Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant and infectious diseases. We have shown in a nonhuman primate HSC gene therapy and transplantation model that the CD34 + CD90 + CD45RAcell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. Here we establish the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy.Current alternative HSC-enrichment strategies purify CD133 + cells or CD38 low/subsets of CD34 + cells from human blood products. We directly compared these strategies to 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 + CD34 + 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 in manufacturing and clinical outcome, ultimately improving the safety and feasibility of patient-specific HSC gene therapy.
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