Case Study in AI-Designed Gene Editing

Outline: Modulation of Immune Response by Editing HLA Class I and Class II

One limitation of using iPSCs clinically is immunogenicity caused by Human Leucocyte Antigen (HLA) mismatching, which can reduce the in vivo survival and therapeutic efficacy of these cells.^1,2 The immunogenicity of iPSCs can be reduced by disrupting the genes responsible for immune recognition using StemEdit's advanced gene editing platform. One approach is to knock-out the B2M (β2 microglobulin) and CIITA (major histocompatibility complex [MHC] II transactivator) genes, creating the basis for the generation of hypoimmune cell lines.  

In humans, the B2M protein is required for the presentation of HLA protein A-G on the cell surface, while CIITA is essential for HLA-II transcription.^2,3 Dual knock-out of the B2M and CIITA genes disrupts the presentation of MHC-I/MHC-II proteins in iPSCs, improving their therapeutic potential while maintaining pluripotency.¹

Role of B2M and CIITA in the regulation of the immune response. B2M regulates the activation of effector T cells. B2M knockouts do not activate effector T cells, leading to a reduced immune response. CIITA regulates the expression of the HLA-II complex. Knockout of CIITA leads to a reduced expression of HLA-II, and a reduced immune response.

References

1. Wang X et al. Diminished expression of major histocompatibility complex facilitates the use of human induced pluripotent stem cells in monkey. Stem Cell Research & Therapy 11:334 (2020).
2. Xu H et al. Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell Stem Cell 24 pp566-578 (2019).
3. Deuse et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nature Biotechnology 37 pp 252–258 (2019).

Case Study: Hypoimmune iPSC Generation

HLA Class I and II expression in B2M/CIITA double knock-out iPSCs. HLA expression was measured by flow cytometry in the unedited parental iPSC line and after knock-out of the B2M and CIITA genes. Expression of HLA I and II is suppressed in B2M/CIITA knockout lines. Results indicate that the generation of iPSC for the purpose of suppressing an immune rejection is successful with StemEdit.

(A.) HLA Class I expression was measured in the parental iPSC-derived macrophages by flow cytometry.

(B.) Undifferentiated iPSCs (“Parental iPSCs”) do not express HLA II. Expression of HLA Class II occurs only in cells post differentiation. Expression of the HLA II gene HLA-DR was therefore measured in iPSC-derived macrophages.