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.

造血干细胞 (HSC) 基因疗法具有治愈许多遗传、恶性和传染性疾病的潜力。我们已经在非人类灵长类动物基因治疗和移植模型中表明，CD34 ⁺ CD90 ⁺细胞部分专门负责多系移植和造血重建。在这项研究中，我们展示了这种富含 HSC 的 CD34 子集在慢病毒介导的基因治疗中的转化潜力。替代的 HSC 富集策略包括从人类血液制品中纯化 CD133 ⁺细胞或 CD38^低/- CD34 ⁺细胞亚群。我们直接将这些策略与 CD90 ⁺的分离进行了比较使用具有临床适用性的良好生产规范 (GMP) 级流式分选方案的细胞。我们表明，与 CD133 ⁺或 CD38^低/– CD34 ⁺造血干细胞和祖细胞 (HSPC) 亚群相比，CD90 ⁺细胞选择导致靶细胞减少约 30 倍，而不会影响体内植入潜力。单细胞 RNA 测序证实，与替代选择相比，CD90 ⁺级分中的谱系定向祖细胞几乎完全耗尽。重要的是，纯化 CD90 ^{+ 中的}慢病毒转导效率细胞导致移植的基因修饰血细胞水平高出 3 倍。这些研究应该对制造患者特异性 HSC 基因疗法和基因工程细胞产品具有重要意义。