Generation and characterization of genetically modified pigs with GGTA1/β4GalNT2/CMAH knockout and human CD55/CD47 expression for xenotransfusion studies

Bin Fang; Chunting Wang; Yilin Yuan; Xiaorui Liu; Lili Shi; Lin Li; Ying Wang; Yifan Dai; Haiyuan Yang

doi:10.1038/s41598-024-81730-2

Generation and characterization of genetically modified pigs with GGTA1/β4GalNT2/CMAH knockout and human CD55/CD47 expression for xenotransfusion studies

Sci Rep. 2024 Dec 2;14(1):29870. doi: 10.1038/s41598-024-81730-2.

Authors

Bin Fang^{1

2

3}, Chunting Wang^{1

2}, Yilin Yuan³, Xiaorui Liu¹, Lili Shi⁴, Lin Li^{1

2}, Ying Wang^{1

2}, Yifan Dai^{5

6

7

8

9}, Haiyuan Yang^{10

11}

Affiliations

¹ Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, 211166, China.
² Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, 211166, China.
³ Gcreatene (Suzhou) Biotechnology Co., Ltd., Suzhou, 215000, China.
⁴ Jiangsu Province Blood Center, Nanjing, 210042, China.
⁵ Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, 211166, China. [email protected].
⁶ Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, 211166, China. [email protected].
⁷ Gcreatene (Suzhou) Biotechnology Co., Ltd., Suzhou, 215000, China. [email protected].
⁸ Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, 211166, China. [email protected].
⁹ State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 211166, China. [email protected].
¹⁰ Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, 211166, China. [email protected].
¹¹ Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, 211166, China. [email protected].

Abstract

Pig red blood cells (pRBCs) represent a promising alternative to address the shortage in transfusion medicine. Nonetheless, a major obstacle to their clinical implementation is immunological rejection. In this study, we generated transgenic pigs expressing human CD47 (hCD47) and CD55 (hCD55) in α1,3-galactosyltransferase KO/β-1,4-N-acetyl-galactosaminyl transferase 2 KO/cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO (TKO) pigs using CRISPR/Cas9 technology. Compared to wild-type pRBCs, TKO/hCD47/hCD55 pRBCs exhibit significantly reduced human IgG/IgM antibody binding. Moreover, when transfused into Cynomolgus monkeys, TKO/hCD47/hCD55 pRBCs remained detectable for 2 h post-transfusion, whereas wild-type pRBCs were eliminated within 20 min. This study demonstrates the potential of multi-gene edited pigs to provide immunologically compatible pRBCs.

Keywords: Gene editing; Transgenic pigs; Xenotransfusion.

MeSH terms

Animals
Animals, Genetically Modified*
CD47 Antigen* / genetics
CD47 Antigen* / metabolism
CD55 Antigens* / genetics
CD55 Antigens* / metabolism
CRISPR-Cas Systems
Erythrocytes / metabolism
Galactosyltransferases* / genetics
Galactosyltransferases* / metabolism
Gene Knockout Techniques* / methods
Humans
Immunoglobulin G
Macaca fascicularis / genetics
Mixed Function Oxygenases* / genetics
Mixed Function Oxygenases* / metabolism
N-Acetylgalactosaminyltransferases* / genetics
N-Acetylgalactosaminyltransferases* / metabolism
Swine
Transplantation, Heterologous*

Substances

CMPacetylneuraminate monooxygenase
Galactosyltransferases
CD55 Antigens
CD47 Antigen
alpha-1,3-galactosyltransferase 1, porcine
N-Acetylgalactosaminyltransferases
Mixed Function Oxygenases
beta-1,4-N-acetyl-galactosaminyl transferase 2
CD47 protein, human
Immunoglobulin G

Grants and funding

81970164/National Natural Science Foundation of China