Human CD90+CD34+ cells are phenotypically, transcriptionally and functionally the most refined target for hematopoietic stem cell gene therapy

Hematopoietic stem cell (HSC) gene therapy and editing has the potential to treat and even cure many genetic diseases and cancer. The ability to target gene modification to a more refined HSC-enriched population would improve efficiency and feasibility while potentially decreasing off target effects. We recently described the CD34+CD90+CD45RA- cell fraction as such an enriched HSC population in nonhuman primates (NHPs). We showed that CD34+CD90+CD45RA– cells were exclusively responsible for rapid and robust multilineage engraftment in NHPs. Here we studied human HSC-enriched cell populations combining cell surface markers, high-throughput single cell RNA sequencing (scRNAseq), and functional mouse engraftment studies. We found that only human CD34+CD90+CD45RA– cells were capable of multilineage engraftment and at the same time complete repopulation of the bone marrow stem cell compartment. Combining phenotypical and scRNAseq analysis, we further found that CD34+CD90+CD45RA– cells were nearly entirely depleted of lineage-committed progenitor cells and markedly enriched for transcriptionally most primitive progenitor cells. Most important for clinical applications, CD34+CD90+CD45RA– cells demonstrated the most efficient reduction of target cells compared to CD34+CD38low/– or CD34+CD133+ cells. These studies support the use of CD34+CD90+CD45RA– cells as target cell population for HSC gene therapy and editing approaches in humans.