Abstract
Hypoimmunogenic universal induced pluripotent stem cells (iPSCs) 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 iPSCs for allogeneic therapy. Heterozygous iPSCs were used for guide RNA (gRNA) design and validation to facilitate the knockout (KO) of HLA-A, HLA-B, and HLA-DRA genes. Electroporation of iPSCs using the selected gRNAs enabled the generation of triple-KO iPSCs, followed by single-cell cloning for clone selection. Clone A7, an iPSC with a targeted KO of HLA-A, HLA-B, and HLA-DRA genes, was identified as the final candidate. mRNA analysis revealed robust expression of pluripotency markers, such as octamer-binding transcription factor 4 (OCT4), SRY (sex-determining region Y)-box 2 (SOX2), Krüppel-like factor 4 (KLF4), Lin-28 homolog A (LIN28), and Nanog homeobox (NANOG), while protein expression assays confirmed the presence of OCT4, stage-specific embryonic antigen 4 (SSEA4), NANOG, and tumor rejection antigen 1–60 (TRA-1-60). Karyotype examination demonstrated no anomalies, and three germ layer differentiation assays confirmed differentiation potential. Following interferon-gamma (INF-γ) stimulation, the gene-corrected clone A7 exhibited the absence of 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 (TCM) and effector memory T cells (TEM). In conclusion, Clone A7, a triple KO iPSC clone that demonstrates immune evasion properties, retained its intrinsic iPSC characteristics and exhibited no immunogenicity.