Engineered hypoimmunogenic iPSC-derived mesenchymal stromal cells secreting high levels of functional FVIII ameliorate the hemophilia A phenotype

Background: Cell-based gene therapy represents a promising therapeutic avenue for genetic diseases such as hemophilia A (HA). However, clinical translation is limited by low transgene expression and host immune responses. Methods: Using CRISPR/Cas9, we targeted a codon-optimized B domain-deleted F8 gene along with CD47 into the B2M locus in human induced pluripotent stem cells (iPSCs) and differentiated these cells into mesenchymal stromal cells (iMSCs). The immunogenicity of the engineered iMSCs was tested and the therapeutic efficacy was investigated in immunodeficient mice and in an HA mouse model. Findings: The engineered iMSCs secreted functional FVIII at high concentrations (1271 ng/106 cells/48 h) and exhibited hypoimmunogenic properties both in vitro and in vivo. In immunodeficient mice, both intravenous and intramuscular administration were shown to be effective. Transplantation of engineered iMSCs into HA mice significantly increased plasma FVIII activity and ameliorated the bleeding phenotype. Interpretation: We describe a universally applicable hypoimmunogenic cell-based gene therapy strategy for HA and other genetic diseases by targeting a codon-optimized B domain-deleted F8 gene along with CD47 into the B2M locus in human iPSCs.