Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.
Department of Pediatrics, Harvard Medical School, Boston, MA.
Blood Adv. 2024 Nov 26;8(22):5885-5895. doi: 10.1182/bloodadvances.2024013932.
Hematopoietic stem cell (HSC) transplantation with lentiviral vector (LVV)-transduced autologous cells has proven an effective therapeutic strategy for sickle cell disease (SCD). However, ex vivo culture or proliferative stress associated with in vivo reconstitution may amplify any underlying genetic risk of leukemia. We aimed to minimize culture-induced stress and reduce genomic damage during ex vivo culture and enhance stem cell fitness and reconstitution of SCD CD34+ cells transduced with BCL11A shmiR-encoding LVV. UM171, a pyrimidoindole derivative, can expand normal HSCs during in vitro culture and has been shown to be safe and effective using umbilical cord blood. We examined the effect of UM171 during ex vivo LVV transduction of SCD HSCs. Culture of SCD CD34+ HSCs with UM171 during transduction reduced DNA damage and reactive oxygen species, decreased apoptosis, and was associated with increased numbers of immunophenotypically defined long-term HSCs. UM171 increased the engraftment of LVV-transduced human HSCs in immunodeficient mice and barcode tracing revealed increased clonal diversity of engrafting cells. In competitive transplantation assays, analysis of bone marrow showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions. In summary, exposure of SCD peripheral blood CD34+ cells to UM171 during LVV transduction enhances stem cell fitness. These findings suggest manufacturing of genetically modified HSCs in the presence of UM171 may improve efficacy, safety, and sustainability of gene therapy using ex vivo approaches. BCL11A shmiR-encoding LVV is in clinical trials to treat SCD (NCT03282656), UM171 is in clinical trials to culture umbilical cord blood (NCT02668315).
利用慢病毒载体 (LVV) 转导的自体细胞进行造血干细胞 (HSC) 移植已被证明是治疗镰状细胞病 (SCD) 的有效治疗策略。然而，与体内重建相关的体外培养或增殖应激可能会放大白血病的任何潜在遗传风险。我们旨在最大限度地减少体外培养过程中的应激反应和减少基因组损伤，并增强 SCD CD34+细胞的干细胞适应性和 LVV 转导。UM171 是一种嘧啶并吲哚衍生物，可在体外培养过程中扩增正常 HSCs，并且已被证明使用脐带血是安全有效的。我们研究了 UM171 在 SCD HSCs 的 LVV 转导体外过程中的作用。在转导过程中用 UM171 培养 SCD CD34+HSCs 可减少 DNA 损伤和活性氧，减少细胞凋亡，并与增加免疫表型定义的长期 HSCs 数量相关。UM171 增加了转导的 LVV 人类 HSCs 在免疫缺陷小鼠中的植入，并且条形码追踪显示植入细胞的克隆多样性增加。在竞争性移植实验中，对骨髓的分析表明，在 UM171 存在下转导的细胞始终比在对照条件下转导的细胞具有更强的竞争力。总之，在 LVV 转导过程中使 SCD 外周血 CD34+细胞暴露于 UM171 可增强干细胞适应性。这些发现表明，在 UM171 存在下制造基因修饰的 HSCs 可能会提高体外方法基因治疗的疗效、安全性和可持续性。BCL11A shmiR 编码的 LVV 正在 SCD 临床试验中（NCT03282656），UM171 正在用于培养脐带血的临床试验中（NCT02668315）。
