Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation
Chang Yang
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorYunfang Wei
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorXinglong Li
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorKaixiang Xu
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorXiaoying Huo
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorGang Chen
Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Search for more papers by this authorHeng Zhao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorJiaoxiang Wang
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorTaiyun Wei
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorYubo Qing
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorJianxiong Guo
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorHongfang Zhao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorXiong Zhang
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorDeling Jiao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorZhe Xiong
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorMuhammad Ameen Jamal
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorCorresponding Author
Hong-Ye Zhao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Correspondence: Hong-Ye Zhao ([email protected]) | Hong-Jiang Wei ([email protected])
Search for more papers by this authorCorresponding Author
Hong-Jiang Wei
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Correspondence: Hong-Ye Zhao ([email protected]) | Hong-Jiang Wei ([email protected])
Search for more papers by this authorChang Yang
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorYunfang Wei
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorXinglong Li
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorKaixiang Xu
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorXiaoying Huo
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorGang Chen
Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Search for more papers by this authorHeng Zhao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorJiaoxiang Wang
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorTaiyun Wei
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorYubo Qing
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorJianxiong Guo
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorHongfang Zhao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorXiong Zhang
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorDeling Jiao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorZhe Xiong
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorMuhammad Ameen Jamal
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
Search for more papers by this authorCorresponding Author
Hong-Ye Zhao
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Correspondence: Hong-Ye Zhao ([email protected]) | Hong-Jiang Wei ([email protected])
Search for more papers by this authorCorresponding Author
Hong-Jiang Wei
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China
College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China
Correspondence: Hong-Ye Zhao ([email protected]) | Hong-Jiang Wei ([email protected])
Search for more papers by this authorChang Yang, Yunfang Wei, and Xinglong Li are first co-authors.
Funding: This work was supported by the National Key R&D Program of China (Grant No. 2019YFA0110700) and the Major Science and Technology Project of Yunnan Province (Grant No. 202102AA310047).
ABSTRACT
Background
The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation.
Methods
In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs.
Results
First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation.
Conclusion
These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.
Conflicts of Interest
The authors declare no conflicts of interest.
Open Research
Data Availability Statement
The dataset supporting the conclusions of this article is included within the article and its additional file.
Supporting Information
| Filename | Description |
|---|---|
| xen12881-sup-0001-FigureS1.tif998.5 KB | Figure S1 Immunosuppressive regimen |
| xen12881-sup-0002-FigureS2.tif1.2 MB | Figure S2 Erythrocyte indexes and electrolytes in recipient monkey. A. The numbers of red blood cells. B-C. Hematocrit (B) and hemoglobin concentration (C). D-I. The concentration of sodium (Na) (D), chlorine (Cl) (E), calcium (Ca) (F), magnesium (Mg) (G), phosphorus (P) (H) and potassium (K) (I) in the serum of recipient monkey. |
| xen12881-sup-0003-FigureS3.tif1.1 MB | Figure S3 The liver function indexes of recipient monkey. A-I. The levels of total protein (TP) (A), ailbumin (ALB) (B), total bilirubin (TBIL) (C), direct bilirubin (DBIL) (D), alkaline phosphatase (ALP) (E), total bile acid (TBA) (F), aspartate transaminase (AST) (G), adenosine deaminase (ADA) (H) and alanine transaminase (ALT) (I). |
| xen12881-sup-0004-FigureS4.tif4.2 MB | Figure S4 Silence of hCD55 expression might be due to methylation. A. The protein expression of αGal, hCD55, hTBM and hCD39 gene in GEC porcine kidney tissue was confirmed by immunofluorescence, in which no hCD55 was detected (scale bar = 50 µm). B. Correspondingly, high methylation level in CpG island of hCD55 coding DNA. Each row repsents a repeat. |
| xen12881-sup-0005-TablesS1-S2.docx116.5 KB | Supporting Information |
| xen12881-sup-0006-SuppMat.doc56.5 KB | Supporting Information |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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