Generation of GGTA1 <sup>-/-</sup>β2M <sup>-/-</sup>CIITA <sup>-/-</sup> Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions

Abstract

Background: The generation of glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex (MHC) antigens SLA-I and SLA-II. Methods: We simultaneously depleted GGTA1, β 2M, and CIITA genes using CRISPR-Cas9 technology to generate GGTA1-/-β2M-/-CIITA-/- triple knockout (GBC-3KO) pigs. Mixed lymphocyte reaction (MLR) and BrdU incorporating assays were utilized to quantify the potency of pig cells to stimulate human T cell responses. Skin grafts were determined the immunogenicity of pig tissues in xenogeneic hosts in vivo. Findings: The genotype of GBC-3KO pigs was confirmed by PCR and Sanger sequencing, and the loss of expression of α-Gal, SLA-I, and SLA-II was demonstrated by flow cytometric analysis. Mixed lymphocyte reaction (MLR) assay revealed that splenocytes from GBC-3KO pigs were significantly less effective than WT pig splenocytes in inducing human CD3+CD4+ and CD3+CD8+ T cell activation (measured by CD25 expression) and proliferation. Furthermore, GBC-3KO pig skin grafts showed a significantly prolonged survival (with a median survival time (MST) of 16 days) in immunocompetent C57BL/6 mice, when compared to WT pig skin grafts (MST = 13.5 days). Interpretation: These results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses. Funding Statement: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030000 and XDA16030303), the National Key Research and Development Program (2017YFA0104400, 2016YFA0100202 and 2017YFA0104402), the National Natural Science Foundation of China (31621004, 81570145, 81870091), and the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences (QYZDB-SSW-SMC022). Declaration of Interests: The authors stated: None. Ethics Approval Statement: Bama miniature pigs were raised and cloned at the Beijing Farm Animal Research Center. Human blood was obtained from healthy volunteers. Protocols related to the use of animals and human tissues were approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the First Hospital of Jilin University and the Institute of Zoology of Chinese Academy of Sciences. All the experiments were performed in accordance with the protocols.