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

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 HSC gene therapy and transplantation model that the CD34+CD90+CD45RA− cell 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 CD38low/− 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 CD38low/−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.