Generation of Hypoimmunogenic Hematopoietic Stem Cells and Their Potential Application in HSCT

The mismatch of human leukocyte antigens (HLA) significantly limits the availability of suitable donors, primarily due to the risk of immune rejection in allogeneic hematopoietic stem cell transplantation (allo-HSCT). To improve donor-host matching, we performed precise depletion of HLA gene on human hematopoietic stem cells (HSCs) via CRISPR-Cas9 system. Combining with umbilical cord blood, one of the best optional sources for HSCs, we attempt to create off-the-shelf HSCs that are more accessible and available.

To identify the highly efficient guide RNAs (sgRNAs) capable of addressing the genetic diversity of the Chinese population，we firstly designed a library of sgRNAs targeting HLA-A and HLA-B genes. Finally,screened sgRNAs were found up to 60% HLA-A/B protein knock-down efficiency in HEK293T cell line.

To determine the knock-down efficiency of screened sgRNAs in human HSCs, CD34⁺ HSPCs from umbilical cord blood were obtained and then transduced with RNP by electroporation. The expression of HLA-A/B was detected by flow cytometry after 3 days' ex vivo culture. The knock-down efficiency of HLA-A/B proteins in CD34⁺ hematopoietic stem and progenitor cells (HSPCs) achieved up to 90%.

To investigate whether the functions of HSPCs with HLA-A/B knocked out(KO-HSPCs) were impaired in vitro, Colony forming cell (CFC) and multi-lineage differentiation assay were performed and the results. confirmed that the abilities of colony forming, self-renewal and lineage differentiation were well retained. To assess their engraftment and reconstruction potential in vivo, we transplanted both KO-HSPCs and non-edited HSPCs into NOG mice via tail vein injection. Transplantation studies in NOG mice showed higher engraftment with KO-HSPCs compared with non-edited ones in peripheral blood, BM and spleen, respectively. To examine the immunogenicity of KO-HSPCs, we performed CFSE-labeled T cell proliferation assay and CD107a assay. These results indicated that KO-HSPCs did not activateNK cells but effectively dampened the response of CD8⁺ T cells. Finally, the off-target detection assay was performed to investigate the safety by deep sequencing. All potential off-target sites were considered safety.

Our data demonstrate the feasibility of generating hypoimmunogenic HSCs by precisely editing HLA alleles in cord blood stem cells, thereby increasing accessibility and availability for patients in need of HSCT.

No relevant conflicts of interest to declare.