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 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 gene-engineered cell products.

Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34+-enriched cells

“…This disparity regarding different CFU populations between expansion systems with and without a MSC FL has been previously observed by our group [12]. Interestingly, a small population that contains more primitive cells (CD34 + CD90 + cells) [38] was also kept in both BM-FBS and AT-FBS conditions, which is aligned with the tendency for higher CAFCproducing capacity observed for these conditions. The CAFC assay is a variant of the long-term cultureinitiating cells (LTC-IC) assay that does not require cell replating.…”

Etched 3D-Printed Polycaprolactone Constructs Functionalized with Reduced Graphene Oxide for Enhanced Attachment of Dental Pulp-Derived Stem Cells

“…As the cells expressing the DsRed morphologically appeared healthy, the increasing percentage of non-fluorescent cells may have simply been indicative of a more rapid proliferative capacity of cells that were not virally transduced. Alternatively, the observed subset population of low-expressing CD90 cells, indicated in Table 4, for DMEM/F12-cultured cells, may account for non-transduced cells, as CD90+ populations have been described to promote enhanced transduction potential [50,51]. Furthermore, the multiplicity of infection used for the transduction was determined based on previous transductions of rat MSCs derived from adipose and bonemarrow tissue and was anticipated to have an efficiency above 80%.…”

Safe and EffectiveIn VivoTargeting and Gene Editing in Hematopoietic Stem Cells: Strategies for Accelerating Development

“…35 These findings are consistent with single-cell RNA analyses demonstrating that CD90+ cells are the most highly enriched CD34+ cell subpopulation that can be targeted for gene therapy or gene editing and represents *5% of the overall CD34+ cell population. 36 Current gene editing approaches for SCD focus on reactivation of HbF, either by inactivating the HbF repressor Bcl11a or by recreating mutations associated with hereditary HbF persistence (HPFH). Dr. Kiem's group focused on the 13-nt HPFH deletion in the promoter region of the c-globin gene that also contains the recently identified Bcl11a binding sites.…”