Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy

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 gene therapy and transplantation model that the CD34⁺CD90⁺ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, 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 good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90⁺ cell selection results in about 30-fold fewer target cells in comparison to CD133⁺ or CD38^low/- CD34⁺ hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential <i>in vivo</i>. 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 gene-engineered cell products.