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.