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

Area of Science:

• Stem Cell Biology
• Immunology
• Gene Editing

Background:

• Allogeneic cell therapies are limited by immune rejection.
• Human leukocyte antigen (HLA) expression on donor cells triggers immune responses.
• Induced pluripotent stem (iPS) cells offer therapeutic potential but require immune compatibility.

Purpose of the Study:

• To generate hypoimmunogenic universal iPS cells for allogeneic transplantation.
• To disrupt key HLA genes (HLA-A, HLA-B, HLA-DR alpha) in iPS cells.
• To characterize the resulting iPS cell clone for pluripotency, differentiation, and immunogenicity.

Main Methods:

• CRISPR-Cas9 gene editing was employed to target HLA-A, HLA-B, and HLA-DRA genes.
• Guide RNA validation and electroporation were used for gene knockout (KO).
• Single-cell cloning, mRNA/protein expression analysis, karyotyping, differentiation assays, and immunogenicity testing were performed.

Main Results:

• A triple-KO iPS cell clone, designated A7, was successfully generated.
• Clone A7 maintained pluripotency marker expression and differentiation potential into three germ layers.
• Clone A7 exhibited no HLA protein expression post-interferon gamma stimulation and showed hypoimmunogenicity, evidenced by absent T cell proliferation.

Conclusions:

• Targeted disruption of HLA genes using CRISPR-Cas9 can create hypoimmunogenic iPS cells.
• Clone A7 demonstrates immune evasion properties while retaining essential iPS cell characteristics.
• This hypoimmunogenic iPS cell line holds promise for future allogeneic cell-based therapies.