Generation of hypoimmunogenic universal iPS cells through HLA-type gene knockout

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. This study focuses on improving cell therapy by reducing the immune response to induced pluripotent stem (iPS) cells. Researchers aimed to generate iPS cells that are less likely to be rejected by the immune system by editing specific genes. The team used a tool called CRISPR–Cas9 to remove certain genes, known as HLA-A, HLA-B and HLA-DRA, from iPS cells. By removing these genes, researchers hoped to make the iPS cells less recognizable to the immune system. The study found that the edited iPS cells did not trigger a strong immune response in lab tests. The edited cells maintained their ability to differentiate into various cell types and showed reduced signs of being attacked by immune cells. This research suggests that editing specific genes in iPS cells can make them more suitable for use in therapies without being rejected by the body. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.