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Exp. Mol. Med., Mar 14 2025, | https://doi.org/10.1038/s12276-025-01422-3
Generation of hypoimmunogenic universal iPS cells through HLA-type gene knockout
Juryun Kim 1,6, Yoojun Nam 1,2,6, Doyeong Jeon 1, Yujin Choi 1, SeonJu Choi 1, Chang Pyo Hong 1, Siyoung Kim 1, Hyerin Jung 1, Narae Park 1, Yeowon Sohn 2, Yeri Alice Rim 3,* and Ji Hyeon Ju 1,3,4,5,*
1YiPSCELL Inc., Seoul, Republic of Korea.
2Department of Biohealth Regulatory Science, Sungkyunkwan University, Suwon, Republic of Korea.
3CiSTEM Laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
4Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
5Department of Biomedicine and Health Sciences, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
6These author contributed equally: Juryun Kim, Yoojun Nam.
2Department of Biohealth Regulatory Science, Sungkyunkwan University, Suwon, Republic of Korea.
3CiSTEM Laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
4Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
5Department of Biomedicine and Health Sciences, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
6These author contributed equally: Juryun Kim, Yoojun Nam.
*Corresponding authors: correspondence to Yeri Alice Rim or Ji Hyeon Ju
Abstract
Hypoimmunogenic universal induced pluripotent stemn (iPS) cells were generated through the targeted disruption of key genes, including human leukocyte antigen (HLA)-A, HLA-B and HLA-DR alpha (DRA), using the CRISPR-Cas9 system. This approach aimed to minimize immune recognition and enhance the potential of iPS cells for allogeneic therapy. Heterozygous iPS cells were used for guide RNA design and validation to facilitate the knockout (KO) of the HLA-A, HLA-B and HLA-DRA genes. The electroporation of iPS cells using the selected guide RNAs enabled the generation of triple-KO iPS cells, followed by single-cell cloning for clone selection. Clone A7, an iPS cell with targeted KOs of the HLA-A, HLA-B and HLA-DRA genes, was identified as the final candidate. Messenger RNA analysis revealed robust expression of pluripotency markers, such as octamer-binding transcription factor 4, sex-determining region Y box 2, Krüppel-like factor 4, Lin-28 homolog A and Nanog homeobox, while protein expression assays confirmed the presence of octamer-binding transcription factor 4, stage-specific embryonic antigen 4, Nanog homeobox and tumor rejection antigen 1-60. A karyotype examination revealed no anomalies, and three-germ layer differentiation assays confirmed the differentiation potential. After interferon gamma stimulation, the gene-corrected clone A7 lacked HLA-A, HLA-B and HLA-DR protein expression. Immunogenicity testing further confirmed the hypoimmunogenicity of clone A7, which was evidenced by the absence of proliferation in central memory T cells and effector memory T cells. In conclusion, clone A7, a triple-KO iPS cell clone that demonstrates immune evasion properties, retained its intrinsic iPS cell characteristics and exhibited no immunogenicity.
논문정보
- Research article
- 2025년 03월 (BRIC 등록일 2025-03-18)
- 국내 연구진
- 생명과학 > 세포생물학
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