ABSTRACT Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant and infectious diseases. We have shown in a nonhuman primate (NHP) HSC gene therapy and transplantation model that the CD34 + CD90 + cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. Here we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC-enrichment strategies include the purification of CD133 + cells or CD38 low/- subsets of CD34 + cells from human blood products. We directly compared these strategies to the isolation of CD90 + cells using a GMP-grade flow-sorting protocol with clinical applicability. We show that CD90 + cell selection results in 40-fold fewer target cells in comparison to CD133 + or CD38 low/- CD34 subsets without compromising the engraftment potential in vivo . Single cell RNA sequencing confirmed nearly complete depletion of lineage committed progenitor cells in CD90 + fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90 + cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and genome editing products.

Sort-purification of human CD34+CD90+cells reduces target cell population and improves lentiviral transduction for gene therapy