Development of RAG2 -/- IL2Rγ -/Y immune deficient FAH-knockout miniature pig

Heng Zhao; Weijian Ye; Jianxiong Guo; Jiaoxiang Wang; Deling Jiao; Kaixiang Xu; Chang Yang; Shuhan Chen; Muhammad Ameen Jamal; Zhongbin Bai; Taiyun Wei; Jie Cai; Tien Dat Nguyen; Yubo Qing; Wenmin Cheng; Baoyu Jia; Honghui Li; Hong-Ye Zhao; Qingfeng Chen; Hong-Jiang Wei

doi:10.3389/fimmu.2022.950194

Development of RAG2 ^-/- IL2Rγ ^-/Y immune deficient FAH-knockout miniature pig

Front Immunol. 2022 Aug 9:13:950194. doi: 10.3389/fimmu.2022.950194. eCollection 2022.

Authors

Heng Zhao^{1

2

3}, Weijian Ye^{4

5}, Jianxiong Guo^{1

2}, Jiaoxiang Wang^{1

2

3}, Deling Jiao^{1

2

6}, Kaixiang Xu^{1

2

6}, Chang Yang^{1

2}, Shuhan Chen^{1

2

3}, Muhammad Ameen Jamal⁷, Zhongbin Bai³, Taiyun Wei^{1

2}, Jie Cai^{1

2}, Tien Dat Nguyen^{1

2

6}, Yubo Qing^{1

2

3}, Wenmin Cheng^{1

2

6}, Baoyu Jia^{1

2

3}, Honghui Li^{1

2

6}, Hong-Ye Zhao^{1

2}, Qingfeng Chen^{4

5}, Hong-Jiang Wei^{1

2

3

7}

Affiliations

¹ Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China.
² Yunnan Province Xenotransplantation Research Engineering Centre, Yunnan Agricultural University, Kunming, China.
³ College of Veterinary Medicine, Yunnan Agricultural University, Kunming, China.
⁴ Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (ASTAR), Singapore, Singapore.
⁵ Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
⁶ Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, China.
⁷ Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China.

Abstract

Human hepatocyte transplantation for liver disease treatment have been hampered by the lack of quality human hepatocytes. Pigs with their large body size, longevity and physiological similarities with human are appropriate animal models for the in vivo expansion of human hepatocytes. Here we report on the generation of RAG2^-/-IL2Rγ^-/YFAH^-/- (RGFKO) pigs via CRISPR/Cas9 system and somatic cell nuclear transfer. We showed that thymic and splenic development in RGFKO pigs was impaired. V(D)J recombination processes were also inactivated. Consequently, RGFKO pigs had significantly reduced numbers of porcine T, B and NK cells. Moreover, due to the loss of FAH, porcine hepatocytes continuously undergo apoptosis and consequently suffer hepatic damage. Thus, RGFKO pigs are both immune deficient and constantly suffer liver injury in the absence of NTBC supplementation. These results suggest that RGFKO pigs have the potential to be engrafted with human hepatocytes without immune rejection, thereby allowing for large scale expansion of human hepatocytes.

Keywords: FAH; IL2Rγ; RAG2; immunodeficient; liver damage; pig.

MeSH terms

Animals
Animals, Genetically Modified
DNA-Binding Proteins / genetics
Disease Models, Animal*
Gene Knockout Techniques
Hepatocytes
Humans
Interleukin Receptor Common gamma Subunit / genetics
Liver Diseases*
Nuclear Proteins / genetics
Swine
Swine, Miniature

Substances

DNA-Binding Proteins
Interleukin Receptor Common gamma Subunit
Nuclear Proteins