和IgM结合或补体激活的影响。体外试验表明，四种基因敲除的组合比单独或双重敲除更有效地减少了人的免疫球蛋白和免疫球蛋白M与猪肾细胞的结合。多重转基因背景和GGTA1单独敲除降低了C3b/c和C4b/c补体的激活，进一步的敲除没有显著的额外影响。结论我们发现携带几种异种保护性转基因和异种反应性抗原敲除的猪很容易产生，这些修饰将对异种移植的存活率产生显著的影响。
Background Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are alpha-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.